Autism spectrum disorder (ASD) is a developmental condition that affects approximately four times as many males as females, a strong sex bias that has not yet been fully explained. Understanding the causes of this biased prevalence may highlight novel avenues for treatment development that could benefit patients with diverse genetic backgrounds, and the expertise of sex differences researchers will be invaluable in this endeavor. In this review, I aim to assess current evidence pertaining to the sex difference in ASD prevalence and to identify outstanding questions and remaining gaps in our understanding of how males come to be more frequently affected and/or diagnosed with ASD. Though males consistently outnumber females in ASD prevalence studies, prevalence estimates generated using different approaches report male/female ratios of variable magnitude that suggest that ascertainment or diagnostic biases may contribute to the male skew in ASD. Here, I present the different methods applied and implications of their findings. Additionally, even as prevalence estimations challenge the degree of male bias in ASD, support is growing for the long-proposed female protective effect model of ASD risk, and I review the relevant results from recurrence rate, quantitative trait, and genetic analyses. Lastly, I describe work investigating several sex-differential biological factors and pathways that may be responsible for females’ protection and/or males’ increased risk predicted by the female protective effect model, including sex steroid hormone exposure and regulation and sex-differential activity of certain neural cell types. However, much future work from both the ASD and sex differences research communities will be required to flesh out our understanding of how these factors act to influence the developing brain and modulate ASD risk. Autism is a lifelong neuropsychiatric condition first apparent during early development that is characterized by social and communication deficits and by repetitive behaviors and restricted interests. The severity and variety of symptomatic behaviors, impairments, and abilities that autistic individuals show is vast, leading to the formal conceptualization of autism as a spectrum  . ASDs alIn recent years, interest in investigating sex differences in the autistic phenotype and exploring a potential need for sex-differential diagnostic criteria has grown more widespread , 5. At tThe most striking sex difference in ASD is its prevalence, as approximately four times as many males have a diagnosis of ASD as females . This 4:At face value, this pattern of disparate prevalence suggests the action of sex-differential risk factors for ASD that act to either increase males’ risk and/or protect females. Just a few decades ago, as our conceptualization of ASD shifted from the domain of psychoanalysis to neuropsychiatry and genetics, an assumption that sex-differential risk factors were also biological in nature followed suit , 23. Morare affected by ASD at higher rates than previously thought, but that they are not being diagnosed. If this scenario were true, it would require a careful reexamination of the ASD phenotype, our understanding of which is based on the study of majority male cohorts, as well as our assumptions about sex-differential risk and protection for ASD.Still, despite more widespread focus on characterizing the autistic phenotype and its cause(s), the hypothesis that some aspect of male and/or female biology modulates ASD risk remained. Several research groups proposed the involvement of general sexually dimorphic factors such as X-linkage , 39, impSince males have predominated in studies of the features and phenotype of ASD, it can be argued that diagnostic criteria and instruments for ASD preferentially describe what ASD looks like in a male. The manifestation of ASD in females, then, may not appear to meet diagnostic criteria, which would lead to a smaller number of females being diagnosed and an apparent male bias in prevalence. To better understand the female’s autistic phenotype, studying diagnosed females is informative, but results must be interpreted in light of the caveat that these are the females who are identifiable under potentially male-biased diagnostic criteria. Additional work is required to unpack the possibility and consequences of missed diagnoses in females.Ideally, such work should explore both (1) phenotypic traits in non-diagnosed females and (2) long-term outcomes. In particular, evaluation of females without ASD diagnoses who meet at least a subset of current defining criteria and/or who have other neuropsychiatric or neurodevelopmental diagnoses may be informative for identifying potential gaps in diagnostic criteria where females are likely to fall short. Following such individuals longitudinally will also be required to determine if quality of life  and/or achievement outcomes  are negatively impacted in these females. Findings of poor outcomes would indicate that these individuals stand to benefit from diagnosis and the services and therapies available to autistic patients. This would also motivate revision of the diagnostic criteria for ASD in order to better identify these females.Detailed analyses of ASD prevalence have certainly hinted at the possibility of skewed diagnoses in males compared with females. For example, studies comparing autistic individuals across a range of intellectual ability have shown that the male bias is as high as 9:1 among cases with intelligence quotient (IQ) in the normal-to-high range (frequently termed “high functioning”) but as low as 1.6:1 among cases with intellectual disability –45. ThouAlternatively, it might be the case that females must present with comorbid intellectual disability or a clear syndrome in order to be evaluated for, or receive a diagnosis of, ASD. Given that our current understanding of ASD is based on a body of research from predominantly male cases, it has been suggested that either the diagnostic criteria for ASD, or clinicians’, educators’, and parents’ understanding of these criteria, do not accurately reflect how females present with ASD . In thisa priori assumptions about which individuals are most likely to be affected  of the Centers for Disease Control (CDC) in the USA periodically reports on ASD prevalence across multiple sites nationwide, and these reports show an increase in ASD prevalence over time , 57, 58.Similarly, as awareness and understanding of ASD in females grow, we may begin to see increased rates of ASD in females reported by these prevalence-monitoring surveys. In fact, results from the study of incidence rates in Denmark demonstrate this very pattern, with increasing diagnoses in females leading to a reduction in the male/female ratio from 5.1 in 1995 to 3.1 in 2010; this reduced male bias was most striking for diagnoses of Asperger’s syndrome (8.4 to 3.0) and PDD-NOS (5.7 to 2.8) . AdditioThe recognition of shortcomings in our understanding of ASD in girls has sparked recent interest in studying autistic females, to better characterize the presentation of their symptoms, their cognitive and neuroanatomical phenotype, and how they differ from boys with ASD . Thus faAutistic females in this sample also self-reported higher scores on a measure of ASD symptoms compared with males, suggesting disconnect between their observed and experienced social behavior. This sort of behavior is consistent with the concept of “camouflaging” one’s ASD symptoms by making conscious, concerted effort to learn and emulate social norms. The application of this rote knowledge of social behavior may effectively hide an innate lack of skill in certain interactions, but the frequently monumental effort required to do this often goes unnoticed. In fact, though autistic females may have similar trouble with social communication as autistic boys do early in life, females may have greater social motivation and desire to be liked and engaged with her peers , 63; thiIn addition to camouflaging behavior in high functioning females, studies of older children and adults with ASD are finding that autistic females tend to show reduced levels of restricted interests and repetitive behaviors compared with males. A large study of individuals from 970 families enrolled in the Autism Genome Project (AGP) observed lower repetitive behavior scores from the Autism Diagnostic Interview-Revised (ADI-R) in females . A breakAn alternative possibility is that females have just as many, or just as intense, restricted interests as males, but that these interests occur in different domains. In other words, autistic children’s interests may differ in much the same ways that the interests of male and female neurotypical children differ from one another, on average: while males with ASD might fixate on transit schedules or maps, females might fixate on horses or popular performers . If diagAll together, a tendency toward increased social motivation, ability to consciously mask social impairment, and reduced or potentially non-prototypical restricted interests may cause affected females to not be evaluated or diagnosed , 70. As Though ongoing work to better characterize and identify autistic females is required to quantify the true risk-modulatory impact of sex, current data, including general population screens , 8–10 andiagnosis of ASD). Awareness of this possibility is critical and much additional work is required to address it, but for the purposes of exploring current work on the FPE model as it is commonly interpreted, we will accept the assumption that females are protected from ASD itself. Given this, under the FPE model, the threshold of risk burden that females must carry  or experience , before their neurodevelopment is impacted to the degree that they present with a diagnosable autistic phenotype, is greater than for males.One theoretical model for the relationship between sex and ASD risk is derived from a multiple threshold liability model and is commonly referred to in the field as a “female protective effect”  associated with ASD risk such as 22q11.2 or 16p11.2 deletions. Carriers of the 16p11.2 deletion sometimes do, and sometimes do not, meet diagnostic criteria for ASD, but as a group, carriers show decreased IQ as compared with the general population and with non-carrier family members –81, demoAnalogous to the distribution of genetic risk across families and individuals, the behaviors and cognitive patterns that define ASD have also been shown to follow a quantitative distribution in general population samples from the USA , 84 and The FPE model also makes several key predictions about the