In this review, Pilley et al. examine the impact of different p53 mutations and focus on how heterogeneity of p53 status can affect relationships between cells within a tumor. p53 is an important tumor suppressor, and the complexities of p53 function in regulating cancer cell behaviour are well established. Many cancers lose or express mutant forms of p53, with evidence that the type of alteration affecting p53 may differentially impact cancer development and progression. It is also clear that in addition to cell-autonomous functions, p53 status also affects the way cancer cells interact with each other. In this review, we briefly examine the impact of different p53 mutations and focus on how heterogeneity of p53 status can affect relationships between cells within a tumor. TP53 is the most frequently compromised gene in human cancer  patients carry germline p53 mutations and are consequently highly likely to develop a variety of different cancers at a young age  p53 functions. While this loss of function can clearly contribute to tumor development, the high incidence of missense mutation compared with nonsense mutation or gene deletion has raised the possibility that there is a selective advantage to tumors in maintaining the expression of mutant p53 proteins. Studies comparing the consequences of loss of p53 versus expression of mutant p53 have revealed a number of mechanisms that could explain the selective advantage of mutant p53 expression during cancer development. These can be broadly grouped into dominant-negative effects on coexpressed WT p53 or independent gain of oncogenic functions .CKIa or mutation in Apc, mutant p53 was shown to have the expected oncogenic effect in the distal tissues of the intestine. However, in the proximal intestinal tissues of the same mice, the presence of mutant p53 was tumor-suppressive, a response that was shown to result from differences in the local microbiome. These results were quite unexpected, as such clear regional differences between mutant and loss of p53 have not been noted in other studies. The critical factor necessary to support mutant p53's oncogenic activity was found to be microbiota-derived gallic acid, leading to the possibility that changes in gallic acid levels between different mouse colonies could influence mutant p53-driven tumorigenesis. Nevertheless, while this tumor suppressor role for mutant p53 clearly warrants further investigation, almost all of our understanding of mutant p53 function to date addresses its protumorigenic activities.Despite the vast literature identifying mutations in p53 as contributors to cancer development, a surprising study recently suggested that, in some contexts, mutant p53 could function as a tumor suppressor . Using aTP53  vary between cancers. However, a recent analysis of >10,000 tumors reported that >90% of tumors with TP53 mutations did not retain a WT allele  ability of mutant p53 to restrain the activity of WT p53 could contribute to tumor development when both are expressed in the same cell . In estaT allele . The freT allele . FurtherT allele . On the  the DBD . LFS pat the DBD .Tp53+/− mice retain a functional WT allele  that contribute to tumor development independently of WT p53. The most compelling evidence for mutant p53 GOFs is seen by comparing mice expressing mutant p53 or no p53. While p53-null mice develop mostly lymphomas and soft tissue sarcomas , mice exFurther experimental studies have revealed a variety of cell-autonomous functions of mutant p53s, including cell survival, chemoresistance, metabolic rewiring, inhibition of autophagy and cell death, resistance to proteotoxic stress, increased rates of proliferation, genomic instability, and enhanced migratory and invasive capacity. These activities are discussed in detail in several recent reviews . In geneThere is considerable debate about the importance of DN and GOF activities of mutant p53 . An analTP53 in cancers are not well established, meaning DN activity may be important during tumor initiation but not in the later stages of tumor progression, at least in some cancers. While it may be possible for GOFs to occur in the presence of the WT protein, in certain contexts, GOFs cannot be identified unless LOH has occurred  make up only a fraction of a solid tumor. The rest of the tumor mass comprises the stroma, consisting of a variable proportion of different immune cells, fibroblasts, adipocytes, blood vessels, and nerves. This composition has led to the description of tumors as “pseudoorgans” . While cSeveral studies have shown that loss of WT p53 in normal stromal cells enhances tumorigenesis , 2017, dAlthough stromal cells can provide tumor-limiting functions, it is well established that cancer-associated fibroblasts (CAFs) exhibit activities that promote tumor growth and progression . InteresThe retention of WT p53 in cancer cells can also affect the tumor-stroma interaction, most clearly by supporting the activation of an anti-tumor immune response . In thesAs with WT p53, the presence of mutant p53 in a cell changes the expression of a variety of secreted proteins in a context- and mutation-dependent manner. The profile of molecules secreted by a tumor cell depends on the tumor genetics  but alsomiR-30d, which is able to influence the structure of the Golgi apparatus. The mutant p53-dependent increase in secretion enhanced tumorigenesis and metastasis in vivo  supported invasive behavior and cancer cell survival in response to the inflammatory cytokine TNFα by promoting the secretion of proinvasive molecules such as MMP9 and CXCL10. Interestingly, these cells also showed mutant p53-dependent secretion of the lymphocyte-attracting chemokines CX3CL1 and LTB, suggesting that the increased motility was accompanied by an induction of a potentially tumor-limiting immune response . SimilarAnother area of growing interest is how mutant p53 affects the production of exosomes by cancer cells can impact tumor progression. Exosomes are small extracellular vesicles that can contain nucleic acids, proteins, lipids, and metabolites, allowing communication between cells and tissues . ExosomeMutant p53 had previously been shown to play a role in modifying the structure of the ECM through the retention of the WT ability to repress the expression of TIMP3, an inhibitor of the matrix metalloproteinases , the actTumor-stromal interactions involve cross-talk between multiple different cell types in the TME, where an effect on one stromal compartment can have a subsequent impact on another. Not surprisingly, the p53 status of the tumor can influence these complex interaction networks. For example, tumor cells can induce fibroblasts to secrete IFNβ, which represses the invasive activity of the cancer cells and has potential to modulate the immune response to the tumor. The response to CAF-derived IFNβ is modulated by mutant p53 expression in the cancer cells, leading to increased invasive capacity .There is a growing appreciation that different p53 mutants may have different activities that support and promote cancer development , an obseThe accumulation of postzygotic changes in DNA ensures that no two cells in an individual are likely to be genetically identical . In tumoDrosophila, cell competition describes the ability of cells to compare their fitness levels with that of their neighbours. During cell competition, cells carrying mutations that do not directly affect their viability in a tissue or tumor become disadvantaged when surrounded by fitter “winner” cells and are actively eliminated  are outcompeted by WT cells as a consequence of direct, mechanically induced competition. Again, elevated levels of p53 were found to be inducing elimination of the loser cells, in this instance via a ROCK-p38 mechanical stress sensing pathway  plays an important role in marking cells as losers A. For ex pathway .Drosophila to be critical for maintaining competitive cell fitness, such as scrib and lgl, are orthologs of human tumor suppressor genes  into basal epithelial cells in mouse skin reduced the generation of differentiated daughter cells following keratinocyte division, maintaining a pool of proliferating cells that continued to expand at the expense of their WT neighbors. Interestingly, the resultant increased epidermal thickness and higher cell density in the basal layer of the epidermis was slowly restored to normal once the entire basal layer had been populated with mutant cells, suggesting the imbalance in cell fate had returned to normal . In respIn the mouse intestine, KRAS activation or APC loss, introduced via low level recombination into Lgr5+ stem cells in the base of crypts, gives cells a competitive advantage over WT progenitor cells; in this case, the advantage of KRAS-expressing cells is seemingly in contrast to the loser phenotype seen following KRAS activation in other contexts as described above. However, acquisition of the p53 mutation R172H  was only seen to confer an advantage to cells after the induction of colitis by the administration of DSS, potentially reflecting the ability of p53 mutant cells to deal with colitis-associated reactive oxygen species (ROS) .A similar role of ROS has been seen in murine esophageal epithelium, where exposure to low levels of ionizing radiation (IR) drives the differentiation of cells due to an increase in oxidative stress. In this system, expression of mutant p53 made cells more resistant to ROS, thus protecting them against irradiation-induced differentiation and allowing for the expansion of the mutant, and potentially tumorigenic, clones . The losDespite the broadly consistent ability of mutation in p53 to promote advantage in cell competition, studies on cultured cells have revealed that this can be context-dependent. p53-R273H-expressing MDCK cells were extruded from a monolayer by surrounding cells that were either WT or null for p53. Similar results were obtained from experiments performed with mouse intestinal organoids. Interestingly, however, if p53-R273H was sporadically induced within a monolayer of oncogenic RAS-expressing MDCK cells, there was no evidence of cell death or extrusion, suggesting that oncogenic RAS allowed the p53-R273H-expressing cells to escape the effects of the competition . PreviouTo date, analysis of the effect of mutation in p53 on interactions with surrounding WT cells has focused on p53 point mutations that give rise to mutant protein expression. As described earlier, these mutants