Cancer research has been heavily geared towards genomic events in the development and progression of cancer. In contrast, metabolic regulation, such as aberrant metabolism in cancer, is poorly understood. Alteration in cellular metabolism was once regarded simply as a consequence of cancer rather than as playing a primary role in cancer promotion and maintenance. Resurgence of cancer metabolism research has identified critical metabolic reprogramming events within biosynthetic and bioenergetic pathways needed to fulfill the requirements of cancer cell growth and maintenance. The tumor suppressor protein p53 is emerging as a key regulator of metabolic processes and metabolic reprogramming in cancer cells—balancing the pendulum between cell death and survival. This review provides an overview of the classical and emerging non-classical tumor suppressor roles of p53 in regulating mitochondrial dynamics: mitochondrial engagement in cell death processes in the prevention of cancer. On the other hand, we discuss p53 as a key metabolic switch in cellular function and survival. The focus is then on the conceivable roles of p53 in breast cancer metabolism. Understanding the metabolic functions of p53 within breast cancer metabolism will, in due course, reveal critical metabolic hotspots that cancers advantageously re-engineer for sustenance. Illustration of these events will pave the way for finding novel therapeutics that target cancer metabolism and serve to overcome the breast cancer burden. Nicknamed the “Guardian of the Genome,” p53 (encoded by the TP53 gene) is believably the most extensively studied and most multifaceted tumor suppressor protein identified to date ,2,3. TheOriginally, altered metabolism was deemed to be merely a byproduct of cancer, rather than being considered as playing an intricate role in its support and maintenance. In the last decade, however, the resurgence of cancer metabolism research has increased exponentially. Cancer metabolism is now being viewed as a series of critical metabolic reprogramming events within biosynthetic and bioenergetic pathways, needed to fulfil the requirements of cancer cell growth and maintenance ,12,13,14This review firstly provides a brief understanding of ongoing problems with breast cancer therapy. Next, we provide an overview of the classical and non-classical roles of p53 focusing in on the emerging, critical roles of p53 in metabolic reprogramming. On one hand, p53 engages in mitochondrial cell death processes in the prevention and treatment of cancer, and on the other hand it plays an important role in cell survival and function, hence ‘balancing the pendulum between cell death and survival’. Further, we bring back the discussion to how these p53-reprogramming events may be important in cell survival and provide new avenues for breast cancer therapies.Breast cancer is the most commonly diagnosed cancer and cause of cancer deaths among women worldwide: an estimated 1.7 million cases were diagnosed in 2014, accounting for 25% of all cancers in women . One in Current therapeutics, used alone or in combination, have been successful in the treatment of breast cancer, with favorable increases in overall survival in recent decades . UnfortuTo date, breast cancer research has been heavily focused on exploring the contributions of hyperactive growth and hormone receptor signaling, inherited and sporadic gene mutations, oncogene amplification, loss of tumor suppressor proteins, and cancer heterogeneity in the development and progression of breast cancer. For instance, mutations in the breast cancer genes BRCA1 and BRCA2 as well as the TP53 gene have all been well characterized in breast cancer ,39,40,41In contrast to most cancers, mutations in the TP53 gene are only present in 20–30% of all breast cancers, suggesting that p53 function is compromised ,44 disc. AlongsiWhat key knowledge are we missing regarding the role p53 plays as the ‘guardian of metabolism’ that will help us understand breast cancer development, survival, resistance to treatment, and impact on breast cancer recurrence?The p53 protein is a transcriptional regulator that is stabilized within the nucleus upon DNA damage or oncogenic signaling. p53 regulates genes involved in cell cycle arrest, cell death (apoptosis), DNA repair, and senescence to prevent tumor development and growth ,49,50,51Much of the p53 literature is directed towards the well-characterized canonical tumor suppressor protective roles of p53, which are typically impaired upon mutation or deletion, leading to the production of many of its oncogenic forms ,39,60. OOne of the early identified major functions of p53 is to prevent accumulation of damaged DNA to potentiate genomic stability, thus inhibiting tumorigenesis . This isOne of the most well-recognized canonical p53 programmed cell death pathways is apoptosis. Nuclear p53 regulates genes associated with apoptosis in response to cellular stress signals via the classical mitochondrial apoptotic pathway . The priNecroptosis is a relatively new concept, which is a combination of necrosis and apoptosis-mediated p53 cell death and contributes to immune system regulation, contributing to managing cells during inflammation, tissue injury, and other organismal stresses such as pathogen infection [Necrosis was once regarded as uncontrolled or ‘accidental’ cell death. This idea has been superseded; necrosis is now regarded as a process of irreversible programmed cell death where apoptosis has failed and can also be triggered by p53 ,64,77. Nnfection ,79.The definition of autophagy, from an ancient Greek word, is ‘self-devouring’. Autophagy is an evolutionarily conserved, genetically mapped process that forms the “recycling facility” of the cell. This is a normal process in the cell to maintain homeostasis through programmed protein degradation and turnover of organelles within the cell. This allows the cell to degrade organelles and proteins to functional building units to fuel bioenergetic and biosynthetic processes ; alternaPerhaps one of the most under-researched roles of p53 is dormancy. It has been suggested that dormancy may be viewed as a natural byproduct of evolutionary mechanisms, where dormant cells are present in both healthy individuals and survivors of cancer . The dorp53 is a decision maker in cellular senescence, quiescence, or dormancy. Senescence is defined as a state of irreversible cell cycle arrest in which cells display an inability to proliferate or respond to growth factors ,96,97,98Only discovered in 2012, ferroptosis is a novel non-apoptotic form of cell death, characterized by lethal iron-dependent accumulation of lipid ROS, in a caspase-independent manner . MorpholIn summary, mitochondria are core powerhouses for metabolic reactions that drive cellular reprogramming through diverse pathways and mechanisms. Classical roads in p53 tumor suppression lead to mitochondrial de-regulation or inactivity, whether it be for cell destruction or temporary/permanent inactivation, respectively. Alternatively, p53 is linked to the quality control mechanisms of the mitochondria and its genome, with projected roles in the organelle fusion–fission process  mice displaying acute alterations in mitochondrial morphology and reduced respiratory capacity . This coWhile this review is focused on the functions of the major p53 isoform, some of the smaller alternatively spliced p53 isoforms have been shown to enhance p53 target expression or inhibit p53 wild-type function , especiaAs mentioned, p63 and p73 do exhibit similar effects or share overlapping functions with p53. These include TAp63 and TAp73’s ability to inhibit glycolysis; the function is similar to that of p53, although the mechanisms vary. Both TAp63 and TAp73 have been shown to induce islet amyloid polypeptide to inhibit glycolysis via inhibiting HK2 . The TApBreast cancer metabolism is diverse and very much dependent on hormone fluctuation . MetabolAs mentioned in p53 is commonly silenced in breast cancer by loss of upstream/downstream mechanisms. A key natural, endogenous regulator of p53 is the tumor suppressor p14 alternative reading frame (p14ARF). The p14ARF protein is an upstream positive regulator of p53 through its binding to HDM2 and preventing p53 degradation, thus stabilizing p53 expression and activating p53 function ,167,168 p14ARF is activated in response to adverse environmental stimuli to prevent hyperproliferation of cells to prevent cancer. The upstream p53 regulator, p14ARF, is silenced by methylation, deletion, or mutation in many breast cancers ,172,173.Although mitochondrial dynamics has been observed to be influenced by p53 , there iThis p53-attenuated apoptosis aligns nicely with a series of studies, illustrating that the estrogen receptor (ERα) interacts with p53, leading to an intrusion in p53 downstream signaling targets ,178,179.Increasing evidence supports the notion that dysregulated metabolism confers drug resistance and recurrence in breast cancer . Breast An interesting advancement in our understanding of maladaptive metabolism and cancer came from studies in exercise physiology. Exercise, which alters cellular metabolism, has long been known to reduce breast cancer risk . AerobicThe studies highlighted in this review reveal that p53 has a plethora of functions in normal and cancer metabolism beyond the classical mitochondrial death syndrome. Our major research focus is on understanding hormone-dependent cancer origin, treatment resistance, and recurrence in breast cancer. It is important to understand the role of p53 in the regulation of cancer metabolism as well as its underlying mechanism and how it applies directly to breast cancer. The mitochondrial targets of p53 that are unfolding would provide a platform to further investigate the role of p53 in modulating mitochondrial metabolic pathways. Future studies would include confirming p53 interactions with the aforementioned mitochondrial proteins, along with formulating a cohesive timeline for the changing metabolic phenotypes associated with p53’s evolving role as a master metabolic regulator in normal mammary cells and breast cancer cell evolution. The ultimate goal will be to elucidate the metabolic functions of p53 within breast cancer metabolism, which will reveal critical metabolic hotspots that cancers advantageously re-engineered for sustenance. Full understanding of these events would pave the way for the development of novel therapeutics targeting breast cancer metabolism and eventually new strategies to fight breast cancer.


