{"text": "Pulmonary fibrosis is a progressively aggravating lethal disease that is a serious public health concern. Although the incidence of this disease is increasing, there is a lack of effective therapies. In recent years, the pathogenesis of pulmonary fibrosis has become a research hotspot. p53 is a tumor suppressor gene with crucial roles in cell cycle, apoptosis, tumorigenesis, and malignant transformation. Previous studies on p53 have predominantly focused on its role in neoplastic disease. Following in-depth investigation, several studies have linked it to pulmonary fibrosis. This review covers the association between p53 and pulmonary fibrosis, with the aim of providing novel ideas to improve the clinical diagnosis, treatment, and prognosis of pulmonary fibrosis. Pulmonary fibrosis is a group of chronic, irreversible, and fatal interstitial lung diseases that occur mostly in middle-aged and elderly people . It main\u03b21 (TGF-\u03b21), tumor necrosis factor-\u03b1 (TNF-\u03b1), and platelet-derived growth factor (PDGF) [It is currently accepted that the development and progression of pulmonary fibrosis are attributable to aberrant repair following repeated alveolar epithelial cell (AEC) injuries in response to various stimuli . The injr (PDGF) \u20138. Theser (PDGF) . This ler (PDGF)  . Nine point mutations were found in ten lung tissue samples. Most of the mutations occurred in the central area of the p53 gene . In anot\u03b21, to accelerate the EMT process and the proliferation of lung fibroblasts. This leads to the secretion of more collagen, which further accelerates pulmonary fibrosis. Many preliminary studies of pulmonary fibrosis have utilized this model [Some research studies have addressed the relationship between p53 and pulmonary fibrosis. Of the numerous methods to produce in vivo models of pulmonary fibrosis, the most common method is the injection of drugs, including bleomycin (BLM), amiodarone, and asbestos . A singlis model , 34. Folis model . In otheis model . This fiCell apoptosis is a type of programmed cell death and a key process that regulates homeostasis at the organizational, tissue, and internal environment level . There a\u03b4 and many other proteins that are involved in DNA synthesis to promote DNA synthesis. p21 directly inhibits DNA synthesis by competing with PCNA to bind with other DNA synthesis proteins [Researchers have constructed a mouse model of pulmonary fibrosis using a one-off intratracheal instillation of BLM. The results showed that apoptosis of type II AECs in the lung tissues of mice with pulmonary fibrosis was clearly enhanced, whereas the expression of the p53 protein was markedly increased. In contrast, knockout of the p53 protein effectively attenuated pulmonary fibrosis, and the extent of type II alveolar epithelial cell (AECII) apoptosis was also markedly alleviated, suggesting that the p53 protein probably accelerated the development and progression of pulmonary fibrosis by inducing AEC apoptosis . In a clproteins . Many stproteins . The afoIn addition, some studies have documented that the p53 protein could induce apoptosis in lung fibroblasts and myofibroblasts during the progression of pulmonary fibrosis, which may further delay the progression of pulmonary fibrosis. The effects of astaxanthin on myofibroblast apoptosis were also studied. The results indicated that astaxanthin could induce myofibroblast apoptosis through the activation of the mitochondrial apoptosis pathway. Further studies have confirmed the critical role of the p53 protein in this process . In anot\u03b2-galactosidase , telomeres and telomerase, senescence-related heterochromatin foci, and senescence-associated secretory phenotype (SASP). Among these, \u03b2-gal is detected earliest and used most commonly as a senescence marker. This lysosome-derived enzyme increases lysosomal biosynthesis in senescent cells [Aging is an inhibited state of cell proliferation, in which the cell cycle is arrested in phase G0 or G1 . Morpholnt cells \u201353. It iRecent studies have shown that the aging-related secretory phenotype, telomere injury, epigenetic changes, mitochondrial autophagy injury, and other pathological processes are probably involved in the occurrence and development of cell aging . In an i\u03b2-galactosidase in A549 cells was markedly enhanced, and that of the p53 protein was also clearly increased in a concentration-dependent manner, suggesting that p53 promoted AEC aging and accelerated the progression of pulmonary fibrosis [\u03b1 (INF-\u03b1), and other related cytokines. Such cytokines can bind to the corresponding receptors on the membrane, leading to the activation of aging-related signals by p53 [A mouse model of pulmonary fibrosis was constructed by the administration of BLM to investigate the correlation between p53 and collagen deposition. The results showed that collagen deposition and p53 protein expression in BLM-induced mice was significantly enhanced compared with that in WT mice. To confirm the mechanism underlying AEC aging, A549 cells were treated with BLM. It was found that the expression of the aging-related marker fibrosis . It is rs by p53 .\u03b2-galactosidase was enhanced significantly, indicating obvious aging, in which the aging-related proteins p53, p21, and p16 played a crucial role [The effect of p53 on the regulation of lung fibroblast aging has been confirmed in a clinical study in which lung tissue was collected from patients with IPF and compared with tissues from healthy lung tissue. The results showed that the telomeres of lung fibroblasts in the lung tissue of patients with IPF were markedly shortened, and the expression of ial role . Cyclooxial role , 63. Ourial role . Interleial role   to inhibit nuclear factor E2-related factor 2- (Nrf2-) induced gene expression [Given the broader understanding of p53, it was found that the p53 protein has the dual functions of oxidation and antioxidation, depending on the degree of oxidative stress . In resppression , 77  or small mother against decapentaplegic3 (Smad3) at the carboxyl terminal, and is then translocated into the nucleus by forming a trimer with small mother against decapentaplegic4 (Smad4). After nuclear translocation, small mother against decapentaplegics (Smads) then bind to other transcription factors to regulate the expression of EMT-related genes, leading to the dysfunction of cellular junctions, cytoskeletal rearrangement, and enhancement of cell migration and cell invasion, and eventually to EMT [Recent studies have shown the close correlation between EMT and the development and progression of pulmonary fibrosis . In respy to EMT .\u03b1-SMA), a marker protein for EMT, was significantly enhanced, suggesting that the EMT process had been initiated. Further studies found that p53 probably participated in the entire EMT process. BLM promoted the phosphorylation of Ser15 in the p53 protein, inhibiting the binding of MDM2, a negative regulator, to p53, and promoting the EMT process [\u03b21, and found reduced expression of the p53 protein. It is assumed that p53 inhibits the process of EMT. The difference between studies is probably related to the following factors. Although the two models are in vitro EMT models, they are two different models. Direct disruption of A549 cells with BLM not only leads to EMT but also to pathological processes related to cell injury. However, it still remains unknown which process is dominant. In addition, both experiments were in vitro tests and could, therefore, not simulate the complete internal environment. Therefore, further experiments are required to validate the functional role of p53 in EMT.Yamamoto et al. treated A549 cells with BLM and found that the morphology of A549 cells changed greatly. Meanwhile, the expression of alpha-smooth muscle actin , and pulmonary function tests, as well as bronchoscopy or lung biopsy if necessary . SerologThe establishment of a pulmonary fibrosis model by intratracheal instillation of BLM in p53 gene knockout and WT mice revealed significantly less collagen deposition in the lung tissue of p53 gene knockout mice and significantly reduced pulmonary fibrosis . These oThe weight of the evidence supports the hypothesis that the expression levels of p53 demonstrate a positive correlation with the severity of pulmonary fibrosis. However, p53 has various roles that are specific for different cells and have different mechanisms of action. During the progression of pulmonary fibrosis, an increase in p53 can promote AEC apoptosis, induce aging, aggravate oxidative injury, promote the progression of EMT, and accelerate the development of pulmonary fibrosis in terms of AECs. For lung fibroblasts, an increase in p53 can promote the apoptosis of lung fibroblasts, aggravate oxidative stress, and delay the development of pulmonary fibrosis. Meanwhile, increased p53 can also induce the aging of lung fibroblasts. This diversity makes it difficult to further clarify the regulatory mechanism of p53 in pulmonary fibrosis and to discover the corresponding drug treatment. Thus, for future treatment of pulmonary fibrosis, the targeted expression of p53 in different cells of lung tissues is a likely direction for study future studies which need to further focus on the following points.The data from most of the current studies support the view that damage to AECs initiates pulmonary fibrosis and drives its development. The ultimate pathological change in pulmonary fibrosis is the massive proliferation of lung fibroblasts and copious secretion of collagen. Thus, is the damage to AECs or the proliferation of lung fibroblasts more important in the overall process of pulmonary fibrosis? Although AECs may be the most important, this view is speculative, with a dearth of valid confirmatory evidence.p53 has different mechanisms of action for AECs and lung fibroblasts. This is a challenge for the development of drug therapies for pulmonary fibrosis that target p53. We believe that the current research on the underlying mechanisms of p53 in pulmonary fibrosis has been relatively superficial. Most studies have not addressed the function of the p53 protein. For example, the molecular mechanisms used by p53 to regulate AECs and lung fibroblasts are unknown. Furthermore, the p53 phosphorylation sites used to regulate apoptosis and senescence in AECs and lung fibroblasts are unknown. These issues must be clarified to further understand the role of p53 in the regulation of pulmonary fibrosis.There have been few studies of drugs targeting p53 for the treatment of pulmonary fibrosis. However, many drugs that target p53 have been developed as cancer therapeutics. It is conceivable that these drugs may be beneficial for the treatment of pulmonary fibrosis.Since p53 has different mechanisms of action in different cells, two points are crucial for the development of new drugs targeting p53 for the treatment of pulmonary fibrosis. First, we need to clarify whether the drug primarily targets AECs or lung fibroblasts. Second, we need to identify the signal transduction mechanism through which the drug exerts its effects. Targeting specific cells and specifically modulating p53 expression in those cells could achieve a better therapeutic outcome."}
{"text": "While numerous studies have provided evidence for selection history as a robust influence on attentional allocation, it is unclear precisely which behavioral factors can result in this form of attentional bias. In the current study, we focus on \u201clearned prioritization\u201d as an underlying mechanism of selection history and its effects on selective attention. We conducted two experiments, each starting with a training phase to ensure that participants learned different stimulus priorities. This was accomplished via a visual search task in which a specific color was consistently more relevant when presented together with another given color. In Experiment 1, one color was always prioritized over another color and inferior to a third color, such that each color had an equal overall priority by the end of the training session. In Experiment 2, the three different colors had unequal priorities at the end of the training session. A subsequent testing phase in which participants had to search for a shape-defined target showed that only stimuli with unequal overall priorities (Experiment 2) affected attentional selection, with increased reaction times when a distractor was presented in a previously high-priority compared with a low-priority color. These results demonstrate that adopting an attentional set where certain stimuli are prioritized over others can result in a lingering attentional bias and further suggest that selection history does not equally operate on all previously selected stimuli. Finally, we propose that findings in value-driven attention studies where high-value and low-value signaling stimuli differentially capture attention may be a result of learned prioritization rather than reward. Evidence is rapidly accumulating that a stimulus\u2019s history can result in a lingering attentional bias toward that stimulus or its features  will almost always have a higher priority than selecting a nonthreatening stimulus . The present study further investigates whether the observed high-reward versus low-reward bias on attention can be explained in terms of prioritization rather than being a direct effect of the availability of reward. To this end, the current study aims to investigate whether adopting a top-down set that unequally prioritizes different stimuli can lead to effects of selection history that mirror the effects obtained by high-reward and low-reward signaling stimuli in value-driven capture tasks. Such an observation would provide evidence that attentional modulation due to selection history can be achieved in the absence of reward, but that reward can potentially modulate the behavioral priority of different stimuli. An explanation in terms of \u201cprioritization\u201d could be instrumental in understanding the discrepant findings concerning the necessity of value in selection history.We conducted two experiments to address the aforementioned questions by manipulating the priority of differently colored stimuli in a training/testing paradigm typically used in studies of value-driven attention  were presented in a circular array consisting of otherwise gray shapes. At the start of the training phase, on-screen instructions were shown explaining which colored circle in each of three possible color pairs contained the target stimulus. The initial training phase was used to further consolidate the learned schemes by having participants repeatedly select the prioritized colored circle and to respond to the target presented inside that stimulus. As illustrated in the left panel of Fig. SD = 4.83 years). All participants gave written, informed consent prior to the experiment. All protocols and procedures were approved by the Vrije Universiteit Amsterdam Ethical Committee, in line with the Declaration of Helsinki.We tested 35 participants with normal or corrected-to-normal vision and no reported history of mental illness. The data of three participants were discarded, as their average accuracy scores on either the training or testing task were more than 2.5 standard deviation below the group mean. Of the 32 remaining participants, 22 were female, and participants had an average age of 21.84 years . Prior to the start of both tasks, instructions were presented on-screen, and the experimenter verified that participants fully understood the task. Both tasks were conducted in a dimly lit, sound-attenuated room on a standard PC with a 22-in. monitor. Viewing distance was fixed at 75 cm. MATLAB\u00ae 2016a and Psychtoolbox 3  and two shapes that were presented in different colors . Every shape in the search display contained a gray \u201cT\u201d (11.4 cd/m2) that could be rotated 0\u00b0, 90\u00b0, 180\u00b0, or 270\u00b0. The target was defined as the \u201cT\u201d shape presented inside one of the two colored shapes, depending on the particular priority schemes the participant was shown at the start of the experiment (described in a subsequent paragraph). All stimuli were presented on a black background. Participants were instructed to respond as quickly and accurately as possible to the orientation of the target \u201cT\u201d using the four arrow keys on the computer keyboard . Note that the orientation of all \u201cT\u201ds in the display were randomly chosen with the exception that the \u201cT\u201ds in the two colored shapes were always differently oriented, such that selecting the wrong color for the particular priority scheme always resulted in an incorrect response. After participants responded, accuracy feedback was presented in the center of the screen (\u201ccorrect\u201d/\u201cincorrect\u201d) for 500 ms. Erroneous responses as well as timed-out trials were considered incorrect in this and all further experiments.The left panel of Fig. At the start of the experiment, participants were presented with three color-response schemes see Fig.  and wereThe training phase consisted of eight blocks of 72 trials each , preceded by two practice blocks of 48 trials each. After each trial in the first practice block, participants received feedback about their accuracy and were presented with an illustration of the color-response scheme defining the target for that trial. Pressing the space bar would start the next trial. In the second practice block, participants again received accuracy feedback after each trial, but the color-response scheme was only presented when the participants responded incorrectly.The testing task  = 1.687, p = .194, \u03b7p2 = .052 (see Table t test), t(31) = 38.91, p < .001, confirming that participants had adequately learned the priority schemes.We began our analysis by confirming that priority averaged out over the course of the training task, such that there were no overall differences in response times between the three different priority schemes. We only analyzed nonpractice trials with correct responses . Furthermore, we normalized the response-time data by trimming trials with correct response times that were 2.5 standard deviations above the conditional mean response time , or trials in which the response was faster than 200 ms . All further reaction time analyses are based on the mean response times obtained for each participant and condition. A repeated-measures ANOVA on the mean response times, with color scheme  as the only within-subjects factor, did not yield any significant effects (F < 1) or shape priority (F < 1), nor an interaction between these factors .As every trial in the testing task included two colors from the training task, we compared performance in trials with the target shape presented in a prioritized color to performance in trials with the distractor shape presented in the prioritized color. Practice and erroneous trials  were excluded from the response-time analyses and the resultant data were also trimmed to normality by removing trials with response times that were 2.5 standard deviations above the conditional mean or faster than 200 ms . A repeated-measures ANOVA on the mean response times, with color scheme  and shape priority  as within-subject factors revealed neither evidence of effects of color scheme  = 1.169, p = .318, \u03b7p2 = .036. Again, no differences between target and distractor priority were present for any of the color schemes . Table The same patterns of null effects were observed when the ANOVA was carried out on the accuracy data, shape priority: The results of Experiment 1 provided no evidence that differentially prioritized stimuli have differing effects on attentional capture when presented simultaneously. No differences in response times were observed between trials with the target versus distractor shape presented in a prioritized color. Importantly, priority was only established at the trial level in Experiment 1 and averaged out over the three possible colors throughout the course of the training task. Although these patterns of null effects are not conclusive, they suggest that this sort of situational prioritization at the trial level may not elicit differential effects of selection history. We tested prioritization at the trial level, but not the task level, as a proxy for situational priority relationships in the real world where a given stimulus or feature may be prioritized in one situation but deprioritized in another situation. Although Experiment 1 did not uncover any evidence that this sort of situational prioritization takes places at the task level, we tested the possibility that stimuli with fixed priority relationships throughout the course of both training and testing tasks may elicit effects of selection history in Experiment 2.and the task level .We tested 30 participants with normal or corrected-to-normal vision and no history of mental illness. Two participants (not included in these demographics) were replaced due to technical difficulties during experimentation. Of the remaining 28 participants, 21 were female, with an overall participant age of 21.54 years , meaning color was no longer prioritized by target selection. However, in Experiment 2, only one colored stimulus was present in the testing display on a random 50% of trials , and two colored stimuli were present on the other 50% of testing trials. When only one colored stimulus was presented, it was never the singleton shape target and instead functioned as a pop-out distractor. When two colored stimuli were presented, one was always the target singleton shape, and the other was the salient distractor shape. We included trials with only one color to measure the extent to which attention was captured by colored stimuli with different priorities, independent of the learned color-response contingencies between pairs of colors. We used the trials with two colors to compare how stimuli with fixed priority relationships compete for attentional resources. Every color pair was presented equally often, such that the target was presented in a higher priority color (HPC or LPC) than the distractor (LPC or NPC) on a random 50% of these two-color trials in the testing task and the distractor was presented in the higher priority color compared to the target in the remaining half of testing trials. The testing phase consisted of four blocks of 96 trials , preceded by a single practice block of 30 trials.F = 23.004, p < .001, \u03b7p2 = .460, such that follow-up planned comparisons confirmed that correct response times for Scheme B trials were significantly faster (641 ms) than response times for Scheme A trials  and Scheme C trials , but that response times for Scheme A and Scheme C trials were not significantly different (p = .589). Overall accuracy scores were relatively high (94.70% correct) and well above 25% chance performance, t(27) = 131.833, p < .001. A second repeated-measures ANOVA on the accuracy data also revealed a main effect of scheme on accuracy , F = 20.077, p < .001, \u03b7p2 = .426. Follow-up planned t tests explicated that mean accuracy for trials in each of the three schemes differed significantly, with the most accurate performance occurring for Scheme B . See Table To determine whether the three different priority schemes affected response times in the training task, we first conducted a repeated-measures ANOVA on participants\u2019 nonpractice, correct , 2.5-standard-deviation-normalized response times . There was a main effect of scheme, After confirming that fixed priorities were successfully associated with the three colors in the training task, we next analyzed the testing task data to determine whether the color priorities instilled at the trial and task levels in the training task would carry over to capture attention in the testing task. As in all previous analyses, we only analyzed nonpractice trial correct responses  within 2.5 standard deviations above the participant\u2019s conditional mean . We then conducted separate analyses on response time and accuracy data for trials with only one color  = 3.233, p = .047, \u03b7p2 = .107 . Surprisingly, the difference between high priority and no priority color failed to reach significance (p = .17). There was no significant difference in response times between trials with low and no priority color distractors (p = .26). Accuracy results were in line with the reaction time observations, as indicated by a main effect of priority , F = 5.254, p = .008, \u03b7p2 = .163  or no-priority distractor (p = .018). No difference was observed between trials with a low versus no-priority distractor (p = .96).A repeated-measures ANOVA on participants\u2019 averaged response times for one priority color (distractor) trials revealed a significant main effect of this color , F = 29.102, p < .001, \u03b7p2 = .519, such that participants responded faster to trials in which target was prioritized over the distractor (827 ms) compared with response times for trials in which the distractor was prioritized over the target  = 1.520, p = .228, \u03b7p2 = .053, nor a significant interaction, F = 1.314, p = .277, \u03b7p2 = .046, we nevertheless conducted planned comparisons between the three schemes to quantify any lingering effects of differential prioritization. These comparisons revealed significantly faster correct response times for trials with targets versus distractors presented in the prioritized color for each of the three scheme , suggesting that imbalances in prioritization carry over as lingering effects of selection history.To investigate whether color prioritization in the training task carried over in the form of lingering attentional bias in the testing task, we next conducted a repeated-measures ANOVA, with