generally lose WT p53 function but also have a potential to acquire dominant-negative or gain-of-function activities. Whether any p53 mutants show GOFs that support a “superfit” cell phenotype and how this might be modulated by the transformed state of tumor cells remains to be identified. The functions of WT or mutant p53 required to control cell competition are also not yet established, although the ability of various forms of p53 to control metabolite production, cytokine secretion, and the mechanical properties of the cell may all contribute to this activity.https://www.proteinatlas.org/ENSG00000141510-TP53/pathology). The reasons for this heterogeneity are unclear, but there is evidence to suggest that mutant p53 expression patterns can be affected at both the gene and protein level.While interactions between WT and mutant p53-expressing cells are the most likely to occur during tumor development, it is also possible that tumors contain a mixture of cells that harbor different p53 mutations or with different levels of p53 expression. Immunohistological evidence shows heterogeneous levels of mutant p53 within tumors; for example, the Human Protein Atlas contains multiple images of tumor sections stained for p53, with clear evidence for profoundly different levels of p53 in individual cells in the tumor (TP53 in different subclones of cells. A study of human breast cancers showed that heterogeneity in mutant p53 protein levels was found in the majority of tumors (p53 mutations acquired during tumorigenesis could lead to the formation of subclones with different p53 statuses within a tumor, resulting in areas with WT p53 and areas with no or mutant p53. Subclonal p53 mutations have been observed at varying frequencies in different cancer types, including lung , skin A, and chrf tumors . InteresMDM2 or TRIM71, may provide a genetic explanation for heterogenous mutant p53 expression (TP53, causing a reduction in mp53 protein in some cells, and mp53 staining patterns may differ depending on the presence of the second WT p53 allele (TP53 mutations found that some of them had transcriptional profiles that more closely resembled mutant p53 tumors than other WT p53 tumors. Furthermore, patients with pseudomutant p53 tumors show survival rates more similar to patients with mutant p53 tumors (Various explanations for this heterogeneous expression of mutant p53 have been put forward. Heterogeneity in the expression of genes that affect the proteasomal degradation of mutant p53, such as pression , or chan3 allele . IFNβ, w3 allele . The sta3 allele , leading3 tumors . Taken t3 tumors .While the impact of contacts between tumor cells that differ in p53 status has not been widely considered, emerging evidence suggests these interactions may have a role during oncogenesis. In mouse epithelium, cells expressing the p53-R273H mutant were eliminated from p53-null epithelia, as they were from p53 WT epithelia, clearly indicating that mutant p53 shows functions that are different from loss of p53 and, in this case, lead to the retention of the loser phenotype . An intep53 is well established as a critical node in the cell's stress detection network, and it is therefore not surprising that p53 is implicated in determining cell fate in response to stresses imposed by other cells. The molecular mechanisms of mutant p53 action are still being debated, with the evidence for increased cancer-promoting capacity balanced by recent evidence that, in some tissues, mutant p53 may even function to suppress tumorigenesis . In addi


SNORD118 gene in humans, is an atypical C/D box snoRNA as it promotes rRNA cleavage rather than 2′–O–methylation and is unique to vertebrates. The U8 snoRNA is critical for cleavage events that produce the mature 5.8S and 28S rRNAs of the large ribosomal subunit. Unexpectedly, single nucleotide polymorphisms (SNPs) in the SNORD118 gene were recently found causal to the neurodegenerative disease leukoencephalopathy, brain calcifications, and cysts , but its molecular pathogenesis is unclear. Here, we will review current knowledge on the function of the U8 snoRNA in ribosome biogenesis, and connect it to the preservation of brain function in humans as well as to its dysregulation in inherited white matter disease.Small nucleolar RNAs (snoRNAs) are non-coding RNAs vital for ribosomal RNA (rRNA) maturation. The U8 snoRNA, encoded by the  Ribosomes are essential cellular machinery that translate messenger RNA (mRNA) into proteins. Making ribosomes, known as ribosome biogenesis (RB), is expectedly a highly coordinated and energy-demanding process. In eukaryotes, RB accounts for over 60% of energy used in the cell  and takeThe cascade of primary 47S rRNA processing into the mature ribosomal subunits is multi–step and produces transient pre-rRNA intermediates . These iSNORD118 gene in humans, is classified as a C/D box snoRNA as it contains the conserved C box  and D box (CUGA) motifs, but U8 is unique as it does not participate in 2′–O–methylation of rRNA. Instead, U8 is required for cleavage of immature rRNA to provide 28S and 5.8S rRNAs that comprise the mature 60S subunit. Interestingly, the U8 snoRNA is not found in yeast, as are many other RB trans-acting factors, but is limited to vertebrates. Recently, Jenkinson et al. determined that mutations in the SNORD118 gene/U8 snoRNA transcript cause the rare inherited white matter disorder (IWMD) known as leukoencephalopathy with brain calcifications and cysts  . In. In37]. SNORD118 gene encoding the U8 snoRNA as a cause of LCC . How exactly mutations in the SNORD118 gene and resultant U8 snoRNA are related to the formation of calcifications and cysts remains unidentified. Recently, white matter disease similar to LCC has been observed in a new Alopecia Neuro Endocrine disease patient, also a ribosomopathy [The history of U8 snoRNA discovery and function stretches back to 1984 . It is os, CeR-2 . These uomopathy , suggestS. cerevisiae, which has been the workhorse organism for understanding nucleolar function. Recent work from Cao et al. has found a nucleolar microprotein that regulates biogenesis of the large ribosomal subunit (LSU) in mammalian cells but so far has not found genetic or biochemical evidence of this microprotein in yeast [While we are not sure why the U8 snoRNA is not found outside of vertebrates, evidence is accumulating that there are significant differences between ribosome biogenesis in human cells and the model organism, in yeast . AlthougXenopus laevis oocytes [SNORD118 variants with the signs and symptoms of patient presentation is a much-needed approach to determine the pathophysiology of U8 snoRNA-variants during brain development.To date, functional analyses of U8 in  oocytes ,21,26 or oocytes  has been oocytes , a mouse


Alvinella pompejana, an annelid worm living on the edge of hydrothermal vents in the Pacific Ocean, is an excellent model system for studying factors that govern protein stability. Low intrinsic stability is a crucial factor for the susceptibility of the transcription factor p53 to inactivating mutations in human cancer. Understanding its molecular basis may facilitate the design of novel therapeutic strategies targeting mutant p53. By analyzing expressed sequence tag (EST) data, we discovered a p53 family gene in A. pompejana. Protein crystallography and biophysical studies showed that it has a p53/p63-like DNA-binding domain (DBD) that is more thermostable than all vertebrate p53 DBDs tested so far, but not as stable as that of human p63. We also identified features associated with its increased thermostability. In addition, the A. pompejana homolog shares DNA-binding properties with human p53 family DBDs, despite its evolutionary distance, consistent with a potential role in maintaining genome integrity. Through extensive structural and phylogenetic analyses, we could further trace key evolutionary events that shaped the structure, stability, and function of the p53 family DBD over time, leading to a potent but vulnerable tumor suppressor in humans.The extremophile  TP53 gene is inactivated by mutation in about half of all human cancers  until the bromophenol blue dye migrated 8 cm.For the EMSA shown in Fig. Supporting Informationreproducibility checklist


Changes in the expression of p27 and CDK2 in visceral and subcutaneous white adipose tissue (WAT) biopsies were also analyzed in a human cohort of obesity and type 2 diabetes. p27, but not cdk2, exhibits a lower expression in subcutaneous than in visceral WAT in mice and humans. p27 is drastically downregulated by aging in subcutaneous WAT (scWAT), but not in gonadal WAT, of female mice. Obesity upregulates p27 and cdk2 expression in scWAT, but not in other fat depots of aged mice. In humans, a significant upregulation of p27 was observed in visceral WAT of subjects with obesity. Taken together, these results show a differential adipose depot-dependent regulation of p27 and cdk2 in aging and obesity, suggesting that p27 and cdk2 could contribute to the adipose-tissue depot’s metabolic differences. Further studies are necessary to fully corroborate this hypothesis.Aging usually comes associated with increased visceral fat accumulation, reaching even an obesity state, and favoring its associated comorbidities. One of the processes involved in aging is cellular senescence, which is highly dependent on the activity of the regulators of the cell cycle. The aim of this study was to analyze the changes in the expression of  Aging is a complex process due to the interaction of genetic, epigenetic, environmental and stochastic factors throughout life, which usually comes together with an accumulation of visceral fat, causing obesity ,2,3. It cdk2, p27, ccna and ccne in different adipose tissue depots during aging, as well as in obesity in mouse models. Additionally, the expression of both CDK2 and p27 was also analyzed in vWAT and scWAT depots from normal-weight, overweight and obese subjects with or without type 2 DM.Adipose tissue alterations manifest not only due to aging but also due to a cessation of cell division, which is known as cellular senescence ,20. CellThe experimental groups used in this study represent different life stages. The 2-month-old group would correspond to a young human age, the 6-month-old groups to adulthood and the 18-month-old groups to a late life stage in humans . Total bcdk2, p27, ccne, ccna) among the three different adipose tissue depots (gWAT vs. scWAT