properties and effects of ASD risk factors, each of which can be tested to support or refute the existence of female-differential protection. First and foremost, the FPE model predicts that among diagnosed individuals, females carry or experience greater risk than diagnosed males, on average Fig. . Given tSpecifically, multiple prospective high-risk sibling studies have failed to find a significant effect of proband sex on ASD recurrence rates in later-born siblings , 55. ThoIn contrast to these reports on high risk and population samples, a study of two population twin cohorts in which ASD traits were measured on a quantitative scale showed significantly higher autistic trait scores in the co-twins of affected females than in the co-twins of affected males . “Affectde novo variation) [de novo genetic risk. Since these children are less likely to share these penetrant genetic risk variants with their siblings, this reduces the power to observe significant increases in recurrence in diagnosed females’ siblings.At present, these disparate results have not been fully reconciled. One reasonable possibility is that differences in genetic architecture between the cohorts used in the studies are responsible for the different patterns observed. Specifically, the Carter Effect is dependent on a penetrant contribution from inherited genetic risk variants that are shared between siblings. Individuals from multiplex families are more likely to carry these inherited risk variants than are simplex, or single incidence families, who show enrichment for risk variants observed only in the child that are not inherited from either parent (riation) . Estimatriation) , 55, 56.de novo than inherited , this would reduce the observed recurrence in siblings of female probands. It may be that affected females who currently escape diagnosis are more likely to carry inherited, or common, genetic risk profiles, and identifying these females may improve power to observe significantly higher recurrence in siblings of female versus male probands. However, despite these potential caveats regarding statistical power, the fact that studies of millions of children from national registries do not observe a Carter Effect remains a conundrum.Bias against the identification of female cases by male-tailored diagnostic instruments may also contribute to this loss of power. If diagnosed females represent a subset of all affected females, and if the key genetic risk variants carried by diagnosed females are more frequently As discussed above, observing a Carter Effect in a sex-biased condition requires inference about the underlying genetic risk. Today, we can observe most genetic variants directly, including single nucleotide variants (SNVs) and large CNVs or structural variants. In the genetic risk space, then, we can directly test the same prediction of the FPE model that is associated with the Carter Effect: do autistic females carry greater risk than autistic males?de novo variants in simplex families do find direct evidence of greater risk burden in diagnosed females at the genetic level. Early work on CNVs in families from the SSC observed a trend toward higher frequency of de novo CNVs in autistic females compared with males [de novo CNVs impact a significantly greater number of genes than those in affected males. A recent analysis of CNVs in the combined SSC and Autism Sequencing Consortium samples was sufficiently powered to observe a statistically significant increase in both the number of genes hit by CNVs and in the frequency of de novo CNVs in females [In fact, risk gene discovery studies of th males , 14. The females .de novo SNVs overall [de novo indels in SSC cases did observe a significantly increased rate of de novo frameshift indels in females [de novo loss-of-function SNVs in genes associated with ASD risk in females [de novo SNVs in females overlaps significantly with the genes disrupted in affected males with low, but not high, IQ [Early exome sequencing studies on the same cohort reported an analogous trend, with a higher rate of  overall , or excl overall  or gene  overall , in fema females . Later a females . A simulhigh, IQ , demonstde novo loss-of-function SNV, dnLoF, or de novo CNV, dnCNV) than cases with higher IQ [de novo risk variant rate. In the analysis of dnCNVs and dnLoF in the combined SSC and ASC cohorts, the presence of a dnLoF or dnCNV was associated with an 8-point decrease in nonverbal IQ (NVIQ) in males and an 18-point decrease in females as compared with sex-matched probands lacking dnLoF or dnCNV [de novo risk variants cannot be fully explained by the greater frequency of ascertained females with low IQ in these samples. Taken together, the results of these genetic analyses are in keeping with the prediction from the FPE model that females with autism are subject to greater risk loads than autistic males.A potential concern with these findings of greater genetic risk in diagnosed females is the impact of IQ on females’ ascertainment in study cohorts. As discussed, females with cognitive impairment may be more likely to be diagnosed with ASD than those with normal-to-high IQ. Irrespective of sex, ASD cases with low IQ are more likely to carry an identifiable genetic risk variant  responsible for such a female protective effect. To accomplish this will require both targeted approaches, like the Autism Sisters Project launched by the Autism Science Foundation which will collect data from unaffected sisters of autistic siblings , as wellde novo variant detection and association approaches, we now know that while there are several ASD-associated genes on the X chromosome, the majority of risk genes are autosomal [Over the years, several preliminary theories for ASD’s sex-differential risk have been proposed and explored. The first, most straightforward hypothesis was that, like many conditions that only affect males, ASD is X-linked . In thisutosomal . Loss-ofp) had greater social and executive functioning skills than females with only a maternal X  [A second hypothesis also invokes the X chromosome, proposing that a single gene on the X that is imprinted and paternally expressed is responsible for females’ protection. Since only females have a paternal X chromosome, males do not express this gene and therefore do not experience its protective properties . This hy . Skuse aA third, genetics-based hypothesis proposes that protection in females is conferred by variation at a single genetic locus, whether on the X chromosome or an autosome. This hypothesis was suggested in response to work characterizing quantitative ASD traits in multiplex families; these studies found that scores from the SRS followed a nearly normal distribution across affected and unaffected males, but that females’ scores showed a bimodal distribution split between diagnosed and non-diagnosed individuals , 101. SiBeyond genetic variation, other proposed mechanisms feature a role for sex steroid hormones during neurodevelopment. The best known is the extreme male brain (EMB) theory, which was proposed by Simon Baron-Cohen and conceptualizes the cognitive-behavioral phenotype of ASD as an amplification of male-typical interests, skills, and behaviors . The theSince typically developing males, in general, fall closer to the autistic phenotype on these scales than females, Baron-Cohen proposed that testosterone exposure during fetal development may contribute to ASD risk, particularly for autistic girls . Insofarfemales, then one would reinterpret the sex-differential risk variant distribution to mean that it is males who require less deleterious genetic variants to present with ASD. In this case, the pertinent question is not what mechanism protects females, but what factor or mechanism sensitizes males to this class of less penetrant  risk variants? Since neither males nor females as a group represent the prototypical human, it is not clear which frame of reference is the “correct” one or the one most likely to facilitate discovery of the mechanisms that mediate sex-differential ASD risk. For now, research in this area should consider the possibilities of both female-specific protective factors and male-specific risk factors when designing and interpreting studies.Only once a specific mechanism is implicated will we know which is the driving force behind setting males’ and females’ liability thresholds. Until then, whether one considers male-specific risk or female-specific protective factors to be key depends on the baseline of one’s frame of reference. For example, it is straightforward to say that females tend to require more deleterious genetic variants before they present with ASD, and therefore females are protected against lesser variants. This assumes though that the scale of variants’ deleteriousness is based on their impact in males, since females are framed as coping with variants that would typically  be penetrant. If the deleteriousness of risk variants is instead normalized to their impact in In their subsequent work to investigate the biology behind the EMB theory, Baron-Cohen and colleagues have performed several studies of the relationship between fetal testosterone levels and phenotypes later in life. In a sample of 235 majority non-autistic children whose mothers had undergone amniocentesis during gestation, the authors observed a significant positive relationship between fetal testosterone levels and quantitative measures of ASD traits . If we cMost recently, by making use of national registry data from Denmark, Baron-Cohen and colleagues were able to test for a relationship between fetal testosterone levels and ASD status in the same individuals . By linkAlso, though the testosterone findings from this analysis are compelling, the elevation of related hormones in addition to testosterone suggests that the pathways that translate early hormone exposure to an autistic phenotype are multifactorial. That cortisol was one of the elevated hormones suggests that these ASD risk pathways may involve stress responses. Whether the elevation in cortisol results from a response to stress or is a consequence of increased testosterone or other confounding phenomena is not known, and will require further investigation.N = 26) and without (N = 25) CAH showed no difference in AQ scores. Despite the small sample size, this finding suggests that, insofar as prenatal exposure to testosterone above a certain threshold shifts individuals’ autistic trait measures toward the diagnosable end of the distribution, females are similarly impacted by testosterone exposure as males.What also remains unclear from each of the above studies of fetal