shape priority  and scheme  on participants\u2019 averaged response times in the two-color trials. Importantly, each display in the two-color trials always consisted of one shape presented in a color that previously had a higher priority than the color of the other shape. On half the trials, the target shape singleton was presented in the higher priority color and the distractor in the lower priority color, and vice versa on the remaining trials. The ANOVA resulted in a main effect of shape priority, F, 54 = 29F = 7.438, p = .011, \u03b7p2 = .216. No significant effect of scheme was observed, F = 1.739, p = .185, \u03b7p2 = .061, nor did the interaction between scheme and shape priority reach significance, F = 2.520, p = .090, \u03b7p2 = .085  and scheme , revealed a main effect of shape priority, indicating that participants responded more accurately when the target was prioritized over the distractor (85.0% correct) as compared with trials in which the distractor was prioritized over the target (81.9% correct), F1, 7 = 7.438Experiment 2 confirmed that effects of selection history can be observed when stimuli are prioritized in a fixed manner at both the task and trial levels. Effects of prioritization were already apparent in the training task, where participants responded faster and more accurately for Scheme B trials compared with Scheme A or C trials. Although these results could potentially be caused by selection history effects, they are at least equally likely caused by differences in ambiguity concerning which stimulus a participant has to respond to. In Scheme B, there is the least ambiguity about which stimulus contains the target because the target was always presented in the high-priority color  and the distractor was always presented in the no-priority color (the color that never required a response). Given that high overall accuracy in the training task suggested the color schemes were all well learned (like in Experiment 1), we instead turned to the testing data to further quantify the effects of fixed prioritization. The testing phase data showed clear lingering effects due to selection history, such that a stimulus with a higher versus lower priority had more influence on attentional allocation. This pattern was observed for all paired levels of prioritization . Therefore, the results of Experiment 2 suggest that fixed prioritization influences attention even when the prioritized feature is no longer task relevant.F = 20.227, p < .001, \u03b7p2 = .259. As expected, there was a significant interaction between shape priority and experiment, F = 15.158, p = .001, \u03b7p2 = .207. Follow-up planned comparisons showed no significant difference between target and distractor prioritization in Experiment 1 , but a significant difference between target and distractor priority in Experiment 2 , confirming that the main effect of shape priority was driven by the fixed priority relationships established in Experiment 2. A similar interaction between shape priority and experiment was observed for the accuracy scores, indicating that the difference between target and distractor priority in Experiment 2 , was larger than the same effect in Experiment 1 . These results once more confirm that situational priority does not lead to observable differences in terms of selection history, whereas fixed priority does.We also conducted a cross-experiment mixed-design ANOVA, with shape priority as a within-subjects factor and experiment as a between-subjects factor on the averaged response-time data for trials in both experiments with two colored stimuli, to assess potential differences between selection effects due to situational (Experiment 1) versus fixed (Experiment 2) prioritization. We observed a main effect of shape priority, such that participants made faster correct responses when the shape singleton target was presented in the higher priority color (887 ms) compared with correct response times for trials with the distractor presented in the higher priority color (908 ms), We investigated whether stimuli prioritized during an initial training task would lead to attentional biases due to selection history in a subsequent testing task where priority was no longer enforced or task relevant. Indeed, results suggest that previously prioritized stimuli can lead to attentional capture. However, whether capture occurs depends on the nature of the priority relationships. Significant effects of attentional capture by previously prioritized stimuli were observed only in Experiment 2, when the prioritized stimuli shared a fixed priority relationship such that throughout the experiment, different colors had a ranked priority that never changed. Experiment 2 showed that under such fixed circumstances, when a single colored stimulus was presented as one of the distractors in the testing task, attentional capture was observed such that correct response times were slower and accuracy decreased when the distractor was presented in the HPC compared with when it was presented in the LPC. When both the target and distractor were presented in prioritized colors, response times were biased by the higher priority stimulus such that participants were faster when the target versus distractor was presented in the higher priority color. On the contrary, when stimuli were only situationally prioritized at the trial level in Experiment 1, there were no corresponding effects of capture during the testing phase. Furthermore, the significant interaction between shape priority and experiment in the cross-experiment analysis offered further support for the proposal that fixed, but not situational, priority induced lingering effects of selection history.The present findings add to our understanding of the attentional phenomenon of selection history. A number of attentional effects have been retroactively linked to selection history, such as intertrial priming , which led to the exclusion of three participants in Experiment 1. Alternatively, we could have chosen to use a less strict criterion and exclude only those participants that scored below chance level. Using a chance-level criterion would not have led to the exclusion of the three participants in Experiment 1. Nonetheless, a recalculation of all statistical tests that included the data of the three discarded participants did not change any result and conclusion drawn here.Finally, throughout the current study, theoretical comparisons between learned prioritization and value-driven attentional capture have been put forward. The current study provides support for recent studies that have shown that the administration of reward is not a necessity for observing lingering effects of stimulus history on attentional selection (Grubb & Li, Grubb and Li  further"}
{"text": "Clopidogrel is increasingly being used for the secondary prevention of ischemic stroke according to the updated guidelines on acute stroke management. Failure to achieve a drug response is referred to as clopidogrel resistance. Similarly, a higher activation of platelets during clopidogrel therapy\u2014high on-treatment platelet reactivity\u2014is equivalent to a reduced effectiveness of a therapy. Clopidogrel resistance is considered to be a common and multifactorial phenomenon that significantly limits the efficacy of antiplatelet agents. The aim of the current study is to review the latest literature data to identify the prevalance and predictors of clopidogrel high on-treatment platelet reactivity among stroke subjects and to establish the potential impact on clinical outcomes and prognosis. Clinical databases were searched by two independent researchers to select relevant papers on the topic, including all types of articles. Several important predictors contributing to clopidogrel resistance were identified, including genetic polymorphisms, the concomitant use of other drugs, or vascular risk factors, in particular nonsmoking and diabetes. Clopidogrel high on-treatment platelet reactivity has a negative impact on the clinical course of stroke, worsens the early- and long-term prognoses, and increases the risk of recurrent vascular events. Platelet function testing should be considered in selected stroke individuals, especially those predisposed to clopidogrel resistance, for whom an improvement in the efficacy of antiplatelet therapy is essential. This particular group may become the greatest beneficiaries of the modification of existing therapy based on platelet function monitoring. Clopidogrel is one of the most widely used antiplatelet agents worldwide. It has an established position as an antagonist of platelet function, leading to a reduced risk of recurrent ischemic events. It plays a primary role in the prevention of coronary heart disease in the field of cardiology. Due to the updated guidelines on stroke, this drug is also increasingly being used for the secondary prevention of ischemic stroke . It mighUnfortunately, knowledge of the role of clopidogrel resistance in ischemic stroke is not well established. In contrast, there are numerous reports on coronary heart disease that encourage individual or personalized treatments based on platelet function testing ,7. ThereABCB1 gene [CYP2C19) (responsible for approximately 50% of active metabolites) and the cytochrome P450 3A4 gene (CYP3A4) (39.8% of active metabolites) [P2RY12 gene encodes the P2Y12 receptor and GPIIIa gene\u2013GPIIb/IIIa complex, a biologic activity target and effector of clopidogrel [Clopidogrel is a pro-drug and is inactive in vitro. It must be biotransformed in the human body after oral ingestion to become operational. Antiaggregative properties are produced by an active shortlisting metabolite generated by the cytochrome P450 (CYP)-dependent pathway in the liver. This is contradictory to other antiplatelet agents, such as aspirin or ticagrerol, which are active from the beginning and do not need to undergo transformation. The inhibition of platelets is a result of the irreversible binding of P2Y12 receptors. Adenosine diphosphate (ADP) activates platelets by binding the two protein receptors on the platelets (P2Y1 and P2Y12), leading to ADP-mediated platelet aggregation with the glycoprotein IIb/IIIa (GPIIb/IIIa) complex pathway. As an ADP receptor inhibitor, clopidogrel prevents platelet aggregation, but it does not interfere with arachidonic acid metabolic pathways . There abolites) . The P2Rpidogrel . Geneticpidogrel .There are several platelet function assays that can be performed to assess the platelet reactivity. The most commonly used are turbidimetric aggerogometry , moderately high shear stress platelet function analysis , light transmission aggregometry , vasodilator-stimulated phosphoprotein phosphorylation assay , and impedance aggregometry . Most platelet function assays measure the level of platelet aggregation in response to a variety of agonists. A higher activation of platelets during clopidogrel therapy high on-treatment platelet reactivity is equivalent to a reduced effectiveness of a therapy. Different assays use specific cut-off points to identify clopidorel resistance or high on-treatment platelet reactivity, e.g., VerifyNow has more than 230 P2Y12 reactive units (PRU); Multiplate has more than 47 Units, PFA-100 has less than 106 s of closure time; LTA has an aggregation rate of >70% or >46%; VASP has a platelet reactivity index of greater than 50% ,17. SomeThe prevalence of clopidogrel resistance in ischemic stroke patients is variable and depends mainly on the method used for the assessment of platelet reactivity and the cut-off value used. In ischemic stroke research, the most frequently used technique to measure platelet aggregation is LTA. Prevalence rates fall into fairly wide ranges (16.83% to 50%) ,18,21. IThe response to the drug may be influenced by genetics or by clinical and pathophysiological factors .CYP2C19 polymorphisms. Recent studies have demonstrated that carriers with one or more CYP2C19 loss-of-function alleles (2* or 3*) are at a higher risk of clopidogrel nonresponsiveness due to having lower levels of its active metabolite [CYP2C19 loss-of-function allele, the percentage of individuals with clopidogrel resistance is estimated to be 71.7% which is significantly different from the prevalence of this phenomenon in noncarriers\u201432.1% [CYP2C19 2* or 3* carriers compared with noncarriers , and Liu et al. [CYP2C19*2 allele carriers is higher than that of noncarriers (odds ratio (OR) = 2.366; p = 0.014). However, it is worth emphasizing that carriers of CYP2C19 loss-of-function alleles were found to be significantly more likely to have clopidogrel high on-treatment platelet reactivity, but not an increased risk or higher recurrence of vascular events [CYP3A4. The CYP3A4*1G variant is considered to be a protective factor in clopidogrel resistance among ischemic stroke patients. It is estimated that the most frequent mutant allele is associated with greater liver metabolic activity, leading to a higher concentration of active clopidogrel-related substances, thus increasing platelet inhibition. Liu demonstrated that among CYP3A4*1G carriers, the estimated risk of clopidogrel resistance is significantly lower compared with that of noncarriers  [ABCB1 gene TT variant and the wild-type CC genotype have been associated with a higher risk of recurrent vascular events during clopidogrel treatment, but these findings were reported among coronary heart disease subjects [ABCB1 gene in ischemic stroke subjects. However, the above studies were limited by a single locus assessment and small sample sizes. Polymorphisms of the P2RY12 and GPIIIa genes have been shown to be associated with clopidogrel-dependent platelet reactivity [P2RY12 TT+CT carriers are more likely to have a higher platelet reactivity following a clopidogrel treatment than P2RY12 CC carriers, and Liu et al. [P2RY12 H2 haplotype compared with those with the H1 haplotype, but this result did not reach statistical significance. Yi et al. [GPIIIa AA carriers and AG+GG carriers differed significantly in terms of the incidence of platelet activity on clopidogrel therapy (p = 0.042). It has been noted that interactions between genes may have a greater impact on platelet activity than the effect of a single gene polymorphism. Moreover, it has been underlined that clopidogrel resistance may not be attributed to single genetic variants at individual loci alone but may be conditioned by multiple gene\u2013gene intracations. Yi et al. [CYP2C19, P2RY12, and GPIIIa variants are independently associated with the efficacy of clopidogrel. Furthermore, the synergistic effect of genes interactions may contribute not only to platelet activity but also to stroke prognosis. Yi et al. [p = 0.003). The molecular mechanisms responsible for the above characteristics still remain unclear.As mentioned above, many different genes are involved in the biotransformation of clopidogrel into the active substance. Their polymorphisms may interfere with the above process, leading to a lower amount of active metabolites, resulting in a reduced drug efficacy. The most common gene variant described in the literature related to clopidogrel resistance concerns tabolite . It is etabolite . Fu et ars\u201432.1% . Similarrs\u201432.1%  demonstrr events ,29,30,31subjects . In contsubjects  and Yi eactivity ,35,36. Au et al.  reportedCYP3A4 complex, while others, such as pravastatin or fluvastatin, do not. The related data on coronary heart disease are controversial. Some studies have not reported a negative interaction of statin coadministration (regardless of the pathway of biotransformation) with clopidogrel effectiveness in subjects undergoing coronary stenting [p = 0.25). To the best of our knowledge, there is currently no study indicating a significant negative impact of statin coadministration on the inhibitory effect of clopidogrel on platelet function in ischemic stroke patients.Most drugs are metabolized in the liver via similar enzyme pathways to the one that produces clopidogrel. Thus, the coadministration of several drugs may competitively reduce the effectiveness of each of them. The most commonly used concomitant drugs in the secondary prevention of stroke are the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) and proton pump inhibitors, a protocol that is used to reduce the risk of developing gastrointestinal complications from antiplatelet agents. Most statins, including atorvastatin, lovastatin, or simvastatin, are extensively metabolized by the stenting . In contstenting  demonstrstenting ,14,21,40stenting  reportedCYP2C19, the coadministration of omeprazole or esomeprazole may be associated with a reduced efficacy of platelet inhibition by clopidogrel [p = 0.02), but these factors were not significantly correlated in a mutlivariate analysis (p = 0.10). Yi et al. [p = 0.71). In contrast, Su et al. [p < 0.001). Overall, based on the above inconsistent findings, proton pump inhibitors that are not involved in the CYP2C19 pathway, such as pantoprazole, ranitidine, or famotidine, should be administered, if clinically warranted, in combination with clopidogrel to avoid competition in terms of the mechanism of action and to provide effective antiplatelet therapy [Data on drug\u2013drug interactions involving proton pump inhibitors and clopidogrel seem to be more conflicting. Several studies have shown that, due to the potent inhibition of pidogrel . Moreovepidogrel . Data onpidogrel  demonstri et al.  noted thu et al.  demonstr therapy .Many reports in the literature refer to the relationships of established risk factors for the development of vascular diseases, including stroke, with the antiplatelet effect of clopidogrel. Several studies have revealed that, in ischemic stroke subjects, clopidogrel high on-treatment platelet reactivity is significantly associated with diabetes ,14,22,40p = 0.002) than nonsmokers and, additionally, the incidence of clopidogrel resistance is significantly lower in smokers . Zhang et al. [Many interesting insights have resulted from studies on the influence of smoking on platelet reactivity during clopidogrel therapy. Many researchers have described the smokers\u2019 paradox, which is that smokers experience better antiplatelet effects following clopidogrel treatment than nonsmokers, resulting in stronger platelet inhibition, in particular, in patients with coronary heart disease . Kang etg et al.  concludeg et al. . The expg et al. . Other rJeon showed that clopidogrel resistance in ischemic stroke patients is significantly asssociated with a higher white blood count and C-reactive protein (CRP) level . SimilarPrevious studies have shown that aspirin resistance may be associated with a large vessel disease background in ischemic stroke patients . Reportsp < 0.001). In particular, recurrent ischemic stroke was more strongly associated with the clopidogrel-resistant subgroup than the clopidogrel responders . However, the incidence of myocardial infarction and death due to vascular causes did not differ significantly between the above groups. Furthermore, in a multivariate regression analysis, the clopidogrel high on-treatment platelet reactivity was found to be the most significant independent risk factor for recurrent ischemic events . In another study, also with a 6 month follow up period, in clopidogrel resistant subjects, a significant association with adverse clinical outcomes in all patients (p = 0.023) and in those with recurrent ischemic stroke (p = 0.021) was demonstrated, compared with clopidogrel responders [p = 0.83) or sudden death of vascular origin (p = 0.82), but it was shown to be an independent predictor of adverse events . A survival analysis by Fu et al. [p = 0.04). Furthermore, clopidogrel high on-treatment platelet reactivity was considered an independent predictor of recurrent clinical events . However, data on individual vascular events are lacking. Rao et al. [p = 0.784, myocardial infarction p = 0.223; vascular death p = 0.752) between clopidogrel-resistant and sensitive subgroups [The main goal of antiplatelet therapy as part of secondary prevention is to decrease the risk of recurrent ischemic events after the onset of ischemic stroke. The most common event is another recurrent stroke or transient ischemic attack, but stroke subjects are also at a higher risk of myocardial infarction (MI), sudden cardiac death, acute limb ischemia, or systemic embolism. Previous studies regarding aspirin resistance have demonstrated a significant, independent association between high on-treatment platelet reactivity and a higher risk of recurrent vascular events in stroke subjects . The datsponders . Similaru et al.  conducteo et al.  only fole p = 0.0 was demop = 0.029). However, only patients with large vessel disease as a cause of stroke were included in this study. Yi et al. [p = 0.001). At the same time, there was no significant difference in the clinical condition assessed with the NIHSS on the day of enrollment (acute phase of stroke) between clopidogrel responders and nonresponders . Conversely, Jeon [p = 0.015). Qiu et al. [p = 0.001). Yi et al. [p < 0.001). Subjects with clinical deterioration had significantly higher levels of platelet aggregation than patients without deterioration (p < 0.001). A regression analysis showed that clopidogrel resistance was an independent predictor of early neurological deterioration . These data underscore the significant influence of adequate platelet inhibition by clopidogrel on the clinical course of stroke and both early and late prognoses.Another very important issue related to the effective action of an antiplatelet agent is the reduction in the severity of ischemic stroke and the improvement of the prognosis and recovery. Therefore, it seems that clopidogrel resistance may significantly worsen the clinical and functional condition of stroke subjects and contribute to a poor prognosis and clinical outcome. Lee et al.  demonstri et al.  investigly, Jeon  showed tu et al.  indicatei et al.  investigp = 0.484). Similarly, Yi et al. [p = 0.992; p = 0.753). In another study, regarding late complications, Yi et al. [p = 0.95) and intracranial hemorrhage (p = 0.92). The above results suggest that the sensitivity of platelets to clopidogrel is not a risk factor for either early or late intracranial bleeding.Intracranial hemorhage, such as gastrointestinal or urinary bleeding, may be one of the adverse effects of antiplatelet therapy that could worsen the clinical condition and recovery. Some researchers have investigated whether the degree of platelet inhibition could contribute to symptomatic bleeding. Lee et al.  showed ti et al.  reportedi et al.  followedp = 0.01), while not increasing the risk of sudden death or bleeding. Unfortunately, both studies included subjects based only on high on-treatment platelet reactivity, without differentiating them by additional determining factors which, in view of the above considerations, constitutes a limitation. Additionally, it should be emphasized that, compared to coronary artery disease, the capacity to modify antiplatelet treatment in stroke is significantly limited, as current guidelines recommend, apart from clopidogrel, only aspirin or dipyridamole.Due to the lack of support by large, randomized, multicenter, clinical trials, the assessment of platelet reactivity in ischemic stroke patients is not currently recommended for routine use. However, numerous significant relationships concerning multiple effects on the course of stroke suggest that, in some clinical situations, the determination of the degree of platelet inhibition may be useful for modifying or intensifying the efficacy of treatment. Based on the provided data, this group should especially include subjects with recurrent ischemic stroke or other vascular events, nonsmokers with long-term and advanced diabetes, and carriers of genetic variants promoting clopidogrel resistance, if available. Platelet function monitoring and confirmation of high platelet activity should be a sufficient reason to switch the antiplatetet agent to improve the effectiveness of secondary prevention treatment. Reports on therapy modification based on platelet function testing are negligible and inconclusive. In a retrospective study, Depta et al.  showed tThis comprehensive review included papers regarding connections between clopidogrel and platelet reactivity, in particular, among ischemic stroke subjects. Clinical databases were searched by two independent researchers. English studies of all types, especially clinical trials, orginal research, observational, cross-sectional and cohort studies, and reviews were included. The various stages of proper selection of data from the literature are presented in In summary, clopidogrel resistance in ischemic stroke patients was presented as a common and important phenomenon with a multifactorial background. Similar to aspirin resistance, it has a negative impact on the clinical course of stroke, worsens the early- and long-term prognoses and increases the risk of recurrent vascular events. This highlights the importance of effective antiplatelet therapy in preventing the occurrence of stroke and the growing role of platelet function monitoring to identify nonresponders and support personalized and improved treatments. Specific features of ischemic stroke patients who are particularly at risk of developing clopidogrel resistance have been identified, and these should be considered and used as indicators of platelet reactivity testing to ensure the maximum efficacy of antiplatelet therapy."}