vs. iBAT) in young CT mice. Regarding p27  decreased in adult (6-month-old) and in aged (18-month-old) CT mice in comparison to young CT (2-month-old) mice. In iBAT, adult CT mice showed a slight non-significant increase in the p27 expression compared to young CT mice that was totally reversed in aged CT mice  male DIO mice, observing an upregulation of p27  and cdk2  mRNA levels exclusively in scWAT. In the case of ccna and ccne expression, no differences were observed in the context of obesity, except for an increase in ccna mRNA levels in iBAT of aged DIO female mice  and compared with aged CT mice. The expression of p27 A and cdk27  in scWAT  with normal-weight, overweight, obesity and obesity with type 2 DM. The upregulation observed on p27 and CDK2 was compared between both fat depots, showing a lower expression of p27 in scWAT when compared to vWAT  between the different fat depots, as well as their regulation during aging and obesity and their potential implications in these physiological/pathophysiological situations. It is well known that a chronic consumption of HFD produces adipocyte hyperplasia and hypertrophy, which increases simultaneously the degree of inflammation and the chance of developing obesity-associated comorbidities [In this study, we characterized the differential expression of several cell cycle regulators . More importantly, this observation was also found in scWAT from humans. These findings suggest that the lower expression of p27 in scWAT vs. vWAT could partially account for the metabolic differences between both white fat depots. In this context, it has been described that subcutaneous preadipocytes present a higher proliferation rate [p27 within this depot. Studies in p27 knockout mice have shown that the lack of p27 leads to increased fat pad size because of adipocyte hyperplasia [p27 in scWAT as compared to vWAT could be a mechanism for favoring fat accumulation within the subcutaneous depot more than in the visceral fat pads. Furthermore, ccne was also reduced in scWAT vs. gWAT and iBAT of young and aged CT mice.First, the differences on the expression of ion rate ,44, whicerplasia . This sup27. For instance, a dramatic decrease of the expression of p27 in scWAT was also observed due to aging. These changes seem to be depot-specific since they were not observed in gWAT. A more moderate reduction in cdk2 mRNA levels was also observed in scWAT but not in gWAT of aged mice. This suggests a possible association between p27 and cdk2 and the multiple alterations occurring in scWAT with aging [p27 expression observed in scWAT of aged CT mice could also contribute to the increased size of this fat pad in old mice as compared to those of young and adult mice. Some studies have suggested that p27 could play a role in cellular senescence during aging [p27 and cdk2 during aging in adipose tissue depots. Our current data suggest that neither p27 nor cdk2 gene expression levels could be considered as markers for the increased cellular senescence during aging in adipose tissue. However, it should be considered that the subcellular location of p27 is critical for its function. It has been proposed that nuclear p27 promotes quiescence, apoptosis, and senescence, while cytoplasmic p27 enhances cell survival and autophagy. Aging has been related with a greater expression of nuclear p27 in several cell types [Aging also induced relevant depot-dependent changes in th aging . Our datng aging . To the ll types . Therefop27, the senescence markers p21 and p57 [p21 was found to be increased in the gWAT of old female mice [Conversely, unlike  and p57 ,48 were ale mice  and to bale mice .p27 and cdk2 exclusively in scWAT, suggesting a likely involvement in the regulation of the expandability of this depot in obesity [cdk2 nor p27 expression. This result suggests a possible implication of p27 and cdk2 on the expansion of scWAT and not gWAT in obesity, which could be due to the physiological difference of both depots [p27 in scWAT of aged obese mice might make difficult the hyperplasia and fat accumulation in this depot, favoring unhealthy fat accumulation in vWAT. In agreement with our data, expression of p27 was reported no to suffer changes due to obesity in gWAT; however, at the protein level, an underexpression in 30-week-old obese mice, fed with HFD for 26 weeks, was observed [p27 mRNA, our data revealed no changes on the expression of p57 nor p21 in the aged DIO group in any of the depots. However, previous studies have suggested a link between p21 and obesity by promoting adipose tissue expansion during high fat feeding [p57 during development protects against age and diet-induced obesity [Our data have also revealed that the long-term high fat feeding during the process of aging provoked a significant increase of the expression of  obesity ,51. No ch depots . The higobserved . In cont feeding . Concern obesity .cdk2 in aged BAT, highlighting the importance of studying the potential role of p27 and cdk2 in the lowering of BAT activity occurring with aging [p21 and p57 in iBAT of aged CT mice as compared with young CT mice, suggesting a potential role of these cell cycle inhibitors in iBAT affectation during aging. Our current data also revealed a marked increase in ccna mRNA levels in iBAT of aged DIO mice as compared to aged CT mice. Moreover, correlation analyses have shown that higher levels of expression of cell cycle regulators such as cdk2 and ccna are associated with bigger iBAT size in aged CT and DIO female mice, suggesting a possible involvement in brown adipocyte hypertrophy and/or hyperplasia.Additionally, it is well known that the activity of BAT is highly affected by aging, being almost non-existent in older people ,56,57 anth aging . We haveccna or ccne mRNA levels in gWAT or scWAT during aging or in response to HFD. This might apparently contrast with a previous study showing an increase of cyclin E in mouse preadipocytes after 6–10 weeks of HFD [p27, cdk2 or ccna/ccne in adipose tissue during aging or obesity are a consequence or cause of these processes. Additionally, although good correlations between mRNA and protein levels for p27 [However, no relevant changes were observed for s of HFD . Therefo for p27 ,66 and f for p27 , data obp27 and CDK2 mRNA was characterized in vWAT and scWAT of apparently healthy overweight/obese subjects or in obese-diabetic subjects and compared to normal-weight healthy women/men. The results showed a significant increase of the expression of p27 in the vWAT of subjects with obesity and obesity with type 2 DM. However, this increased expression of p27 was not observed in the gWAT of mice. This could be attributed to several factors such as the differential regulation in both species [p27 tended to be upregulated in the overweight group and in the obese with type 2 DM patients, which agrees to that observed in the scWAT of DIO mice. Similarly, CDK2 was also slightly increased in human scWAT in obesity which was also observed in the same depot of DIO mice. Together, these results suggest that CDK2 and p27 could have a critical role in WAT development, especially on scWAT.If the regulation of cdk2 and p27 during aging or in obesity is relevant for the development and function of adipose tissue depots, these should be evolutionary conserved between different species . For tha species , the dif species . Regardip27 and CDK2 in scWAT was associated with bigger scWAT depot in humans. These positive associations were also observed in mice, pointing to a possible role in the expansion of the scWAT depot. Additionally, no association was found between age and the expression of p27 and CDK2 in neither of the depots. However, this could be a consequence of the limited number of subjects in each age range within the cohort. In order to corroborate this result, a larger cohort with enough individuals representing each age range would be necessary. Human obesity is affected by several factors, such as environment, lifestyle, onset and duration of obesity and genetic background, while obesity can be induced in mice under controlled conditions avoiding the influence of most of these factors [Another interesting finding of the current study was the observation that high expression of  factors ,72. This factors ,74, wher factors ,76. Besi factors , althoug factors . For thi factors ,80,81,82p27 and CDK2 expression in WAT, the analysis was also performed separately for men and women, showing both men and women similar trends in p27 and CDK2 expression. However, the correlation analyses revealed that higher levels of CDK2 in scWAT positively associated with triglycerides and BMI in men but not in women. Conversely, the significant positive association between p27 and CDK2 expression in scWAT and the size of this fat depot found in the whole cohort was not observed when the cohort was divided in men and women, probably because of the limited number of subjects within each group with body fat measurements by CT scans. Furthermore, the human cohort was formed by individuals within a wide range of age, making it difficult to establish associations between the expression of p27 and CDK2 and age. Finally, the studies were performed with whole adipose tissue, for both human and mice, which apart from mature adipocytes contains several cell types included in the stroma-vascular fraction , which could be an additional confounding factor in understanding the observed results. Therefore, it will be of interest to carry out future studies analyzing the expression of p27, cdk2 and associated cyclins both in isolated adipocytes and the stroma vascular fraction of the different depots of adipose tissue.It has been shown that male and female mice have different susceptibility to develop obesity . FurtherTaken together, the results obtained suggest an important transcriptional regulation of p27 and cdk2 in adipose tissue during aging and obesity. However, further studies are necessary to better characterize if these changes are the cause or consequence of these processes, as well as to discern the potential role of these cell cycle regulators within adipocyte metabolism, and the susceptibility to regulate obesity and aging.n = 8), adult CT , aged CT  and aged DIO . At the endpoint, body composition was measured by magnetic resonance technology  in non-fasted mice. After overnight fasting, mice were sacrificed and the different adipose tissue depots  were collected, weighed, and placed in liquid nitrogen