testosterone and later phenotypes is whether fetal testosterone acts to skew risk toward males simply because males are more likely to be exposed to higher levels of testosterone or instead whether fetal testosterone preferentially impacts males because females are protected from its effects via some other process. This question is perhaps best addressed by studying the relationship between testosterone levels and ASD-associated phenotypes specifically in females, to determine how testosterone exposure impacts this presumably protected group. Individuals with congenital adrenal hyperplasia (CAH) have a deficiency in the enzyme 21-hydroxylase which causes their adrenal glands to produce abnormally high levels of testosterone, providing a unique population in which to examine the effects of testosterone in females. One study of 34 women with CAH and 24 of their unaffected relatives found that females with CAH scored significantly higher on the Autism Spectrum Quotient (AQ), a self-report questionnaire that measures individuals’ autistic traits . CAH femN = 415) reported symptoms of steroid hormone irregularities such as irregular menstrual cycles or precocious puberty, that are frequently associated with PCOS [That testosterone may also affect females’ ASD risk is further supported by reports linking ASD and polycystic ovary syndrome (PCOS), another condition associated with increased testosterone production. In one study, a higher than expected proportion of females with ASD  from males with ASD and their unaffected brothers. Genes differentially expressed between siblings showed significant enrichment for genes involved in cholesterol metabolism and androgen biosynthesis pathways , and a sRORA), is a transcription factor that is involved in a sex steroid hormone expression regulatory pathway. Specifically, estrogen and androgen receptor binding sites have been found upstream from RORA’s transcription start site and its expression in SH-SY5Y cell culture increases in response to treatment with 17-β estradiol (E2) but decreases in response to dihydrotestosterone (DHT) [RORA binding sites have been identified upstream from CYP19A1 (aromatase), an enzyme that converts testosterone to estradiol, and there is a positive relationship between RORA and CYP19A1 expression, including coordinate reduction of both RORA and CYP19A1 protein in post mortem ASD frontal cortex [RORA and target expression is stronger in males [RORA to sex-differential biology. It remains unclear as to whether RORA acts as a primary risk factor for ASD, or whether its dysregulation in ASD is a downstream consequence of altered androgen or estrogen expression. Regardless, this deep characterization approach for investigating ASD-associated molecular pathways will be important for understanding the mechanistic links between ASD and sex-differential biology.Complementary analyses of differential methylation in LCLs and gene expression in the ASD brain identified two candidate genes with elevated methylation in ASD and decreased expression in the ASD frontal cortex and cerebellum . One of ne (DHT) . RORA bil cortex . Severalin males , furtherpost mortem cortex, and results from exome sequencing and coexpression network analyses in ASD, to determine how risk genes or related pathways overlap with sex differences in the human brain [Genome-wide screening approaches will also be important for identifying additional molecular and cellular pathways involved in ASD and sex-differential biology. Unlike candidate gene approaches, genome-wide methods such as exome, genome, or even RNA sequencing have the potential to provide an unbiased survey of the key biological processes involved in ASD. A recent study applied such a tactic, using a genome-wide survey of sex-differentially expressed (sex-DE) genes in the adult and fetal human an brain . If ASD-post mortem autistic brain relative to similarly male-skewed control groups [In fact, this analysis did not find any evidence for sex-differential expression of ASD-associated risk genes as a class. Instead, the results showed significant overlap between genes with higher expression in males  and genel groups , 115. Thl groups , 117. Tol groups –120, hypl groups , 122, anl groups , 124, anLooking forward, given the small number of independent samples in each tested data set, it is imperative that this observation of male-skewed expression of astrocyte and microglial genes be replicated in independent, well-powered data sets. If this pattern does reflect biological truth, it opens up the next round of questions regarding the cause of this sex-differential gene expression and its mechanistic relationship with ASD etiological pathways. For example, is the higher expression of genes associated with astrocyte and microglial functions in males the result of sex differences in cortical cell type composition, i.e., do males have a greater number of microglia than females? Do males’ astrocytes and microglia express marker genes at higher levels? If so, what sex-differential regulatory mechanisms are responsible for directing these differences in expression levels? Or, are other cell types in males more likely to show ectopic expression of astrocyte and microglial marker genes?Given the overlap at this particular functional and cellular junction with gene expression patterns observed in ASD brain, the answers to these questions are likely to be pertinent for understanding ASD risk and for identifying potential therapeutic targets. Rigorous experimental work to pursue this line of questioning or others that may emerge from high-throughput, bioinformatics approaches would certainly not only be relevant to ASD but would also advance our knowledge of the sex-differential development, structure, and function of the human brain more generally. Concerted and collaborative efforts between ASD clinicians, epidemiologists, geneticists, and neuroscientists with expertise in sex differences will be required to facilitate progress toward better understanding of the processes governing sex-differential neurobiology, ASD biology, and the ways in which they intersect with each other to increase risk for, or protect against, ASD.RORA, and sex-differential functioning of astrocytes and microglia may participate in sex-differential risk mechanisms.ASD is a condition with a striking male bias in prevalence that remains largely unaccounted for. Though the field is beginning to appreciate that a diagnostic bias against the identification of affected females may contribute, careful prevalence screens that do a better job of identifying affected females so far indicate that ASD may remain approximately twice as prevalent in males. Further investigation is imperative to determine if there is a population of affected, currently undiagnosed females who would benefit from diagnosis and treatment. Additionally, studies of phenotypic differences in autistic males and females show that females present with fewer restricted interests and repetitive behavior symptoms and greater social motivation. It remains to be determined whether these differences result from sex-differential socialization or expectations or whether they are rooted in sex-differential neural circuitry or activity. Studies of genetic risk variants in ASD patients are increasingly consistent with the actions of female protective and/or male risk factors, and investigation of putative molecular and cellular factors suggests that fetal testosterone and/or other steroid hormones, a regulatory pathway involving the gene Though each of these observations serves to chip away at the gaps in our understanding of the interactions between sex-differential biology and ASD risk pathways, each implicated sex-differential factor has been associated with ASD in relative isolation. Much work remains to flesh out the mechanistic pathways that connect sex-differential biological factors to differences in ASD risk and prevalence. Work that taps into the expertise of the sex differences research community will be critical to accomplish this goal.In particular, as our list of ASD genes grows, it will be useful to generate and curate similarly robust knowledge of the genes that are involved in sexually dimorphic biology, including genes with sex-differential expression, genes that are regulatory targets of estrogen, androgen, and other sex steroid hormone receptors, and genes that are regulated by transcription factors translated from sex-linked genes. Spatiotemporal information about sex-differential expression and regulation across developmental time, brain regions, and cell types would also increase the utility of such resources by allowing researchers to pinpoint critical points of intersection between sex-differential regulatory pathways and the etiological mechanisms of ASD or other conditions.It will also be useful to work toward advancing our understanding of gross and fine-scale neurobiological sex differences in brain regions outside of the hypothalamus, a region that has received much experimental attention due to its role in reproductive behavior and robust morphological sex differences. However, ASD and other neuropsychiatric disorders with sex-differential prevalence or presentation largely involve other regions and circuits, and an awareness of where and to what degree sex differences exist throughout the brain will facilitate our understanding of the root causes of these differences. In addition to focused study by sex differences researchers, the newly implemented mandate from the US National Institutes of Health that requires scientists across diverse fields to include sex as a variable in preclinical research may further build on our knowledge of sex effects in a wider array of biological systems. This increased attention to, and awareness of, sex in biological research at all stages will also increase the value of generating a foundational understanding of sex differences in human brain development and function.Ultimately, the aim of research in this area is to develop a comprehensive understanding of sex in the brain, in development, and in risk mechanisms for ASD. This understanding will be critical, as sex is a potent modulator of ASD risk, and therefore knowledge of the pathways that link sex differential biology to the ASD phenotype may offer key targets for effective, well-tolerated therapeutics. In general, a mutual awareness of the approaches used and findings from sex differences research and of work on ASD genetics and neurobiology will facilitate this intended progress toward making a tangible positive impact on the lives of individuals with autism and their families.