{"text": "The p38 MAPK pathway is well known for its role in transducing stress signals from the environment. Many key players and regulatory mechanisms of this signaling cascade have been described to some extent. Nevertheless, p38 participates in a broad range of cellular activities, for many of which detailed molecular pictures are still lacking. Originally described as a tumor-suppressor kinase for its inhibitory role in RAS-dependent transformation, p38 can also function as a tumor promoter, as demonstrated by extensive experimental data. This finding has prompted the development of specific inhibitors that have been used in clinical trials to treat several human malignancies, although without much success to date. However, elucidating critical aspects of p38 biology, such as isoform-specific functions or its apparent dual nature during tumorigenesis, might open up new possibilities for therapy with unexpected potential. In this review, we provide an extensive description of the main biological functions of p38 and focus on recent studies that have addressed its role in cancer. Furthermore, we provide an updated overview of therapeutic strategies targeting p38 in cancer and promising alternatives currently being explored. Mitogen-activated protein kinase (MAPK) cascades are signaling components that show a high degree of conservation throughout evolution and play a key role in converting extracellular stimuli into a broad range of cellular responses. All MAPK signaling cascades consists of a three-tiered module of protein kinases: MAPK kinase kinases  at the top, MAPK kinases  in the middle, and MAPKs at the bottom .In mammals, three major MAPK cascades have been described. The ERK1/2 pathway is activated mainly by mitogens and has been shown to be upregulated in many human tumors. In contrast, the Jun N-terminal kinase (JNK) and p38 pathways are activated mostly by environmental and genotoxic stresses and are therefore, also generically known as stress-activated protein kinases or SAPKs. The JNK and p38 signaling pathways show a certain degree of redundancy in their actions; however, the extent of crosstalk between them and their implications in cell physiology regulation depends on the cellular type, tissue, and organism. This review outlines current understanding of p38 MAPK family members and their involvement in tumor development, as well as the strategies to treat cancer based on targeting p38.The four p38 MAPKs are encoded by distinct genes: p38\u03b1 MAPK14), which comprises two different spliced variants; p38\u03b2 (MAPK11); p38\u03b3 MAPK12); and p38\u03b4 (MAPK13) [; and p38, which cSeveral mouse models genetically targeting p38 have been produced. Of note, p38\u03b2, p38\u03b3 and p38\u03b4 knockout mice do not have developmental defects. Moreover, of these three models, only p38\u03b4 knockout mice display a clear phenotype. In this regard, these animals are protected against high-fat diet-induced insulin resistance through the regulation of PDK1 activity and insulin secretion. This observation, therefore, indicates that this MAPK isoform plays a key role in the regulation of glucose homeostasis ,8. MoreoFull activation of most protein kinases requires phosphorylation on a flexible motif known as the activation loop. In particular, p38 activation takes place by the dual phosphorylation of Thr and Tyr on the Thr\u2012Gly\u2012Tyr motif located on the activation loop found on the kinase subdomain VIII . Dual phThe p38\u03b1 isoform can be activated by noncanonical mechanisms see . One of The strength and dynamics of p38 pathway signaling are key elements in determining proper cellular responses . MoreoveTo date, a full characterization of the p38 phosphoproteome has not been performed . All p38Upon phosphorylation-mediated activation, p38 can in turn phosphorylate both cytoplasmic and nuclear proteins. In order to have access to nuclear substrates, activated p38 transiently accumulates and translocates to the cell nucleus. Since MAPKs do not have nuclear localization or nuclear export motifs, the molecular mechanism by which translocate into the nucleus is not fully known. Given that p38\u03b1 can genetically complement a budding yeast strain lacking the p38-homologous Hog1 MAPK , initialFinally, p38 signaling ends upon the removal of the phosphates from the Thr\u2012Gly\u2012Tyr motif. Several Ser/Thr phosphatases, such as PP2A and PP2C, have been described to remove the threonine phosphate on the activation motif. The action of these phosphatases leads to the appearance of Tyr monophosphorylated p38 forms that lack kinase activity . In addip38 is activated by a wide range of environmental stimuli , genotoxic and DNA damaging agents , inflammatory cytokines, PAMPs (pathogen-associated molecular patterns), and DAMPs (danger-associated molecular patterns) .p38 is a multitasking kinase that regulates multiple cellular functions, including cell proliferation, differentiation, stress response, apoptosis, and cell migration and survival, among others, by interacting with a plethora of substrates . Part ofThe contributions to physiology of p38 have been addressed, mostly by using inhibitors that block kinase activation, as well as by using genetically engineered mouse models. Since mice lacking p38\u03b1 die before birth, some of the strategies to overcome this include the generation of p38\u03b1 tissue-specific conditional knockouts, deletion of upstream or downstream proteins of the MAPK pathway like MKK3/6, MSK1/2, MK2, or WHIP1, and generation of dominant negative p38\u03b1 forms .The ablation of p38\u03b1 leads to an increased proliferation of hematopoietic progenitor cells through the regulation of Epo expression , as wellOf particular importance is the capacity of p38 to control cell cycle progression, which is crucial for cellular homeostasis. p38 regulates proliferation at both G1/S and G2/M phases of the cell cycle by activating checkpoint responses .Exposure to osmotic stress, reactive oxygen species, DNA damage, and prosenescence stimuli activates p38 signaling, thereby leading to the induction of the G1/S checkpoint. p38 activity promotes the downregulation of cyclin D1, which is essential for S phase transition. This downregulation is achieved by stimulating ubiquitin-dependent degradation of cyclin D1  and inhiNotably, and contrary to what is commonly reported, p38 can enhance proliferation. However, this effect has been observed only in hematopoietic cells and in some cancer cell lines . Such anThe role of p38 in cancer has been extensively studied. Several reports have shown that p38 functions as an antitumorigenic factor see . Its rolThe importance of the p38 pathway in the regulation of the immune response has attracted considerable attention in the context of carcinogenesis. Immune cells can strongly modulate tumor progression by secreting cytokines and chemokines. Indeed, cancer and inflammation are tightly associated, and some chronic inflammatory diseases, particularly those occurring in the gastrointestinal tract, correlate with a higher risk of cancer development . p38 regThe role of p38 in promoting cell differentiation has also been reported to be relevant for its tumor inhibitory activity. In rhabdomyosarcoma cells, expression of either MKK3 or MKK6 reactivates the pathway, leading to terminal differentiation of cancer cells . DeletioAdditionally, the pro-apoptotic activity of p38 is related to its ability to inhibit tumor growth. This is particularly evident in cases where the effects of chemotherapeutic drugs rely on p38 activation . AnotherDespite numerous studies that provide experimental evidence of the antitumorigenic role of p38, many others demonstrate that this kinase promotes cancer by enhancing survival, migration, or resistance to stress and chemotherapeutic agents in tumor cells  see Fig. p38\u03b1 waIn contrast to the p38\u03b1 tumor-suppressing role, p38\u03b3 is required for Ras transformation in a phosphorylation-independent manner. In this regard, the expression of p38\u03b3 is selectively induced in colon cancer cells harboring mutated Ras and is required for proliferation ,97, therCompared to other genes that present recurrent mutations in several cancer types, the p38 gene does not show systematic loss-of-function mutations in common human malignancies. In total, results obtained from the sequencing analysis of a pool of 1500 samples revealed that the frequency of p38 mutations  in human cancers is lower than 1% . This inThe stage of tumor development could be a strong component governing the function of p38 in tumorigenesis. Cancer is a highly complex pathology in which genetic and metabolic rearrangements occur in every step. Therefore, tumor requirements are completely stage-dependent. In general, experimental evidence indicates that low p38 activity impairs tumor formation and growth during early stages of the disease, while more advanced tumor stages can benefit from higher activation of the pathway . An exteCancer is not simply a collection of cells that develop independently from their surroundings. The relevance of the tumor microenvironment as an active component of cancer development has gained special attention in recent years, although it is still a largely unexplored field. Extensive communication between cancerous and nontransformed cells takes place through a continuous flux of cytokines, chemokines, growth factors, and extracellular enzymes and determines tumor survival and progression . A recenTumor cells can detach from the initial tumor mass to migrate and invade other tissues, severely affecting homeostasis. This invasive process, known as metastasis, is by far the highest risk factor in cancer and is estimated to account for about 90% of deaths in cancer patients . There ip38 might also be involved in tumor metastasis via other mechanisms. For instance, p38 reduces the expression of the extracellular glycoprotein fibulin 3, which regulates cell migration . Indeed,As stated above, abundant experimental evidence shows that p38 can exert pro-oncogenic functions in various types of cancer. This finding opens up the possibility of developing cancer therapies based on small inhibitory compounds. Seven clinical trials targeting p38 for the treatment of cancer are currently underway see . One exaAlthough many efforts have been channeled into developing effective p38 inhibitors for clinical practice, almost all the trials performed to date in patients have failed, and no compounds have yet been approved. The appearance of systemic side effects in the heart, liver, and nervous system caused by such small molecule inhibitors accounts for the failure of many of these trials . These sA second approach to improve the effectiveness of cancer treatments involves targeting other kinases downstream the p38 pathway, thus limiting the response to only a subset of p38-mediated responses. For instance, the modulation of MAPKAPK2 (MK2), which is a direct downstream kinase of p38 that regulates several processes, including apoptosis, cell cycle, and stress response to oxidizing agents. MK2 regulates the expression of pro-inflammatory factors and responds to DNA damage by inducing the G2/M checkpoint response, thereby contributing to tumor growth and invasiveness in several cancer types ,118,119.S249E/T252E mutant acts as a super-repressor and blocks cyclin expression, preventing cell proliferation in all the cancer cell lines tested so far  and leading to reduced tumor formation in a mouse xenograft model of triple-negative breast cancer (TNBC) [The retinoblastoma (RB) protein emerges as another interesting potential candidate and a therapeutic target downstream of p38. RB is essential for the proper modulation of the G1/S cell cycle transition, and its inactivation contributes to deregulated cell proliferation, which is a hallmark of cancer, thus leading to tumorigenesis . As mentr (TNBC) ,73,83. Fr (TNBC) . In thisr (TNBC) , so patiDespite the limited success of medicinal chemistry to design a drug that targets p38 with a clear therapeutic effect, this stress-activated kinase or its downstream components remain an attractive candidate for the development of new therapies. Indeed, there is still very limited knowledge about some basic aspects of the functionality of the p38 pathway. For instance, p38 isoform-specific biological functions remain a mystery. Specifically, p38\u03b4 and p38\u03b3, which have unique expression patterns and perform a different set of functions, have received less attention than p38\u03b1 and p38\u03b2.p38 has classically been considered a tumor suppressor gene, in spite of the lack of mutations in the genomic sequence in human cancers. However, there is now a large collection of publications supporting a tumor promoter role for p38 and thus in disagreement with its former classification as a prominently anticancer protein. In particular, the role of p38 in metastasis is gathering attention exponentially, since advanced tumorigenic stages still represent a major health challenge worldwide. Nevertheless, even with the increasing body of evidence and studies available, a clear picture of the contribution of p38 to tumorigenesis is still missing. In this regard, many cancer-related studies lack an isoform-dependent approach and hence their translational potential is limited. In this regard, the way each p38 isoform, especially \u03b4 and \u03b3, contributes to oncogenesis is not well understood and the results can often be controversial. Moreover, the large versatility of the stress-activated pathway is probably another important handicap to the effectiveness of therapies. It is evident that the factors mentioned above have hindered the development of more effective and safer drugs for specific malignancies.A deeper understanding of the p38 pathway will translate into better therapeutic strategies for the treatment of cancer, with overall higher success rates. Nevertheless, and considering what has been explained above, it should be taken into account that, while p38 inhibition-based therapies efficiently block tumor growth, they might simultaneously facilitate malignant initiation in other tissues subjected to oncogenic stimuli. As discussed in the previous section, other therapeutic options are also being explored intensively, with the aim of overcoming this hurdle. One particular alternative involves the combination of existing drugs with p38 small inhibitors, while another seeks to target upstream or downstream regulatory factors within the pathway. The rationale behind the latter is that a focus on other regulatory proteins might bypass the undesirable pleiotropic effects caused by p38 inhibition. In particular, recent studies have been devoted to MK2 kinase downstream of p38, and to RB, a p38-dependent transcriptional regulator of cell proliferation. Remarkably, experiments targeting these two proteins have shown promising results in vivo, thereby reflecting their exceptional potential for clinical applications."}
{"text": "The nuclear transcription factor p53, discovered in 1979, has a broad range of biological functions, primarily the regulation of apoptosis, the cell cycle, and DNA repair. In addition to these canonical functions, a growing body of evidence suggests that p53 plays an important role in regulating intracellular redox homeostasis through transcriptional and nontranscriptional mechanisms. Oxidative stress induction and p53 activation are common responses to chemical exposure and are suggested to play critical roles in chemical-induced toxicity. The activation of p53 can exert either prooxidant or antioxidant activity, depending on the context. In this review, we discuss the functional role of p53 in regulating chemical-induced oxidative stress, summarize the potential signaling pathways involved in p53's regulation of chemically mediated oxidative stress, and propose issues that should be addressed in future studies to improve understanding of the relationship between p53 and chemical-induced oxidative stress. A number of cellular systems have been identified to contribute to ROS generation, including plasma membrane, cytosol, peroxisomes, mitochondria, and endoplasmic reticulum. Mechanistically, ROS generation is mainly due to excessive stimulation of NAD(P)H oxidases or the oxidative energy metabolism in mitochondria [An imbalance in the oxidation reduction (redox) system in favor of oxidants is known to cause oxidative stress, a condition that is characterized by the overproduction of reactive oxygen species (ROS) and/or decreased antioxidative capacity , 2. Typichondria .Oxidative stress has been shown to contribute to many pathological conditions, such as cancer \u20136, cardiThe major transcriptional factors involved in redox regulation include Nrf2, Nrf1, p53, and FoxO , 28\u201330. Oxidative stress can be activated in response to certain chemicals and has been demonstrated to play a critical role in their toxicity , 35\u201338. Cisplatin is a first-line therapeutic drug for certain types of cancer; however, its nephrotoxicity is a major impediment to its clinical application . It has \u03b1 could ameliorate triptolide-induced apoptosis by suppressing ROS accumulation in primary cardiomyocytes H9c2 cells [Similarly, in the case of doxorubicin (DOX), a topoisomerase II inhibitor widely used in chemotherapy due to its efficacy in combating a wide range of cancers, the induction of cardiotoxicity is a major safety issue, with oxidative stress the most probable mechanism for its cardiotoxic effect . Using wc2 cells .Colistin, also known as polymyxin E, could be the first choice in the treatment of infections caused by multidrug-resistant Gram-negative bacteria ; howeverIn addition to the side effects of therapeutic drugs, p53-mediated oxidative stress has also been noted in toxicant-induced toxicities. Patulin, a mycotoxin produced mainly by Aspergillus and Penicillium, is commonly found in moldy fruits and their derivative products , and it \u03b1 (PFT\u03b1) significantly reduced the expression of oxidative markers and glycerol-induced renal tubular injury in rats. These findings indicate that p53-mediated oxidative stress can contribute to glycerol-induced kidney injury.Glycerol is a simple polyol compound often used in the food, medical, and pharmaceutical industries and in personal care preparations . However\u03b1 treatment, silibinin could not induce ROS generation in the p53-mutated human epithelial tumor A431 cell. These findings further verify the crucial role of p53 in silibinin-induced ROS generation [Silibinin is a major active constituent of silymarin, and many studies have reported on the involvement of ROS and p53 in silibinin-activated pathways \u201352. In Hneration .In contrast to the prooxidant characteristics of p53, its antioxidant activity has also been reported to occur in chemically induced oxidative stress, as summarized in \u03b1, knockdown of p53 expression with antisense oligonucleotide, and p53 knockout, leads to more severe liver injury by APAP. Conversely, the activation of p53 by its activator, nutlin-3a, resulted in ameliorated APAP-induced hepatotoxicity. These results clearly suggest that p53 plays a novel protective role in APAP-induced liver injury. Moreover, it was revealed that the protective effect of p53 on APAP-induced hepatotoxicity is attributed to its ability to inhibit the activation of JNK, a key mediator in APAP-induced oxidative stress [Acetaminophen (APAP) is one of the most commonly used analgesic drugs and is usually safe at the appropriate therapeutic doses. However, excessive doses can induce hepatotoxicity, and APAP overdose has become the leading cause of liver injury in the United States and most European countries , 54. Huoe stress , 37, (Hu1-Methyl-4-phenylpyridinium (ion) (MPP+) is a dopamine neurotoxin, which can induce parkinsonism . MPP+ isThe antioxidant function of p53 has also been found in the oxidative stress-related pathology of cardiovascular diseases. Oxidative stress induced by free fatty acids (FFA), for example, is considered to be a contributing factor to the metabolic syndrome-associated development of cardiovascular diseases. Exposure of FFA palmitate to human endothelial cells (ECs) caused the transcriptional inactivation of p53 via the inhibition of its acetylation, which, in turn, led to increased ROS generation and endothelial damage . These iGlucose, which is a simple sugar that provides the body with its primary source of energy, has been found to lead to p53 inactivation and ROS accumulation in human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC). Wu et al.  concludep53 is also involved in oxidative stress induction caused by nitric oxide in vascular smooth muscle cells (VSMC). Popowich et al.  reportedRecent research has identified the activities of p53 in the regulation of ROS, along with the upstream and downstream regulators responsible for p53 antioxidant and prooxidant functions. Numerous studies have shown, for example, that p53 can exert prooxidant activity to promote oxidative damage through the regulation of its transcriptional targets, such as p53-inducible genes (PIGs), NCF2/p67phox, a cytosolic subunit of the NADPH oxidase enzyme complex , 62, p66P66shc, a splice variant of p52shc/p46shc, is a cytosolic adapter protein that transduces mitogenic signals from activated receptors to Ras . p66shc \u03b1 downregulates p66shc expression in vivo, a finding which was consistent with the results obtained in the cell culture model.The role of p66shc in chemically induced p53-mediated oxidative stress was examined in a study by Yuan et al.  in whichIn 1997, Polyak et al.  used theIt has been shown that mycotoxin patulin induces p53-mediated oxidative stress in kidney cells . To inveThe GPXs are a group of selenocysteine-containing antioxidant enzymes , of whicThe mitogen-activated protein kinase (MAPK) cascades are evolutionary conserved intracellular signal transduction pathways. They are involved in the transmission of mitogenic signals from the cell surface to regulatory targets and also regulate multiple cellular processes, including proliferation, differentiation, and cell death \u201379. To dIt is well established in APAP-induced liver injury that APAP induces the rapid production of the reactive metabolite NAPQI, followed by the depletion of glutathione (GSH) and generation of ROS, consequently resulting in the activation of JNK , 84. TheAs a family of evolutionarily conserved proteins, Sestrins can suppress reactive oxygen species and provide cytoprotection against oxidative stress \u201387. ThreSestrins have been widely reported to link p53 with redox regulation , 65, 88.Glutaminase (GLS), which is the initial enzyme in glutamine metabolism, has two isoenzymes, namely, GLS1 and GLS2 . GLS2 de2O2 induces GLS2 mRNA significantly in HCT116 p53+/+ cells but not in p53\u2212/\u2212 cells, thus revealing that H2O2 induces GLS2 mRNA levels in a p53-dependent manner. It has also been found that p53 can regulate GLS2 basal expression levels in HepG2 cells. Moreover, H2O2 accumulates more ROS in HCT116 p53\u2212/\u2212 cells than in p53+/+ cells, while silencing GLS2 can also increase ROS accumulation in HCT116, H1299, and HTB-15 cells. These findings demonstrate that p53 regulates cellular antioxidant defense through the modulation of GLS2 expression.GLS2 has also been reported to act as a mediator of p53's role in energy metabolism and antioxidant defense , 91. TreAs has been described above, p53 has the ability to exert either prooxidant activity to mediate chemically induced apoptosis or antioxidant activity to protect cells from chemically induced cytotoxicity. Therefore, p53 is considered a reasonable target through which to suppress chemically induced oxidative stress and toxicity , 43, 60.As mentioned above, p53 is an important transcription factor, and it transcriptionally regulates multiple downstream targets that are involved in regulating various biological processes such as cell cycle, apoptosis, and DNA repair. Targeting p53 could offer protection against chemical-induced oxidative stress and toxicity, but meanwhile, it might affect physiological functions of p53 to produce certain side effects. To avoid these unwanted influences, the manipulation of p53 needs to be optimized so that the basal p53 activity can be maintained to enable its physiological functions unaffected. For example, p53 was activated by PAT, which induced ROS generation and cytotoxicity . TitratiOxidative stress can lie either upstream or downstream of p53 activation, which has been found capable of exerting either prooxidant activity to mediate chemically induced apoptosis or antioxidant activity to protect cells from chemically induced cytotoxicity. The determinants of this paradoxical role of p53 in the regulation of oxidative stress remain elusive. It is generally believed that, under physiological conditions and the conditions of mild stress, p53 exerts antioxidant activity to inhibit ROS generation and protect cells from oxidative DNA damage, while under conditions of severe stress, activated p53 enhances oxidative stress and promotes cell death. Nonetheless, this speculation cannot account for all stress cases. p53 is activated in APP-treated liver cells, for example, and APAP treatment has been found to cause severe oxidative stress followed by severe liver injury. However, activated p53 is reported to play an antioxidant role in this stressed condition, which suggests the involvement of additional factors in determining the prooxidant or antioxidant function of p53.Evaluation of the influence of the source or type of ROS on the pro- or antioxidative activity of p53Assessment of the role of levels of p53 activation in response to chemical exposure in its pro- or antioxidative functionDetermination of whether the cell type is a contributing factor for the controversial function of p53 in regulating oxidative stressInvestigation of the novel mechanisms underlying p53-regulated oxidative stress in response to chemical exposureFurther investigations into the activity of p53 should address the following issues:A better understanding of the factors prompting the activity of p53 as either prooxidant or antioxidant will further the development of this important cell tumor antigen as a precisely utilized target to manage the oxidative stress-mediated toxic responses to chemical exposure."}