before being stored at −80 °C. The main study was carried out in 7-week-old female C57BL/6J mice obtained from Harlan Laboratories . Mice were kept under controlled conditions of temperature (21 ± 2 °C), light/dark cycles (12 h/12 h) and humidity (50 ± 10%). After 10 days of acclimation, mice were divided in the following experimental groups: a control group (CT), fed with standard laboratory diet and a DIO group fed with HFD. The standard diet provided 20% proteins, 67% carbohydrates, and 13% lipids , and the HFD provided 45% fat, 20% protein and 35% carbohydrates . Two-month-old mice were fed ad libitum with either of the two diets and were sacrificed at different time points, organizing the study in the following groups: young CT  and an HFD-fed group (DIO) (n = 5). Animals were fed these diets ad libitum for 3 months. At the endpoint, scWAT depots were collected, weighed and placed in liquid nitrogen before being stored at −80 °C. For the male mice study, C57BL/6J mice (7 weeks-old) were obtained from Harlan Laboratories and housed in the same conditions of temperature . Standard diet was the same than in the female study. HFD was provided by Research Diets with a distribution of macronutrients of 60% of kcal from fat, 20% from carbohydrates, and 20% from protein. After a 7 d acclimation period, C57BL/6J male mice were split into 2 groups: a control group (CT) that was fed a standard diet  in accordance with the EU Directive 2010/63/EU.2, n = 10), overweight , obesity  and obesity with type 2 DM . The study was approved by the Aragon Ethics Committee of Investigation (20/2014) with the informed consent of all the participants, and by the Research Ethics Committee of the University of Navarra (protocol 2019.153). For this study, 60 adult participants were selected from the cohort (50% women and 50% men in each group). Subjects were divided into 4 groups according to their BMI and the presence or not of type 2 DM. Additionally, vWAT and scWAT biopsies were obtained during elective surgeries and stored in the Biobank of the Aragon Health System at −80 °C in cryopreservation tubes until RNA extraction. The study with human samples was carried out in vWAT and scWAT biopsies from the FATe cohort . BMI wasPlasma determinations of glucose, triglycerides, cholesterol, insulin, and HbA1c were performed at the Clinical Biochemistry Department in the Miguel Servet University Hospital  using state of the art analyzers. All analyses were in compliance with the requirements for quality and competence (ISO 15189:2012) for medical laboratories.2.As described elsewhere , the vishttps://www.ncbi.nlm.nih.gov/tools/primer-blast). Primer sequences are shown in 36b4 and β-ACTIN were used as housekeeping genes in the mouse and human study respectively. Relative expression of the specific genes was determined using the 2−∆∆Ct method [RNA from mouse and human samples was extracted with TRIzol™ reagent , according to the manufacturer’s instructions. RNA quality and concentrations were measured by Nanodrop Spectrophotometer ND1000 . RNA (1–5 µg) was then incubated with DNase I  for 30 min at 37 °C and reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit  according to the manufacturer’s instructions in a Touch PCR system . Real-time PCR was performed using the Touch Real-Time PCR System . The expression of genes was determined using Power SYBR Green PCR Master Mix (BIO-RAD) or TaqMan master mix (Applied Biosystems). SYBR Green primers were obtained from published studies and tested with Primer-Blast software . The comparison among the different groups was performed with one-way ANOVA or Kruskal–Wallis test followed by post hoc test for multiple groups-comparisons after testing the normality with Shapiro–Wilk tests. Student’s t test was used for comparisons between two groups. Correlation analyses were carried out by Pearson or Spearman correlation test for variables following parametric or non-parametric distribution respectively.


REE was measured by indirect calorimetry and body composition by air displacement plethysmography. Statistically significant differences in REE/fat-free mass (FFM) regarding obesity or gender were not observed. Absolute REE increases with age (p < 0.001), but REE/FFM decreases (p < 0.001) and there is an interaction between gender and age (p < 0.001) on absolute REE showing that the age-related increase is more marked in boys than in girls, in line with a higher FFM. Interestingly, the effect of obesity on absolute REE is not observed in the 8–10 year-old group, in which serum leptin concentrations correlate with the REE/FFM . In conclusion, REE/FFM is not affected by obesity or gender, while the effect of age on absolute REE is gender-dependent and leptin may influence the REE/FFM in 8–10 year-olds.In children and adolescents, obesity does not seem to depend on a reduction of resting energy expenditure (REE). Moreover, in this young population, the interactions between either age and obesity or between age and gender, or the role of leptin on REE are not clearly understood. To compare the levels of REE in children and adolescents we studied 181 Caucasian individuals (62% girls) classified on the basis of age- and sex-specific body mass index (BMI) percentile as healthy weight ( The prevalence of obesity among children and adolescents has increased dramatically in the last decades ,2,3,4. OObesity has a multifactorial nature resulting from an imbalance between energy intake and expenditure during an extended time period . Daily tOverweight and obesity are consequences of excess of calories intake and/or low levels of energy expenditure . HoweverLeptin is an adipokine mainly produced by adipose tissue in proportion to the amount of fat mass, being involved in the regulation of food intake, glucose and lipid homeostasis, reproduction, angiogenesis, and blood pressure, among others ,30. CircPrevious studies have shown that, similar to what happens in adults, REE expressed in absolute terms is increased in children and adolescents with obesity, but in most of them there are no statistically significant differences when REE is adjusted by FFM ,42,43,44n = 38), 11–13 (n = 50), and 14–17 years (n = 93). The experimental design was approved by the Hospital’s Ethical Committee responsible for research (protocol 2020.236). Informed consent was obtained from all parents or guardians and from all participants over 12 years old. Children under 12 years willingly agreed to participate in the study.To compare the levels of REE in children and adolescents with overweight and obesity we studied 181 Caucasian individuals (62% girls) from an age range of 8–17 years . Volunteers were recruited from children and adolescents attending the Department of Paediatrics at the Clínica Universidad de Navarra, Spain for conventional check-up. Each child and adolescent was classified on the basis of age- and sex-specific body mass index (BMI) percentile as with normal weight (BMI < 85th percentile), with overweight (BMI ≥ 85th and < 95th percentile), or with obesity (BMI ≥ 95th percentile)  based on®, Life Measurements, Concord, CA, USA) and converted to a body composition estimate using the Lohman equation. Data for estimation of body fat by this plethysmographic method has been reported to agree closely with the traditional gold standard hydrodensitometry (underwater weighing) [Body weight was measured with a digital scale to the nearest 0.1 kg, and height was measured to the nearest 0.1 cm with a Harpenden stadiometer . BMI was calculated as weight in kg divided by the square of height in meters. Waist circumference was measured with a non-elastic tape at the midpoint between the iliac crest and the rib cage. Body fat and FFM were estimated by air displacement plethysmography (Bod-Podeighing) . Blood peighing) .®, Diagnostic Products Corp., Los Angeles, CA, USA). An indirect measure of insulin resistance was calculated using the homeostatic model assessment (HOMA). Total cholesterol and triglyceride concentrations were determined by enzymatic spectrophotometric methods . High-density lipoprotein (HDL-cholesterol) was quantified by a colorimetric method in a Beckman Synchron® CX analyzer . Low-density lipoprotein (LDL-cholesterol) was calculated by the Friedewald formula. High-sensitivity C-reactive protein (CRP) was measured using the Tina-quant® CRP (Latex) ultrasensitive assay . Thyroid-stimulating hormone (TSH) concentrations were measured by an electro-chemiluminescence immunoassay (ECLIA) using Roche Elecsys® E170 immunoassay analyzer (Roche). Leptin was quantified in a subsample of 113 children and adolescents by a double-antibody radioimmunoassay method ; intra-and inter-assay coefficients of variation were 5.0% and 4.5%, respectively. Blood samples were collected after an overnight fast in the morning in order to avoid potential confounding influences due to hormonal rhythmicity. Plasma glucose was analyzed by an automated analyzer (Roche/Hitachi Modular P800) as previously described , with qup value lower than 0.05 was considered statistically significant.Data are presented as mean ± standard error of the mean (SEM). Differences between groups were analyzed by ANOVA followed by Fisher’s LSD (Least Significant Difference). Two-way ANOVA was used in order to analyze the interaction between age and gender or age and the degree of obesity. CRP concentrations did not fulfill the normality criteria and were therefore logarithmically transformed. We used crude Pearson’s correlation coefficients to test the statistical relation between REE and REE/FFM with any other variable. The calculations were performed using SPSS 23  and GraphPad Prism 8 . A p < 0.001). Waist circumference was significantly higher in the subjects with either overweight or obesity (p < 0.001). Blood pressure was within the normal range in all groups, exhibiting a small though significant increase in the overweight group, being further increased in the obese group compared to the healthy weight group (p < 0.001). Children and adolescents with obesity exhibited normoglycemia, but showed insulin resistance as evidenced by the increased insulin concentrations (p = 0.006) and HOMA values (p = 0.004). Circulating concentrations of triglycerides were increased (p < 0.001), while HDL-cholesterol was decreased (p = 0.019) in the group with obesity. Patients with obesity exhibited increased circulating concentrations of CRP as compared to both groups with either normal weight or