The fundamental metabolic decision of a cell, the balance between respiration and fermentation, rests in part on expression of the mitochondrial genome (mtDNA) and coordination with expression of the nuclear genome (nuDNA). Previously we described mtDNA copy number depletion across many solid tumor types . Here, we use orthogonal RNA-sequencing data to quantify mtDNA expression (mtRNA), and report analogously lower expression of mtRNA in tumors  across a majority of cancer types. Several cancers exhibit a trio of mutually consistent evidence suggesting a drop in respiratory activity: depletion of mtDNA copy number, decreases in mtRNA levels, and decreases in expression of nuDNA-encoded respiratory proteins. Intriguingly, a minority of cancer types exhibit a drop in mtDNA expression but an increase in nuDNA expression of respiratory proteins, with unknown implications for respiratory activity. Our results indicate suppression of respiratory gene expression across many cancer types.DOI:http://dx.doi.org/10.7554/eLife.21592.001 Are the mitochondria of tumors characteristically different from those in normal human tissues? Several recent reports have described somatic changes to the mitochondrial genome (mtDNA) in tumors, which have expanded our knowledge of cancer genetics as well as basic mitochondrial biology . In our A drop in mtDNA copy number can decrease the expression of critical (and necessary) proteins of the electron transport chain (ETC)/ATP synthase. Respirometry experiments indicate that cells typically harbor an excess of mitochondrial respiratory capacity, implying that a modest decrease in ETC protein levels will not impact the basal respiration rate of the cell . HoweverThus, there remains a gap between the observation of mtDNA copy number changes and their propagation to a drop in respiration. Linking measurements of mtDNA copy number directly with respiratory flux remains difficult: flux studies of comparable sample size to contemporary genomic investigations are not available. However, one can lay a partial bridge between respiration and mtDNA ploidy by examination of mRNA expression of mtDNA (mtRNA). Mitochondrial DNA serves as the template for the transcription of 13 protein-coding genes, each of which is a critical integral membrane subunit in the electron transport chain or ATP synthase. A synchronous change (increase or decrease) in both mtDNA copy number and mtRNA expression in tumors compared to normal tissue can therefore be used as complementary evidence for a change in respiratory flux, which can then be evaluated experimentally.Here, we use RNA-sequencing data of TCGA tumors to expand upon our prior study of mtDNA copy number variation in cancer . In parthttp://firebrowse.org), the abundance of the 13 proteins encoded in mtDNA are not reported there. Therefore, to quantify expression from mtDNA, we re-aligned TCGA RNA sequencing data from thousands of the same tumors analyzed in our prior study of mtDNA copy number, and retained for analysis sequencing reads aligning to the mitochondrial chromosome. Estimates of mtRNA expression , confounded our estimates of mitochondrial transcription. Previous work has shown that NUMTs are, in the vast majority of cases, integrated into intergenic/intronic regions of the nuclear genome, and are unlikely to be transcribed . NeverthAcross all tissues, mtRNAs were highly transcribed. However, we observed substantial differences in the abundance of any given mitochondrial (MT) transcript from one tissue to the next  . For exaIn , we obseBreaking the general trend, one cancer type, KICH , had higher levels of mtRNAs in tumors compared to normal tissues. The over-expression phenotype was notably large: each of the 13 genes had greater than 2-fold over-expression in tumors compared to normal tissue . Importaincreased mtRNA levels in KICH tumors is likely to counterintuitively reflect a drop in respiration. As noted before, experimental respirometry measurements must be made to confirm this hypothesis.The role of mitochondrial dysfunction, and of a mitochondrial accumulation phenotype, in chromophobe renal cell carcinomas has been appreciated for some time . Two recInterestingly, we also found differences in the tendency for any one mtDNA-encoded gene to be differentially expressed across cancer types, which likely arises from the molecular details of mitochondrial transcription. The mitochondrial genome is transcribed in a polycistronic fashion, with all mRNAs and tRNAs on a strand transcribed simultaneously. Following transcription, tRNAs are excised from the transcript, and the majority of the remaining mRNAs are polyadenylated . PolycisWe also evaluated the extent to which mtRNA levels were associated with clinical features , using available clinical data from the TCGA consortium . Among tMore interestingly, we identified five cancer types  in which higher mtRNA expression levels were associated with increased overall survival. Three of these cancer types , showed similar associations in our prior analysis using mtDNA copy number  can control the rate of transcription, while yet others can modulate mtRNA stability and degradation . Thus, i−5) positive correlation across all or nearly all mtDNA-encoded genes  the non-parametric Spearman correlation between mtDNA copy number and mtRNA levels (in RSEM counts), for all samples with available mtDNA copy number and mtRNA expression estimates , and to a lesser extent with mtDNA copy number (Spearman For one cancer type (kidney clear cell), the TCGA consortium  used reve 0.003) .Interestingly, correlations between mtDNA and mtRNA were not necessarily homogeneous between tumor and normal samples from the same tissue. Upon closer examination of samples from KICH , we founS. cerevisae found that the primary mode of regulation was through rapid translational, rather than transcriptional, synchronization . The remaining roteins) . The proroteins) . Intrigunization . This leTo see if the nuclear and mitochondrial genome experience analogous changes in respiratory gene expression in the course of tumorigenesis, we examined the differential expression of mtOXPHOS and nuOXPHOS subunits for all 13 cancer types with available expression data for both tumor and normal tissues . We calculated a differential expression (DE) score for each cancer type, which captured the tendency for a set of genes to be down-regulated (corresponding to a score of −1) or up-regulated (a score of +1) in tumors compared to normal tissue. In 9/13 cancer types, we observed agreement in DE scores between mtOXPHOS and nuOXPHOS subunits . More interestingly, we found that four cancer types  have opposite patterns of differential expression in mtOXPHOS and nuOXPHOS genes. For example, in BRCA, while nearly all mtOXPHOS genes are down-regulated in tumors compared to normal tissue (mirroring the mtDNA copy number depletion in BRCA), nearly all nuOXPHOS genes actually had increased mRNA levels.S. cerevisae to a non-fermentable carbon source led to rapid induction of transcriptional changes in nuOXPHOS, but not mtOXPHOS. Instead, rapid redistribution of mitochondrial ribosomes supported fast adapation to respiration (e.g. via an increase in translation efficiency of Complexes III and IV). Together, these two studies suggest that the examination of translational activity (e.g. by ribosome profiling) may be useful tools for examining an imbalance in mtOXPHOS and nuOXPHOS transcription.For a functional electron transport chain, subunits of each OXPHOS complex must assemble with compatible stoichiometries in a process that requires coordination of individual subunit protein levels . Taken tThe purpose of this study was to compare cancer-associated changes in mitochondrial transcript levels with changes in mtDNA copy number as reported in In tandem with our prior study , this woWhile changes in mtDNA copy number and mtRNA expression cannot be used as surrogates for changes in respiratory flux, several of the cancer types examined here are driven by genetic changes affecting mitochondrial respiration, suggesting that changes in mtRNA levels may reflect bona fide changes in respiration. For example, clear-cell renal cell carcinomas are driven by homozygous loss of VHL, which leads to activation of hypoxia inducible factor (HIF), and subsequently increased transcription of glycolytic enzymes . The sigFurthermore, while our data suggests that several cancer types may decrease mitochondrial respiration, mitochondrial metabolism provides several other important services to the cell. In some cases, respiration may be critical to these processes; for example, aspartate biosynthesis is heavily reliant on electron acceptors produced by mitochondrial respiration, and respiration-deficient cells become limited by the availability of aspartate . Beyond lignancy .When comparing the differential expression of mtRNA  to changes in mtDNA copy number , we found two cancer types (LUAD and KICH) had opposite changes in the two measures. To highlight a single example, lung adenocarcinomas were the only cancer type to accumulate mtDNA copies compared to normal tissue. Using mtRNA data, however, these tumors had lower mtRNA levels of 6/13 MT genes. We can speculate as to the source of this inconsistency: it may be that our inference from sequencing data of mtDNA copy number or mtRNA expression was erroneous for these tumor types. While this is a possibility, we would remind readers that the same analytical pipeline was applied to all cancer types, the majority of which had features consistent with our expectations, e.g. correlation of whole-exome and whole-genome estimates of mtDNA copy number in α,ERRα) . A more C. elegans claimed that it promotes longevity via activation of the mitochondrial unfolded protein response (UPRMT), a transcriptional response preserving mitochondrial function in the face of stress  protein imbalance in f stress . Intriguactivity . If cancSeveral of our findings discussed above allude to intriguing tissue-specific patterns of variation in mtDNA and mtRNA. Both the accumulation/depletion of mitochondrial transcripts ,4,5 and https://cghub.ucsc.edu, Research Resource Identifier RRID:SCR_003193). When FASTQ files were not available, e.g. for stomach adenocarcinoma, we downloaded aligned sequence reads (in BAM format) and extracted reads from BAM files with the Java program ubu.jar (https://github.com/mozack/ubu) before processing samples using our pipeline. All acronyms for cancer types conform to the TCGA nomenclature . Sample sizes for each cancer type are reported in The input of our analysis pipeline is raw sequencing reads (in FASTQ format). The raw reads of the RNA-seq samples were retrieved from the Cancer Genomics Hub  of 175 regions annotated as mtDNA pseudogenes in GENCODE v19. While these genes are a subsample of the total number of NUMTs in the genome (on the order of 1000 ), they audogenes .As a second quality control measure, we compared estimates of expression counts produced from two computational approaches, RSEM and featureCounts. These methods differ fundamentally in the way that they count reads mapping to multiple regions in the genome (multi-mapping reads). RSEM handles multi-mapping reads using an expectation-maximization procedure, while featureCounts by default simply discards multi-mapping reads. Thus, if a read maps ambiguously to both mtDNA and a NUMT, then featureCounts will ignore this read while RSEM will not. For all 13 mtDNA-encoded mRNAs, we compared (1) the total expression (in counts) as reported by RSEM and featureCounts  and (2) Differential expression analysis was completed using the limma voom package . TMM norDifferential expression score is defined asA score of 1 indicates that all genes in the gene set are overexpressed in tumor compared to normal tissue, while a score of −1 indicates that all genes in the gene set are underexpressed.Genes included in the nuDNA OXPHOS geneset were derived from the MSigDB Reactome geneset file .All data for mtDNA copy number analysis was derived from All data for clinical analysis was downloaded from the Broad Firehose. For survival calculations, Cox regression was used to associate log In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by a Senior Editor and three reviewers, one of