{"text": "In addition, p63 plays a key role as a quality control factor in oocytes and p63 mutations can result either in compromised genetic quality control or premature cell death of all oocytes.The protein p63 belongs to the family of the p53 tumor suppressor. Mouse models have, however, shown that it is not a classical tumor suppressor but instead involved in developmental processes. Mutations in the The p63 gene encodes a master regulator of epidermal commitment, development, and differentiation. Heterozygous mutations in the DNA binding domain cause Ectrodactyly, Ectodermal Dysplasia, characterized by limb deformation, cleft lip/palate, and ectodermal dysplasia while mutations in in the C-terminal domain of the \u03b1-isoform cause Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) syndrome, a life-threatening disorder characterized by skin fragility, severe, long-lasting skin erosions, and cleft lip/palate. The molecular disease mechanisms of these syndromes have recently become elucidated and have enhanced our understanding of the role of p63 in epidermal development. Here we review the molecular cause and functional consequences of these p63-mutations for skin development and discuss the consequences of p63 mutations for female fertility. Failure to maintain cellular homeostasis is a common driver of diseases like cancer and neurodegeneration, and also plays a role in ageing, immunological, and metabolic disorders. Quality control systems are the first line of defense that detect potentially stressful or dangerous conditions and provide the information for the cell to initiate countermeasures. Similarly, proper response to environmental cues and stress controls correct cell fate decisions that are essential for development and homeostasis of tissues and organs. Depending on the type of stress, the cellular response can include, for example, activation of DNA repair, cell cycle arrest, expression of chaperones, inhibition of translation, or activation of an inflammatory response. If the stress cannot be resolved, cell death pathways can become activated. One of the most important integrators of cellular stress response pathways is the tumor suppressor p53 . DNA dam\u2212/\u2212 mouse. The p73\u2212/\u2212 mouse suffers from abnormal hippocampal development, hydrocephalus, chronic infections and inflammation, as well as abnormalities in pheromone sensory pathways [\u2212/\u2212 mouse is even more severe. It is born alive, but dies soon after birth caused by multiple developmental defects that include limb truncations and the lack of a multilayered skin and other epithelial structures. These mouse models are in stark contrast to the p53\u2212/\u2212 mouse which shows mostly relatively mild developmental defects such as ocular abnormalities and defects in tooth formation as well as death of ~20% of female embryos and newborn mice due to defects in neural tube closure [\u2212/\u2212 mice are infertile due to lack of mature sperm cells. In addition, p73 is essential for the development of multiciliated cells as, for example, present in the trachea [\u2212/\u2212 mice [The discovery that p53 has two siblings\u2014called p63  and p73 pathways . The phe closure ,18. Thes closure ,20. Male trachea . Lack of\u2212/\u2212 mice , which iCaenorhabditis elegans [Drosophila melanogaster [Ciona intestinalis [For p63, two different main functions have been identified: p63 is responsible for the development and maintenance of stratified epithelial tissues such as skin ,23,24, a elegans ,28, Drosnogaster ,30, Cionstinalis , or sea stinalis  express stinalis . In mammstinalis ,34. Thisstinalis ,36,37. Istinalis  that elistinalis ,40. Detestinalis . While tstinalis , which istinalis . This acstinalis  and de-pstinalis ,47,48,49stinalis  that abrstinalis  . Insteapromoter . While \u0394promoter , RNAseq promoter ,58,59 aspromoter ,61,62,63\u2212/\u2212 mouse revealed that it suffers from a lack of stratified epithelial tissues [\u2212/\u2212 mouse has demonstrated that the maintenance of this compartment is dependent on the expression of p63 with its homozygous inactivation resulting in a disorganized pseudostratified epithelium that does not undergo full differentiation and expresses simple epithelial markers such as keratin K8/K18. Wild type skin shows a clear gradient of p63 expression with high expression in the basal layer followed by decreasing expression in more differentiated keratinocytes and loss of expression in the upper layers [\u2212/\u2212 mouse. These tissues include esophagus, the proximal portion of the stomach, cervix, urogenital tract, prostate, and breast, as well as structures derived from the same stem cells responsible for these tissues such as mammary, sebaceous, lachrymal, and salivary glands [\u2212/\u2212 mouse, such as the apical ectodermal ridge (AER) on the limb buds, a region of specialized, multilayered epithelium, which is essential for the development of distal limbs, digit patterning, and morphogenesis [The analysis of the p63 tissues ,23. The  tissues ,65 that r layers . In addiy glands ,23. Furtogenesis ,67, and \u2212/\u2212 mouse not showing any obvious epidermal defects [In addition to these squamous stratified epithelial tissues, p63 is expressed also in pseudo-stratified epithelia such as the epithelium of the trachea where p63 is found in the basal cell layer . Interes defects , first r defects .The important role of p63 for the integrity of stratified epithelial tissues is tightly associated with its regulatory role in gene expression. Consistent with in vivo observations, cellular studies have shown essential roles of p63 in cell proliferation, differentiation, and cell adhesion ,72,73 inDifferent from previous expectation that p63 regulates only a few dozens of direct targets, tens of thousands of p63 binding sites  have beeTo coordinate specific transcriptional programs, p63 cooperates with other transcription factors. Cooperation with TFAP2C was observed in a study using ATACseq (Assay for Transposase-Accessible Chromatin using sequencing) experiments and transcriptome data from hESCs undergoing epidermal differentiation . In this\u2212/\u2212 mice. Direct interaction of p63 with epigenetic factors has also been observed in the case of HDAC1/2 which bind to the C-terminal TI domain [The combination of several investigations has shown that p63 is involved in higher order regulatory mechanisms by cooperation with chromatin remodeling proteins. Among these proteins are SATB1 , a proteI domain ,85. ThisI domain ,51,104, I domain .In line with the already mentioned bookmarking role of p63 in binding to enhancers, a pioneer function role of p63 has also been proposed. Comparison of loci to which p63 is bound in keratinocytes with the same loci in other cell types has demonstrated that without endogenous p63 expression these loci are nucleosome-enriched, compact, and hence transcriptionally inactive . In keraIn summary, the data revealed by novel epigenomic technologies have consolidated the master regulatory role of p63 in stratified epithelia.\u2212/\u2212 mouse led to the identification of mutations in p63 being responsible for several human syndromes [The publication of the phenotype of the p63yndromes ,109,110 yndromes ,112. Poiyndromes , which cyndromes . Most ofMutations responsible for the Ankyloblepharon-ectodermal defects-cleft lip/palate  syndrome are found in the C-terminus of p63  includinOther p63-related syndromes\u2014Limb mammary syndrome , Acro-Dermato-Ungual-Lacrimal-Tooth malformations syndrome , and Rapp-Hodgkin syndrome  are similar to either the EEC  or AEC TP63 deletion develop normally [Mutations in p63 that cause the syndromes mentioned above are believed to act via a dominant negative or gain of function mechanism. Haploinsufficiency was thought not to be relevant for p63-caused syndromes. This interpretation was mainly based on the observation that individuals who have a constitutive terminal deletion of the long arm of chromosome 3, which harbors the p63 gene locus do not suffer from EEC like syndromes . This wanormally ,23. In cnormally . The twoThe Ectrodactyly, Ectodermal Dysplasia, and Cleft lip/palate syndrome is the most common and best investigated p63-related syndrome. Biochemical studies have revealed that the missense mutations in the DNA binding domain result in loss of DNA binding capability , which dDue to the master regulator function of p63, loss of DNA binding by EEC-type mutations has drastic effects on the chromatin landscape and transcriptome. Through a combination of ChIPseq and ATAC-seq analysis, it was shown that the entire enhancer landscape is changed in EEC patient keratinocytes ,62,84. WInvestigation of the defects in limb formation has revealed that Dlx5 and Dlx6 are direct targets of \u0394Np63\u03b1 ,126. Bot+/L514F), known from human patients to cause AEC, was created and analyzed [+/L514F epidermis and palate. In particular, the mRNA level of the epidermal specific isoforms Fgfr2b and Fgfr3b were reduced, while the levels of Fgfr1 and Fgfr4 did not show differences relative to wild type or p63+/\u2212 tissues. These results are consistent with the identification of p63 binding sites in Fgfr2 and Fgfr3 loci by ChIPseq. Epidermal cells self-renewing potential was affected both in p63+/L514F and in Fgfr2b\u2212/\u2212 keratinocytes. Interestingly, the Fgfr2b\u2212/\u2212 mouse shows epidermal hypoplasia, tooth and hair defects, as well as cleft palate, strongly phenocopying the AEC syndrome.The Ankyloblepharon Ectodermal defects-Cleft lip/palate (AEC) syndrome is caused by three different types of genetic variations in the p63 gene: (1) Point mutations in the SAM domain, (2) point mutations within the TI domain, and (3) frame shift mutations C-terminal to the oligomerization domain . To gainanalyzed . This muOther transcriptional p63 target genes that were found downregulated by AEC mutations, at least in human cells exogenously expressing AEC mutant p63, are HOPX, GRHL3, KLF4, PRDM1, and ZNF750 ,133, tra+/L514F mouse model [To understand the structural consequences of the L514F mutation in the SAM domain, the structure of the mutant form was determined by NMR spectroscopy and compared with the structure of the wild type domain . No signse model . Analysise model . Interesse model . Aggrega\u2212/\u2212 mouse despite lacking functional p63 in the oocytes and required a later TA-isoform specific knock out mouse [In addition to its role as master regulator of epidermal development, p63 is the master quality control factor in oocytes  ,137,138.ut mouse . This orut mouse ,39,42. PTP63 [Some reports of the effect of p63 mutations on female fertility have started to appear in the literature. Nonsense mutations within the SAM domain  ,140 haveTP63 , leadingNrg1 is a direct transcriptional target of p63 in basal cells of the mammary gland [The p53 protein family is arguably one of the most important protein families for genetic quality control and integration of stress and repair pathways in the cell. The p73 and p63 knock out mouse studies in combination with the identification of several developmental syndromes in human patients have shed a new light on the evolutionary origin of this family. Likely, the original function was that of a genetic quality control factor in germ cells, which are virtually immortal, as germ cells are also the source of the germ cells of the following generation. Strict quality control of the genetic integrity is, therefore, important for all organisms. In the case of p63, this control function of germ cells as the most important stem cell was then transformed into its role as a master regulator of epithelial development. Despite its high sequence similarity to p53, p63 does not seem to have a tumor suppressor activity , which iry gland . As Nrg1"}
{"text": "Epithelial organs are the first barrier against microorganisms and genotoxic stress, in which the p53 family members p63 and p73 have both overlapping and distinct functions. Intriguingly, p73 displays a very specific localization to basal epithelial cells in human tissues, while p63 is expressed in both basal and differentiated cells. Here, we analyse systematically the literature describing p63 and p73 protein\u2013protein interactions to reveal distinct functions underlying the aforementioned distribution. We have found that p73 and p63 cooperate in the genome stability surveillance in proliferating cells; p73 specific interactors contribute to the transcriptional repression, anaphase promoting complex and spindle assembly checkpoint, whereas p63 specific interactors play roles in the regulation of mRNA processing and splicing in both proliferating and differentiated cells. Our analysis reveals the diversification of the RNA and DNA specific functions within the p53 family. The p53-family is a group of proteins, which consists of p53, p63, and p73 ,2. p63 ap53, p63 and p73 proteins are represented by multiple isoforms that regulate each other\u2019s transcription and functions ,9. Full In the full length TAp73\u03b1 and TAp63a, N-terminal TA domain acts as the transcriptional activator whereas C- terminal containing SAM and inhibitory domains repress TA activity ,12. GeneIn particular, DNA-binding domain is the most conserved structural element across the p53-family , leading to identical DNA recognition ,17,18,19Specifically, p63 and p73 play distinct roles in epithelial morphogenesis. The p63 is a major player in skin biology and its role in epidermis was thoroughly investigated during the last two decades ,37,38,39The p63 knockout in mice leads to E1 lethality and pups born with non-differentiated skin ,34. TotaIn contrast, the p73 knockout mice are characterised by runting phenotype, hippocampal dysgenesis and hydrocephalus, sterility, chronic inflammation and infection in the lungs, sinus, and ears . Total pIn the human skin, some keratinocytes fail to divide and become polyploid upon differentiation that can be induced by the DNA damage and is associated with overexpression of Cyclin E ,45. UsinIntriguingly, p73 displays a very specific localization to basal epithelial cells in all human tissue where it\u2019s localization was determined by immunohistochemistry. In contrast, the p63 is expressed in both basal and differentiated cells ,48.The role of p63 in polyploidy has not been evaluated, however, the presence of p63 in differentiating keratinocytes suggests that p63 is not interfering with the polyploidy of these cells. At the same time, p73 suppresses polyploidy and aneuploidy in the absence of functional p53 ,49. AlsoHere, we systematically analyzed the literature describing protein interactions and functions of p63 and p73, trying to reveal their distinct functions that could be a physiological basis for their differential distribution in epithelial organs.What we found is that p73 and p63 cooperate in the genome stability surveillance in proliferating cells with the specific p73 function in the APC/C and spindle assembly checkpoint. Unexpectedly, our survey suggests a distinct function of the p63 in the regulation of mRNA processing and splicing in both proliferating and differentiated cells.http://www.proteinatlas.org ..98].In contrast to the APC/C components, p63 interacting ribonucleoproteins are found in all cells of the basal and differentiated epithelial layers. Consistent with continued presence of the p63 in the differentiated keratinocytes, it is possible to speculate that these interactions regulate differentiation-specific RNA splicing. For example, physical association of the heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB) and p63a via its SAM domain led to a specific shift of FGFR-2 alternative splicing towards the K-SAM isoform essential for epithelial differentiation . In addiAnother case of the p63 mediated RNA regulation is p63 interaction with DHX9 (RNA helicase A) that is highly expressed in differentiated keratinocytes and is known to regulate circRNA formation through binding to the inverted-repeat Alu elements . The cirIn addition, the DNp63a interacts with RNA binding protein FUS , that isThus, direct and indirect evidence suggest that p63 mediated regulation of RNA metabolism influences basal and differentiation specific programs in epidermis ,152,156.The role of p73 in promoting translational elongation is recently revealed , althougThus, direct and indirect evidence suggest that p63-mediated regulation of RNA metabolism influences basal and differentiation specific programs in the epidermis ,152,156.Finally, we address a question if distinct p63 and p73 protein interactions correspond to conserved or diverse protein domains .While p63 binding sites for the p63 interacting complexes are not mapped, a lot more is known about putative p73 interacting complexes B. NotablAberrant expression of the p53 family members protein isoforms underline progression of many cancers by regulating the balance between proliferation and apoptosis ,169,170.First, cluster analysis of the protein interactions by the Metascape obtained from the open source database (Biogrid) highlights a unique role of p73 in the regulation of cell cycle and mitosis and, unexpectedly, suggests a unique role of p63 in the RNA metabolism and related cellular functions. Moreover, literature suggests that a specific p73 isoform, DNp73\u03b2, plays a dominant and yet redundant role in the basal keratinocytes differentiation and related functions, although the effects on polyploidy are yet to be analyzed.There are unresolved questions that need to be answered. While a role of the TAp73\u03b1 isoform in the mitotic checkpoint is established, the role of the DNp73\u03b1 isoform which is expressed in the basal keratinocytes of skin is not clear yet. Other proteins, central to SAC regulation, are expressed in the skin but details of the SAC checkpoint control which is established mostly in the cancer cell lines is likely different in the normal epithelial cells.Surprisingly, transcriptional and post-transcriptional regulation of p63 and p73 via protein\u2013protein interactions are not well understood. Apparently, p63 and p73 isoforms regulate chromatin modifications in a cell specific and context dependent manner. Moreover, the issue is complicated by the fact that p63/p73 isoforms bind to DNA not only as transcription factors, but also as a non-DNA bound component transactivating other transcription factors . Few investigations suggest that differential activity of p63/p73 is complex with each other versus other p63/p73 specific interacting proteins switch TAp73 from anti- to pro-oncogenic activities ,21,71. T"}
{"text": "The analysis of mental and psychological health is a relevant public issue in modern societies. Migration is a process that may have a lasting impact on a person\u2019s mental well-being. In this study, perceived health, emotional intelligence, sociocultural adjustment and the participants\u2019 perceived general situation, not only economical, were analyzed to attest their impact on psychological distress as a measure of mental well-being. Sixty-three migrants from Romania and Ecuador were contacted twice during a 14 month period in a middle-sized Spanish city. Attrition analyses show no significant differences in perceived psychological distress between those who participated only one time or who participated in both waves. Less psychological distress is related to less attention to one\u2019s feelings and higher mood repair in both data waves. Stronger behavioral adjustment is also linked to less distress. Less distress in time 1 led to better perceived health, sociocultural adjustment and a perception of a better general situation in Spain in comparison to their home country in time 2. In general, more attention to negative feelings triggered more perceived psychological distress, whereas mood repair elicited less psychological distress, in time 2. The relevance of understanding the impact of emotional intelligence to health promotion programs with migrants is discussed. International migrants represent approximately 258 million people in 2017, roughly 3.4% of the world population . Consequently, our dataset presents no problems in terms of common method variance in both waves. Internal reliability and all partial correlations when controlling for sex and country of origin are presented in Results reveal that a single factor could not account for the variance in the data [T1 Delta r ranged from 0.29 to 0.51) and T2 (r ranged from 0.50 to 0.59).Results show adequate reliability in all variables . Attentir = 0.28) and more cognitive adjustment problems (r = 0.26). Clarity and mood repair presented no significant results.In addition, in T1 attention correlated significantly with more perceived psychological distress (r = 0.85 and r = 0.84 respectively). Psychological distress correlated with worse cognitive and behavioral adjustment in T1  and T2 , respectively.Behavioral adjustment correlated significantly with cognitive adjustment in both waves  = 5.74, p = 0.016). In addition, there are significant differences in behavioral adjustment between participants without studies  and those with completed secondary studies  (f(63) = 4.24, p = 0.047) in T1, and without  and with university studies in T2  (f(63) =2.81, p = 0.047). Distress in T2 also differs in relation to educational level. Participants without formal education  showed a higher level of perceived distress than migrants who had completed university studies  (f(63) = 2.98, p = 0.039).There are differences in perceived health in T1 by education level. Participants without formal studies  perceived significantly worse health than the participants with primary  and secondary studies , but not with university education  (f(63) = 10.11, p = 0.002) and T2 (f(63) = 6.76, p = 0.002), and in cognitive adjustment in T1 (f(63) = 8.23, p = 0.006) and T2 (f(63) = 5.84, p = 0.019). In all cases, participants from Ecuador showed worse socio-cultural adjustment. Differences were also found in attention to feelings in T1 (f(63) = 7.69, p = 0.007) but not in T2 (f(63) = 0.7, p = 0.793). Participants from Ecuador showed higher levels than Romanians, indicating that they tended to monitor more one\u2019s own moods and emotions.Regarding country of origin, differences were found in behavioral adjustment in T1  comparing those participants with a longer stay in Spain (75 percentile: 9 years), and those with the shortest stay (25 percentile: 5 years) showed that length of stay did not play a role in changing patterns of sociocultural adaptation, perceived psychological distress, perceived health, EI and the perceived general situation.