overweight (p < 0.001). Leptin levels were increased in the overweight and obese groups (p < 0.001).Clinical characteristics of the children and adolescents enrolled in the study are summarized in p < 0.001) in children and adolescents with obesity as compared to the overweight and healthy weight groups (p < 0.05) and obese (p < 0.001) children and adolescents as compared to the healthy weight group (p < 0.001) decreased in females as compared to males (p < 0.001) and 14–17 years (p < 0.001) children and adolescents as compared to the 8–10 years group  and a strong negative correlation with REE/FFM  in the global cohort (p < 0.001), but the differences in REE/FFM were lost (p = 0.070). Similarly, we reanalyzed the data matching by age  finding statistically significant differences in REE (p < 0.001) (p = 0.111) , weight , BMI , fat mass , FFM , waist circumference , HOMA  and HDL-cholesterol , among others. REE/FFM was significantly correlated with height , weight , and fat mass , among others   but not = 0.111) . REE wasg others .p < 0.001, for interaction between age and gender) as can be observed in p < 0.001) and obesity (p < 0.001), with the latter being absent in the 8–10 years group  and, although a slight trend was observed, we found no effect due to age (p = 0.182)  in both boys  and girls . In the global sample, we found a statistically significant positive correlation of serum leptin concentrations with REE , but not with REE/FFM  as can be observed in r = 0.48; p = 0.011) was found in the 8–10 years age group  A. Serum ge group B.The major findings of the present study are that (i) obesity is associated with an increased REE in children and adolescents that is not observed after adjusting REE to FFM; (ii) REE is higher in males but similar to females after adjustment by FFM; (iii) absolute REE increases with age but decreases when referred to FFM; (iv) there is an interaction on absolute REE showing that the age-related increment is more marked in males than in females, which is not observed for REE/FFM; (v) the effect of obesity on REE is not observed in the 8–10 years age range; and that (vi) serum leptin levels correlate with REE/FFM in the 8–10 years age group only.Previous studies in adults have shown that the increased REE observed in obesity is not observed when REE is adjusted by FFM, the major determinant of REE ,25,26,27Gender is another factor that may be contributing to REE variability in children and adolescents. In our hands, absolute values of REE are higher in males than in females, but the differences are lost after adjustment by FFM. Some studies have found a significant influence of gender on REE when FFM is accounted for ,59,60 orData from the present study show that leptin exhibits a positive correlation with REE in children and adolescents in the whole sample, while the association is lost when REE is adjusted by FFM. Interestingly, when the analysis is performed by age groups we observe a significant positive correlation of leptin with REE/FFM in the youngest cohort (8–10 years of age). Several studies have shown that leptin is correlated with TEE in children and adolescents ,69,70, sSome potential limitations of our study should be pointed out. First, our study was conducted in Caucasian subjects and it would need to be determined whether our findings extend to other ethnicities. Second, the sample size of the group of lower age with healthy weight was not particularly big. However, the sample was of enough size in comparison to previous studies and the statistical analyses performed allowed us to detect statistically significant differences among groups. Third, some authors consider that although the normalization of REE to FFM (REE/FFM) is a commonly used method, this approach may be inappropriate when a comparison between lean and obese individuals is made. The logic behind this argument is that skeletal muscle increases with obesity proportionally more than other more metabolically active organs, which also form part of the FFM. Therefore, individuals with obesity tend to have lower REE/FFM than lean individuals because REE does not increase in the same proportion, because their FFM is proportionally less metabolically active than in lean individuals ,76,77. HIn conclusion, our results indicate that obesity is not related to changes in REE when REE is adjusted for FFM. REE/FFM is similar in both genders. Absolute REE increases with age, but decreases when referred to FFM and there is an interaction between gender and age on absolute REE showing that age-related increment is more marked in boys than in girls, in relation to a higher FFM. Interestingly, the effect of obesity on REE is not observed in children aged 8–10 years, which is the only age group in which serum leptin levels are positively correlated with REE/FFM.


In this paper, we reviewed the role of dairy products in dietary zinc absorption. Dairy products can have a reasonable contribution for dietary zinc intake in Western diets, where dairy consumption is high. However, the co-ingestion of dairy products can also improve zinc absorption from other food products. Such improvements have been observed when dairy products  were ingested together with food such as rice, tortillas or bread products, all of which are considered to be high-phytate foods with low inherent zinc absorption. For foods low in phytate, the co-ingestion of dairy products did not improve zinc absorption. Improved zinc absorption of zinc from high-phytate foods following co-ingestion with dairy products may be related to the beneficial effects of the citrate and phosphopeptides present in dairy products. Considering that the main dietary zinc sources in areas in the world where zinc deficiency is most prevalent are typically high in phytate, the inclusion of dairy products in meals may be a viable dietary strategy to improve zinc absorption. While the nutrient content of food products is typically determined on an individual product basis, the nutritional value is, in many, cases difficult to assess on a product basis. This is because in most cases, food products are consumed as meals rather than as single products. As a result, while the nutrient content of the meal can be considered as the sum of the nutrients in the products making up the meal, this is not true for its nutritional value. The combination of different products into a meal can both improve but also reduce its nutritional value. Examples of (non-)complementary different food products can be observed when considering the proteins in different food products . The nutThere are, however, instances where the combination of two food sources impairs the digestibility of proteins. This has, for instance, been observed in the case of egg protein, which, when combined with black tea, was shown to have lower digestibility than without black tea . This efZinc is an essential element because it is required in several cellular processes, such as differentiation, apoptosis and proliferation, which affect growth and development . The aduBecause the human body does not synthesize zinc, the intake of zinc through foods or supplements is required. The population reference intake is 9.4 and 7.5 mg per day for adult (>18 y) males and females, respectively, for those consuming a low phytate diet 300 mg/d), but is notably higher if diets contain more phytate . Among t0 mg/d, bThe absorption of zinc occurs within the small intestine and the absorbed zinc is released in portal circulation through transporters ,33,34. SThe absorption of zinc in the small intestine occurs by both active (saturable carrier-mediated process) or passive transport (unsaturable diffusion-mediated process) mechanisms, with the former occurring more often at a low luminal zinc concentration and the latter at high concentrations . An actiIn general, ~33% of dietary zinc is absorbed in humans ,44, but In countries with a high dairy intake, dairy products contribute significantly to overall zinc intake. For instance, in the Netherlands, >20% of dietary zinc is supplied by dairy products . Dairy c65Zn) and their absorption was determined by measuring the whole-body retention of the isotope. Even though the zinc content of the formulas was similar to, or higher, than that of human milk, zinc absorption from the formulas and bovine milk was found to be significantly lower (p < 0.05) than from human milk  anp < 0.001) increased zinc absorption from bovine milk, i.e., from 21% to 32% [p < 0.05) higher zinc absorption from bovine milk compared to a soy formula (28 vs. 14%) that was high in phytic acid (200 mg/L), which is agrees with the understanding that phytates are powerful inhibitors of zinc uptake [The greater bioavailability of zinc from human milk compared to bovine milk was hypothesized to due to the different chemical composition of the two milks, particularly the different casein:whey protein ratio . This hy% to 32% . Moreove% to 32%  reportedc uptake . Talsma c uptake , using ac uptake .As outlined in p < 0.05) increase (by 62%) in zinc absorption from rice when 90 g of high-phytate rice was consumed together with 600 mL of UHT-treated whole milk, compared to when the same amount of rice was consumed with water. Rosado et al. [p < 0.05) increased zinc absorption by approximately 50% for milk and 68% yoghurt. Sandstrom et al. [p < 0.001) increase in the zinc absorption from whole meal bread with a high concentration of phytates when ingested with milk and cheese, thereby counteracting the negative effect of phytates and facilitating the absorption of zinc. Using a radioisotope technique, Hunt et al. [p < 0.0001) increased the absorption of zinc in adults. From these studies, it thus appears that the consumption of dairy products in conjunction with food products rich in phytate, i.e., rice [Talsma et al.  examinedo et al.  studied m et al.  reportedt et al.  showed te., rice , corn toe., rice  or breade., rice ,57 could4 in gelatin capsules) to 18 subjects orally with breakfast and found that the consumption of 200 mL of milk and 50 g of cheese with breakfast  reduced zinc absorption. It should be noted here, however, that the zinc was provided as a supplement and not via a food product. Sandström and Cederblad [p < 0.05) in zinc absorption from a soybean meal  by the addition of milk (125 mL) and hypothesized that the additional calcium provided by milk could hinder zinc absorption. A non-significant (p > 0.05) reduction in zinc absorption