whom, Chi Van Dang, is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled "Mitochondrial Respiratory Gene Expression is Suppressed in Many Cancers" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife the spectrum of mtDNA alterations relative to normal tissues. Understanding somatic alterations of mitochondrial genome during tumorigenesis is critical for the next generation of cancer therapeutics. Several recent studies based on the TCGA specimens have revealed that mitochondria DNAs are altered in cancers, including mtDNA mutations as well as copy number changes. The authors previously reported that mtDNA copy number depletion in human cancers. In this follow-up study, they analyzed mitochondria gene expression using the TCGA RNA-seq profiles. They presented an expressional landscape of 13 mitochondria genes across >10 cancer types which have normal control specimens from the TCGA, and found that mtRNAs were dysregulated in cancers. This comprehensive mtRNA expression profile could contribute to better understanding both mitochondria biology and tumorigenesis.The manuscript by Reznik et al. reports alteration mitochondrial gene expression amongst human cancers, for which they have previously reported in However, there is a similar recent paper in Nature Communications on this topic by Frezza and colleagues "Tissue-specific and convergent metabolic transformation of cancer correlates with metastatic potential and patient survival".Further, the mitochondrial genomic changes documented in this manuscript do not provide any insight into what advantage decreasing respiratory genes will have on tumorigenesis.Complete loss of the respiratory chain impairs cancer cell proliferation and tumorigenesis. The authors do not observe a complete loss but a suppression of respiratory chain. What advantage does moderate suppression of respiratory chain provide for the cancer cells in vivo?Additionally, two statements are not correct:"Our results provide quantitative evidence for suppression of respiration across many cancer types." Any given cell only utilizes 10-30% of maximal respiratory capacity. Thus, a cell can have large drop in mtDNA without respiration being affected. The authors don't have any direct measurements that examine respiration."On the other hand, mtDNA depletion will not necessarily lead to a drop in respiration". If cells have complete mtDNA depletion then they will be respiratory deficient. mtDNA encodes for critical subunits of the respiratory chain.Other omissions that detract from the paper include the following issues:1) Is there clinical significance of mtRNA dysregulation in cancers? E.g. are the expression levels of mtRNAs correlated with disease stage/grade, patients' age, outcome and other clinical factors?2) A detailed information for sample numbers of each cancer type needs to be provided, especially for the corresponding control specimens. Relatively small number of normal specimens may create biases for analyses. [Editors’ note: the author responses to the first round of peer review follow.][…] However, there is a similar recent paper in Nature Communications on this topic by Frezza and colleagues "Tissue-specific and convergent metabolic transformation of cancer correlates with metastatic potential and patient survival".The reviewers are correct in noting that a recent paper by Gaude and Frezza describes an association between aggressive disease and downregulation of mitochondrial gene expression across many cancers. While the Gaude paper complements our own, the two analyses are completely distinct. Gaude and Frezza use the expression of nuclear-DNA-encoded mitochondrial genes as a surrogate for respiratory gene expression. In contrast, our manuscript is the first (and to our knowledge the only one to date) to quantify and analyze expression of the mitochondrial genome. Publically available data from the TCGA consortium (used by Gaude and Frezza and in dozens of other manuscripts) does not quantify expression of mitochondrial DNA; mitochondrial genome expression data is in fact discarded prior to expression quantification.We realized that we had not done a sufficiently good job in emphasizing the difference between other publications examining the expression of metabolic genes in cancer samples. We have created a new subsection and included additional text to clarify the novelty of our study with respect to prior work (“Quantifying Expression of mtRNAs Across Cancers). More importantly, because data for mtRNA expression is not available through the Broad Firehose data portal for TCGA data, we have included estimates of mtRNA expression across all 6,614 samples investigated as a supplementary resource with this publication .Furthermore, our analysis in Further, the mitochondrial genomic changes documented in this manuscript do not provide any insight into what advantage decreasing respiratory genes will have on tumorigenesis.Complete loss of the respiratory chain impairs cancer cell proliferation and tumorigenesis. The authors do not observe a complete loss but a suppression of respiratory chain. What advantage does moderate suppression of respiratory chain provide for the cancer cells in vivo?Additionally, two statements are not correct:"Our results provide quantitative evidence for suppression of respiration across many cancer types." Any given cell only utilizes 10-30% of maximal respiratory capacity. Thus, a cell can have large drop in mtDNA without respiration being affected. The authors don't have any direct measurements that examine respiration."On the other hand, mtDNA depletion will not necessarily lead to a drop in respiration". If cells have complete mtDNA depletion then they will be respiratory deficient. mtDNA encodes for critical subunits of the respiratory chain.The reviewer raises two critical and related points.We agree that we did not adequately explain our results in the context of physiological mitochondrial respiration. Respirometry experiments can be used to estimate the difference between the basal oxygen consumption rate (OCR) of a cell and the maximal OCR upon decoupling of oxygen consumption from ATP synthesis (e.g. using an uncoupler such as FCCP). These kinds of experiments frequently  demonstrate that cells harbor excess respiratory capacity. The implication, therefore, is that a reduction in functionally respiratory mitochondria in a tumor will not necessarily reduce the basal respiration rate of the tumor. However, in cases where the reduction in respiratory capacity is sufficiently large (e.g. large or complete mtDNA depletion), the basal respiratory rate of the cell will drop, because mtDNA encodes necessary components of the respiratory chain. These changes are now noted throughout the Introduction. We further emphasize in the discussion in subsection “Questions and Future Directions” that a drop in mtRNA expression does not necessarily indicate a drop in respiratory flux, but suggest that because of a consistent decrease in mtDNA copy number, mtOXPHOS expression, and nuOXPHOS expression in 5 cancer types, future experiments may initially focus on these cancers when investigating changes in respiration.We further agree with the reviewer that a critical question remaining from our study is the selective advantage provided by a potentially moderate suppression of respiration. We are unable to perform in vivo or in vitro experiments to measure how suppression of mtRNAs affects respiratory flux and proliferation/malignancy. Indeed, an adequate investigation into the quantitative link between mtRNA levels and respiratory flux is a project in its own right. Nevertheless, we agree it is important to understand how potential changes in respiratory capacity enable tumors to survive selective pressures. To do so, we used three approaches.In the first (Results section), we utilized established cancer biology to discuss how the observation of changes in mtRNA expression may arise from known tumorigenic driving mechanisms in two cancer types, clear-cell renal cell carcinoma (KIRC) and chromophobe renal cell carcinoma (KICH). In both of these cancer types, we propose that changes in mtRNA are an adaptive response to “driver” genetic mutations and upstream changes in cell signaling. For example, VHL inactivation in KIRC leads to activation of HIF which upregulates glycolytic genes and suppresses OXPHOS gene expression. The signature of HIF activation is present not only in gene expression data, but also in metabolomic data published by us , and suggests that KIRC tumors may experience a bona fide drop in respiration. In particular, KIRC tumors show a distinct metabolomic signature in TCA cycle metabolites, with those metabolites upstream of SDH , i.e. citrate, cis- aconitate, and succinate, exhibiting increased levels in tumors  and those metabolites downstream of SDH exhibiting decrease levels. One explanation (but perhaps not the only) for this partitioning of changes in metabolite levels is a decrease in respiration and SDH activity, which would induce a metabolic bottleneck and the accumulation (depletion) of metabolites upstream (downstream) of SDH.In the second approach , we leverage the reviewer’s suggestion to discuss our results in light of available clinical data (Comment 1 below). We find that down-regulation of mtRNAs is significantly associated with decreased survival in 5 cancer types, including adrenocortical carcinomas and low grade gliomas. While this analysis does not speak to the mechanism by which changes in mtRNA expression are linked to a selective advantage, it does indicate that a drop in mtRNA expression is relevant, and is associated with more aggressive disease in some cancer types.In the final approach, we felt it was also important to emphasize that mitochondria (and their metabolic “services”) are required for tumor cells to remain viable and malignant. In subsection “Questions and Future Directions, we briefly discuss the essential metabolic roles of mitochondria beyond respiration .Other omissions that detract from the paper include the following issues:1) Is there clinical significance of mtRNA dysregulation in cancers? E.g. are the expression levels of mtRNAs correlated with disease stage/grade, patients' age, outcome and other clinical factors?We agree with the reviewer that, in light of the analysis in Gaude et al. (see our first response above), it would be interesting to analyze our results alongside clinical data. We have now analyzed our results with respect to clinical data provided by the TCGA, including age, pathological stage, and overall survival. In particular, we associated the expression of each mitochondrial mtRNA with overall survival (using Cox regression) and age and pathological stage (using non-parametric Spearman correlations). These results are described in 2) A detailed information for sample numbers of each cancer type needs to be provided, especially for the corresponding control specimens. Relatively small number of normal specimens may create biases for analyses.This information is now available in