\u03c72 = 157.48; RMSEA = 0.06; CFI = 0.91). Sex and country of origin were defined as covariates due to the minor differences found in previous comparisons.An SEM using the Maximum Likelihood Estimation method was used. The model is presented in The model presented is composed only by statistically significant paths. For this reason, the clarity dimension has been removed from both waves. Results show that perceived psychological distress in T1 is affected by both dimensions of EI (attention to feelings and mood repair), and the behavioral dimension of socio-cultural adjustment in T1. The lower the attention to feelings, the higher the mood repair, and higher the behavioral adjustment in T1, the less the perceived psychological distress. Perceived psychological distress in T1 had an effect on perceived health, on both dimensions of socio-cultural adjustment and on the perception of the general situation in T2. Less perceived psychological distress in T1 triggered better perceived health and better socio-cultural adjustment in T2, and also the perception of a better general situation in Spain compared with their home country.Moreover, both dimensions of EI in T2 had an effect on perceived psychological distress in T2. The greater the attention to feelings, the more the perceived psychological distress, and more mood repair implied less perceived psychological distress.With regard to country of origin, migrants from Ecuador, in comparison to those from Romania, stated a higher level of perceived psychological distress in T2. This result is consistent with the higher level of cognitive and behavioral adjustment problems mentioned by these same participants.There is stability of results and scores after a 13\u201316 month period regarding psychological distress, adjustment and perception of one\u2019s own situation. These results support those presented by Jasinskaja-Lahti, Horenczyk, and Kinunen , statingEI and sociocultural adjustment have an impact on perceived psychological distress, confirming our first hypothesis. Whilst clarity is not related to mental well-being, repair is a positive predictor of mental well-being. This result is in line with previous data  which shIn line with hypothesis 2, whilst being able to regulate negative moods and prolong positive ones is linked to better perceived mental well-being, focusing on one\u2019s mood and emotions is an important predictor of negative mental well-being. Paying too much attention towards emotion is linked to ruminative strategies (guilt), avoidance, loss of control, and health problems . FocusinThis is an interesting result for migrant populations that may have to ruminate or avoid their feelings in order to understand new situations and environments. Learning how to adequately regulate emotions  is a positive well-being factor which should be included by professional practitioners in health promotion programs to avoid enhancing negative outcomes .Country differences in attention to one\u2019s mood and emotions may be explained by the higher collectivistic scores found in Ecuador in comparison to Romania . CollectPsychological distress plays an important role in health and psychological adjustment more than a year later. Those participants who stated they were more distressed were also those who afterwards would state that their general health was worse and that they were in general, not only socioeconomically, worse off in Spain in comparison to their home country. These results confirm that positive mental well-being has implications in the long term, and that creating positive contexts for migrants to interact with and manage their lives  may have a lasting and positive effect on mental well-being.First, the number of participants is scarce; although most participants stated that they would have answered the follow up study if still living in the city, due to the nature of the labor market and its need for mobility many participants had moved to another city or were out of town for long periods of time. Nevertheless, attrition results show that participants who answered in both waves were not significantly different in their responses to those who answered only in T1. In fact, the only statistical difference in attrition rate was between men and women. As in other studies conducted with small sample sizes using either a qualitative or quantitative methodology, results must be interpreted with caution so as to not overextend and generalize conclusions to other social groups or situations. Although well designed research with a small sample size can yield important results it may also produce type II errors by interpreting as non-significant results which truly are significant. Nevertheless, various authors ,59 have Second, this research is based upon follow-up and self-reported data recorded over a relatively short time span. As can be the case in general self-reports, results could be influenced by social desirability responding, although this is not necessarily always something negative . The resThird, only two different migrant groups were included in the study. Moreover, we could not conduct separate SEMs to test for group differences. Nevertheless, there were no relevant sex or country of origin differences in any variables under consideration. Although the sample size is small, and we must be cautious with the over-generalization of results, controlling the sex and country of origin allows us to stress the validity of the results, their alignment with other emotion related studies, and their importance as a novel, and overlooked, approach to the analysis of acculturation well-being and public health issues over time.Due to the lack of published studies on the focus of this research, this study can be an addition to the scarce literature on acculturation, emotions and mental well-being and health for a number of reasons. The results of this study can lead to a series of recommendations in health programs and interventions with migrant populations similar to those studied in this research. First, it is necessary to develop longitudinal or follow-up studies that allow us to analyze the relationship between migration and mental well-being as a process. Research designs which aim to inform public health policies should stress the longitudinal analyses of variables and the nature of possible changes. Furthermore, the results from this study could be applied within a public health context. The training of migrants in EI can be beneficial and necessary as it will help them overcome possible social obstacles and present less psychological and cultural distress, as noted in a recent study . MoreoveA second important aspect of this study is the inclusion of variables neglected in most previous studies. The analysis of how emotions and their regulation have a significant impact on migrants\u2019 health has important consequences in the development of programs aimed towards reducing mental distress. Adjustment to one\u2019s new culture, both in terms of learning new cultural codes  and learning how to act in different situations, is an important factor in well-being. In stressful situations, people tend to pay excessive attention to their negative emotional states which, in turn, can lead to more distress. Hampton, Peter, Corus and Brinberg  mentioneThird, although the study comprises a relatively small sample, the strength of the paths in the SEM, as well as its theoretical background, and the alignment of results with previous studies conducted with different populations, point to the relevance of including emotion-related variables in the explanation of migrants\u2019 mental well-being due to the persistent impact of these factors. Looking after migrants\u2019 health is cost effective, facilitates their social and personal adaptation, and enhances more adequate public health interventions. As a result, studying and improving the health of all individuals and collectives within society should be an aim of research and interventions conducted within the social and medical sciences."}
{"text": "The median survival under treatment was 4.82 years. Patients with a more restrictive disease (FVC < 80% pred.), with a rapid progression (FVC decline >10% pred. p.a.), previous smokers and patients > 60 years of age seemed to profit more from pirfenidone treatment. Conclusions: We report the effectiveness of pirfenidone in a European \u201creal world\u201d IPF cohort with outcome data extending up to 9 years. Global analyses demonstrated a positive effect of pirfenidone on the decline of the lung function over time. Survival was dependent on Gender\u2013Age\u2013Physiology (GAP) score and age prior to therapy.Background: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic pulmonary disease with rising incidence. In this study the effectiveness of pirfenidone, as measured by longitudinal change in individual slope of forced vital capacity (FVC) prior to and after initiating pirfenidone treatment, was evaluated in IPF patients recruited into the European registry for idiopathic pulmonary fibrosis (eurIPFreg). Secondary variables were the evaluation of the change in individual slope of diffusion capacity of the lungs for carbon monoxide (DLco), the Borg dyspnea scale, and six-minute walking distance (6MWD), as well as survival analyses. Results: Data of 122 eurIPFreg patients, who had at least two pulmonary function tests (PFTs) prior to or under treatment with pirfenidone, were analyzed by calculating slope-changes. The global analysis revealed an average slope change of +1.48 \u00b1 0.28 (% per annum (p.a)) after start of treatment ( Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually fatal fibrotic pulmonary disease with rising incidence, being associated with an economic healthcare burden . The natAs fibrosis advances and further impairs pulmonary physiology, affected patients experience a high burden of symptoms. Progressive dyspnea is the hallmark symptom of IPF and leads to significantly impaired exercise capacity. Patients also commonly experience non-productive cough and fatigue. Due to symptoms and the resultant impact on physical, social, and emotional well-being, patients with IPF suffer from decreased health-related quality of life (HRQL) ,5. A preInternational consensus guidelines recommend that the diagnosis of IPF should be made on a multi-disciplinary level, calling for the exclusion of known causes of ILD and the presence of a usual interstitial pneumonia (UIP) pattern on high resolution computed tomography (HRCT) or lung biopsy . In accoThe natural course of IPF is quite heterogeneous. IPF usually progresses gradually, but acute exacerbation of IPF may occur at any stage in the clinical course . GreaterHence, it is necessary to identify reliable prognostic factors for the risk of lung function decline and the response to medical therapies in a non-selected patient cohort. Data from patient registries, collecting longitudinal data on IPF patients, have already helped to improve the understanding of the clinical characteristics, the impact that the disease has on their quality of life and survival, as well as current practices in diagnosis and management . The datPrior to the development of pirfenidone and nintedanib, there had been little therapeutic innovation in IPF treatment for several decades, and treatment options had provided very limited values in terms of either disease progression or physical performance. Pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone), was the first drug to be approved for use on IPF patients . It is aCommercial release of pirfenidone in the European Union (EU) was granted based on the evidence from the two multinational pivotal phase III trials, PIPF-004 and PIPF-006  as well as a third supportive phase III study, conducted in Japanese patients ,20. The 0) and required the existence of at least two PFTs prior to and two PFTs after the onset of therapy. This is a retrospective secondary data use study to evaluate the effectiveness of pirfenidone in IPF patients. The primary endpoint was assessment of the treatment response, defined as change in annual rate of FVC decline after the start of pirfenidone treatment (t0);pts. with limited functional impairment (>80% predicted FVC at tpts. with stable disease or slow disease progression before treatment (<10% decline of predicted FVC p.a.);pts. with progressive disease prior to treatment (>10% decline in FVC p.a.);pts. progressing after start of treatment (>10% decline in FVC p.a.);pts. with different smoking history ;pts. younger and older than 60 years;pts. with Gender-Age-Physiology (GAP) composite scores I, II and III;Further primary objectives were to assess the effectiveness of pirfenidone, as measured by change in individual slope of FVC prior to and after initiating pirfenidone treatment in IPF patients (pts.) recruited into the eurIPFreg as well as in the following subgroups: 0) of DLco (% predicted), Borg dyspnea scale (grades 0\u201310), and six-minute walking distance test ;change in individual slope prior to and after baseline  and the European IPF Biobank (eurIPFbank) were launched in November 2009 in the frame of the European IPF Network under the FP7 program to better explore the pathogenesis and natural course of IPF, also in order to facilitate translational research in biomaterials from IPF subjects . Both, eOn a local level, each patient\u2019s IPF diagnosis was evaluated in a multidisciplinary discussion including at least chest physicians, pathologists and radiologists on the basis of the respective ATS/ERS/JRS/ALAT guidelines. The registry had no explicit exclusion criteria, thereby reducing selection bias. The clinical data were collected at the time of enrolment and in intervals 3 to 12 months thereafter . The patient questionnaire included the patient\u2019s demographics, a detailed medical history making use of the WHO classification, complaints as well as report of co-morbidities . After eFollow-up data were acquired in a similar way, making use again of patient and physician questionnaires including additional information on intermittent respiratory infections, working status, transplantation or any changes in the medication, also such events as death. Biological materials such as blood, bronchoalveolar lavage fluid (BALF) and tissue samples as well as exhaled breath condensates and electronic Nose (eNose) profiles were centrally recorded and managed in the centralized European IPF Biobank (eurIPFbank) located in Giessen, providing materials for further clinical research.The study cohort was recruited between November 2009 and May 2018 from the following sites: Universities of Giessen and Marburg Lung Center, Germany, including the nearby Agaplesion Lung Clinic Waldhof-Elgershausen, as well as from Competence Center for Rare Pulmonary Diseases of Hopital Bichat in Paris, France.Data were collected from IPF patients with at least two subsequent records without antifibrotic treatment and by at least two records under pirfenidone treatment.Individual analysis: slopes and slope-changes were taken as estimated from each individual patient.Global analysis: the data from all patients were subtracted from the respective baseline values and then pooled. The pooled data was used to fit a global segmented regression model.The data were analyzed in two different ways:0). Baseline values of all quantitative variables were interpolated using natural spline fits with two degrees of freedom through the available data. If only a single value was given, then this value was used as the baseline value. The baseline GAP score and Borg scale were taken to be the next available score value closest to the baseline date, but maximally one record earlier or later.Baseline was defined as the value at the date of the first pirfenidone treatment . Survival analyses were performed using Cox proportional hazards models. Effects estimated in the \u201cglobal\u201d analysis with p < 0.001 were considered statistically significant. In the other analyses, a p < 0.05 was regarded as significant.The changes in the key variable (change in FVC % predicted per year) were analyzed with segmented regressions by patient. The break-point was fixed at baseline thttps://www.R-project.org). The baseline GAP score and Borg scale were taken to be the next available score value closest to the baseline date, but maximally one record earlier or later. Data of subsequent visits after a change in the treatment or after transplantation was disregarded.All continuous and ordinal variables were shown as mean and standard deviation; categorical variables were separated into tables by number and percentage of the whole cohort (in %). All analyses were carried out with the statistics software R 3.4 (0 (first pirfenidone treatment). The values at t0 predicted by the segmented regression model were taken as the baseline response value for the respective patient.Survival analyses were performed using Cox proportional hazards models. Time-series data were analyzed with segmented regression models fixing the break-points at t0 are displayed in 0 was 64.5 \u00b1 17.5% of the predicted value, the mean FVC at t0 was 63.0 \u00b1 18.3% of the predicted value, indicating an already advanced state of the disease.In the current analysis, change in FVC slopes pre/post treatment was assessed in 122 IPF subjects under pirfenidone treatment. The demographics and baseline lung function values at tIn our cohort, bronchoscopy was performed in 91 cases; the bronchoalveolar lavage (BAL) differential revealed elevated neutrophil (15.5 \u00b1 17.9%) and eosinophil (4.5 \u00b1 4.6%) counts in face of normal lymphocyte (10.2 \u00b1 11.7%) and reduced macrophage counts .0, after t0, and the difference of the slopes after and before t0) were obtained. Distribution of individual slope changes for patients with different numbers of PFTs (2\u201310) is shown in In the individual analysis, the slopes for each patient were calculated in an individual fashion on at least two PFTs prior to and after initiation of treatment, so that 122 statistically independent estimates for the slopes . The data regarding the treatment effect displayed a wide range between further FVC decline of 5-percentage points (%) p.a. and an improvement in FVC values up to 2% p.a. .The individual analysis was modified by taking at least three PFT measurements prior to and after pirfenidone treatment. The data were available for 106 patients. When doing so, there were also no significant changes between pre and post treatment initiation (0 were \u22122.84 and \u22121.36% predicted p.a. (p < 0.001), respectively. The slope change at t0 was found to be +1.48 \u00b1 0.28 (p < 0.001). Our results hence show a reduction in the annual FVC decline of roughly 50% under treatment with pirfenidone.After setting baseline and performing individual FVC slope analysis individually as detailed above, all FVC data were corrected for the baseline values and analyzed together employing a global segmented regression, hereby disregarding the statistical dependencies of the patients\u2019 recordings. In this analysis, the slopes and slope change were determined in a single segmented regression model using all data of all 122 patients and 2040 FVC values together . The sloFor the subgroup analysis, the data were used as in the global analysis, but the segmented regression model included the interaction of the slope coefficients with the co-variable of interest, allowing testing of the null hypothesis that the slope differences in the subgroups are zero as well as the difference in slope-differences between the subgroups.0 (n = 22) as compared to patients with a relevant reduction in FVC at baseline . Both patient subgroups showed a reduction in the FVC decline in response to treatment, indicated by a positive value for the slope change. However, in patients with preserved baseline lung function (right part of right panel), this did not reach significance (p = 0.17), whereas in patients with a relevant reduction in baseline FVC, this change did reach statistical significance (p < 0.001). The slope changes in both subgroups were found to be statistically different (p < 0.001).Data from patients with and without limited functional impairment showed a negative FVC slope before t0 . However0 (n = 98). In response to treatment, there was a significant (p < 0.001) reduction in the FVC decline in this patient sub-population, resulting in a positive value for the slope changes at t0  of less than 10% per year prior to treatment showed a slope of ~\u22124% before t0 approximating 18% (n = 24). In response to treatment, there was a significant (p < 0.001) reduction in the FVC decline in this patient sub-population, resulting in an impressive slope change of around 16% at t0. The change in the slope-differences in response to treatment between both subgroups was not found to be statistically significant (p = 0.345).Data from patients with progressive disease prior to treatment, defined as a decline of the FVC (in % pred.) of more than 10% prior to treatment showed a negative FVC slope before t0), defined as an annual FVC decline of >10% pred., showed an FVC decline of ~3.2% pred. prior to treatment (n = 28), as compared to an FVC decline of ~2.4% pred. in patients who did not deteriorate after initiation of treatment . Accordingly, patients with a deterioration after initiation of treatment showed a massive and highly significant (p < 0.001) worsening in the FVC slope , as compared to the similarly significant (p < 0.001) slight improvement in the FVC decline in those patients stable upon initiation of pirfenidone treatment . The change in the slope-differences in response to treatment between both subgroups was found to be statistically significant (p < 0.0001).Data from patients with progressive disease after initiation of treatment (tn = 84) showed a somewhat less prominent decline in FVC prior to treatment (left part) as compared to never smoking IPF subjects . In current or former smokers, therapy with pirfenidone resulted in a significant (p < 0.001) reduction in FVC decline (right part of right panel). In never smoking IPF subjects the slope change was less prominent and it did not reach statistical significance . There was a significant difference between both groups with regard to the response to treatment (p < 0.001).IPF patients in these two cohorts showed an FVC decline prior to treatment regardless of their smoking habits. However, current or former smokers with IPF (n = 93) showed a more prominent decline in the FVC prior to initiation of treatment  as compared to patients younger than 60 years . Response to treatment was statistically significant (p < 0.001) in case of the patients above 60 years of age  and resulted in a reduction in the FVC decline. In patients under 60 years of age, there was also an attenuation of the FVC decline, which, however, it did not reach statistical significance . Response to treatment was statistically significant between the two groups (p = 0.022).IPF subjects older than 60 years (n = 26) did not show a meaningful change in the FVC slopes, whereas GAP II patients (n = 51) showed a weakly significant reduction in FVC decline (p = 0.04), and GAP III patients (n = 26) showed a highly significant reduction in FVC decline (p < 0.001). There was a significant (p = 0.007) difference in the treatment responses between GAP I versus GAP II patients.FVC decline prior to initiation of treatment was found to be dependent on GAP stages , respectively. The slope change at t0 was found to be +3.51 \u00b1 0.63 (p < 0.001). Although the individual slope analysis did not reveal any significant treatment effects, the global analysis revealed a significantly reduced DLco decline under pirfenidone. The slopes before and after tp = 0.019; n = 43).The global analysis showed an increasing Borg dyspnea grade prior to initiation of treatment and a significant attenuation of the dyspnea score in response to treatment with pirfenidone (p < 0.001).The global slope analysis showed a decline of 28 m per year in the 6MWD prior to the initiation of treatment . Upon inAdditionally, and in order to rule out a bias based on the drop out of deceased patients during the treatment period, we performed a subgroup analysis of the IPF cohort, who survived at least 24 months after beginning of pirfenidone therapy. In comparison to the whole IPF cohort, we saw similar pirfenidone effects on declines in FVC, DLco, 6MWD as well as improvement of the Borg scale. 0. The event (death) was only accepted if the patient died at the end of the valid treatment sequence. Dates of transplantation, changes in treatment or lost-of-follow ups were censored. The median survival under treatment was 4.82 years . The Kaplan\u2013Meier curve is shown in Survival time was analyzed starting from tp = 0.002).As shown in p = 0.03).As shown in p = 0.014) impact of an FVC more than 60% versus less than 60% predicted on survival. The results are shown in When analyzing the impact of FVC at the timepoint of treatment initiation, we did observe a weak, albeit significant ; the decline remains about constant and even increases again after a few years (so 5\u20136). The decline from the year before to the year after the start of therapy seems significant, but the p-value for the mean difference of these values (year \u22121 to year +1) was p = 0.12. Although the data spread wide, despite all the effort to extract dependent variances, treatment with pirfenidone seemed to be presumably equally effective throughout the whole treatment time.To determine the mean changes per year in FVC, the FVC values per patient were fitted to a loess curve by a re-descending M estimator with Tukey\u2019s bi-weight function and a span of 1 (year) and 12 iterations, showing a curve through the time course of the FVC values, predicted for tThe mean values and standard errors of the mean FVC slope decline per year (liters) are presented in 0  assessing efficacy of pirfenidone in IPF have provided important insights into the clinical course of IPF and the response to treatment. It should, however, be kept in mind that patients recruited into these studies represent a rigorously selected population and do therefore not necessarily reflect the characteristics of IPF subjects seen in clinical routine. Demographics and baseline measures of disease status of our study cohort are consistent with mild-to-moderate physiologic impairment and comparable to those in ASCEND and CAPACITY trials ,23,24. E0 approximating 18% and, as response to treatment, a significant (p < 0.001) reduction in the FVC decline, resulting in an impressive slope change of around 16% at t0.With regard to the analysis of change in FVC, patients with a more restrictive disease (FVC < 80% pred.), with a rapid progression (FVC decline > 10% pred. p.a.), previous smokers and patients > 60 years of age seemed to profit more from the pirfenidone treatment, in that the treatment elicited effects in these subpopulation appeared to be more profound. As an example, patients with progressive disease prior to treatment showed a negative FVC slope before tIn our \u201creal world\u201d analysis of response to treatment with pirfenidone, GAP I patients did not show a meaningful decline prior to treatment and an only modest, non-significant change under treatment; GAP II patients showed a much more prominent FVC decline prior to, and a weakly significant reduction in FVC decline under treatment. Finally, GAP III patients showed the highest FVC decline prior, and a highly significant reduction in FVC decline under treatment. There was a highly significant difference in the treatment (FVC) responses between GAP I versus GAP III patients, whereas differences between GAP I and GAP II patients were not significant. Costabel et al. published data on pirfenidone treatment in 187 patients with more advanced disease, and 409 patients with less advanced disease; it was shown that longer-term pirfenidone treatment resulted in a similar rate of lung function decline and safety profile in patients with more advanced versus less advanced IPF .Based on our individual slope analyses, the response to pirfenidone demonstrated a marked heterogeneity, hence rendering assessment of the individual treatment effect on disease progression quite challenging. The individually determined FVC slopes displayed a wide range between a further decline of 5% p.a. and an improvement of up to 2% p.a. After