TP53 mutation is more frequent in these cancers among US males than females, with poorest survival correlating with its mutation. Second, numerous X-linked genes are associated with p53, including vital genomic regulators. Males are at unique risk from alterations of their single copies of these genes. High expression of X-linked negative regulators of p53 in wild-type TP53 cancers corresponds with reduced survival. Third, females exhibit an exceptional incidence of non-expressed mutations among p53-associated X-linked genes. Our data indicate that poor survival in males is contributed by high frequencies of TP53 mutations and an inability to shield against deregulated X-linked genes that engage in p53 networks.The disproportionately high prevalence of male cancer is poorly understood. We tested for sex-disparity in the functional integrity of the major tumor suppressor p53 in sporadic cancers. Our bioinformatics analyses expose three novel levels of p53 impact on sex-disparity in 12 non-reproductive cancer types. First,  There is disproportionally high cancer prevalence in males. Here, the authors analyse the tumour suppressor p53 in sporadic cancers, highlighting a higher incidence of its mutation in males. Males are further disadvantaged by a failure to shield against the expression of damaged X-linked genes in p53-networks. These factors likely contribute to sex-disparity. Cancer sex disparity is evident across multiple non-reproductive, sporadic cancers1. Male lifestyle has been blamed for greater exposure to carcinogens4, however, it is likely that both environmental and genetic factors influence cancer incidence. Emerging molecular and physiological peculiarities that appear to affect disparity in animal studies, prompted NIH to demand preclinical evaluation in both males and females5. Intriguingly, not all animals develop cancer, and in elephants, protection of both sexes is attributed to multiple copies of the TP53 gene6. The lack of cancer in elephants also argues that p53 can override an impact of hormones on cancer sex disparity. Critically, the single TP53 gene copy is outstanding as the most commonly altered gene in human cancers7. Further, in a compound mutant p53 mouse model that we generated, males developed more aggressive cancers and reduced lifespan than females8. This triggered us to investigate the connection between p53 and cancer sex disparity in non-reproductive cancers.Cancer incidence and death rates are higher in males than females9, which promote temporary arrest to facilitate repair, or permanent growth inhibition, including senescence, apoptosis10 and ferroptosis11. P53 also targets core-regulatory molecules in metabolic pathways , immune responses14 and aneuploidy15. Remarkably, the activities attributed to p53, include the four key processes recently identified through gene expression analyses to link to cancer sex disparity: (1) immune response, (2) apoptosis and cell cycle, (3) metabolism-related and (4) DNA-repair and p53-pathways 2. In healthy individuals, inherited genetic variants in the standard p53-pathways were more frequent than for all other pathways and linked to cancer exclusively, and not other diseases16. These findings highlight the importance of p53 and its autosome partner proteins as the greatest natural deterrent against cancer. Whether these p53 functions are distinct between cancers in males and females remain unanswered.Tumour suppressor p53 is a transcription factor that crucially integrates stress signals into protective cellular responses. Stresses such as DNA damage, trigger activating-modifications in p5317. Breakdown in p53 function is attributed almost equally to TP53 gene mutation7, or excessive levels of its key negative regulators: MDM2 and MDM418. TP53 mutation not only strips away its tumour-suppressive capacities19 but also confers neomorphic, cancer-promoting properties, referred to as gain-of-function . Discrepant rates of cancer and related mortality between males and females provoked us to investigate whether the tumour-suppressive ability of p53 is equally competent between the sexes. In this study of non-reproductive cancers, we identified three novel layers of risk for cancer sex disparity that are critically affected by p53 status and subject to the function and expression of its X-chromosome interactors.P53 activities and expression are normally tightly regulated, and a universal signature of human cancers is the loss of effective p53 tumour-suppressor activity21. To explore this disparity, we examined whether mutation of the tumour-suppressor gene TP53 differs in frequency in the human population, between male and female somatic cancers. This human sex-disparity study was prompted by our discovery of a link between p53-functionality and cancer aggression in male mice8.The most common somatic cancers are more frequent in malesTP53 mutation between males and females is currently not feasible as: (1) it is an age-dependent parameter, which requires sampling across multiple age-brackets; (2) sampling every organ is impractical and (3) even with the advances in detecting circulating tumour DNA, the current technology lacks sufficient sensitivity.Directly measuring the relative general population cancer risk for TP53 mutation incidence between male and female cancers. First, the National Cancer Institute’s Surveillance, Epidemiology, and End Results program (SEER data21) provides the comprehensive cancer incidence for the overall US population. This cannot be obtained from the second data set, The Cancer Genome Atlas (TCGA22). Rather, TCGA offers the rates of cancer-associated TP53 mutations (or pathogenic mutations) from random cancer sampling across the sexes, but does not claim to be representative of population incidence. The pathogenic definition is built on the observation that most cancer-associated TP53 mutations occur in its DNA-binding domain and cause interruption of p53 function10 (as defined in detail in the Methods section). From TCGA, we considered all TP53 exome mutations with functional impact to be pathogenic. Specifically, we examined our hypothesis that TP53 mutation frequency is distinct between the sexes by applying a probability calculation for 13 non-reproductive cancers. Explicitly, this represents all the cancers with at least five TCGA cases in all four groups: female mutant TP53, female wild-type (wt) TP53, male mutant TP53 and male wt TP53. Our calculations show for the first time that males have a higher probability of developing cancers with TP53 mutations than females, for the majority of non-reproductive cancers: subsequently referred to as the 12 disparity cancers  are disrupted in cancer, either by TP53 mutation or by malfunction in this p53 network. The possibility that the p53 network is not equally functional between males and females is unexplored. We chose to search for connections between p53 and the X chromosome because: (1) numerous tumour-suppressor genes are encoded on the X chromosome26, but are under-studied for connection to p53 and sex disparity; (2) X-chromosome genes are not yet incorporated into established p53 networks (eg. KEGG16); (3) X-chromosome analysis is frequently avoided in sporadic diseases because X-inactivation (Xi) silencing in females complicates data interpretation (reviewed in refs. 28) and (4) males are at greater disease risk than females from X-chromosome mutations (reviewed in ref. 29).The normal strict control of the tumour suppressor p53 and its multiple downstream effector pathways  database tool31. A reproducible p53-STRING set of 90 genes was defined (with confidence limits ≥ 0.3) from among the ~800 coding genes on the X chromosome . At the whole-exome level, only minor differences in the total number of gene mutations per patient  were evident between the sexes in the disparity cancers, excluding the Y chromosome . This can be seen for all cancers together in Fig. p-value ≈0; Supplementary Data 32, which are largely pseudoautosomal (reviewed in ref. 33) with a few exceptions3. The identity of the X-chromosome copy that is silenced by Xi cannot be deduced from TCGA exome data. Equally, the X-chromosome gene mutations that are expressed cannot be identified from exome sequencing. To identify which X-chromosome exome mutations were expressed in male and female cancers, it was necessary to search for the corresponding mutations in their mRNA.To search for evidence of sex disparity in the p53-STRING set, we analysed TCGA data for somatic exome mutations (DNA) and their expression (mRNA); as outlined in Fig. ome Fig. . In contVariant allele frequency (VAF) is a commonly used metric employed to identify  somatic variants at the DNA level. To date, a measure for RNA expression of DNA mutations has not been formally assigned. To fill this gap, we created a novel concept: RNA-mutated allele frequency (RMAF), which we define as the mRNA equivalent of VAF. Explicitly, RMAF