Editorial on the Research TopicNETosis 2: The Excitement ContinuesMollerherm et al.) as well as in other myeloid cells like eosinophils and basophils . Leukotoxic hypercitrullination and deficient mitophagy can initiate mitochondrial DNA expulsion (Konig and Andrade). These processes can be discriminated from canonical NET formation . This editorial summarizes the collection of articles of the research topic “NETosis 2, the excitement continues.”Chromatin externalization to form extracellular traps has first been described in neutrophils  but can asophils . DNA extormation . Howeverormation . FurtherYousefi and Simon). Some investigators question the notion that NETs form by cytolysis. They argue that it seems inconsistent with neutrophil “containment” processes . Furthermore, the term “NETosis” has been criticized by some researchers who prefer the term “NET formation.”There is ongoing discussion whether the neutrophils are still viable when they release their chromatin and if they release chromatin at all. There are reports that claim the cells mainly externalize mitochondrial DNA  the inducer and its concentration determine, at least in part, these processes as well as the content of the NETs. For purposes of this review, we will use the term NET formation rather than NETosis to include the various forms of this cellular processes as described by various research groups.Surveying the published data, including those published in this special issue, we want to summarize the current knowledge (I) Depending on the stimulus, NET formation can be a lytic death process eventually leading to disintegration of the neutrophil or a process where neutrophils remain migratory and impermeable to ionic dyes; (II) these processes have been observed Maueroder et al.). The neutrophil granule-derived enzyme peptidylarginine deiminase type 4 has been proposed to be involved in the formation of NETs and its pharmacological inhibition suppresses chromatin decondensation and NET formation following certain types of stimuli . In response to hyphae of Candida albicans or immobilized β-glucan neutrophils that are associated with the extracellular matrix rapidly form NETs independent of their ability to perform an oxidative burst (O’Brien and Reichner). The pathway of immune complexes to induce NET formation involves cross-linking of the receptor for IgG Fc fragments FcγRIIIb, the TGF-β-activated kinase 1, and activation of MEK/ERK signaling . NET formation is also triggered by interaction of neutrophils with platelets  and by a complex interplay between neutrophils and bacterial lipopolysaccharides (LPS). Both “suicidal” and “vital” NET formation depend on the bacterial origin of the LPS and are modulated by the presence or absence of platelets . NETs generated in the presence of propylthiouracil display a disordered phenotype which renders them resistant to treatment with DNaseI, and have been proposed to favor the generation of ANCAs (Soderberg and Segelmark). During gestation, the capacity to form NETs increases and involves chorionic gonadotropin, G-CSF, and estrogen .NET formation is favored by the higher pH at the borders of inflamed areas . Furthermore, Raloxifene, a selective modulator of the receptor for estrogen prevents NET formation induced by PMA .The increased capacity to form NETs during gestation is controlled by the inhibitory activity of progesterone. This hormone reduces the nuclear translocation of the neutrophil elastase leading to decreased NET formation . These proteins may either be derived from the neutrophils’ granules, from the vicinity of the NETs or from bystander cells. Neutrophils are endowed with a “tool kit” for alternative complement activation. They secrete properdin and deposit C3-derived complement fragments on NETs and on NET-bound bacteria . Since NETs are endowed with a potent toxic and proteolytic armament they need regulatory components to limit tissue injury. Hence, the secretory leukocyte protease inhibitor SLPI is bound to NETs. However, SLPI-NETs carry a certain risk; interacting with plasmacytoid dendritic cells, they induce type 1 interferon in vitro, cytokines that have been associated with the development of various autoimmune conditions . Extracellular histones are cytotoxic to endothelial cells and thus may contribute to septic pathology and death. The NET component long pentraxin PTX3 protects against histone-mediated cytotoxicity .The NET backbones mainly consist of chromatin with modified, often citrullinated, histones and is overall similar in most NETs reported. However, there are considerable differences in their protein cargo . Antibodies against posttranslational modified, citrullinated histones are pathognomonic for patients with rheumatoid arthritis (Neeli and Radic). In contrast to humans, some autoimmune mice are tolerant to citrullinated histones , a finding that highlights differences in PAD4 expression in human versus mouse antigen-presenting cells.Histones are endowed with bactericidal and fungicidal activities. This comes at the prize of bystander cytotoxicity toward endothelial cells. Thus, extracellular histones are partially responsible for tissue damage and septic death. To dampen overwhelming histone toxicity, NETs can be decorated with the long pentraxin PTX3 that limits histone-mediated tissue damage . Patients with malaria show circulating NET-binding P-ANCA (anti-MPO) and to a lesser extend C-ANCA (anti-PR3) which does not react with NETs. This supports the notion that NETs are involved in the etiopathogenesis of this, often fatal, disease . Excessive NET formation in Balb/c wild type mice leads to generation of MPO-ANCA production in vivo . Importantly, the autoantibody binding to NETs may not only contribute to immune pathology but may aid in the clearance of NETs (Soderberg and Segelmark).Deimination is a physiological process that is amplified at sites of inflammation. However, only individuals with genetic predispositions for rheumatoid arthritis develop antibodies against deiminated proteins (ACPAs) . When exploring intravascular NET formation in disease, the triad neutrophils, platelets, and endothelium should be analyzed . For in vitro and ex vivo assays, the medium composition has to be precisely reported and strictly controlled during the entire assay and not just at its beginning . Detection of deiminated histones is often used as an indication of NETosis but it should be stressed that the current methods for detection of citrullinated proteins require further optimization .In general, NETs can be detected by staining of DNA and its co-localization with granular and modified nuclear proteins in web-like, spiky or cloudy structures, exceeding the size of a neutrophil. In neutrophil-rich areas like inflamed synovium or densely infiltrated tissues, these NETs tend to aggregate , 5 and fBilyy et al.; Biermann et al.].NETs generated at high neutrophil densities often aggregate and form extended matted structures , 5 decorKimball et al.). As in SLE, in ANCA associated vasculitis, the degradation pathways of NETs are perturbed. Due to low DNaseI activity, the degradation of intravascular NETs is slow leading to an increased amount of circulating NETs (Soderberg and Segelmark). This phenomenon may also contribute to the pathogenesis of severe malaria . Similarly, NETs are discussed to contribute to the thrombotic pathologies observed in patients with cancer .Intravascular NETs have pro-coagulant activities associated to several pathologies . In an animal model of paw edema by injection of nanodiamonds into wild-type mice and in those with deficient capacity for oxidative burst and NET formation (Ncf1** mice), the inflammatory response resolves in the former and becomes chronic in the latter as a result of the failure to dampen the neutrophil-driven inflammation . Importantly, ANCA in general are reported to drive immune complex-mediated pathologies but they may also aid in the clearance of circulating NETs or NET remnants (Soderberg and Segelmark).After bone marrow transplantation, the capacity to form NETs is reportedly reduced for up to 200 days . NETs are involved in a plethora of pathological conditions including autoimmunity, atherosclerosis, cancer, etc. . Excess neutrophil recruitment to the alveolar space and NET formation in lungs reportedly cause inflammation and asthma . NETs can easily expand in the pulmonary alveoli and cause lung injury. DNA disintegration by DNase, neutralization of NET proteins, anti-histone antibodies, and protease inhibition may alleviate NET-associated pathologies (Porto and Stein). Raloxifene, a selective modulator of the estrogen receptor inhibits the NET-based killing of the leading human bacterial pathogen MRSA . Furthermore, it was recognized that viruses induce NETs and target these for immune evasion (Schonrich and Raftery). Harming of the blood–brain barrier and of neural cells by NETs was observed in humans with Alzheimer’s disease and in a murine model of this disabling disease .Though NETs have initially been described as mechanism of bacterial defense, they must be considered as double-edged swords of innate immunity (Konig and Andrade). The synthetic peptide P140/Lupuzor™ selectively modulates chaperone-mediated autophagy but not NET formation in sensu stricto . As high fat diet reportedly increases the formation of NETs dietary intervention and reduction of fat intake may be beneficial in NET-associated disorders .NETs are discussed as a source of the citrullinated autoantigens pathognomonic for patients with rheumatoid arthritis and for the DNA observed in tissues and in the circulation of patients with SLE. Consequently, PAD4 is proposed to be a potential therapeutic target for these chronic inflammatory rheumatic diseases , the early inflammatory responses to Sendai virus infection , and the disseminated intravascular coagulation observed in severe forms of malaria . As already established for patients with cystic fibrosis, the clearance with recombinant human DNaseI of the NET-associated DNA, the neutralization of NET-borne proteins using anti-histone antibodies, as well as inhibitors for NET-bound proteases are discussed as therapeutic options for pulmonary diseases involving alveolar NETs in Porto and Stein.Targeting NET formation as well as NET-associated chromatin decondensation may delay the pathogenesis of Alzheimer’s disease (in vivo and in vitro have been carefully defined. It is the hope of the authors that the combined efforts presented here will contribute to further shape the consensus in the field, energize efforts to understand NET biology, and lead to novel therapies for major human disorders that present with abnormal NET release or impairments in NET degradation.From the papers included in this Research Topic, it is evident that the field of NET research is now more mature and sophisticated than even just a few years ago. Physiological and disease conditions that induce abundant NET formation are now firmly established and experimental methods for detection of NETs All authors wrote and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Assessment of the cortical role during bipedalism has been a methodological challenge. While surface electroencephalography (EEG) is capable of non-invasively measuring cortical activity during human locomotion, it is associated with movement artifacts obscuring cerebral sources of activity. Recently, statistical methods based on blind source separation revealed potential for resolving this issue, by segregating non-cerebral/artifactual from cerebral sources of activity. This step marked a new opportunity for the investigation of the brains’ role while moving and was tagged mobile brain/body imaging (MoBI). This methodology involves simultaneous mobile recording of brain activity with several other body behavioral variables , through wireless recording wearable devices/sensors. Notably, several MoBI studies using EEG–EMG approaches recently showed that the brain is functionally connected to the muscles and active throughout the whole gait cycle and, thus, rejecting the long-lasting idea of a solely spinal-driven bipedalism. However, MoBI and brain/muscle connectivity assessments during human locomotion are still in their fledgling state of investigation. Mobile brain/body imaging approaches hint toward promising opportunities; however, there are some remaining pitfalls that need to be resolved before considering their routine clinical use. This article discusses several of these pitfalls and proposes research to address them. Examples relate to the validity, reliability, and reproducibility of this method in ecologically valid scenarios and in different populations. Furthermore, whether brain/muscle connectivity within the MoBI framework represents a potential biomarker in neuromuscular syndromes where gait disturbances are evident  remains to be determined. Whether human locomotion is driven primarily by supraspinal or subcortical mechanisms within the central nervous system (CNS) is object of long-lasting debates. In mammalians, muscle activation requires CNS inputs either dependently  or indepThe nerves convey the stimulus of volition to the muscles … and the whole machine is thus put in motion under the guidance of the will”  from the human scalp . Since t2Hb) and has the advantage of being less prone to movement artifacts contamination. However, using fNIRS in this context has some limitations as well. To name a few, fNIRS is not able to provide information about