modifying the individual analysis by taking at least three PFT measurements prior to and after pirfenidone treatment (106 patients), there was still no visible significant treatment effect, although post treatment slopes were shifted to a lesser decline. This was reinforced by the finding that the intraindividually assessed slope changes in response to treatment were not substantially different if much more than two PFT\u2019s were present for the slope analysis.0, where the event (death) was the end point . Our outcome analysis demonstrated a median survival under treatment of 4.82 years . In contrast to the substantial treatment effect on FVC seen in GAP III patients, patients at GAP stage III vs. GAP stage I (p = 0.001) showed a worse survival. When analyzing the impact of the FVC at the timepoint of treatment initiation, we did observe a weak, albeit significant impact of an FVC more than 60% versus less than 60% predicted on survival. Similarly, patients with a progressive disease (FVC decline > 10% per year) prior to therapy, although showing a striking increase in FVC, still showed a significantly higher mortality under treatment.Survival time was analyzed starting from tn = 43). The authors presumed that efficacy of pirfenidone in IPF patients with severe lung function impairment may diminish after 6 months of treatment [n = 307); this decline was even less than that noted in the ASCEND trial, in which mean decline from baseline in FVC was \u2212235 mL in the pirfenidone group over 52 weeks [n = 502) of Japanese IPF patients, Bando et al. reported a mean decline of FVC of \u221230 mL and of \u2212158 mL in the first and second year of therapy, respectively [n = 91) [0  and treatment with pirfenidone for at least 6 months, Tzouvelekis et al. showed a tendency towards a reduction in functional decline as compared to the 6-month period before treatment initiation, but failed to show any benefit after one year of treatment .Based on a Cox regression analysis, we additionally performed a risk score analysis adjusted for the FVC decline and GAP stage prior to the initiation of pirfenidone treatment. The data show that the higher the GAP score and the higher the FVC decline, the higher the risk of death under treatment. Fang et al. showed that IPF patients with severe baseline PFTs DLco < 30%) were three times more likely to develop disease progression or acute exacerbation or death than the IPF subjects with DLco > 30% . The med% were thp = 0.002).Noble et al. analyzed pooled data from the multinational pirfenidone trials ASCEND and CAPACITY and found that treatment with pirfenidone for 1 year resulted in clinically meaningful reduction in disease progression in patients with IPF; analysis of outcomes at 1 year demonstrated that pirfenidone reduced the proportion of patients with a. \u226510% decline in FVC % pred. or death by 43.8% and increased the proportion of patients with no decline by 59.3% (95% CI 29.0\u201396.8) compared with placebo . Antoniop = 0.0107); treatment-emergent all-cause mortality (p = 0.0094); idiopathic-pulmonary-fibrosis-related mortality (p = 0.0029). Consistent with the pooled analysis, meta-analyses for all-cause mortality at week 52 also showed a clinically relevant and significant risk reduction in the pirfenidone group compared with the placebo group [Nathan et al. showed in the pooled analysis that at week 52, the relative risk of death for all four mortality outcomes was significantly lower in the pirfenidone group than in the placebo group in the pooled population , or rapid progression (FVC decline > 10% pred. p.a.) seemed to profit more from this therapy. Our results provide additional information as to the effectiveness of pirfenidone treatment on IPF disease progression and on treatment response in dependency of IPF-subgroups. Patients with a higher GAP score and higher age prior to treatment are more likely to experience a worse outcome despite treatment with pirfenidone. Our data highlights the important role of large real-world data registries to document and analyze changes in IPF management."}
{"text": "Generation of mice lacking p73/TAp73 revealed a plethora of developmental defects, with very limited spontaneous tumors arising only at a later stage. Concurrently, novel TAp73 target genes involved in cellular growth promotion that are not regulated by p53 were identified, mooting the possibility that TAp73 may have diametrically opposite functions to p53 in tumorigenesis. We have therefore comprehensively evaluated the TAp73 target genes identified and validated in human cancer cell lines, to examine their contextual relevance. Data from focused studies aimed at appraising if p53 targets are also regulated by TAp73\u2014often by TAp73 overexpression in cell lines with non-functional p53\u2014were affirmative. However, genome-wide and phenotype-based studies led to the identification of TAp73-regulated genes involved in cellular survival and thus, tumor promotion. Our analyses therefore suggest that TAp73 may not necessarily be p53\u2019s natural substitute in enforcing tumor suppression. It has likely evolved to perform unique functions in regulating developmental processes and promoting cellular growth through entirely different sets of target genes that are not common to, and cannot be substituted by p53. The p53-related targets initially reported to be regulated by TAp73 may therefore represent an experimental possibility rather than the reality. Identification of p73 as a structural homolog of p53 fueled early studies aimed at determining if it was capable of performing p53-like functions. This led to a conundrum as  The returned articles were further filtered by language to include English-based results only. From those articles, publications that met the above criteria for TAp73 target genes were considered relevant to the purpose of this review and were further grouped either as individual gene studies or genome-wide studies. We would like to highlight that studies describing other TAp73 transcriptional targets may have not been included in this analysis due to the nature of the search terms used for article identification. Nonetheless, we believe that the analysis provide a general overview that is representative of the TAp73 targets identified thus far. Since p73 was first identified as a structural and functional homolog of p53 ,39, its www.p53.iarc.fr). More than half were without a functional p53: 8 cell lines are devoid of p53 expression  and 29 expressed mutant p53, and three cell lines, namely, HEK293T which was used in 7 studies, NHEK in 1 study, and Hela in 6 studies, are considered functionally inactive for p53 due to the overexpression of SV40 T antigen or genes of human papilloma virus , MDM2, and BAX were the top 3 genes reported in multiple studies: 19, 12, and 12 studies respectively, as they were often used as positive controls. Firefly luciferase-based promoter assay was the most common method used to validate the regulation of target gene transcription upon TAp73 overexpression. The potential TAp73 binding sites were explored mainly by searching for typical p53REs in the proximal promoter regions, with the assumption that the binding sites would be evolutionally conserved and thus similar for TAp73 and p53. The majority of the 81 target genes contain a predicted p53RE in their genomic regulatory regions, of which 33 were validated.The target genes derived from the 71 individual focused studies that met our criteria are listed in https://amp.pharm.mssm.edu/Enrichr/), signaling pathways that emerged were DNA damage response, cell cycle arrest, and p53/p63/p73 networks (https://david.ncifcrf.gov/) (data not shown). It is noteworthy that many of these studies were aimed at identifying the target genes with intent, i.e., known p53 targets were evaluated to determine if they were also regulated by TAp73. Therefore, it is not surprising that the emerged pathways are similar to p53-regulated pathways and that these target genes are similarly regulated by p53. When these TAp73 target genes were pooled together for Gene Ontology (GO) term analysis using the online gene set analysis tool Enrichr , contained wild-type p53 but differed in the expression of the MLH1 gene that was shown to be necessary for TAp73 activity ,121.Among these studies, two analyzed the effects of endogenous TAp73 on promoter binding upon TNF-\u03b1  or hydroNine out of the twelve articles provided a full or partial list of genes identified and included data on the validation of the targets. Incidentally, none of the target genes identified were common across all nine studies , likely APOC3, C11orf21, GZMA, RPS27, and RRAD were identified twice in separate studies, and genes such as CDKN1A (p21) and MDM2 were identified thrice  and promoted the Warburg effect via stimulating the expression of phosphofructokinase 1 (PFKL), both in human cancer cell lines as well as E1A+Ras-transformed mouse embryonic fibroblasts [Other studies also found TAp73 to support cellular proliferation by enhancing cellular metabolism. TAp73 was shown to enhance the pentose phosphate pathway by directly activating the expression of roblasts ,28. ThesCxcl2, Ereg, and Vegf-c [hTERT, CylcinB1, and Caspase-2S have also been noted [A role for the p73 proteins in hypoxia has also been well investigated. TAp73 was noted to inhibit angiogenesis by destabilizing HIF-1\u03b1 via a protein\u2013protein interaction, independent of its transcription activity, and thus indirectly led to the suppression of pro-angiogenic genes such as d Vegf-c ,126. On d Vegf-c ,45. Whild Vegf-c ,45. Simien noted ,72,85.These data collectively demonstrate that TAp73 can have a distinct role in promoting cancer cell growth. TAp73\u2019s ability to activate a unique and ever-expanding universe of transcriptional targets that do not converge with the canonical p53-like targets, often as a result of a collaborative co-existence with other transcription factors, highlight a more promiscuous function of TAp73 that is dependent on and dictated by the cellular contexts.Since its discovery over two decades ago, much effort has gone into deciphering the role of TAp73 in cellular growth control and in proving that TAp73 can be a substitute for p53, being able to perform similar functions through the transactivation of overlapping target genes. With this focused end goal in mind, many of the early studies were based on the \u201cintended search and confirm\u201d philosophy, thus unsurprisingly uncovering many common TAp73/p53 targets. However, both unbiased genome-wide studies and studies based on cellular phenotypes led to the revelation of many novel TAp73 targets that were associated with its role in cellular growth promotion, which is consistent with it being rarely mutated but over-expressed in human cancers.+/- mice [The current analyses of TAp73 target genes collectively highlighted several points worth pondering . Firstly+/- mice ,128, togNext, based on the primary observation that TAp73 is often overexpressed without being mutated in human cancers, a series of studies have now demonstrated that TAp73 expression can indeed be induced by growth-inducing conditions. Consequently, TAp73 has been suggested to be able to promote cellular growth and survival, mainly through the activation of target genes that appear to be context-dependent, and hence non-canonical. Importantly, it is emerging that these novel genes are often transactivated by TAp73 in collaboration with other transcription factors that promote cellular survival in various ways. These observations, together with the fact that many TAp73 target genes that regulate the developmental phenotypes noted in the absence of p73/TAp73 do not have a typical p53RE, indicate the promiscuity of TAp73 in being able to be adept in converging contextual signals to enable cellular survival. An interesting and yet often overlooked feature of these studies highlighting a pro-growth role for TAp73 is that most of the experiments were initially performed in MEFs that have been transformed, from which the target genes were identified and confirmed in human cancer cell lines. Importantly, these MEFs contained wild-type p53. Hence, it is tempting to speculate that in normal contexts where p53 is present, the primary role of TAp73 is context-dependent, being involved in regulating developmental genes at the early stages and for cellular proliferation and survival thereafter. Moreover, as a target of the key cell cycle regulator E2F-1 , it is nIn summary, we conclude that TAp73, though being a structural homolog of p53, is not the latter\u2019s natural functional substitute. In its own right, TAp73 has evolved to perform unique functions in regulating developmental processes and cellular growth, through entirely different mechanisms and through different sets of target genes that are not common to and cannot be substituted by p53. While TAp73 can perform p53\u2019s functions, the opposite is not true. Thus, the p53 target genes identified to be regulated by TAp73 may indeed represent a virtual reality."}
{"text": "To compare the differences in the clinical course of acute appendicitis between early elderly (60-79 years) and late elderly patients (\u226580 years).The sample consisted of 177 patients aged over 60 that underwent surgery at the emergency service with the diagnosis of acute appendicitis between January 2010 and May 2018. Patients\u2019 data were retrospectively obtained from electronic records. Patients that had undergone appendectomy or negative appendectomy or had an appendix tumor were excluded from the study. The patients were divided into two groups by age; early elderly (60-79 years) and late elderly (\u226580 years).Of the 177 patients included in the study, 162(91%) were 60-79 years old and 15 (9%) were over 80. A statistically significant difference was found between the early and late elderly groups in terms of perforation, requirement for intensive care (p\u00a0=0.001), red cell distribution width (p\u00a0=0.025), the Clavien-Dindo classification (p\u00a0=0.020), and the Charlson comorbidity index (p\u00a0=0.005). The mean hospital stay was four and 11 days for early and late elderly groups, respectively. Multivariate analysis revealed that age alone was an independent factor with a statistically significant effect on mortality ).In particular, in the elderly patients over 80 years old, the clinical manifestation of acute appendicitis at hospital admission is in the form of perforation. Therefore, in this age group, a careful, precise and prompt diagnosis is crucial. Acute appendicitis (AA) is one of the most common surgical pathologies in the emergency service . It occuIn geriatric patients, the most common specific perioperative complications are general complications, such as postoperative wound infection, pneumonia, and cardiovascular problems . The difPatientsThis study was planned in Ankara Numune Training and Research Hospital General Surgery clinic. A total of 93 males (52.5%) and 84 females (47.5%) patients over 60 years of age that underwent surgery at the emergency surgery service with the diagnosis of AA between January 2010 and May 2018 were included in the study. Local ethics committee approval has obtained for the study . The patients with AA were retrospectively identified based on the electronic records of the patients. The diagnoses of AA have confirmed by final pathology reports for all 177 patients. Inclusion criteria were, all patients; over 60 years of age, underwent appendectomy between mentioned dates, have confirmation for the diagnosis, and have adequate medical records. Exclusion criteria were having undergone appendectomy and negative appendectomy as part of another surgical procedure or having an appendix tumor. The patients who were confirmed to have undergone appendectomy only due to AA were included in the study. The patients were divided into two groups according to age; early elderly (60-79 years) and late elderly (\u226580 years).DataAfter obtaining the approval of the ethics committee for the study, the data of 177 patients over 60 years of age obtained from the records were evaluated. The electronic records were screened for details of treatment, surgical notes, and pathology reports. The data obtained from medical records consisted of age, sex, symptoms, physical examination, laboratory findings, imaging, appendicitis type (perforated or non-perforated), postoperative complications, length of hospital stay, and in-hospital mortality. The comorbidities of the patients were examined according to the Charlson comorbidity index (CCI), and their clinical, perioperative and pathologic findings were graded according to the American Association for the Surgery of Trauma (AAST) classification. Postoperative morbidity was defined as presence of wound infections, ileus, intraabdominal abscess or pneumonia complication, and requirement of intensive care. The treatment applied to the complications was classified according to the Clavien-Dindo classification (CDC).Statistical analysisp\u00a0value ofAll statistical analyses were undertaken using SPSS Windows version 12.0 .\u00a0 The numerical data were given as median  and compared using the Mann-Whitney U test when the normality assumption was violated. The categorical variables were expressed as percentage (%) and compared with the chi-square or Fisher\u2019s exact tests. Statistically significant variables were assessed by multivariate logistic regression. A\u00a0p=0.016), intensive care requirement (p=0.001), mortality (p=0.002), red cell distribution width (RDW) (p=0.025), CDC (p\u00a0=0.020), and CCI (p = 0.005). However, there was no statistically significant difference between the two groups in terms of sex, complication , white blood cell (WBC) count (103\u00a0\u03bcl), neutrophil count (103\u00a0\u03bcl) and AAST grading. The mean hospital stay was four (IQR=2-5) and 11 (IQR=2-6) days in the early and late elderly groups, respectively (p=0.154) (p<0.001).Of the 177 patients included in the study, 162 (91%) were 60-79 years old (early elderly) and 15 (9%) were over 80 years of age (late elderly). There were 87 men (53%) and 75 women (46%) in the former group and six men (40%) and nine women (60%) in the latter group, with the median ages of 66 years (min 60-max: 79) and 85 years , respectively. A statistically significant difference was found between the early and late elderly groups in terms of perforation (p=0.154) . As summAA is one of the most prevalent surgical phenomena in all age groups and is increasingly seen in the elderly population . GeriatrIn this study, the perforation rate was found to be 2% and 66% in the early and late elderly groups, respectively. This shows that perforation was statistically significantly increased in the patient group aged 80 years or older compared to the patients that were 60 to 79 years old. This higher rate among the late elderly patients can be attributed to their low pain threshold, higher frequency of living alone, presence of multiple co-morbidities, and AA not being considered in initial diagnosis compared to the early elderly patients. Furthermore, the older patients generally have fewer physiological reserves, a higher rate of comorbidities, and a greater possibility of having had previous abdominal surgery, which all increase their surgical risks compared to the younger patient groups.p\u00a0= 0.005). Mortality was also higher in patients aged 80 years and over. However, the 2% mortality rate in the early elderly group underlines the importance of considering AA for early diagnosis and immediate surgical treatment in the over-80 patient group. In this study, the need for postoperative intensive care in the late elderly group was found to be statistically significant. This may be associated with comorbidities or a result of late diagnosis and perforation.Tehrani et al. showed that patients who had undergone appendectomy for perforated appendicitis had higher complication and mortality rates than those having had appendectomy without peritoneal closure . AnotherIt has been reported that some surgeons hesitate to perform surgery on octogenarians and choose to monitor them until their symptoms become more pronounced, but this delays diagnosis and increases the possibility of perforation . Other rp\u00a0= 0.000).In the literature, age was shown to have an independent effect on mortality and postoperative morbidity . BlomqviCurrent studies indicate that elderly patients have a longer hospital stay than younger patients following appendectomy, and the average of this duration is longer than one week . In thisGeriatric patient groups are usually difficult to manage and require a multidisciplinary approach in terms of diagnosis and treatment. A highly sensitive and effective diagnostic test for AA is very desirable in elderly patients in order to avoid high risks associated with surgery and possible complications due to delayed diagnosis. In the literature, RDW has been shown as a good indicator of systemic inflammation and mortality in elderly patients . The RDWet al., [Huive\u00a0et al.,  detectedThe limitation of this study includes the retrospective design, and consequently the data being based on existing electronic records of the patients.In conclusion, the anamnesis of elderly patients is often incomplete, complex, and atypical or has different clinical features and multiple comorbidities. Especially in the octogenarian and nonagenarian patient groups, the presentation of AA in clinical practice is in the form of perforation. Therefore, these patients\u2019 groups require a more careful, meticulous and rapid diagnosis."}
{"text": "To display microRNA155 (miRNA155) expression in different entities of Beh\u00e7et\u2019s disease (BD), and to find out if expression is affected in more than one of disease status than another, either phenotypically, according to HLA B51 expression, presence of family history, or patients\u2019 age.Thirty BD patients (13 of which were HLAB51 positive) and 15 healthy subjects\u2019 samples were obtained. White blood cell miRNA155 expression in both types of samples was estimated.Results showed that there is a degree of relation between decrease of miRNA155 expression and different disease aspects, and also, that miRNA155 has an inverse relation with the patients\u2019 ages.MiRNA155 might be used as a measure of disease of different phenotypes, and that any manifestation of the disease can happen when the expression level decreases. White blood cell (WBCs) miRNA155 expression in both sample types was estimated through RNA extraction, reverse transcription, amplification, and then detection by small RNA assays.Figures 1 and 2).MiRNA155 expression levels were decreased non-significantly not only in the whole patients group compared to the control group, but also decreased non-significantly in all disease clinical presentations except for those not having eye problems who significantly had a decreased expression from the control group p= 0.013, and from patients with ocular involvement (p=0.041) .Figure 3): this may be explained by decreased disease activity with increasing age. These findings prove what was mentioned in several studies, that miRNA155 decreases with increase of BD activity, such as Hassouna et al.5Also, there was no significant difference between miRNA155 expression in patients having positive family history of BD (7 patients) and those who did not (p=0.081). MiRNA155 expression appeared to be directly proportional to age   through IL13 receptor alpha targeting .9 A study showed that high level of MiRNA155 was found in Tregs in a FOXP3-dependent way, and Treg homeostasis is disabled in miRNA155 insufficiency. When miRNA155 is missing in Treg, miRNA155 target of cytokine signalling 1 expression suppression increase with diminished responses to IL2, which is an imperative Treg homeostasis controller this was found by Lu et al.10 Moreover, it was discovered by Ceppi et al. that miRNA155 down-regulates lipopolysaccharide induced inflammatory pathways through inhibition of monocyte-derived DCs.11The results shown are coherent with researches telling that increase in miRNA155 is related to decreased expression in some cytokine, eg, interleukin (IL)1, IL 6 and IL 17, as is shown in Zhou et al.12 Another research found that T cells miR-155 lack in mice did not lead to advancement of extreme autoimmune encephalomyelitis (EAE) and has a diminished IL-17 generation which is a critical cytokine for EAE pathogenesis.13A few other researches revealed an inverse result to ours, that miRNA155 expression is expanded in activity of BD, and that MiR-155 focusing on Ets-1 controls Th17 response and that concealment of miR-155 diminishes pathogenic IL-17-expressing T cells, as was seen in the study of Na et al.14 found that mice lacking in miRNA155 have impedance in Th17 differentiation and in turn are protected from joint inflammation initiated by collagen.Moreover, Bl\u00fcml et al.The opposite findings to our research results shown in some other studies may be due to ethnic differences, and different activity levels of the disease in the involved patients from one study to another. Also, several studies  were built on animal research, which cannot be relied on. Thus, there is a controversy about miRNA155 role in the pathogenesis of BD disease.MiRNA155 might be used as a measure of disease state. Any manifestation of the disease can happen when the expression level decreases."}