was observed by Flanagan et al. [p > 0.05) on zinc absorption when 400 mL of reconstituted non-fat dried milk per day (200 mL with breakfast and 200 mL with dinner) was consumed with basal diets consisting of normal foods, selected to provide the U.S. Recommended Dietary Allowances. Contrary to earlier studies where dairy products improved zinc absorption from primarily phytate-rich foods, it appears that for other foods this was not that case. As outlined in Pecoud et al.  administederblad  reportedn et al.  when 210n et al.  also fou−6 vs. 0.32 g/100 mL), a finding that was also observed in concentrations of zinc ions in solution (1.6 × 10−7 vs. 0.017 mol/L). Dietary zinc can interact with dietary phytate in the intestinal lumen  [From H 6–7.4) , formingH 6–7.4) . The preH 6–7.4) . In otheH 6–7.4)  a phytat6 L/mol (site 1) and 8 × 104 L/mol (site 2) [4 L/mol) than phytate. Citrate is the main low molecular weight zinc ligand in human milk and is known as a zinc absorption enhancer [The stability and solubility of the zinc/phytate complex depends on various factors such as the pH value, the molar ratio and the presence of other compounds in the solution . Phytate(site 2) . On the enhancer ,28. Humaenhancer , which cenhancer . As a rep > 0.05) on zinc absorption after the addition of a high amount of calcium to high phytate meals. Several studies reported that a phytate x Ca:Zn molar ratio above 200 is needed to hamper zinc absorption [Several hypotheses have been posed for improved zinc absorption from phytate-rich foods when consumed in combination with dairy products. It has been hypothesized that this improvement in zinc absorption from phytate-rich foods by co-ingesting dairy products may be due to a higher proportion of phytate-bound calcium in the gastrointestinal tract, thereby blocking phytate binding sites and allowing more zinc to become available for absorption . Howeversorption ,71.One suggested explanation for the improvement of zinc absorption by dairy products is that amino acids  or peptides, that become liberated during the digestion of milk proteins, bind zinc in the intestinal lumen at intestinal pH and form complexes, thereby increasing both their bioavailability and solubility ,72. Thesp < 0.05) zinc absorption in humans after the addition of CPPs to a rice-based cereal  that was low in phytate (20 mg per serving), but no significant effect (p > 0.05) on zinc absorption occurred when CPPs were added to whole grain cereal  that were high in phytate (198 mg per serving). Hansen et al. [p > 0.05) on zinc absorption after the addition of different amounts of CPP to bread-based meals  (Other studies suggest that the increase of zinc absorption by dairy products could be due to casein phosphopeptides (CPPs), which are phosphorylated peptides that are released when casein is digested and can bind zinc, thereby preventing it from binding to phytate ,46,73. Rn et al.  found nof water) . Three dFrom the results explored above, it is clear that dairy products play a dual role with regard to the absorption of dietary zinc. Both provide a source of dietary zinc, which in general has good bioavailability. However, in addition to this, they also modulate the absorption of zinc from other food sources. Such effects are arguably not unique to only dairy products or zinc, and warrant further investigation and the consideration of the bioavailability of nutrients at a meal level rather than an individual product level. Considering that areas where zinc deficiencies are most prominent are also those where zinc is often ingested via high phytate products, dietary recommendations on the inclusion of dairy products, or products with a comparable ability to modulate zinc absorption in meals, can present significant advantages.


In accordance, enhanced p53 protein turnover due to elevated expression of the critical p53 E3 ligase MDM2 or MDM2/MDMX is found in many human cancers. Likewise, the HPV viral E6 protein-mediated p53 degradation critically contributes to the tumorigenesis of cervical cancer. In addition, growth-promoting signaling-induced cell proliferation is accompanied by p53 downregulation. Animal studies have also shown that loss of p53 is essential for oncogenes to drive malignant transformation. The close association between p53 downregulation and carcinogenesis implicates a critical role of basally expressed p53. In accordance, available evidence indicates that a reduced level of basal p53 is usually associated with disruption of homeostasis, suggesting a homeostatic function mediated by basal p53. However, basally expressed p53 under non-stress conditions is maintained at a relatively low abundance with little transcriptional activity, raising the question of how basal p53 could protect homeostasis. In this review, we summarize the findings pertinent to basal p53-mediated activities in the hope of developing a model in which basally expressed p53 functions as a barrier to anabolic metabolism to preserve homeostasis. Future investigation is necessary to characterize basal p53 functionally and to obtain an improved understanding of p53 homeostatic function, which would offer novel insight into the role of p53 in tumor suppression.Apart from mutations in the  Of note, despite sharing a high degree of structural similarity with MDM2, MDMX lacks the NLS and is a predominantly cytoplasmic protein  . Among sNonetheless, p53 cytoplasmic distribution prevents it from binding to DNA, equivalent to functional p53 inactivation. Indeed, cytoplasmic p53 accumulation is found in a subset of human cancers . While tIn addition to the change in the p53 protein abundance, p53 activity can also be regulated via post-translational modifications. For example, by counteracting against p53 acetylation, which is necessary for its transcription activity, deacetylation of p53 by HDAC such as histone deacetylase eight diminishes p53 transcription activity . AnotherIt is well documented that p53 is typically growth inhibitory, or p53 is usually incompatible with increased cell proliferation. Based on this notion, induction of cell proliferation would predict a reduction in p53 activity/level. Zwang et al. reported that mitogen such as EGF-induced proliferation in normal human mammary epithelial cells was mediated by two temporally separable waves of growth signals during which induction of metabolic pathways is associated with downregulation of antiproliferative genes . Many ofIn agreement with the fundamental importance of metabolism in cell growth, induction of cell proliferation is contingent upon metabolic reprogramming from catabolic to anabolic metabolism. In accordance with its function in growth inhibition, p53 typically antagonizes anabolic pathways while stimulating oxidative phosphorylation. Available information indicates that p53 mainly regulates cellular metabolism in a transcription-dependent fashion . The basThe studies together suggest a model in which basally expressed p53 can keep anabolic metabolism in check to maintain homeostasis. In accordance, cell growth signals disable this p53-mediated metabolic constraint to induce anabolic metabolism, promoting cell proliferation. Therefore, further investigation is warranted to explore how basally expressed p53 keeps anabolic pathways under control.Like cell proliferation that depends on anabolic metabolism, T cell activation represents another typical process involving a metabolic switch from catabolic to anabolic metabolism. Wang et al. demonstrated that metabolic reprogramming from the TCA cycle to the anabolic pathways, including glycolysis, pentose-phosphate, and glutaminolysis, is coupled with T cell activation . This swStudies have also uncovered an important role of p53 in B cell activation and expansion . During The inflammatory response is energy-consuming process and relies on anabolic programs. For instance, in response to LPS stimulation, macrophages undergo metabolic reprogramming from oxidative phosphorylation to glycolysis via activation of the mTOR-HIF1α pathway , resultiMacrophage is one of the major cell types that contribute to the inflammatory responses. Depending on stimuli, macrophages can be induced into different functional states, for instance, M1 or classically activated macrophages and M2 or activated macrophages, according to the simplified classification method. M1 macrophages are pro-inflammatory that is characterized by the release of inflammatory cytokines , reactive oxygen species (ROS), and nitrogen species, whereas M2 macrophages, in contrast, participate in the anti-inflammatory response to facilitate wound healing and tissue repair. Importantly, M1 and M2 are intimately linked to and controlled by distinct metabolic programs . StimulaIn support of the role of p53 in inflammation, Li et al. reported that loss of p53 stimulated whereas activated p53 impeded M2 macrophage polarization . Using aMdmxS314 A was blocked. Of significance were the observations that impediment of p53 decline was associated with mitigation of the suppression of immune responses as reflected by increased immune cell tumor infiltration and enhanced macrophage M1 polarization compared with that in wild-type mice.In tumorigenesis, tumor cells can substantially impact surrounding cells to shape the tumor microenvironment (TME) that promotes cancer progression. The dynamic interactions between tumor cells and immune cells have been widely reported. However, how p53 participates in regulating the tumor immune microenvironment is only beginning to be investigated. A recent study by Wang et al. showed that implanted mammary carcinoma cells acted on their surroundings in the host to induce an immunosuppressive microenvironment facilitating tumor growth . A contrMdmxS314 A mice was coupled with a significant delay in tumor growth. Thus, the study implicates that tumor cells can induce an immune suppressive microenvironment by downregulating p53 in peritumor cells, suggesting a role of basal p53 in the maintenance of the immune response, However, further studies will be necessary to understand how basally expressed p53 preserves immune homeostasis.Moreover, the improved immune response in Within the context of the tissue microenvironment, p53 was reported to play a role in maternal reproduction by controlling the expression of basal as well as inducible level of leukemia inhibitory factor (LIF), a cytokine critical for implantation .Within tissues, cell-cell interactions are