brain structure for anatomical reference, the separation of the hemodynamic changes originating either from cerebral tissue or extra-cerebral tissues/structures is difficult  for the assessment of functional brain activation during human bipedalism , 15. fNIifficult , and finifficult  and alloifficult .Electroencephalography allows the direct measurement of post-synaptic neuronal activity, with very high time resolution but low spatial resolution. However, in recent years several improvements to overcome the spatial resolution limitation of EEG have been made. Enhanced EEG source reconstruction techniques –23 in coThe application of statistical methods to segregate cerebral and non-cerebral sources  from the EEG recordings  through This aforementioned development has kicked-off a new research field, tagged as mobile brain/body imaging (MoBI) , 36, thaA MoBI set-up allows an undefined number of body sensors to record human movement behavior concurrently with brain activity . That is, the essence of the MoBI approach would require not only a mobile brain recording device but also simultaneous (and thus precisely triggered in time) several other measurement devices, in order to explore key aspects of the human locomotion linked to brain behavior. An example of such mobile wearable body sensors could be wireless surface electromyography (sEMG), 3D motion capture, inertial measurement units (IMUs), foot plantar pressure measurement systems, and/or eye tracker devices. A more comprehensive reading on wearable sensors options for MoBI studies can be found elsewhere .An important step in combining EEG with wearable body sensors has been performed by using sEMG. A recent MoBI study firstly showed that the motor cortex is actively committed in driving human locomotion such as walking . This waThe promising potential of using wearable sensors in combination with brain imaging devices, investigating the communication between supraspinal and subcortical sites with concurrent kinematic events, is shown by recent studies involving peripherally attached  IMUs coupled with magnetoencephalography. These studies have shown promising results in the sense that using such combined techniques  is capable to parallel CMC in assessing the neuronal communication between brain and muscle(s) –61. CKC A comparable measure of CMC, referred to as “intramuscular coherence” or “EMG-EMG coherence”, takes into consideration the common synchronized oscillatory drive to a pair of sEMG placed over the same muscle . IntramuIt can be reasoned that CMC reliability as measured during human locomotion tasks  is also promising; however, thorough investigations of the psychometric properties of the measurement approach are needed to substantiate or refute this assumption. We are currently far from having precise and accurate measurements of neuronal communication during human bipedal motion and available results from studies employing CMC (or EMG–EMG) techniques should be interpreted with prudence. The reproducibility and reliability of CMC measurements during human bipedal motion is an important issue not yet clarified on both group and individual levels and should be topic of future studies.Another issue that needs to be resolved relates to the consistently reported efferent cortical role during the gait cycle , 65, 66.Finally, future studies on CMC should consider to both establishing the reproducibility and reliability of CMC measurements as well as disentangling the causality in the connectivity measures , 66, possarcopenia (the hallmark of aging (It has been shown that CMC has the potential to distinguish the presence of neuromuscular disorders such as in upper motor neuron disease . Howeverrcopenia . Sarcopercopenia –81. Sarcrcopenia . Mountinrcopenia . Indeed,of aging , able toof aging . Combiniof aging .In conclusion, the last two decades have opened a new and fascinating “door” on the motor control research field investigating neuronal communication in human locomotion. This field of research is still in its fledgling state, however, it is promising for revealing some aspects of the brain’s role in human locomotion. To increase the chances for replication both on group and individual human levels, studies using MoBI approaches in humans should specify parameters of the tests used, e.g., the exact procedures and instrumentation used, the duration of testing, and applied algorithms used for data analysis . An impoFG has developed the conception and design of the manuscript under the lead of EB. FG drafted a first version of the manuscript. EB substantially revised the manuscript. Both FG and EB have read, revised, and approved the final version of the manuscript.The authors declare that the research has been conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Research suggests that one physiological effect of concussion is a disruption in regulation of autonomic nervous system control that affects the balance between parasympathetic and sympathetic output. While changes in heart rate after concussion have been observed, the nature of the heart rate change during progressive exercise has not been well evaluated in acutely symptomatic patients. Additionally, little is known about the relationship between HR and RPE in this population.We compared changes in heart rate and perceived effort during graded treadmill exertion in recently concussed patients to elucidate the effect of brain injury on cardiovascular response to exercise. Resting HR, HR on exercise initiation, and changes in HR and RPE during the Buffalo Concussion Treadmill Test (BCTT) were compared on two test visits: When patients were symptomatic (acute) and after recovery. Results were compared with the test-retest results obtained from a control group consisting of healthy, non-concussed individuals.Patients had a significantly lower HR at onset of exercise when acutely concussed as compared to when recovered and reported greater perceived exertion at every exercise intensity level when symptomatic, despite exercising at lower workloads, than when recovered. Sympathetic response to increased exertion was not affected by concussion - HR increased in response to exercise at a comparable rate in both tests. These differences observed in response to exercise between the first BCTT and follow-up evaluation in initially concussed patients were not present in non-concussed individuals.Our results suggest that during the acute phase after concussion, acutely concussed patients demonstrated an impaired ability to shift from parasympathetic to sympathetic control over heart rate at the onset of exercise. Changes in the autonomic nervous system after concussion may be more complex than previously reported. Continued evaluation of autonomic regulatory effects in the acute phase after concussion is warranted. There is increasing interest about the effects of acute concussive injury on the capacity of the central nervous system to appropriately regulate peripheral functions such as cardiovascular rhythm, metabolism and temperature regulation and how this may relate to prolonged recovery from concussion –4. StudiAlthough some literature supports autonomic consequences of brain injury, there are discrepancies that make it difficult to generalize these findings. For instance, some studies suggest increases in parasympathetic influence  in the very early stages after acute injury ,13. SimiTo comprehensively test the influence of concussion on autonomic function, we conducted a prospective, controlled evaluation of cardiovascular function in recently concussed patients.We conducted an IRB-approved prospective study on patients visiting a concussion clinic over an 18-month period 2014–2016) for treatment of acute concussion. Recently concussed patients  were selected for the study if they were symptomatic  [014–2016 We evaluated HR and RPE changes during graded exercise on the Buffalo Concussion Treadmill Test (BCTT)  in patieThe BCTT has previously been demonstrated to have good inter-rater and test-retest reliability . During Timing of the BCTT was controlled to minimize potential confounds from pre-exercise activity. Participants completed the test at approximately the same time of day at each visit after spending at least one hour in the medical clinic before testing. This reduced the possible influence of caffeine, other drugs, or physical or cognitive exertion immediately before testing. All participants were instructed to refrain from new medications between testing sessions, and to report any major changes to their daily lifestyle.Resting HR and HR and RPE at each time point during exercise were compared between groups using two-sample t-tests. Age, body mass index (BMI), and resting HR were also compared between groups using two-sample t-tests. For both groups, two repeated-measures analysis of variance (RMANOVA) were performed to examine both the change in HR over time and changes in RPE over time, both as a factor of group (acute or control). Age was considered as a covariate in each analysis of HR change but did not contribute significantly to the model. Significance was defined as a p-value <0.05 and all statistical analyses were done using SPSS.For acutely concussed patients, resting HR did not differ significantly when participants were symptomatic when compared with same participants after recovery . At the start of exercise (time 0), HR was significantly lower when participants were acutely concussed than when they were recovered (p=0.015); however, the relative increase in HR seen during each successive increase in exercise intensity (increased HR corresponding to an increase in exercise exertion) was not significantly different when patients were acutely concussed as compared to when they were recovered .In contrast, the discrepancy between predicted and observed rating of exertion (RPE) was consistently higher when patients were acutely symptomatic. RPE was consistently rated higher for comparable work in symptomatic participants versus when they were recovered, despite relatively similar HRs during exertion and comparable level of physical work p<0.01; . Age andFor control participants, there were no significant differences between HR at initiation of exercise or relative change in HR with exertion between visits , nor anyTo our knowledge, this is the first study to evaluate cardiac performance during progressive aerobic exercise in acutely symptomatic concussed individuals. The evaluation of cardiovascular response to exercise as measured during treadmill testing in patients with acute concussion symptoms revealed a significant reduction in HR at the point of exercise initiation when compared with the same patients at the time of recovery. While the typical HR rise seen with increasing workload  was obseCareful consideration of the literature reveals subtle indices of a parasympathetic system that is slow to withdraw in cases of acute concussion. La Fountaine et al.  demonstrRegulatory abnormalities are likely multifaceted and many involve more subtle disturbance of autonomic pathways than previously suggested. A more sophisticated understanding of cardiovascular-autonomic dysregulation after concussive injury is imperative for the evaluation and treatment of concussion. Current therapies for concussed patients, including some which utilize biofeedback for retraining of the autonomic system after injury , while wWhile this study provides evidence for autonomic dysregulation after acute concussion, it is limited in its lack of additional measures of autonomic function. Further investigation into the time-course of autonomic disturbance after injury is warranted and should include additional variables of interest such as blood pressure, temperature, heart rate variability, and cardiac output. In addition, the present study does not directly provide evidence for the viability of providing therapeutic interventions for recently concussed, symptomatic individuals that may directly target disruptions in autonomic function.Future study into the benefit of rehabilitating the autonomic nervous system as a focus for recovery from concussion is warranted. Future research is also needed to determine if these results can be reliably extended to populations beyond our sample group of adolescent athletes.