is a measure of the relative frequency with which a mutation in DNA is expressed in mRNA, relative to the total expressed mRNA (refer to Methods).We predicted that for the X chromosomes, the RMAF of females would be lower than for males. This was founded on the expectation that for males, all mutations from their single X chromosome can be expressed. For healthy females, expectation of lower RMAF was founded on the prediction that mutations are evenly spread across the two individual X chromosomes, and that X inactivation is completely random. However, in contrast to healthy females, in cancers we could not presume that this would be the case. This led us to formerly test RMAF across the 12 disparity cancers for females and males.p-value ≈ 0). To tease this further apart, we interrogated which specific gene mutations were expressed or not, in male and female cancers.We found that in female cancers, more than half of their X-chromosome exome mutations were not transcribed into their corresponding mRNA . This is displayed for the 12 disparity cancers altogether in Fig. These discoveries define our second cornerstone that: the X chromosome encodes vital protein regulators of genomic fidelity that are linked to p53 function; and males are at higher risk of expressing mutations of these genes due to their obligatory mono-allelic expression, in contrast to the chromosomal choice in females.Given the lower incidence of non-reproductive cancers in females, we hypothesised that they are preferentially protected from the expression of cancer-risk gene mutations, compared with males. As we were unable to test this in healthy people, we examined data from cancer biopsies, aware that we were searching for remnants of a broken protective system. We compared the expression of X-chromosome mutations between cancers of females and males, where X-chromosome exome mutations that were not detected at the mRNA level  only in females.The incidence of NEMs reached significance only in females .In the disparity cancers, mutations in 268 genes (~1/3 of the X-linked coding genes) were significantly less expressed in cancers of females than males. GO analysis of these 268 genes identified pathways of protein acetylation (including histone acetylation); histone modification and negative regulation of both organelle organisation and microtubule depolymerisation . GO analysis of these 50 genes identified their involvement in fundamental pathways: histone modifications , protein localisation (to chromosome and telomeric region), regulation of telomere maintenance, and cell cycle DNA replication as key pathways  were numerous , and statistical significance was defined by an adjusted 34). Literature mining led us to identify 11 negative regulators of wt p53 among the p53-STRING set: UBE2A (RAD635), MAGEA236 and UTP14A37, CDK1638, PPEF139, HUWE140, CUL4B41, DDX5342, NOX143, PMSD1044 and TAF146. We tested the relative expression levels of these genes in tumours and their matched normals, for each cancer type separately, with additional stratification according to wt TP53 status and patient sex. We undertook these studies on 11 of the 12 disparity cancers, but excluded LGG, due to a lack of matched normal tissue.We chose to focus on the X-linked negative regulators of wt p53. We postulated that high expression of these negative regulators could reduce wt p53 activity, in an analogous manner to high levels of the MDM proteins, which have proven to be oncogenic , MAGEA2  and UTP14A (which binds p53 and promotes its degradation37) for each cancer, in a wt TP53 context. In this individual cancer approach, we did not further stratify for patient sex to avoid diminishing statistical power, which is a risk associated with too few patients per cohort. We identified significantly reduced survival in a total of five cancers types that expressed high levels of one of these three genes , we tested whether their expression levels could be linked to the measurable biological outcome of survival in cancers with wt nes Fig. .KDM6A, DDX3X  and UBA1 were significantly differentially expressed among the p53-STRING set. For comparison, we extended this test to the healthy samples of the 1000 genomes project, which are lymphoid in origin. Among these, statistically significant differential expression (DE) was identified between the sexes for 13 of the p53-STRING set genes . All 13 genes were expressed at higher levels in females than in males , however, the picture is far from clear. Guided by our previous study demonstrating sex disparity in a compound mutant p53 mouse model8, together with the unprecedented incidence of TP53 mutations in human cancers, we tested the contribution of p53 to cancer sex disparity. We identified three levels of connection between p53 and sex disparity in somatic non-reproductive cancers. For the first time, our statistical analyses predict a significantly higher frequency of cancers with mutated TP53 in males than females, for 12 common sporadic cancers, across the US population (as determined through the integration of TCGA and SEER data). The exception is KIRC, which was more common in males than females, but with exceptionally few TP53 mutations. Notably, in KIRC, although TP53 mutation is infrequent, poorest survival corresponds with high levels of MDM2, which emphasises the importance of evading p53 tumour suppression also in this disease48. This outlier dismisses a direct correlation between cancer prevalence and TP53 mutation as its explanation. These findings are in line with somatic TP53 mutations accumulating preferentially in tissues arising from the embryonic layers of the ectoderm and the endoderm, in contrast to germline TP53 mutations which at least for non-reproductive cancers, preferentially occur in tissues derived from the mesoderm23, which includes the kidney.The well-documented, higher incidence of lethal, non-reproductive cancers in males than females is attracting a growing interest into its underlying causes . It is also relevant to note that TCGA is comprised of primary disease, regardless of whether originating from resections or biopsies, sampled from metropolitan centers. SKCM is the only exception, but most of these tumours are wt TP5350. The nature of these samples may have contributed to an underestimate of TP53 mutation incidence in the population, as metastatic disease is frequently associated with mutation of this tumour suppressor (reviewed in ref. 51) and non-metropolitan disease is frequently more severe52. In sum, the incidence of TP53 mutation in the US population may be even higher than our calculations predict, with males at greater risk than females.It is pertinent to our study that the ethnic population composition of TCGA and SEER correlate remarkably closely. The verified TCGATP53 mutation is exacerbated by patient sex. This is in line with the specific instance of glioblastoma astrocytes, in which components of the p53 and RB pathways were recently reported to contribute to sex disparity54. This could result from a number of causes. One possibility is that male non-reproductive tissues (pre-cancer) may be more exposed to carcinogens and/or prone to spontaneous pathogenic TP53 mutation than their female counterparts. Alternatively, females may have a better tumour-suppressive capacity due to a greater ability to clear or restrain cells with TP53 mutations, compared with males. Testing these hypotheses will require prospective sampling for TP53 mutations in large data sets from healthy populations, across multiple tissues, with temporal follow-up. Currently, this information is unavailable. Significantly, among the disparity cancers, the occurrence of TP53 mutation corresponded with the worst survival outcomes. This suggests that TP53 mutation is an overriding oncogenic event. Indeed, many of the regulatory and auto-regulatory loops with p53 are operative only in the context of wt p5355. As males have higher cancer incidence and as we demonstrate, a greater frequency of TP53 mutations than females, this is a likely contributor to their poor cancer survival statistics. While this study is restricted to the US population, similar statistics would be expected across the western world.Our findings indicate that tissue-specific vulnerability to somatic 56). Accordingly, X-linked inherited diseases manifest predominantly in males, frequently at a young age. Notably, with our colleagues we recently identified that p53 drives Xi in females during development57, and its absence in females is fatal, largely associated with neural tube defects58. In contrast, most cancers arise from somatic mutations in the aging population, where we predicted alternative mechanisms must account for lower cancer incidence in females. We uncovered a novel p53-X-chromosome network  corresponded with poorer survival in five different cancers  of the 11 p53-STRING set genes reported to negatively regulate