regulated by a host of mechanisms to preserve homeostasis. In addition to cell-intrinsic mechanisms to eliminate cells that contain unrepaired damages or are suboptimal, cells can also sense their neighbors to determine relative fitness, which constitutes an important mechanism to eliminate comparatively weaker cells, a process described as cell competition. Ample evidence indicates that cell competition is involved in various processes such as development, tissue homeostasis, and tumorigenesis. Cell competition to eliminate damaged and unhealthy cells are expected to yield positive and beneficial outcomes. It is, however, also conceivable that competition may contribute to tumor development. In accordance, studies have shown that malignant cells acquire various mutations to gain growth advantages in competition with neighboring normal cells . While dGiven the homeostatic function of p53 and the well-established role of cell competition in preserving tissue homeostasis, it is probably not unexpected that p53 has been reported in the regulation of cell competition. Bondar et al. reported that a moderate increase in p53 induced by treatment with radiation at a low dose of 1Gy was associated with a loser phenotype in hematopoietic stem and progenitor cells (HSPCs) . The autLike the modest p53 induction by treatment with low-dose radiation, a genetic method-induced mild increase in p53 also resulted in a less competitive status in both embryos and adult cells. Zhang et al. generated haploinsufficiency of Mdm2 and Mdm4 mice where p53 was slightly elevated but had little effect on growth . HoweverThe finding that a moderate increase in p53 resulted in a less fit status would predict that reduced p53 level/activity might be associated with a more fit status. Indeed, in a study of embryonic development, knockdown p53 rendered embryonic stem (ES) cells a competitive advantage resulting in the replacement of wild-type ES cells when they were co-injected into the mouse embryo . While t. The high level of p53 in the cytoplasm may keep it poised in response to potential stress. Indeed, DNA damage triggered by X-ray or UV irradiation induces p53 redistribution to the nucleus leading to p53 activation and subsequent induction of p53 target genes that promote ESCs differentiation , which can be established by introducing reprogramming factors  into somatic cells. iPSCs display the ability of self-renewal and differentiation into many cell types, a feature like embryonic stem cells. Somatic cells undergo transitions in gene expression profile, epigenetic status, metabolic characteristics, and cellular morphology . Ample eIn line with the p53-mediated barrier function, the function of p53 in iPS is suppressed usually via a mode of post-translational modifications, which include ubiquitylation, acetylation, phosphorylation, methylation, or sumoylation of specific residues of p53 . Lee et An additional type of modification frequently involved in the regulation of p53 in human ES cells is the acetylation of a lysine residue in the p53 protein. It was reported that despite being distributed in the nucleus of human ES cells, p53 is transcriptionally inactive because the 120/373 lysine residues are not acetylated. Although Sirt1 can maintain the non-acetylated status, a NAD-dependent deacetylase induced transcriptionally by Oct4 , some lyThus, it is clear that p53 activity is attenuated or inactivated in stem cells, which appears necessary to allow stem cells to replicate. The inactivation of p53 in stem cells can result from either a deficiency in p53 transcriptional activity or post-translational modifications on the p53 protein that result in an inactive p53 protein. Collectively, these studies suggest that p53 controls the transition between cell self-renewal and differentiation. p53 restricts the ability of somatic cells to undergo reprogramming into iPSCs.The importance of metabolic regulation during the reprogramming to pluripotency has been well documented . RelativAmple evidence has shown that there is a very dynamic cross-talk between metabolic pathways and epigenetic programs. Cells continuously modify their metabolic programs and activities in response to nutrient availability, extracellular signals, and reprogramming/differentiation cues. Many intermediary metabolites can function as cofactors for epigenetic enzymes that catalyze histone methylation and acetylation reactions, contributing to the regulation of gene transcription. This cross-talk between intermediary metabolism and epigenetics has been demonstrated as central mechanisms by which metabolic pathways are engaged in stem cell fate determination . PluripoA proper stress response is critical for maintaining homeostasis. When encountered with different levels of stress, cells have to determine the fate between survival and death. In response to excessive stress that is destructive to genome integrity and other cellular structures, cells must sense the intensity of damage and rapidly activate responses such as cell cycle arrest, DNA damage repair, senescence, or apoptosis if the damage is unrepairable. However, living cells or organisms are often exposed to temporary and low levels of stress in our daily lives. In response to such transient and mild stress, inciting cellular senescence or cell death would not make sense economically. Under such conditions, cells must finely tune their response to the perturbation based on stress level. Abundant evidence indicates that p53 is one of the key players in regulating the stress response . The impAvailable information indicates that mild stress can induce an adaptive response, an evolutionally conserved defense mechanism to preserve homeostasis . EvidencStudies have shown that the adaptive stress response is primarily mediated by a modest increase in ROS as treatment of cells with an antioxidant such as N-acetyl cysteine could mainly diminish the adaptive response . The rolHomeostasis, a property crucial for normal physiology, is maintained by coordinated actions of diverse cellular processes and pathways. As a process fundamental to all biological functions, metabolism is intimately involved in regulating every facet of biological processes, which contributes to maintaining homeostasis. The basally expressed p53 safeguards homeostasis by keeping anabolic metabolism in check, which functions as a barrier to cell proliferation and governs numerous anabolism-dependent processes. In line with this notion are the observations that induction of many anabolism-driven processes is accompanied by a decline in nuclear p53 level/activity. While the ability of p53 to antagonize against Myc, HIF1α, NF-κB, or mTOR likely contributes to restraining anabolic metabolism , p53-med


Our data support ΔNp73 levels contained in extracellular vesicles as such a non-invasive and premature biomarker for the early diagnosis of colorectal cancer.The survival of colorectal cancer patients largely relies on the stage at diagnosis. The identification of early and non-invasive biomarkers to be used in screening programs for the diagnosis of the disease at the premalignant stage is mandatory. The aim of this study is to validate in plasma-derived extracellular vesicles secreted by malignant cells the diagnostic potential of well-known tumor-associated genes, ΔNp73, TAp73, and Δ133p53, in healthy subjects (n = 29), individuals with premalignant lesions (n = 49), and colorectal cancer patients (n = 42). Extracellular vesicles’ ΔNp73 levels were already significantly high in subjects with premalignant lesions. Δ133p53 levels were statistically increased in colorectal cancer patients compared to the other two groups and were associated with patients’ survival. Remarkably, TAp73 mRNA was not detected in any of the individuals. The evaluation of ΔNp73, Δ133p53 and CEA sensitivity, specificity and AUC values supports ΔNp73 as a better early diagnosis biomarker and CEA as the best to identify advanced stages. Thus, low levels of CEA and a high content of ΔNp73 may identify in screening programs those individuals at higher risk of presenting a premalignant lesion. In addition, Δ133p53 emerges as a potential prognosis biomarker in colorectal cancer.The early diagnosis of colorectal cancer is a key factor in the overall survival of the patients. The actual screening programs include different approaches with significant limitations such as unspecificity, high invasiveness, and detection at late stages of the disease. The specific content of extracellular vesicles derived from malignant cells may represent a non-invasive technique for the early detection of colorectal cancer. Here, we studied the mRNA levels of ΔNp73, TAp73, and Δ133p53 in plasma-derived extracellular vesicles from healthy subjects ( Colorectal cancer (CRC) is the third most common tumor diagnosed and accounts for more than 10% of all tumor types worldwide. It is estimated that CRC is the fourth leading cause of cancer-related death, with around 600,000 deaths reported annually .CRC arises due to the accumulation of genetic alterations following a stepwise process from normal colonic epithelium to premalignant lesions (adenomatous and serrated polyps) and invasive cancer . The ideIn recent years, liquid biopsy, referred to as the isolation of cancer-derived components from biological fluids, has been studied in several clinical scenarios, including cancer screening . These cTP73, a member of the TP53 family, is altered in most human cancers and has prognostic implications [TP73 translates into different variants with opposing functions: TAp73 and ΔTAp73  [TP73 has been reported in premalignant lesions of other tumor types [TP53 variants have been also described and attracted during the last few years the interest of the scientific community due to their putative oncogenic properties and association with cancer progression [The expression of ications . TP73 tr ΔN’p73) . In pati ΔN’p73) ,16,17 an ΔN’p73) . Finallyor types ,20,21,22gression ,24,25.TP53 variant Δ133p53 may function as an oncogene by acting as a dominant-negative inhibitor of other p53 family members and transactivating a specific set of pro-tumoral effectors [Thus, similar to ΔNp73, the N-terminal truncated ffectors ,27,28. Affectors ,30 and fffectors  and moduffectors . In addiffectors . Δ133p53The deregulated expression of ΔNp73 and Δ133p53 in different tumor types and its association with cancer patients’ outcome support them as potentially good