Animal research over many decades has established that stress and stress hormones have a profound influence on the brain, primarily on the hippocampus, amygdala, and prefrontal cortex (PFC) , hand–eye coordination, mental rotation, and spatial memory , and an impulsive responsiveness for an arithmetic task under acute stress  ; whereas risk perception about imagined risky situations increased under acute stress . On the brain level, the effect of acute stress is reflected in decreased amplitude of the P2 ERP component  time-locked to the onset of multiplication formulas during an arithmetic task ; and decreased neural efficiency during a well- trained perceptual task, i.e., more involvement of brain activation in primary and secondary somatosensory cortices is required to produce optimal behavioral performance .Behavioral results in this research topic showed the detrimental effects of acute stress on executive functions  suggested that chronic perceived stress could be one of the modulating factors influencing neuroplasticity and behavioral benefits from cognitive training. Two participants with a history of stroke and a difficulty with working memory (WM) underwent a course of auditory WM training (n-back task) for 6 weeks with training conducted 5 days each week. Only the participant who had lower level of perceived chronic stress demonstrated improvements on n-back performance after training; while neural activation in the fronto-parietal regions critical for WM was decreased for this participant, suggesting increased neural efficiency of WM. There was no such effect, however, for the participant who had higher levels of perceived chronic stress. These results highlight the need to evaluate perceived chronic stress when studying neuroplastic changes in people with stroke. Nevertheless, this conclusion needs to be replicated in future studies with a larger sample size.The relationship between chronic stress and impaired cognition has also been well reported in the literature , a mental arithmetic task , time pressure when performing an arithmetic task  and the Paced Auditory Serial Addition Test  brain state under stress Arnsten, .JW wrote this manuscript. JY revised this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, many studies to-date rely on the use fluorescent dyes or genetically encoded probes that enable visualization of a structure or cell population of interest, but do not illuminate the complexity of the surrounding tumor microenvironment. Here, we show that multi-modal microscopy combining 2-photon fluorescence with CARS can begin to address this deficit, enabling detailed imaging of the tumor niche without the need for additional labeling. This can be performed on live tumor-bearing animals through optical observation windows, permitting real-time and longitudinal imaging of dynamic processes within the tumor niche.The use of confocal and multi-photon microscopy for intra-vital cancer imaging has impacted on our understanding of cancer cell behavior and interaction with the surrounding tumor microenvironment  The dependency on complex tumor / host interactions for cancer progression has driven the need to exploit advanced imaging technologies such as confocal and multi-photon laser scanning microscopy (MPM) to image deep in tumor tissue in living animals. The increasing spatial and temporal resolution of these microscopy techniques, together with the improved imaging depth offered by multi-photon excitation, has led to studies of exquisite detail at both the cellularIncreasingly 2-photon fluorescence (2-PEF) is being combined with ‘label-free’ non-linear optical methodologies, including Second and Third Harmonic Generation  for intra-vital cancer imaging. This enables visualization of a range of structures deep within tumor tissue, including fibrillar collagen,Since its resurrection in 1999 by the Xie group at Harvard, Coherent Anti-Stokes Raman Scattering microscopy (CARS) has emerged as a promising ‘label-free’ imaging modality that can be applied to biomedical samples. CARS is a form of fast-acquisition Raman microscopy that generates image contrast using the Raman active vibrational frequency of given chemical bonds, and can be tuned over the Raman spectrum to generate image contrast from a wide variety of chemical bonds present within biological samples. The CARS process is described in more detail elsewhere.−1 which has been associated with the lipid asymmetric CH2 stretch, 2,930 cm−1 which has been associated with the protein rich CH3 stretch, −1 which has been associated with the water OH stretch. −1  resulted in a combined image akin to a pseudo H & E stain , and nonlinear interferometric vibrational imaging (NIVI), has been reported to enable identification of cells with different morphological features −1, together with 2-photon excited GFP fluorescence and SHG arising from the collagen matrix (Supplementary Movie 1). The images highlighted a number of interesting features that could be discerned by the co-generation of a CARS image. Firstly, Red Blood Cells (RBC) within tumor infiltrating blood vessels and the endothelial cells layering the wall of the vasculature were readily visualized , with the aim of label-free identification of tumor tissue and malignant cancer cells. Fig. S1 and Supplementary Movie 7). Using GFP-labeled SCC tumors immediately following surgical resection from the mouse, we acquired CARS, 2-PEF, and SHG images from a variety of regions at the tumor margins , nerve cells . CARS imaging at the invasive edge revealed an area of intense tumor / host interaction, supporting the recognized role that host cells play in assisting this process. At this point we do not know the identity of the host cell/s identified using CARS. It has been shown that tumor cells are extensively involved in re-modeling the extracellular matrix.It has also previously been reported that host cells, such as macrophages and stromal fibroblasts, can assist tumor cell invasion and intravasation by a variety of mechanisms.Supplementary Video 4). Movement of the tissue was kept to a minimum as the window was clamped in place, but breathing and other mouse movement can cause flickering artifacts across sections of the image. We focused on blood flow as an example of a highly dynamic process occurring within tumors, and asked whether CARS could provide a quantitative measure of relative blood flow, so permitting sub-classification of blood vessels. Time-lapse imaging of blood flow within GFP-labeled SCC tumors established under optical windows revealed considerable heterogeneity in the rate of blood flow, with vessels displaying different blood flow speeds branching from each other (Supplementary Movies 4 - 6), and using image thresholding we calculated the area occupied by RBCs over the total area of the blood vessel as a measure of hematocrit (the percentage of RBCs in the blood) .We next addressed whether CARS could be applied in vivo using dorsal skinfold optical observation windows, enabling dynamic measurements at moderate laser powers that do not compromise tissue viability. We found that image quality was slightly lessened when using windows, although features such as endothelial cells remained visible . These benefits are further enhanced by the added flexibility of being tunable to produce images from across the Raman spectrum, yielding detailed morphological and structural information similar to that attained from haematoxylin and eosin staining routinely used in histological examination of fixed tissue. We conclude that multi-modal CARS / 2-photon imaging represents a very promising method for imaging structure / function relationships within cancer tissues and at exposure to modest laser power.Third Harmonic Generation is a valuable technique to identify tissue structures in tumor environments, and has been instrumental in identifying that cancer cells exploit a variety of structures such as adipose cells, nerves, and muscle as tracks along which to invade and migrateThe application of multi-modal CARS imaging to the real-time study of cancer in vivo has the potential to impact in a number of important areas that remain poorly understood. Using this approach we have observed cancer cells projecting structures, akin to what others have termed invadopodia or filopodia, through the perivascular collagen network to contact the endothelial cells lining the vasculature. These invasion-associated structures are known to be rich in matrix metalloproteinase activity, and drive local degradation of the extracellular matrix (ECM).Tumor vasculature plays a vital role in not only sustaining tumor growth, but also in enabling tumor cell dissemination and ultimately metastasis. Properties including blood flow rate and hematocrit will likely have effects on local tumor cell behavior, through for example influencing local oxygenation or through increased shear forces affecting survival of intravasating cancer cells. Imaging these properties in conjunction with local tumor cell behavior may help us understand the influence of blood flow characteristics and their local impact on cancer cells and even activity of cancer driver pathways. Indeed, measurements of cancer driver pathway activation, such as Src,In summary, here we provide evidence that CARS has enormous potential when combined with 2-photon microscopy in imaging the internal environment in tissue and tumors, including in living animals. In the future, an exciting possibility is that the successes of Raman based techniques in identifying immune cell types,