p53, exposed a higher level of expression in cancers with wt ues Fig. . This prers Fig. . These f59, we did not expect that all cancers would be impacted equally by these genes. It is expected that in different cancer types and for each individual cancer case, different combinations of genes will be involved, with different permutations of deregulated expression and/or mutations. A particular permutation would be influenced by the status of TP53, whether these X-linked genes are either positive or negative regulators/effectors, and further whether these genes themselves have undergone mutation.As each cancer develops along a different trajectory of mutationsTP53 status is generally a good prognostic marker for response to conventional genotoxic drugs due to their reliance on wt p53 activation (reviewed in ref. 60). In the context of high expression of negative regulators of wt TP53, as exposed in our study, p53 activation is likely to be ineffective, leading to poor response, and consequently to poor survival. Our findings suggest clinical pertinence, where altered protein expression of negative p53 regulators such as these offer potential value as prognostic markers . Also when overexpressed, these genes represent targets for therapy in these male dominated cancers. A specific example is HDACi trichostatin A plus etoposide administered to treat cancers with elevated MAGEA247. Our study lays a foundation for developing rational strategies to treat cancer patients on the basis of expression levels of these regulators in the context of TP53 mutational status. The greater prevalence of these cancers in males, predicts disproportionate application of these tools for prognosis and treatments between the sexes.An interesting translational angle arises from this data. Wt 64. At the other extreme, elimination of KDM6A is proposed as an independent marker of prognosis for pancreatic cancer65. DDX3X protects against replication stress through its regulation of DNA damage repair66. Pertinently, a peculiar male vulnerability to DDX3X mutation has just been reported for melanoma67, a disease where TP53 is predominantly wt. In sum, the cancer protective potential of KDM6A and DDX3X in normal females preferentially, is emphasised by our data, which also reinforces their particular mutation risk in male cancers. UBA1 is also essential for proper response to replication stress and DNA damage68. Specifically, UBA1 localises to DNA breaks and mediates ATR/Chk1 signalling69. A vital ancestral role for Uba1 is supported by its highly conserved counterpart in Drosophila, which is implicated as a tumour-suppressor gene. Apoptosis is defective in the absence of Uba1 in Drosophila70, consistent with weak alleles of Uba1 protecting from cell death and strong Uba1 alleles driving cell cycle arrest71.In contrast to these cancer risks, in females, both in the cancer-matched normal TCGA samples and the 1000 genomes project, we identified elevated expression of a number of p53-STRING set genes that are noted for tumour-suppressive capacity. Their higher levels in females are predicted to afford them a more robust innate cancer protection compared with males, who were identified with inherently lower expression. The most prominent of these are candidate tumour suppressors; KDM6A (lysine demethylase 6 A), DDX3X  (dead-box RNA helicase 3) and UBA1. Notably, KDM6A inhibits EMT by antagonising TGF-β induced genes, which are instrumental in this process72. The exceptional abundance of female NEMs among the p53-STRING gene set, across the 12 disparity cancers, suggests that this phenomenon is not simply random . MED12, a key component of the pre-initiation complex that promotes p21 transcription75; HUWE1, an E3 ligase of p53 that regulates DNA damage response to UV damage76; and ATRX, that works with p53 to mediate DNA repair77. Breaching these defence barriers in females during cancer onset is predicted from the identification of expressed mutations among the p53-STRING set in cancer samples. Intriguingly, GO analysis of cancer EMs, including many of the p53-STRING genes, exposed distinct pathway disruption between females and males.Our third novel link of p53 to sex disparity entails the unique protection of females from expression of mutations from a set of X-linked genes connected to its function. In marked contrast to the males, most X-chromosome exome mutations in the female cancers were not detected at the mRNA level. This phenomena is distinct from the X-chromosome loss identified in solid tumours with aneuploidydom Fig. . The p53dom Fig. . We arguTP53 mutation is greater than females, across 12 disparity cancers in the US population. Poorer survival outcomes for patients with mutant TP53 cancers is consistent with TP53 status contributing to cancer sex disparity. Second, we uncovered a set of X-linked genes encoding proteins that interact with p53. Due to the distinct mode of X-chromosome expression between the sexes, males are peculiarly vulnerable to the consequences of X-linked gene alterations compared with females. These two levels are overlaid by a third, exclusively female barrier that restricts expression of X-linked somatic gene mutations, particularly for the p53-STRING set. We propose that the combination of all three levels amplify the impact of p53 on cancer sex disparity and that disruption of this network defines a disproportionate male cancer risk.Our studies discovered three levels of connection between p53 and cancer sex disparity. First, the frequency of males with Patient sex is currently not a major dictator of therapeutic choices. Our findings expose differential molecular sensitivities between the sexes in cancers. They offer scope for explaining sex discrepancy in treatment efficacy. Importantly, we predict that male and female cancers are likely to benefit from distinct treatment options, cancer surveillance and prevention. While our study focuses on cancer, it exposes a potential defence phenomenon for females against other sporadic diseases and syndromes caused by X-chromosome mutations.22 patient data were accessed and processed for analysis guided by the approach of Yuan et al.2. Briefly, for proper statistical analysis, TCGA cohorts must be appropriately adjusted for cofounding factors. This was achieved using a re-weighting system based on propensity scores calculated using the algorithm developed by Rosenbaum and Rubin78 and adapted by Yuan et al.2. Specifically, TCGA clinical data  for the 12 cancer types were obtained from The Broad Institute GDAC Firehose79. Initially, propensity scores based on patient sex were calculated using logistic regression. Then, using these scores, a matching weight scheme was performed to re-weight the samples80. This scheme was adopted to balance the propensity scores and in turn the covariates. A strict checking loop was implemented throughout this process to perpetuate continuous revision until all covariates were balanced between males and females. The weights calculated by the propensity scores algorithm were used in a number of statistical models to compare and assess significance for the different molecular data  using patient sex as the independent variable.TCGA79 and analysed in R. Kaplan–Meier survival curves were generated using the top and bottom medians of gene expression, and differences in survival distributions between medians were tested using a log-rank test.In order to assess the prognostic value of the expression of the p53-STRING set genes among the disparity cancers, normalised gene expression and survival data from the TCGA data set were accessed using The Broad Institute GDAC Firehose81; annotated mutations were analysed. Mutations classified as “3’UTR”, “5’UTR”, “Frame_Shift_Del”, “Frame_Shift_Ins”, “Missense_Mutation”, “Nonsense_Mutation”, “Splice_Region” and “Splice_Site” were used to focus on mutations with the potential of having a more profound functional impact. Patients with available sex annotation and <15,000 somatic mutations were analysed.For these non-reproductive cancers, a full list of exome level somatic mutations was downloaded from Genomics Data Commons Data Portal and GDAC Firehose, more precisely, the Oncotatorhttp://www.cancergenomicscloud.org/). For each X-chromosome DNA mutation, we defined a new metric: the RNA-mutated allele frequency (RMAF), an equivalent to variant allele frequency (VAF) commonly used to call mutations at DNA level , a wrapper of htslib and samtools82. Based upon the relative RMAF distributions in the male and female cancer samples, we established two thresholds in an effort to account for tumour purity and heterogeneity and classified mutations with an RMAF greater or equal to the threshold of 0.75 as EMs and those less or equal to 0.2 as NEMs.To assess whether DNA mutations in the X chromosome were transcribed into RNA, accounting for Xi, we classified mutations into two kinds: NEMs and EMs, and tested