cancer early diagnostic candidates to further explore in plasma-derived EVs. The content of tumor suppressors in EVs as tumor biomarkers has been poorly explored; therefore, TAp73, which has previously been found overexpressed in different tumor types but controversially associated with patients’ outcome , could pn = 29), individuals with premalignant lesions (PL) (n = 49), and CRC patients at different stages of the disease (n = 42) in order to explore their potential role in the early diagnosis of CRC through a non-invasive approach.With this background, we evaluate here the mRNA levels of ΔNp73, TAp73 and Δ133p53 in plasma-derived EVs from healthy subjects . Blood samples were obtained with the corresponding informed written consent. Plasma samples were collected from 29 healthy individuals with negative colonoscopy, 49 subjects with premalignant lesions (low- and high-grade adenomas) and 42 CRC patients diagnosed at different stages of the disease. Sample size varied from 3–5 mL of plasma for our experimental workflow. Among these samples, 15/29 of control samples, 19/49 of adenoma samples, and 9/42 of CRC samples were additionally used to study their humoral response in a previous work . The cliPlasma-derived EVs were isolated by a modified and adapted protocol of sequential ultracentrifugation previously described in . Identif8 and 109 particles/mL. Three repeated measurements of 60 s were taken per sample and the mean value was used to determine particle number. The temperature of the laser unit was controlled at 25 °C. NTA software measured the size distribution (ranging from 10 to 1000 nm) and concentration (particles/mL) of nanoparticles.The NanoSight NS500 instrument equipped with a 405 nm laser  was calibrated using Silica Microspheres beads before measurements. Samples were diluted 1/1000 in PBS in order to obtain a particle concentration between 10TM Exosome RNA Amplification kit  following the manufacturer’s instructions. cDNA synthesis was performed with the Transcriptor First-Strand cDNA Synthesis Kit . Quantitative real-time PCR was performed in a LightCycler 2.0 instrument using Fast Start DNA MasterPlus SYBR Green I kit (Roche) as previously described in [RNA from resuspended EVs was extracted by SeraMirribed in . Primer https://www.r-project.org/; accessed on: 1 October 2020), and the corresponding area under the curve (AUC) and the maximized sensitivity and specificity values were calculated using the R packages ModelGood and Epi. The cut-off value for the survival analysis was selected upon ROC curves for disease recurrence or death in patients with localized CRC and death for the whole cohort of patients with CRC. The optimal cut-off value was determined using the Youden’s J index. For ΔNp73, we selected 0.76 and −0.83 as the cut-off values for disease-free survival (DFS) and overall survival (OS), respectively. For Δ133p53, we selected 2.93 and 1.99 as the cut-off values for DFS and OS, respectively. ΔNp73 and Δ133p53 mRNA levels were classified in low and high levels accordingly. We used the log-rank test to compare DFS and OS according to ΔNp73 and Δ133p53 mRNA levels. Kaplan–Meier analyses were used to estimate medians. A Cox proportional-hazards model was used to estimate hazard ratios and 95% confidence intervals. The Spearman rank-order correlation test was used to examine the relationship between autoantibodies and EVs mRNA of the different p53 family isoforms. p-values ≤ 0.05 were considered statistically significant. Statistical analysis was performed with SPSS software (version 24).To normalize the results, qPCR data were related to the number of particles of each sample. Then, these data were modified with log10 transformation. The D’Agostino–Pearson and Shapiro–Wilk tests were used to assess the normality of the data distribution. Subsequently, the Kruskal–Wallis and U-Mann–Whitney tests were used for statistical analysis. The receiver operating characteristic (ROC) curves were constructed with the R program  A, 64.1% Finally, we decided to compare the potential of ΔNp73 and Δ133p53 versus CEA to discriminate among the three groups. CEA AUC values for discriminating healthy subjects from PL subjects were 52.9% and 85.7% (sensitivity 100% and specificity 85%) from healthy subjects and CRC . As prevNext, we have evaluated the EVs’ mRNA content of these isoforms at the different stages of the disease. Non statistical significative differences were observed for Δ133p53. However, ΔNp73 was observed to be increased at stage II and IV, although it did not reach statistical significance at this later stage .n = 38) and 16 (37.2%) deaths were observed in the whole cohort (n = 42). At 3 years, 63% , 47 to 78) of the patients with localized disease remained alive without disease recurrence and 69%  remained alive in the whole cohort.In the CRC cohort, the median follow-up was 36.2 months . During this period, 15 (34.9%) events (recurrences or deaths) were observed among patients with localized disease (p = 0.009) in favor of the high Δ133p53 group. In the whole cohort of CRC, patients with low Δ133p53 levels did not reach the median OS and patients with high Δ133p53 levels had a median OS of 50 months , with a HR of 0.23 .Survival analysis did not reveal a correlation between ΔNp73 EV content and DFS and OS A,B. Howen = 15; PL, n = 19; CRC, n = 9). Here, the correlation between the autoantibody levels and the ΔNp73 and Δ133p53 EV content was evaluated. We have observed a negative correlation trend in ΔNp73α seroreactivity and ΔNp73 EV levels in patients with CRC    in a sub = 0.09) .CRC screening is currently performed by different techniques which present significant limitations. On one hand, the fecal occult blood test shows a lack of specificity and a low sensitivity for early stages and premalignant lesions. On the other hand, colonoscopy, although with high specificity and sensitivity, is highly invasive and adherence of the general population is relatively low. Therefore, the detection of soluble biomarkers from different body fluids, known as liquid biopsy, as a non-invasive technique emerges as a promising approach for the early diagnosis of the disease. In this sense, the analysis of the cargo of plasma EVs released by the tumor and by premalignant lesions in apparently healthy subjects is of great interest and remains largely unexplored.TP53 family members in human cancers and its association with a poor prognosis of the patients have been described. Thus, ΔNp73 upregulation is associated with shorter survival in hepatocellular carcinoma [TP53, it has been described as overexpressed in many human tumors including CRC and associated with poorer survival [TP53 variant, Δ133p53, has attracted during the last few years the interest of the scientific community due to its putative oncogenic properties. Δ133p53 may act as a survival factor [The overexpression of different arcinoma , neuroblarcinoma , lung caarcinoma , gastricarcinoma , acute parcinoma , and colarcinoma . ΔNp73 harcinoma . Interessurvival ,42,43,44l factor , inhibitl factor , interfel factor ,29, indul factor , and prol factor ,28, suchl factor , the JAKl factor , pro- anl factor  and EMT l factor . Remarkal factor . In the present study, we report statistically significant high levels of ΔNp73 in plasma EVs in both subjects with CRC and with PL compared with healthy individuals. The comparison of AUC values, specificity and sensitivity addressed by ROC curves between ΔNp73 in EVs and CEA levels in plasma in the three groups of study clearly revealed a much greater potential of ΔNp73 to identify individuals with PL. Our findings highlight the plausible usefulness of ΔNp73 to the detriment of CEA as a non-invasive biomarker for the detection of the disease at the premalignant stage, probably playing a key role in the initiation of the tumor. It is noteworthy that EVs’ cargo can be uptaken by different cells of the cancer microenvironment, leading to a pro-tumoral stroma ,45,46. IWe expected low levels of TAp73 in the EVs from PL individuals and CRC patients compared to healthy subjects. Surprisingly, TAp73 was not detected in any of the groups evaluated in the current study. These findings suggest that packaging and secreting tumor suppressor mRNAs in EVs may not provide a selective advantage to the tumor. The latter has to be confirmed since this is the first study evaluating the EV content of TAp73. In addition, many studies analyze the pro-tumoral cargo of EVs and its function in tumor progression and conditioning of the premetastatic niche, but few evaluate its diagnostic and prognosis biomarker potential ,47.To the best of our knowledge, this is also the first report evaluating the presence of Δ133p53 in EVs in CRC. Here, we observed higher levels of Δ133p53 in plasma-derived EVs from CRC patients than those from healthy subjects and individuals with PL. Fujita K et al. have previously reported the status of Δ133p53 in tissues from healthy, PL and CRC subjects . AlthougWe have observed that those CRC patients at stage II and IV of the disease present higher levels of ΔNp73. These findings are similar to those previously reported  regardinNo correlation was observed between ΔNp73 EV content and DFS and OS in our series of CRC patients. This is in contrast with previous results in which we observed a trend with survival . The smaTP53 family members to this aim, we have recently described a significant difference in the seroreactivity for ΔNp73 between CRC patients and subjects with PL versus healthy controls [In our attempt to identify biomarkers for the early detection of CRC and the plausible usage of the controls . Interescontrols ,55. ThisThis is the first report that studies the implication of ΔNp73, TAp73 and Δ133p53 content in plasma-derived EVs as early diagnosis biomarkers of CRCs, using for this purpose healthy subjects, individuals harboring premalignant lesions and CRC patients at different stages of the disease. Our data support the potential role of EVs’ ΔNp73 content to detect premalignant lesions as the earliest stage of the disease. Interestingly, although Δ133p53 cannot detect the disease at the premalignant stage, this isoform in EVs emerges as a potential prognosis marker in CRC. Further investigation could establish a combination with other non-invasive biomarkers and improve the actual screening programs of the disease, for instance, the combined levels of CEA in plasma and ΔNp73 in EVs as we describe here.