{"text": "Saliva may contain useful biomarkers which provide information about animal welfare using convenient and non-invasive sampling methods. In addition, the development of automated techniques of measuring analytes in saliva provides advantages from the technical point of view since they are cost-effective, reliable, and replicable. In this study, 21 salivary analytes measured by automated assays were tested as potential biomarkers of lameness, one of the most prevalent diseases in dairy cows producing significant economic losses. As a result, total esterase (TEA) showed increases in saliva in a group of 11 cows with lameness, decreasing when the lameness was solved after a specific treatment consisting of a hoof trimming and a medical treatment. In addition, TEA activity correlated with the severity of the lameness. Further studies using a larger population of cows with different causes of lameness and severity should be performed to determine the potential of TEA as a biomarker of lameness in cows.p = 0.140), days in milk (DIM) (p = 0.780), and body condition score (BCS) (p = 0.074). Initially, 21 biochemical analytes were determined in the saliva of six cows with lameness at the diagnosis time (T0) and twenty days after hoof trimming that successfully solved the lameness (TF). This exploratory study only showed significantly higher values in lipase (Lip) and total esterase (TEA) at T0 compared to TF . When both analytes were measured in the additional five lame cows and the results of all the animals of the lame group (n = 11) were compared with the healthy group (n = 11), only TEA showed higher activities in the group of lame cows than healthy cows (p = 0.004). TEA was positively correlated with both NRS and LSS . In conclusion, this study showed that cows with lameness in our experimental conditions had higher TEA values than healthy cows, and these values decreased after treatment. This is a pilot study, and further studies using a larger population of cows with lameness due to different causes and severity should be performed to determine the potential of TEA as a biomarker of lameness in cows.The possible changes in a panel of 21 salivary analytes on a population of cows with lameness before and after treating lameness by hoof trimming were analyzed. Then, the analytes that showed significant changes were studied in a larger population of cows with lameness and compared with healthy cows For this purpose, two groups of cows were made by a specialized veterinarian. One consisted of healthy cows with no external signs of diseases and no hematological or biochemical abnormalities, and showing no signs of lameness according to the numerical rating system of severity ; and the other composed of cows showing only lameness with a NRS of 3.1 \u00b1 0.87 and a lesion scoring system  of 3.3 \u00b1 0.89. Both groups did not differ in parity ( Lameness is, together with infertility and mastitis, one of the most prevalent diseases in dairy herds producing significant economic losses, reporting mean prevalence of lameness in dairy cows to be 31.6% in UK . It has In addition to the economic effect, lameness is recognized as a major welfare problem in cows. Cortisol has been extensively employed as a stress biomarker in lame cattle since acute pain induces activation of the hypothalamus-pituitary-adrenal (HPA) axis ,6,7, altSaliva rather than blood has been rising in interest in recent years as biologic fluid for the evaluation of welfare, stress, and disease in animals, since saliva can be easily and repeatedly collected in a large number of samples even at short-time intervals in a non-invasive way, without producing pain, and no need for specialized staff ,10. In tThe study hypothesizes that lameness in cows can produce changes in selected analytes in saliva and that possibly some of these analytes could reflect improvements in lameness after treatment. Therefore, this study aimed to evaluate changes in a panel of 21 salivary analytes in cows with lameness exploring the changes in these analytes after the lameness was solved.Twenty-four Holstein-Friesian dairy cows from a commercial dairy herd located in the southeast of Spain  were initially selected for this study. Information about the parity, days in milk (DIM), body condition score (BCS), and milk yield is described in The cows were fed with a total mixed base-ration offered once daily ad libitum at 08:30. From \u221250 days relative to calving, they received a far-off diet , and once calving the cows received a lactation diet . Water intake was available ad libitum. All the cows included in this study were housed in free-stalls  but with straw, and they were checked daily and milked two times a day.Two groups of cows were made. The lameness group (L group) was initially composed of twelve cows identified by a specialist veterinarian (PJV-M) for showing signs of lameness using a numerical rating system (NRS) based on a 5-point scale, in which a score of 1 represented a no-lame animal and 5 represented a severely lame animal  . The othOnce lameness was detected in the L group, one day later the cows were subjected to hoof trimming to characterize and treat the lameness. At that moment, two hoof-trimmers trained examined each hoof and recorded the presence of lesions by a 4-point lesion scoring system (LSS) according to Flower et al.  . Otherwise, the H group was sampling one day after the L group was sampling at T0 and TF, respectively. The samplings were made in the milking parlor right after the milking, after removing the milkers, and while the nipples were post-dipped, at the moment when the sampler had the opportunity to do it. The sampling procedure was always performed in the first milking between 07:00 and 12:00. All cows were sampled in a similar period of the day at each sampling time, with differences lower than two hours.g for 10 min at 4 \u00b0C. Saliva and plasma specimens were stored at \u221280\u00b0C (less than five months) until analysis.First, saliva samples were collected by introducing a sponge clipped to a flexible thin metal rod into the cow\u2019s mouth to be chewed. The sponge was then placed in collection devices . Just after saliva sampling, blood was obtained by venipuncture in vena caudal using a sodium heparin device . Both saliva and blood devices were stored in ice until arrival at the processing laboratory (less than 2 h), where they were then centrifuged at 3.000\u00d7 Enzymes: aspartate aminotransferase (AST), alanine aminotransferase (ALP), \u03b3-glutamyl transferase (gGT), lipase (Lip), alpha-amylase, lactate dehydrogenase (LDH), creatine kinase (CK), butyrylcholinesterase (BChE), total esterase (TEA), and adenosine deaminase (ADA).Metabolites: creatinine, urea, uric acid, total bilirubin, triglycerides, glucose, and lactate.Proteins: total proteins and albumin.Minerals: phosphorus and total calcium.Hormones: cortisol.The salivary analytes evaluated in the chemistry profile were:This chemistry profile was carried out on an automated chemistry analyzer  and adapted to saliva, as previously reported . SalivarIn our study, the salivary chemistry profile was applied in an exploratory population of six cows from the L group before (T0) and after (TF) the treatment selected randomly to evaluate possible differences in the salivary chemistry profile. Then, the salivary analytes that showed significant changes and pCor were measured in the additional 5 lame cows (n = 11) and the healthy group (n = 11).p = 0.140) and DIM  were observed, although a tendency was in BCS . Therefore, a mixed linear model study was performed using the BCS as a covariate in the salivary analytes\u2019 results previously selected after the exploratory study. An unpaired Student\u2019s t-test (2-tailed) was also performed in the milk yield at T0 in both groups to assess possible differences.Previous the statistical analysis, data were checked for normality using the Shapiro-Wilk test, and those showing no normality were previously log-transformed to normalize it. The parity, DIM, and BCS at T0 in both groups were evaluated by an unpaired Student\u2019s t-test (2-tailed) to guarantee no differences between groups. No differences in parity  and a power of 80%. It showed that a minimum number of 10 animals per group would be needed to get appropriate results for the selected analytes at different times. The two-way ANOVA of repeated measures followed by Sidak\u2019s multiple comparisons tests were then used to assess whether significant changes existed between the 11 cows from the L group and the 11 cows from the H group.For salivary analytes evaluation, a paired Student\u2019s t-test (2-tailed) was performed to assess the possible significant differences between T0 and TF in the exploratory population (six lame cows). In those analytes that showed significant changes, a stand-alone power program for statistical testing (G-Power)  was emplSpearman or Pearson r, depending on whether the data were non-normally or normally distributed, were calculated in the selected salivary analytes in the global population to evaluate the correlation between them and the pCor concentrations, NRS, and LSS. An r-value between 0.90 to 1 was considered to have a very high correlation, 0.70 to 0.90 high correlation, 0.50 to 0.70 moderate correlation, 0.30 to 0.50 low correlation, and less than 0.30 little if any correlation .p \u2264 0.05 were selected to indicate significance in all analyses.All statistical analyses were performed using the commercial statistics package GraphPad Prism 8 . Values of p = 0.028).Lower milk yield was observed in the L group compared to H group .p = 0.014), with higher concentrations in the L group (2.41 \u00b1 1.09 \u00b5g/dL) than H group (1.30 \u00b1 0.86 \u00b5g/dL) at T0 (p = 0.05). No significant differences between L and H groups at TF  were observed (p = 0.45).Cortisol in plasma showed changes between groups .When salivary analytes were evaluated in the exploratory population before and after lameness treatment , only Lip = 0.004) and TEA  showed no change between times in the H group , but higher activities at T0 than TF  in the L group (p = 0.050). The mix linear model study showed no significant effect of the BCS in Lip (p = 0.728) and TEA (p = 0.429) results.Lip  between them. TEA activity showed a low positive correlation with the NRS  and the LSS , while Lip only with the NRS . No correlations were observed between TEA (p = 0.723) and Lip (p = 0.936) in saliva with pCor.Correlations results are showed in In our study, changes in salivary analytes in cows with lameness were evaluated. The lameness severity showed a scoring mean of 3, considered as moderate . This deIn this trial, an extensive salivary chemistry profile was initially applied in lame cows before and after a treatment consisting of hoof-trimming. The evaluation of an extensive chemistry profile in saliva has been used in other species, such as horses , and it No relationship was observed between TEA with pCor, probably because of the different release source, being TEA related to the SNS activation, while pCor related to the HPA axis. In a study made in horses, no correlation between an analyte related to SNS, such as sAA, and sCor when a painful disease was observed . The difThis study has various limitations. Although previous publications evaluating lame cows used a similar number of animals than in this report ,33, it sIn conclusion, the research hypothesis of our study was confirmed, and lameness in cows produced changes in selected analytes in saliva, that reflect improvements in lameness after treatment. Namely, cows with lameness in the conditions of this study showed increases in TEA activities in their saliva compared to healthy cows, which decreased when a specific hoof treatment was performed. This report should be considered a pilot study, and further studies in a large population with cows with lameness due to different causes and severity should be performed to determine the potential of TEA as a biomarker of lameness in cows. If the TEA is confirmed as a biomarker in these additional studies, point-of-care assays that allow the measurement of this analyte quickly and easily on-farm conditions could be developed and applied in routine by farmers or under the coordination of Dairy Herd Improvement associations."}
{"text": "Increasing evidence has revealed that the initiation and progression of breast cancer are greatly affected by the immune environment. Neutrophils are the most abundant leucocytes in circulation and act as the spearhead in inflammation, including in breast cancer. Circulating neutrophils are closely related to the prognosis of breast cancer patients, and tumor-infiltrating neutrophils have varied functions at different stages of breast cancer, such as antitumor or tumor-promoting neutrophils, which are termed N1 and N2 neutrophils, respectively. In this review, we will discuss the utility of circulating neutrophils for predicting prognosis and therapeutic efficacy and the underlying mechanisms of their chemotaxis, the dynamic regulation of their antitumor or protumor functions and their different spatial distributions in tumor microenvironment. Finally, we also discuss the possibility of targeting neutrophils as a therapeutic strategy in breast cancer. AlthougNeutrophils can present both antitumorigenic (\u201cN1\u201d) and protumorigenic (\u201cN2\u201d) phenotypes in various cancers or specific circumstances. The term neutrophil in several studies also includes both mature neutrophils and myeloid-derived suppressor cells (MDSCs). MDSCs are described as a subset of neutrophils with immunosuppressive functions that express CD11b and Gr1 , 10 and To avoid confusion, we mainly focus on the biological function of mature neutrophils and related therapeutic strategies for targeting them in BC. We provide a comprehensive review of the prognostic value of circulating neutrophils and the mechanisms of how tumor-associated neutrophils (TANs) exert antitumor or tumor-promoting functions in BC, and in the end, we also discuss the potential of targeting neutrophils as a therapeutic strategy in cancer.Tumors can be thought of as wounds that will not heal and are characterized by chronic inflammation. Neutrophils are the most rapidly responding immune cells to inflammation, and many studies have found that the NLR is closely related to the prognosis and treatment response in patients bearing BC , 13. A rp = 0.012)  and the NLR eight consecutive times before and after chemotherapy found that patients who died had lower ALC and higher NLR values than patients who remained alive throughout the treatment course; additionally, among the patients who died, a steady increase in the NLR over the baseline measurement was observed at subsequent time points . Another= 0.012) . A low N= 0.012) , 31.p < 0.001) but not the PLR (p = 0.104) was a significant indicator for both DFS and OS  was in the group with an NLRhigh/PLRhigh profile  and decreased tumor-associated lymphocytes but was not significantly related to CD66b+ infiltrating neutrophils . Therefo+CD4+ and \u03b3\u03b4 T cells were the major sources of IL-17  usually acts as a damage-associated molecular pattern that is released by dying cells or stressed cells to initiate inflammation and was later found to be an important chemoattractant for neutrophils . Epithelin vivo , can alsin vivo . Meanwhiin vivo . It is nin vivo , 81. In in vivo , 83. Addin vivo .In addition, similar to lymphocytes and macrophages, neutrophils are more likely to localize in tumors of triple-negative breast cancer (TNBC) than to tumors of other BC subtypes . Recentl2O2), tumor necrosis factor (TNF)-\u03b1 and NO, and that TANs are more likely to obtain a protumorigenic phenotype with tumor progression (lowCD54high) that was able to stimulate T cell proliferation and IFN-\u03b3 release, which suggested a pro-inflammatory rather than immunosuppressive state of TANs in early-stage lung cancer (2O2 to cytotoxic hypochlorous acid (HOCI)  , 89. Recd (HOCI) .2+ channels and mediated by transient receptor potential cation channel, subfamily M, member 2 (TRPM2) expression on tumor cells (http://gepia2.cancer-pku.cn/#index), active NOX1, catalase and SOD were also increased in the membrane of cancer cells, forming a complex mechanism by which tumor cell apoptosis induced by ROS is prevented  and CD56bright (protumor) subsets, and CD56bright NK cells are enriched in the tumor microenvironment and draining lymph nodes  . A mouseBecause of the lack of direct information from previous publications, we tried to determine the abundance and subtype of TANs in human BC via the CIBERSORT-LM7 deconvolution algorithm based on mRNA expression datasets  . Our anaChemotherapy and radiotherapy are integral parts of BC treatment that can influence the immune microenvironment. Anthracycline and cyclophosphamide-based chemotherapy regimens are still widely used in BC treatment , 145. Itin vivo  but not microbeam radiation therapy (MRT) induced a substantial increase in TAMs and TANs, and increased levels of CCL2  were also observed in tumors subjected to CRT . In addiin vivo . Furtherin vivo \u2013158. Howin vivo .Since the majority of studies have revealed a protumor function of neutrophils in BC, targeting neutrophils as a therapeutic strategy has been investigated. In mouse mammary tumor models, depletion of neutrophils with anti-Ly6G antibodies resulted in diminished tumor formation and lung metastasis . In addiIFN-\u03b2 and TGF-\u03b2 are the cytokines that are most often reported to modulate the switch between N1 and N2-like neutrophil polarization. Steven et al. first revealed that TGF-\u03b2 blockade significantly increased the influx of antitumor neutrophils and activated CD8+ T cells in a BC mouse model . ThereafA schematic picture depicting the dual role of neutrophils in BC . Here, wYS and CN wrote the manuscript. CZ and WZ edited the manuscript. YS, WZ, and SP collected the related literatures. HH, JJ, and WC finished the tables and figures. TZ provided the feedback and guidance. All authors read and approved the final 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."}
{"text": "To investigate the association of prenatal alcohol exposure (PAE) and early neurodevelopment in the first 2 years of life, adjusting for maternal socio-demographic and psychosocial factors, in the Drakenstein Child Health Study (DCHS), a South African birth cohort study.n = 260) and 24 months of age (n = 734). A subset of alcohol-exposed and -unexposed children was included in this analysis at age 6  and 24 months . Multiple hierarchical regression was used to explore the associations of PAE with motor and language development.The DCHS comprises a population-based birth cohort of 1143 children, of which a subsample completed the Bayley Scales of Infant Development-III (BSID-III) at 6  functioning after adjusting for maternal socio-demographic and psychosocial factors at 6 months of age only. No significant effects were found in either receptive or expressive communication and cognitive outcomes at either time points.PAE was significantly associated with decreased gross motor [odds ratio (OR) = 0.16, 95% confidence interval (CI) = 0.06\u20130.44, PAE has potentially important consequences for motor development in the first 2 years of life, a period during which the most rapid growth and maturation occur. These findings highlight the importance of identifying high-risk families in order to provide preventive interventions, particularly in antenatal clinics and early intervention services. PAE had a significant impact on motor functioning after adjusting for variety of socio-demographic and psychosocial factors at 6 months of age, but not on cognitive or language development.The findings of this study highlight the importance of identifying psychosocial risk factors, particularly in antenatal clinics and early intervention services in a South African context.The BSID-III tool assesses general ability of a given task but may have low sensitivity for detecting minor developmental impairments especially during infancy.Language impairments are very subtle in the early years and may be more difficult to identify impairments than in other domains.A small sample size may have reduced the power of the study, and findings may not be generalisable to other populations.et al., et al., et al., et al., et al., et al., et al., et al., Prenatal alcohol exposure (PAE) has been recognised as a major global public health concern. A recent study estimated 9.8% of mothers consumed alcohol during pregnancy and 4.3% were heavy drinkers  is a pattern of often irreversible physical and mental birth deficiencies , a South African birth cohort study.et al., et al., et al., et al., This study formed part of the DCHS, a multidisciplinary birth cohort study investigating the early determinants of child health , whereas the full cohort was invited to participate at 24 months (n = 734) making a larger sample available. At 6 and 24 months of age, a subset of infants and toddlers were selected whose mothers reported moderate-to-severe levels of alcohol consumption and for whom BSID-III data were available. Of the 260 infants, 52 were exposed to alcohol at 6 months, and of the 734 toddlers, 92 were exposed to alcohol at 24 months. The unexposed group comprised 104 at 6 months and 184 at 24 months. Unexposed control children were randomly matched for maternal education and clinic site in a 1:2 ratio , every effort was made to ensure a safe, anonymous, confidential and supportive environment. Translation of the measures from English to Afrikaans and isiXhosa included a standard forward and back-translation process , marital status and HIV status  and psychological variables (PTSD and depression) administered antenatally. Maternal smoking was assessed using the ASSIST  were presented for normally distributed data. Medians [interquartile range (IQR)] were presented for data that were not normally distributed and for all BSID-III scores. For comparisons between alcohol-exposed and -unexposed children, chi-squared tests were used for categorical variables, while p = 0.001) and 24 months of age . There were no significant differences between the exposed and unexposed groups regarding infants\u2019 birth weight and BMI.The maternal and child socio-demographic and psychosocial characteristics are presented in Table\u00a0p = 0.006; fine: median scores = 11.5 (IQR = 10.0\u201313.0) vs. 13.0 (IQR = 12.0\u201315.0), respectively, p = 0.001)]. At 24 months, there were no significant differences, although there remained a trend towards impairment for fine motor functioning in exposed children . There were no significant differences in language and cognitive functioning at 6 or at 24 months.Table\u00a0p = 0.001] and fine motor functioning  after controlling for BMI, SES, and smoking and psychological variables at 6 months of age. BMI was significantly associated with both gross  and fine motor functioning , while SES was significantly associated with gross motor functioning  at 6 months.Table\u00a0F(4) = 3.98, p = 0.002, adj R2 = 0.10), and the R2 showed that the amount of variance increased from 5%  to 14% after adding PAE into the model (Appendix F(4) = 3.66, p = 0.004, adj R2 = 0.13), and the R2 showed that the explained variance accounted for an extra 9% (0.04\u20130.13) in fine motor functioning (Appendix The final model explained a significant amount of variance in gross motor functioning (This study comprehensively assessed motor, language and cognitive functioning in a population-based cohort over the first 2 years of life. The findings of our study indicated that PAE is associated with both gross and fine motor functioning at 6 months of age, even after adjusting for maternal socio-demographic and psychosocial factors. While PAE was not associated with receptive and expressive communication nor cognitive performance at either time point in this group, there remained a trend towards significance for poorer fine motor functioning at 24 months of age.et al., Our findings demonstrate that PAE is associated with deficits in motor functioning across the first 2 years of life. This is consistent with previously reported cohort studies in preschool age (Fried & Watkinson, et al., et al., In our cohort, PAE was not found to be associated with receptive or expressive communication or cognitive functioning at the age of 24 months. Previous studies have reported impairments in language and cognitive functioning in toddlers between the ages of 12 and 24 months (Fried & Watkinson, Additional limitations deserve consideration. Firstly, the substudy comprised a small sample size which may have limited the power to detect differences between the groups. Secondly, despite assurances of confidentiality, some women may have chosen not to disclose or minimise reporting alcohol use to the research teams and the low reported alcohol consumption may therefore represent an element of response bias. Thirdly, the BSID-III tool measures general ability in completing a given task but may have low sensitivity for detecting minor developmental impairments especially during infancy. Further, although this tool has been validated for use in South Africa, this study may not be generalisable to other populations.A large proportion of very young children in LMICs do not reach their developmental potential due to a wide variety of socio-demographic and psychosocial factors that may impact early developmental outcomes. Our study, reporting the association of PAE and early motor functioning, is one of only a few studies that have additionally addressed important potential psychosocial confounders which frequently co-occur with alcohol use in these communities. These findings highlight the importance of identifying high-risk families in order to provide preventive interventions, particularly in antenatal clinics and early intervention services."}