differences in numbers between males and females. For each cancer type, we combined the exome somatic mutation list with the corresponding TCGA RNASeq BAM files using the Cancer Genomics Cloud platform by Seven Bridges . We calculated a statistic for each of the 561 genes with NEMs, using the propensity score weightings which, under the null hypothesis: no association between a patients’ sex  and no association with the status of the expression of the specific gene mutation (expressed versus not) would be asymptotically distributed as chi-squared on one degree of freedom. By combining these statistics across all the genes and test for differences in NEMs numbers between the p53-STRING set and other X-chromosome genes, we collected the associated chi-scores: the signed square roots of the chi-squared statistics, with the sign determined in the same way for all genes. We assigned a plus sign if the proportion of NEM in females was larger than the proportion of NEM in males; and the converse was discriminated with a minus sign. These chi-scores should be asymptotically distributed as standard normal  under the null hypotheses, for both the p53-STRING set and other genes. We tested (Wilcoxon rank test) the difference in distribution between these two groups and found that p53-STRING set genes host significantly larger number of NEMs compared with other genes in the X chromosome.After quantifying NEMs and EMs, we applied the propensity score algorithm and tested for significant differences in NEMs and EMs numbers between male and female patients. We applied a weighted chi-squared test and highlighted genes that had adjusted 79. Reads were converted to counts per million (CPM). Lowly expressed genes (fewer than 5 CPMs) were discarded. CPM counts were adjusted and normalised by library size using the R package edgeR83. By accessing the clinical data and mutation lists, two groups were analysed: Wt-TP53 Tumours and Mt-TP53 tumours , each one stratified by patient sex. In addition, RNAseq count data from 465 healthy lymphoblastoid cell lines  was downloaded from the 1000 Genomes project (https://www.ebi.ac.uk/arrayexpress/experiments/E-GEUV-1)84. Normalisation was also performed using edgeR83. By accessing the available clinical data two groups were analysed: males and females. Differences in gene expression between these groups were computed with the R package Limma85, and significant changes were inferred by obtaining p-values and by adjusting for multiple hypothesis testing (Benjamini–Hochberg adjustment), false discovery rates were calculated. Propensity scores were also considered in the differential expression analysis.Raw gene-level counts for each cancer type were downloaded from The Broad Institute GDAC FirehoseTP53 tumours), the corresponding gene lists ranked by fold change were analysed for enrichment of negative X-linked p53 regulators using the fgsea package in R86. Normalised enrichment scores (NES) and adjusted p-values (adjustment by FDR) were used to highlight the enrichment of these genes in wt-p53 tumours.To further investigate the role of p53-STRING genes in the disparity set, after performing differential expression analysis  analysis of the gene sets was performed in ClueGO Cytoscape. The following parameters were selected: GO tree interval between levels 3 and 8; GO terms with at least three proteins and 4% of proteins; a kappa score of 0.4 and only GO terms with a 31 was adopted for analysis of X-linked genes that link to TP53 . TP53 was coincidently included in the gene input, to define association. Parameters of selection included: textmining, experiments co-expression, gene-fusion, and co-occurrence. We ignored genes that sorted into a confidence score <0.3.Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database toolSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5


Tp53 compromise therapeutic response, due either to the dominant-negative effect over the functional wild-type allele; or as a result of the survival advantage conferred by mutant p53 to which cancer cells become addicted. Thus, targeting mutant p53 represents an effective therapeutic strategy to treat over half of all cancers. We have therefore generated a series of small-interfering-RNAs, capable of targeting four p53 hot-spot mutants which represent ~20% of all p53 mutations. These mutant–p53-specific siRNAs (MupSi) are highly specific in silencing the expression of the intended mutants without affecting wild-type p53. Functionally, these MupSis induce cell death by abrogating both the addiction to mutant p53 and the dominant-negative effect; and retard tumor growth in xenografts when administered in a therapeutic setting. These data together demonstrate the possibility of targeting mutant p53 specifically to improve clinical outcome.Mutations in  A decade of pan-cancer genome sequencing efforts have resulted in the identification of a large number of genomic alterations across almost all cancer types , 2. ThisAn ideal drug against a mutated protein would therefore be one that will only affect the functioning of the mutant form, without any effects on the WT version. However, up till now and to our knowledge, no drugs or molecules have been generated that are capable of such high specificity. A very recent report has identified a much more potent inhibitor of mutant c-Kit, with dramatically reduced effects on the WT proteins . NonetheTp53 (hereinafter referred to as p53) occur with the highest frequency [p53 can occur almost on all of its 393 residues [p53 in the germ line lead to cancer predisposition, as exemplified in the Li−Fraumeni syndrome, and in many model organisms [p53 have been associated with poor response to therapy, due often to the dominant-negative (DN) effects of the mutant protein over the remaining WT protein, which could be ameliorated by reducing the expression of the mutant form [Among the mutated genes in cancers, mutations in the tumor suppressor gene requency , 2, 11, requency , 13. Mutrganisms –18. In aant form –25. Finaant form –32. Howeant form , 32.From the therapeutic perspective, mutant p53 would therefore be expected to be the prime target to treat cancers. However, the abysmal lack of enthusiasm to develop reagents to target mutant p53 stems from its persona as an “undruggable” transcription factor, which has hampered progress for decades . Moreovemutant p53-specific siRNAs (referred to as MupSi), and demonstrate their ability in selectively silencing the expression of the intended mutant p53 forms, without cross-reactivity against other mutants or against the WT protein. Furthermore, these siRNAs have been used to demonstrate the amelioration of the DN activity of mutant p53 over the WT form, thereby sensitizing tumor cells to therapeutic treatment. Moreover, they also abrogate the addiction of cancer cells to mutant p53 for survival, leading to cell death of tumor cells expressing mutant p53. Finally, we show that these siRNAs can be used as therapeutic agents in patient-derived xenografts, and are capable of retarding tumor growth in vivo without resulting in any side effects or organ toxicity. Together, these data demonstrate that mutation-specific siRNAs can be routinely generated, and this approach could therefore be expanded to target the other major tumor suppressors and oncogenes that are altered in various pathological conditions.Small-interfering RNAs (siRNA) have been developed for many targets to silence their expression successfully , 34, andp53 alleles, without having an impact on WT p53 expression. To this end, we generated a library of a large number of siRNAs, by performing sequence walks such that the position of the mutant nucleotide was varied with respect to the entire siRNA strand. All the siRNAs were transfected in a series of H1299-based isogenic cell lines which stably expressed the various p53 mutants, or the temperature-sensitive (TS) WT p53 [We embarked on generating siRNAs that will be capable of only silencing the mutant ) WT p53 , and datWe therefore evaluated the efficacy and specificity of the selected siRNAs on a panel of 17 different cancer cell lines that express either WT or the various p53 mutants  or heterozygous for mutant p53 (p53+/R248W) [+/R248W cells but not in p53+/− HCT and RKO cells, indicating specificity  . Transfeity Fig. . Concomills Fig. . Moreoveted Fig. , collect+/− vs. p53+/R248W cells in untransfected and scrambled shRNA transfected: 50.9 and 50.3 vs. 14.9 and 11.3; in HCT cells: 61.1 and 51.2 vs. 32.1 and 28.6), highlighting the DN effects   Fig. . These dp53, indicating that the specific shRNAs are effective in silencing the expression of the respective mutant p53 in vivo during tumor growth  expressing the scrambled or the respective mutant-specific shRNAs. Cancer cells which express the various p53 mutants and were transiently infected with viral particles expressing the scrambled shRNA grew to a large volume over time, whereas cells expressing the respective mutant p53-specific shRNAs were markedly retarded in growth in vivo Fig. . Immunohwth Fig. .Fig. 7Mu3, mice were treated twice weekly with scrambled siRNA or mutant p53-specific siRNA that was delivered intravenously in nano-liposomes, which have been shown to effectively deliver to tumors [In the next model, we examined if the growth of R249S mutant expressing, patient-derived triple-negative breast cancer tumors (PDX) can be influenced by the siRNAs utilized in a therapeutic treatment protocol. In essence, PDX tumors were grown orthotopically, and when they reached 170 mmo tumors , 43. TreThe results presented here demonstrate that siRNAs that are highly specific and capable of distinguishing one nucleotide change can indeed be regularly generated, and highlight their utility in targeting four p53 hot-spot mutants. These four p53 mutants account for about 20% of all p53 mutations found in cancers , and tarThe era of precision medicine has promoted the development of many drugs that are specific for the highly active mutant forms of proteins, and has resulted in huge clinical benefits, as in the case of leukemia with Bcr-Abl translocations (treated with Imatinib); and lung cancers with EGFR mutations (gefitinib), amongst others , 46. WhiIn this respect, we have recently developed monoclonal antibodies against three of the above described four hot-spot p53 mutations , as partp53, supporting the notion that with sufficient screening, nucleotide-specific siRNAs can be generated and evaluated in clinical trials, as has been demonstrated with the mutant keratin-specific siRNAs to treat the skin disorder Pachyonychia Congenita [siRNAs have been generated successfully to silence gene expression and has been extensively used in research, and also been translated to the clinical setting , 49. Mosongenita .We have chosen mutant p53 to demonstrate the ability to generate nucleotide-specific siRNAs, as it is the most mutated gene across all cancers. Importantly, not all p53 mutants behave similarly , and thuIn conclusion, we have demonstrated the generation and characterization of siRNAs that are extremely specific for the various p53 mutants that are highly represented in human cancers. These data could be translated directly for clinical evaluation with the appropriate delivery mechanisms, some of which are in clinical trials . Furtherp53 alleles generated in our screen was used as a positive control for pan-p53 targeting. Control scrambled siRNA had no bio-informatically predicted sequence target in the human genome and was used as a negative control.A large library of siRNAs was designed to target p53 hot-spot mutations , out of which eight were shortlisted for the four mutants for further characterization (si-1−8). One siRNA against all BamHI at the 5′ and HindIII at the 3′). The coding region for each hairpin is nested within a single oligonucleotide (upper oligo: 5′-AAGCTTTN [cells. Clones with the shRNA insert were selected and purified before transfection.shRNA target sequences were designed to be homologous to the siRNA sequences afore-described. The pRetro-Super vector contains a human H1 polymerase-III (pol-III) promoter for shRNA expression , 53. EacAAGCTTTN –29 (sensAAGCTTTN –29 (antiAAGCTTTN –29 (sensAAGCTTTN –29 (antiSupplemental TextSupplemental Figure and Table


Dear Sir,89Zr-immunoPET” in EJNMMI Zr4+ chelators” is no more correct.During the manuscript review process, DFO*-NCS was made commercially available at ABX . Therefore, the following statement in the introduction “However, in the absence of an improved, clinically applicable chelator, there is room for more efficient [The stability assays were performed in PBS at pH 7.4.The amounts of DFO and EDTA used for the challenge experiments were calculated based on the amount of chelators.DFOcyclo* was used as a racemate in the experiments reported in the article.89Zr]Zr-DFOcyclo*-trastuzumab was compared with [89Zr]Zr-DFO-trastuzumab, of which the results are depicted in Fig. 4c. Once we observed an improved in vivo stability of [89Zr]Zr-DFOcyclo*-trastuzumab, a subsequent in vivo study was performed to investigate its stability compared with [89Zr]Zr-DFO*-trastuzumab as well . Although aimed at similar experimental conditions, the use of a freshly thawed cell line and new mice could have caused heterogeneity between the two experiments , which could explain the slight differences in tumor uptake, blood kinetics, and uptake in other organs.The results depicted in Figs. 4c and 5a are based on two different sets of experiments. Initially the in vivo stability of [Following the publication of our article entitled “Direct comparison of the in vitro and in vivo stability of DFO, DFO* and DFOcyclo* for n EJNMMI , readers


Results: The response rate was 91%. There was no significant correlation between patients’ preferences and their race, age and gender (p > 0.05). The majority of participants (74.3%) would prefer to use a DD in their next session (p < 0.001). This preference negatively correlated with the time required to place a DD and the duration of the current visit (p < 0.001). While most of those who would prefer to use a DD in their next visit were pleased with how it was placed in the current session (76.6%), most of those who would not do so (66.7%) were uncomfortable. Overall, the highest proportion of participants (40.2%) reported that prevention of instrument swallowing was the most important advantage of DD isolation (p < 0.001). Conclusions: Overall, DD isolation for RCT is generally well accepted by patients regardless of their country of origin, gender, education and awareness of its advantages. Patients’ safety was the most attractive advantage for patients to the application of the DD. The time required to place the dental dam and first visit experience in placing the DD affect patients’ future preference.Background: A number of factors  may influence patients’ preferences regarding dental-dams. In general, patients accept placing it and that it must be used for teeth isolation during endodontic procures for the sake of patient safety and infection control. Objectives: The aim of this study is to investigate preferences and experiences of patients using dental-dam (DD) isolation during root canal treatment (RCT) and to explore influencing factors among the residents of Madinah Munnawara, Saudi Arabia. Methods: Following an ethical approval and a pilot study, a self-administrated questionnaire was distributed to 305 patients attending endodontic clinics at the Taibah University College of Dentistry (TUCOD) over six months. Patients voluntarily participated in the study after understanding the methodologies and signing a consent form. They were asked to fill out a questionnaire on their experiences and preferences in placing the DD during RCT. Data were analyzed using the Chi-square test at  The dental-dam (DD) has been an ideal tool for tooth isolation and a standard of care in dentistry, especially during root canal treatments (RCT) . HistoriIn addition, the DD promotes better cross-infection control for both patients and the dental team , especiaDespite these advantages, DD isolation during root canal treatment is still not adopted in dental practice in many countries ,15,16,17□Would there be any significant difference between patients who would prefer placing a DD in a future visit and those who would not?□Could patient acceptance be influenced by factors such as DD application time and length of the treatment session with the DD in place?□Which policy do patients prefer  to increase the use of DDs in dental practice?A recent study has shown slight improvement in using DDs in Saudi dental practices  comparedConsequently, the study aimed to test the following null hypothesis: There would be no significant differences between the proportion of patients who would prefer using DD in the future and that of patients who would not.This observational descriptive study was ethically approved by the Taibah University College of Dentistry Research and Ethics Committee (TUCDREC) and conducted between January and June 2017. A pilot self-administrated questionnaire  was distp < 0.001)  was significantly greater than those who would not (18.4%) (< 0.001) . There wp = 0.115) (p = 0.530). Significantly, more patients (31.1%) were 21–30 years old (p < 0.001); with no significant difference between males and females (p = 0.057). Overall there was no correlation between participant age and their preference in using a DD in the next visit (p = 0.07). The proportion of male participants (45.2%) was not statistically significant from that of females (54.8%) (= 0.115) . In addip < 0.001) (p = 0.090). Whilst the highest proportion had a Secondary School degree (48.6%), the lowest had completed University Postgraduate Study (4.4%), followed by University and Primary School degrees . These differences were statistically significant (< 0.001) . There wp < 0.001) (p < 0.001). Patient preference on using a DD in the next visit significantly correlated with classification of the treating clinicians (p < 0.001). The vast majority of patients treated by consultants (93.2%) would prefer to use a DD in the next visit, which was significantly greater than those treated by undergraduate or postgraduate students (61.2% and 66.7% respectively).The proportion of participants who would prefer using a DD in the next session and were treated at the KFGH (95.7%) was significantly greater than those who would do so and were treated at TUCOD clinics (74.4%) (< 0.001) . While tp < 0.001), with no significant differences between the proportion of those who would prefer to use a DD in the next visit (46.8%) and those who would not (39.1%) (p = 0.416)  of patients reported that prevention of instrument inhalation or ingestion is the main advantage of DD use (= 0.416) . The lowp < 0.001), with a significantly greater proportion of those who would prefer to use a DD in the next visit (77.9%) compared to those who would not (52.2%) (p < 0.001) (p = 0.001). Overall, the highest proportion of patients (72.3%) needed up to one minute to place the DD by the treating clinician (< 0.001) . Overallp < 0.001). The highest proportion of those who would prefer to use a DD in the next visit were pleased with how it was placed in the current session (76.8%). By contrast, the highest proportion of those who would prefer not to use a DD in the next visit were uncomfortable with the placing of the DD in the current session (70%).Significantly, most participants (65.7%) were pleased with the placing of the DD in the first (current) visit . Overallp < 0.001), with a significantly greater proportion of those who would prefer to use a DD in the next visit (71.2%) compared to those who would not (48%) (p < 0.001)  reported that the duration of the current session with a DD in place was reasonable (< 0.001) .p < 0.001) (either in detail (39.9%) or briefly (33.2%) (p = 0.189)). However, this did not affect participant preferences on placing a DD in the next visit (p = 0.568)  reported that the dentist explained the reasons for using a DD (= 0.568) .p = 0.002). Whilst the highest proportion of those who would not prefer placing a DD in the next visit and believed that the DD should be optional (up to patients) (52%), the lowest of those who would prefer placing a DD reported the same policy for DD placement (17.6%) (p < 0.001).The highest proportion of patients reported that placing the DD should be optional (up to dentists) (41.2%), followed by those who believe it should be mandatory (33.1%) or optional (up to patients) (23.9%) . These dSurvey studies can provide valuable information about preferences, opinions, attitudes, experiences, and demographics of participants. However, such studies should be carefully conducted, so that results are representative and can be generalized ,30. SurvThe proportion of male participants (45.2%) was not statistically significant from that of females (54.8%), which reflects good study sampling. In addition, there was no significant difference between males and females regarding age groups, although the highest proportion of them 31.1%) were 21–30 years old. There were significant differences among participant level of education. While the highest proportion had a Secondary School education (47%), the lowest proportion of them had a University Postgraduate Studies degree (4.4%). Nevertheless, results showed that patient gender, age, country of origin and education level did not have any significant impact on their preferences on placing DDs. In contrast, Stewardson and McHugh’s  results .1% were The highest proportion of participants in our study (65%) were pleased with the placing of a DD during the session, after which they completed the questionnaire. These findings are consistent with those obtained in previous studies ,27,28. OOur results showed that the proportion of participants who would prefer using a DD in the next session and were treated at KFGH (95.7%) was significantly greater than those who would do so and were treated at TUCOD clinics (74.4%). This may confirm our assumption, as indicated earlier, that different treatment environments can affect patient attitudes and preferences. Those treated by undergraduate students were the least likely to prefer using a DD in the next visit. On the other hand, the vast majority of those who were treated by endodontic consultants (93.2%) would prefer using a DD in the next visit. While the highest proportion of patients at TUCOD were treated by undergraduate students, the highest proportion of patients at KFGH were treated by endodontic consultants. However, the clinician providing RCTs may be another environment factor that may affect patient preferences. Therefore, these results should be carefully interpreted, and such a conclusion should be drawn with caution.It was obvious that the experience of placing a DD in the first session did have an impact on patient overall attitudes. The highest proportion of those who would not prefer to use a DD in the next visit were uncomfortable with the current experience of DD placement. We assumed that a thorough explanation of the advantages of using a DD may positively affect patient preferences. However, the results did not show such an impact whatsoever, with only 27.4% of patients confirming that the dentist did not explain why a DD would be used. One possible reason for such a result is that the majority of patients (81.9%) would prefer to use a DD in the next visit in the first instance. These findings, however, contradict with those of Kapitan et al. [This study revealed how patient experiences and attitudes towards placing a DD, whether acceptance or rejection, reflected on their opinion regarding the length of the current session time. The majority of those who would prefer to use a DD in the next session (72%) thought that the current session duration was reasonable. By contrast, most of those who would not prefer to use a DD in the next session (52%) thought that the current session was long. These results were inconsistent with those of previous studies where the duration of the treatment while the DD was in place had no influence on patient preferences on DD placement ,27,28.Patient safety is usually the first priority in medical and dental fields. The current study reflected this fundamental concept as the highest proportion of participants 46.8%) reported that the most important advantage of using a DD is the prevention of accidental swallowing of files, although this had no significant influence on the patients preferring to use a DD in the next visit. It is likely that patients who were better informed tend to cooperate more, even if they were not satisfied with the application of the DD. In earlier studies, patients indicated that a DD was beneficial to both dentists and the patients ,27. The .8% reporA previous study investigating the frequency of DD use in Saudi dental practice revealed that the majority of participants (90.7% endodontists and 74.1% general dental practitioners suggested a mandatory use policy of DDs in dental practice . An almoThe discrepancies between the current study and previous studies are based on specific aspects of patient acceptance of placing a DD during RCTs. This validates the conduction of our study and confirms our assumption, in the first instance, that different dental profession environments may affect the preferences, perceptions and opinions of patients on using DDs. However, there are certain limitations; this study only included patients attending the governmental sector clinics. Therefore, conducting further research including private sector patients is recommended. In addition, we believe that including additional centers from Middle-Eastern and European countries may show the real impact of various environmental, economic and cultural factors on patient preferences. Nevertheless, this study added further proof that patients, overall, have no objection towards the placing of a DD during RCTs. Hence, further research is needed in an attempt to close the gap in using DDs between the undergraduate training stage and post-graduation practice. Within the limitation of the current study, it can be concluded that patients generally accepted having a DD placed and were willing to undergo RCT procedures under DD isolation for future visits. Patient safety remains the most attractive advantage for patients to the application of DD use. Patient factors such as race, age, gender and level of education did not have any remarkable impact on patient preference in placing a DD during root canal treatment. Additionally, the time required to place a DD, first experience in placing a DD, treating clinician and sector of dental services seem to be the key factors influencing patient preferences.


Astrocytic glycogen represents the only form of glucose storage in the brain, and one of the outcomes of its breakdown is the production of lactate that can be used by neurons as an alternative energetic substrate. Since brain metabolism is higher in wake than in sleep, it was hypothesized that glycogen stores are depleted during wake and replenished during sleep. Furthermore, it was proposed that glycogen depletion leads to the progressive increase in adenosine levels during wake, providing a homeostatic signal that reflects the buildup of sleep pressure. However, previous studies that measured glycogen dynamics across the sleep/wake cycle obtained inconsistent results, and only measured glycogen in whole tissue. Since most energy in the brain is used to sustain synaptic activity, here we employed tridimensional electron microscopy to quantify glycogen content in the astrocytic processes surrounding the synapse. We studied axon-spine synapses in the frontal cortex of young mice after ~7 h of sleep, 7–8 h of spontaneous or forced wake, or 4.5 days of sleep restriction. Relative to sleep, all wake conditions increased the number of glycogen granules around the synapses to a similar extent. However, progressively longer periods of wake were associated with progressively smaller glycogen granules, suggesting increased turnover. Despite the increased number of granules, in all wake conditions the estimated amount of glucose within the granules was lower than in sleep, indicating that sleep may favor glucose storage. Finally, chronic sleep restriction moved glycogen granules closer to the synaptic cleft. Thus, both short and long wake lead to increased glycogen turnover around cortical synapses, whereas sleep promotes glycogen accumulation. The human brain has remarkably high energy demands, accounting for ~20% of the body metabolism . All animal procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and facilities were reviewed and approved by the IACUC of the University of Wisconsin-Madison and were inspected and accredited by AAALAC. Mouse protocol number: M005697.Homozygous B6.Cg-Tg(Thy1-YFP)16Jrs/J transgenic mice of either sex (4 week old) were used in this study. Sleep and synapse dynamics, including the analysis of perisynaptic astrocytic processes (PAPs), have been extensively characterized in this mouse strain  mice were killed during the light phase after 6–7 h of sleep and at the end of a long period of sleep ; (2) spontaneously awake  mice were killed during the dark phase after ~7 h of wake and at the end of a long period of wake ; (3) acutely SD (n = 3) mice were sacrificed during the light phase after 8 h of SD enforced by introducing novel objects and new bedding whenever the animals appeared drowsy. This method has been validated in previous studies using EEG-implanted mice  mice were subjected to Mice of the same age and comparable weight (~12 g at p25) were randomly assigned to four experimental groups Figure : (1) sleTo avoid potential tissue damage and inflammation mice were not implanted with EEG electrodes. Behavioral states in S and W mice were estimated by quantifying motor activity using continuous video-monitoring with infrared cameras. As previously described , glued on the tip of a metal pin, and coated with silver paint to minimize specimen charging during imaging.Quantification of glycogen content and analysis of synaptic ultrastructure were performed in the same image dataset previously used to characterize the dynamics of PAPs in S, SD, W and CSR mice , and a backscattered electron detector (SBF-SEM). Image series were processed and analyzed using TrakEM2, a FIJI plug-in ) − 1), where 13 is the average number of GR per chain, 2 is the branching degree, L is the length of one tier  equipped with 3ViewNon-parametric statistics were used for the analysis. Kruskal-Wallis test followed by Dunn’s test was used for multiple comparisons. Correlation analysis was performed with the Spearman test. Alpha was set to 0.05. No outliers were detected, and all the data points were included in the analysis.n = 308, ROIs from 3 mice), spontaneously awake , acutely sleep deprived  or chronically sleep restricted , with W (p = 0.01), SD (p < 0.0001), and CSR (p < 0.0001) showing higher densities than S  and W , indicating that after sleep and spontaneous wake small-medium synapses could count on higher number of glycogen granules than large synapses. However, this was no longer the case as the time spent awake increased: after SD the negative correlation was only a trend  and it disappeared after CSR , indicating that after extended wake glycogen granules were equally distributed to all synapses . We also assessed whether the presence of SER was related to the density of glycogen granules within each experimental group. This was the case in S mice (p = 0.016), while all wake conditions showed comparable (p > 0.05) densities of glycogen granules between SER+ and SER− synapses . We also tested whether, within each experimental group, the presence of a presynaptic mitochondrion was related to the density of glycogen granules. In S mice the density of glycogen granules trended to be higher in synapses lacking the presynaptic mitochondrion (p = 0.05). However, this was not the case during waking: in all wake groups  synapses with or without a mitochondrion showed a comparable density of glycogen granules , W , SD  and CSR  mice. Quantitative analysis revealed an effect of behavioral state (p < 0.0001) and post hoc comparisons found that granule size was larger in S mice than in W (p < 0.0001), SD (p = 0.04), and CSR (p < 0.0001) animals. Differences in size were also found across wake groups, with greater granule size in W than in SD (p < 0.0001) and CSR (p < 0.0001) mice, and greater in SD than in CSR (p < 0.0001) mice  and pairwise comparisons showed that S had higher estimated number of GR than W (p < 0.0001), SD (p < 0.04), and CSR (p < 0.0001). Comparisons within the wake groups were also significant, with the number of GR progressively diminishing with time spent awake . Post hoc comparisons confirmed that this distance was shorter in CSR mice relative to S (p = 0.03), W (p = 0.001) and SD (p = 0.0001) mice. All other pair-wise comparisons were not significant , suggesting small and large granules were uniformly distributed within the PAPs in this condition. By contrast, a modest but significant negative correlation was observed in CSR  are more subjected to turnover, whereas larger granules function as a reserve that can be used during high intensity exercise , in CSR the astrocytic coverage negatively correlated with the average distance . Whether glycogen granules can move within the astrocytes is currently unknown, but it is well established that astrocytic peripheral processes are extremely motile (Bernardinelli et al., in vitro studies found that glycogen content in astrocytes is reduced in the presence of glucocorticoids (Tombaugh et al., Glucocorticoid stress hormones are known to affect cerebral metabolism and, specifically, glucose uptake and utilization (Landgraf et al., Ppp1r3c, the gene coding for PTG, was strongly upregulated after both spontaneous wake and short SD relative to sleep (Bellesi et al., Ppp1r3c expression was also significantly higher after SD than after spontaneous wake (p = 0.03; W raw expression: 10.2 ± 0.17, 4 h SD raw expression: 10.6 ± 0.35; Bellesi et al., In their original hypothesis, Benington and Heller  proposedBesides size, granule shape could be similarly important. Abnormal shape could reflect alterations of the granule structure, such as an inhomogeneity of chain growth owing to aberrant glycogen branching, which can lead to formation of insoluble aggregates (Roach et al., MB designed and performed the experiments, analyzed the data, and wrote the manuscript. LV performed the experiments and carried out the acquisition of the EM images. SK analyzed the EM images. GT and CC designed the experiments and wrote 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.