{"text": "For more than three decades, numerous studies have demonstrated the function of p53 in cell cycle, cellular senescence, autophagy, apoptosis, and metabolism. Among diverse functions, the essential role of p53 is to maintain cellular homeostatic response to stress by regulating proliferation and apoptosis. Recently, adipocytes have been studied with increasing intensity owing to the increased prevalence of metabolic diseases posing a serious public health concern and because metabolic dysfunction can directly induce tumorigenesis. The prevalence of metabolic diseases has steadily increased worldwide, and a growing interest in these diseases has led to the focus on the role of p53 in metabolism and adipocyte differentiation with or without metabolic stress. However, our collective understanding of the direct role of p53 in adipocyte differentiation and function remains insufficient. Therefore, this review focuses on the newly discovered roles of p53 in adipocyte differentiation and function. The multifunctional protein p53  is a key regulator of cell proliferation and apoptotic response to exogenous or endogenous cellular stresses . Under vIn the absence of cellular stresses, mouse double minute 2 (MDM2), an E3 ubiquitin ligase, regulates p53 levels via proteasomal degradation pathways . When ceAdipose tissue plays an important role in the regulation of systemic energy balance and glucose homeostasis. For example, adipose tissues regulate energy storage, lipid metabolism, glucose homeostasis, and energy expenditure . The epiOn the other hand, the main function of WATs is energy storage in the triglyceride form. WATs are generally located subcutaneously throughout the body and constitute as much as 20\u201330% of the body weighty of normal humans. WAT progenitor cells are differently developed according to location; subcutaneous white adipose tissues develop before birth but visceral white adipose tissues develop after birth ,27. HoweIn this review, we discuss the important role of p53 and highlight the key molecules that are regulated by p53 during adipogenesis. We also discuss the role of p53 in regulating metabolism in adipose tissues. Finally, we identify important outstanding questions and issues pertaining to the role of p53 in adipogenesis that remain to be addressed.p53 is a regulator of cell survival and proliferation in response to multiple stress signals against abnormal cell growth and tumorigenesis. Apart from its role as a tumor suppressor, p53 has been involved in the regulation of differentiation of mesenchymal cells that can suppress white adipocyte differentiation ,33,34. WCarm1 and reduced its protein expression  variant of p53 who were administered a high-fat diet developed severe obesity and metabolic dysfunction than mice expressing a proline 72 variant . In lineBATs are located around the central organs and are essential for whole-body energy homeostasis, particularly energy expenditure ,64. HencUcp1 transcriptional activity [Foxc2 in mouse preadipocytes, which increased the expression of cyclin E and inhibited that of p53 and p27 in preadipocytes (A recent study performed by Molchadsky et al. demonstrated that p53 is a positive regulatory factor that maintains proper brown adipocyte maturation in vitro and prevents the development of obesity because of a high-fat diet in the mouse model . Loss ofactivity ,74. Overactivity . In vivoactivity . This phactivity . Foxc2 iactivity ,77. Gan ipocytes .On the other hand, Hallenborg et al. showed that depletion of p53 in mice protected them against high-fat diet-induced obesity . Loss ofPRDM16 expression via interaction with PRDM16 or binding to the PRDM16 promoter [p53 has been known to be involved in the aging process of WATs . The totpromoter ,47. HoweOver the past four decades, the roles of p53 have been extensively studied because the mutation or inhibition of p53 initiates tumorigenesis ,81,82,83Heretofore, p53 role in adipogenesis, especially white adipocytes, is a negative regulator of adipogenesis. However, the role of p53 in negative regulation of adipogenesis under exact molecular upstream and downstream networks remains unknown. Generally, p53 is considered a stress response protein. WATs have been considered to be simple energy storage tissues containing lipid droplet organelles but they also have endocrine functions and secrete several factors such as adipokines . The assOne of the major unsolved questions is the direct role of p53 in brown adipogenesis. Previously performed studies have reported contrasting results regarding the role of p53 in brown adipogenesis. Inhibition of p53 suppressed brown adipogenesis in Prdm16-overexpressed myogenic C2 cells in vitro . On the The other mechanism by which p53 regulates cellular differentiation involves its chromatin-binding activity or interaction with specific protein partners. As described above, p53 affects adipogenesis dependent cell types in vitro. These results suggest that promoter remodeling following p53 binding to chromatin occurs at different sites in different adipogenic stages or binding with different protein partners in each cell. BATs and WATs share many essential adipogenic regulatory transcriptional factors including PPAR\u03b3 and CEBP\u03b1. If p53 regulates promoter remodeling in the same way, there is no different response to adipogenic development regardless of the cell type. p53 interacts with chromatin response to diverse signaling and regulates transcription of specific target genes with or without co-binding factors ,91,92. SThe role of p53 in tumorigenesis has been extensively studied because the activation of p53 kills cancer cells . Many re"}
{"text": "ARF is a tumor suppressor encoded by the CDKN2a locus that is frequently inactivated in human tumors. P14ARF protein quenches oncogene stimuli by inhibiting cell cycle progression and inducing apoptosis. P14ARF functions can be played through interactions with several proteins. However, the majority of its activities are notoriously mediated by the p53 protein. Interestingly, recent studies suggest a new role of p14ARF in the maintenance of chromosome stability. Here, we deepened this new facet of p14ARF which we believe is relevant to its tumor suppressive role in the cell. To this aim, we generated a monoclonal HCT116 cell line expressing the p14ARF cDNA cloned in the piggyback vector and then induced aneuploidy by treating HCT116 cells with the CENP-E inhibitor GSK923295. P14ARF ectopic re-expression restored the near-diploid phenotype of HCT116 cells, confirming that p14ARF counteracts aneuploid cell generation/proliferation. P14 ARF which has a potent tumor suppressor activity. Loss of the CDKN2a locus is the second most frequent copy number alteration that characterizes human tumors and cancer cell lines. One of the most well-defined functions of p14ARF protein is to suppress aberrant cell proliferation in response to oncogene insults by activating the transcription factor p53 that, in turn, triggers the expression of many apoptosis inducers and cell cycle inhibitory genes [ARF binds the p53-specific E3 ubiquitin ligase Mouse double minute 2 homolog (Mdm2) and keeps it in the nucleolus. This event prevents the interaction between Mdm2 and p53 by blocking the transport to the cytoplasm of p53 and its degradation by the proteasome Mdm2-mediated [The alternate open reading frame (ARF) gene was originally identified as an alternative transcript of the CDKN2a locus on human chromosome 9p21 that encodes the nucleolar protein p14ARF, p53, and MDM2 genes were more tumour prone when compared to mice lacking only p53 and MDM2 [ARF is highlighted by the effect of its loss in mouse models: mice were highly prone to develop tumors and mostly died during their first year of life. In addition, p14ARF-null mouse embryonic fibroblasts (MEFs) can overcome the senescence crisis and proliferate continuously, and they also can be easily transformed by the Ras oncogene [ARF is a tumor suppressor it is not surprising that most human cancers  are characterized by its inactivation mainly due to its promoter hypermethylation, homozygous deletions and mutations  that was the result of chromosome mis-segregation [ARF is involved in the cell response to the aneuploid phenotype triggered by the impairment of different types of proteins, namely MAD2 and DNMT1 [ARF can respond either by arresting the cell cycle and entering senescence if necessary or by inducing apoptosis [ARF is critical to halt aneuploidy. To this regard, we recently demonstrated that when CENP-E is reduced by 50% and aneuploidy is consequently induced, HCT116 cells, where p14ARF is not expressed [ARF induction. HCT116 cells could finally recapitulate IMR90 cell behaviour and recover from aneuploidy once they ectopically re-express p14ARF [ARF is involved in blocking the proliferation of aneuploid cells. However, we only analysed the effects of p14ARF ectopic expression in a heterogenous population of HCT116 cells expressing different levels of p14ARF and we did not investigate on the mechanism that allows the elimination of the aneuploid cells.On the other hand, there is some evidence that ARF, especially its small mitochondrial isoform (smARF) , also haand MDM2 . In addiand MDM2 . Moreoveand MDM2 . The imponcogene . Consideiewed in . Due to iewed in ,11. ARF regation ,13. Theyregation . On the nd DNMT1 . In partpoptosis ,16. Altoxpressed , are nots p14ARF . These eARF cDNA integrated in the genome specifically at \u2018TTAA\u2019 sites by the PiggyBac transposon vector system (ePB) (ARF c-DNA and the vector carrying the transposase enzyme (hyPB). Transfected cells were then selected with the antibiotic blasticidin for ten days and then single colonies were picked, analysed and one positive clone was chosen for further investigation. In the engineered HCT116 cells (HCT116 ePB cells), p14ARF cDNA expression is under the control of the TET-On technology that allows activation of gene expression by administration of doxycycline. By using a low dose of doxycycline (10ng/mL) we obtained the consistent expression of p14ARF  A,B. Brief p14ARF C\u2013E. Reve16 cells C. Moreov protein D,E. Follgression F,G. ARF induction/re-expression , these cells became aneuploid (50%). However, when doxycycline was washed out and thus p14ARF was expressed, the percentage of aneuploid cells was reduced from 50% to 22% [ARF could partially reduce aneuploidy of CENP-E-depleted HCT116 cells from 73% to 56% [ARF ectopic re-expression restore the near-diploid phenotype of HCT116 cells. These results confirm that p14ARF counteracts aneuploid cell generation/proliferation. It is well established that p14ARF regulates cell cycle arrest and apoptosis, however it remains unclear if p14ARF uses one or both mechanisms to fight aneuploidy [ARF after treatment with GSK923295 (ARF was expressed. This made us think that apoptosis could likely be the way through which p14ARF induces the clearance of the aneuploid cells. We hypothesized thus that this could be the mechanism through which p14ARF helps the maintenance of chromosomal stability. Hence, to identify the presence of apoptotic cells, we performed acridine orange and ethidium bromide staining which discriminates live, apoptotic, and necrotic cells. By this mean we observed 25% of apoptotic cells in HCT116 cells expressing p14ARF following GSK923295 treatment. These results strongly suggest that p14ARF triggers the apoptotic pathway to halt aneuploid cell proliferation.We then induced aneuploidy in these engineered HCT116 cells by treating them for 72 h with the CENP-E inhibitor GSK923295 . GSK9232pression A,B. As e% to 22% . In addi% to 56% . Therefoeuploidy ,14,21. TSK923295 C\u2013F. PropARF mutations. Though, p14ARF role in cancer as tumor suppressor is vastly demonstrated. Noteworthily, it has been noticed that patients with mutations in p14ARF have high risk of developing melanoma and pancreatic cancer [ARF has a fundamental role in fighting cell transformation and suggest that, due to the high frequency of deregulation in cancer, this role could be played at early stages of cell transformation. We think that this could be due to p14ARF ability in counteracting aneuploidy. Through this mechanism numerical chromosomal stability would be preserved in normal cells. This is well-known during development of Drosophila melanogaster that indeed uses apoptosis to eliminate aneuploid cells . In addic cancer ,25. Furtc cancer . AltogetARF was firstly identified as an alternate transcript of the CDKN2a locus, which is the second most frequently mutated site in cancers after p53. Its tumor suppressor role as regulator of the cell cycle and apoptosis has been indeed well established. Classically, a hyperproliferative signal that results from an oncogenic insult triggers ARF activation. In this brief report, by confirming previous results, we suggest that p14ARF can also respond to the onset of aneuploidy identification, thus providing an additional function as a sentry of the euploid set of chromosomes. Remarkably, aneuploidy, i.e., an abnormal number of chromosomes, is a hallmark of virtually all tumors. We observed that the absence of p14ARF worsens the aneuploid phenotype of the cells, however it was generated. These cells, indeed, cannot either block their cell cycle or enter apoptosis, and therefore, as a matter of fact, the possibility to impede the proliferation of aneuploid cells is denied. The maintenance of aneuploidy can undoubtedly contribute to malignant transformation. The presence/restoration of p14ARF, instead, allows cells to counteract aneuploidy, most probably by sentencing the aneuploid cells to apoptosis. In fact, this mechanism of aneuploid cell elimination by apoptosis has been observed during development. It is conceivable that this function of ARF is an important part of its role as a tumor suppressor. Furthermore, it is generally established that gene copy number correlates with gene expression. It is not too daring to think that the imbalance of gene expression that is induced by aneuploidy could then generate a signal resembling the hyperproliferative stress typically sensed by p14ARF. This, in turn, could activate p53-dependent apoptosis. In conclusion, through this evidence, it is clear that p14ARF not only responds to oncogene stimuli or to DNA damage, but also to an \u201caneuploidy signal\u201d and protects the cells from the maintenance of such aberrant and potentially dangerous condition. P14"}
{"text": "A reduction in the magnitude of corneal astigmatism of \u22651 D was observed in 52.8% of eyes. No significant changes were observed in corneal endothelial density (p = 0.317). Significant changes were found in the anterior vertical coma component (p = 0.002) as well as in the spherical aberration of the posterior corneal surface (p = 0.004). Only two relevant complications were described: one corneal microperforation with penetration of the ring segment into the anterior chamber , and a case of ring extrusion . ICRS implantation in children keratoconus eyes allows a reduction of corneal astigmatism, irregularity, and aberrations, leading to a significant visual improvement.The short-term safety and efficacy of intracorneal ring segment (ICRS) implantation in keratoconus eyes of children are investigated in this study. A retrospective interventional case series study including a total of 33 keratoconus eyes (age 8 to 17 years) that had undergone ICRS  implantation was conducted. Information about visual, refractive, pachymetric, corneal topographic and aberrometric, and corneal endothelial changes during a 3-month follow-up were extracted and analysed. A significant improvement was observed in logMAR corrected distance visual acuity ( Keratoconus is a degenerative corneal disease that is commonly manifested during puberty, with the possibility of progressing until the third or fourth decade of life . This coAlthough keratoplasty is a potential solution in the most advanced keratoconus cases, both penetrating and deep lamellar keratoplasty are not exempt from complications such as rejection of the donor button, vascularization and infectious diseases . In chilRetrospective interventional case series including a total of 33 keratoconus eyes (21 men and 12 women) that had undergone ICRS implantation using the femtosecond laser technology for the creation of the tunnels. All cases included were treated and followed at Clinica de Ojos Larco Vision , with all surgeries performed in the period from May 2015 to July 2018. Inclusion criteria were keratoconus diagnosis according to the standard criteria that consider the presence of the following signs: anterior corneal topographic asymmetric bowtie pattern, KISA \u2265 100, and one or more biomicroscopic keratoconus signs, such as Fleischer ring, significant corneal thinning, Vogt striae, conical protrusion on the cornea at the apex or anterior stromal scar [A complete preoperative ophthalmological examination was performed in all cases including measurement of uncorrected (UDVA) and corrected distance visual acuity (CDVA), autorefraction , cycloplegic and manifest refraction, slit lamp evaluation of the anterior segment, fundus evaluation, pupillometry, specular microscopy for corneal endothelial cell density analysis  and corneal topographic, pachymetric and aberrometric evaluation with the Scheimpflug imaging-based system Pentacam . Postoperative examinations were performed the day after surgery, at 1 week, 1 month, and 3 months after surgery. In the last postoperative visit, a complete eye examination identical to the preoperative exam was performed. The importance of not rubbing the eyes and maintaining the anti-allergic treatment was remarked to all patients and their relatives, with the additional recommendation of attending to eye controls every 3 months.All patients received indications and anti-allergic treatment prior to surgery and it was maintained indefinitely.All surgical procedures were performed by the same experienced surgeon (PL) under topical anaesthesia . The femtosecond laser platform FS 200  was used to create the corneal incision and tunnels. The tunnels were created at 70% of corneal depth ensuring that at least 100 \u03bcm of corneal tissue was present under the tunnel. Keraring segments  were implanted in all cases, with a selection of the number of segments to implant as well as their diameter (SI5 or SI6), arc length and thickness according to the manufacturer\u2019s nomogram and the scotopic pupil size measured. https://www.imim.cat/ofertadeserveis/software-public/granmo/. Assuming a paired test in a same group for detecting significant differences in CDVA, the statistical power was 84.0% for the detection of a minimum difference of \u22120.16 logMAR (minimum change in CDVA observed at 3 months postoperatively), considering an alpha error of 0.05, the sample of 33 eyes, and the standard deviation of differences in CDVA at 3 months after surgery (0.31 logMAR).The statistical power associated to the sample size selected was calculated using the online calculator, p-value of less than 0.05 was considered as statistically significant.Statistical analyses were performed with a commercially available software package . Normality of data samples was evaluated by means of the Kolmogorov-Smirnov test. When parametric analysis was possible, the Student t test for paired data was used for comparisons between preoperative and postoperative visits, whereas the Wilcoxon ranked sum test was applied to assess the significance of such differences when parametric analysis was not possible. The Pearson or Spearman correlation coefficient was used to assess the level of correlation between preoperative corneal astigmatism and the clinical outcomes obtained depending if the condition of normality could be assumed or not. For all statistical tests, a A total of 33 eyes of 33 patients with mean age of 14.9 years  were enrolled. The sample was comprised of 21 males (63.6%) and 12 females (36.4%). A total of 15 right (41.7%) and 21 left eyes (58.3%) were included. The model of ring segments SI5 was implanted in a total of 16 eyes (44.4%), whereas a total of 20 eyes (55.6%) were implanted with the SI6 ring segments. In all cases except in 12 eyes, two ring segments were implanted , with the following distribution in terms of arc length: 90\u00b0 in 11 eyes , 120\u00b0 in 18 eyes , 150\u00b0 in 15 eyes , 160\u00b0 in 9 eyes , 210\u00b0 in 4 eyes , and 340\u00b0 in 3 eyes . Concerning ring segment thickness, the distribution of cases was as follows: 150 \u03bcm in 3 eyes , 200 \u03bcm in 14 eyes , 250 \u03bcm in 12 eyes , and 300 \u03bcm in 31 eyes .p \u2265 0.066). However, a significant improvement was also observed in logMAR CDVA (p = 0.005). Concerning keratometric readings, a statistically significant reduction was observed in K1 (p = 0.001), K2 (p < 0.001), and KM (p < 0.001). Consequently, a significant reduction was observed in the magnitude of corneal astigmatism (p < 0.001), with 11.1% (4/36) and 69.2% (18/26) of eyes with magnitude of anterior corneal astigmatism of 3 D or less before and 3 months after surgery, respectively (p < 0.001) (p = 0.647).ectively . A total< 0.001) . Concernp = 0.002) as well as the spherical aberration of the posterior corneal surface (p = 0.004). No significant changes were observed in the remaining aberrometric components (p \u2265 0.200). A poor although significant correlation was found between preoperative coma RMS and the change induced in this parameter with surgery . No correlation was found between the preoperative magnitude of spherical aberration and the change induced in this parameter with surgery .p = 0.317), with mean preoperative and 3-month postoperative values of 2795.08 cell/mm2  and 2624.50 cell/mm2 , respectively.Concerning safety and complications, only two relevant complications were described, one corneal microperforation with penetration of the ring segment into the anterior chamber , and a case of ring extrusion  . No signIn the last two decades, advances in keratoconus management have been focused on avoiding corneal transplantation . Althougp = 0.001). Alfonso et al. [Concerning the refractive outcomes, a reduction in magnitude was observed in sphere, cylinder and SE in most of patients, but not reaching statistical significance in the overall sample. This is consistent with the variability of ICRS outcomes reported in the peer-reviewed literature according to the corneal topographic phenotypic profile and the level of severity of the disease ,25,26. Ro et al.  reportedo et al.  also repThe change induced at 3 months postoperatively in the magnitude of anterior corneal astigmatism was strongly correlated with the preoperative magnitude of anterior corneal astigmatism. This correlation was negative which indicated that less reduction of corneal astigmatism was achieved in those cases with lower levels of baseline corneal toricity. This suggests that no more undercorrection was present in cases of high levels of corneal astigmatism. However, despite the greater astigmatic corrective effect achieved in very toric corneas, the impact of potential misalignments of the meridian of correction is higher, as demonstrated in previous series . In any The analysis of changes in corneal aberrations in the current series revealed that statistically significant changes were found in the vertical coma component of the anterior corneal surface as well as in the spherical aberration of the posterior corneal surface. There was a trend to a reduction of primary coma RMS that did not reach statistical significance, as has been reported in other studies evaluating the corneal aberrometric outcomes after KeraRing implantation . The maiFinally, the safety of the procedure was confirmed with the gain of lines of CDVA and the presence of only two relevant complications, a corneal microperforation with penetration of the ring segment into the anterior chamber and a ring extrusion. These complications have been also reported in series with adult patients . In our As limitations of the study, it should be noted that no validated questionnaires were used to assess and characterize changes in symptomatology. This should be investigated in future series evaluating the impact of ICRS implantation in children with keratoconus. Likewise, the results of this technique in a longer follow-up period should be reported to confirm the long-term safety of the procedure. Despite these limitations, this article adds new information in terms of tomographic and corneal aberrometric changes to the currently available peer-reviewed literature ,14,33 abIn conclusion, the implantation of ICRS in children is a safe procedure, which is not exempt from complications like the two reported cases, related to manipulation of the eyes, despite having been warned not to do it and that they were solved without problems. The case of the migration of the ICRS to the anterior chamber had a previous history of micro perforation 3 months before the implantation of the ICRS. Possibly, the combination with CXL is necessary in most cases to avoid keratoconus progression, but more studies are still needed to confirm this and to evaluate the safety and efficacy of this combination. Abreu et al.  reported"}
{"text": "Investigation of protein expression in cancer cells is an important part of the diagnostic process. Increasing knowledge about expression of different proteins has been exploited for prognostic assessments and in some cases also for selection of treatment. The p53 protein has proven important in development of various cancers, and the expression of this protein and its signaling pathway is therefore of interest when examining cancer patient samples. Here, we present mass cytometry as a tool for detection of p53 expression. Mass cytometry allows for measurement of up to 50 parameters per sample with single cell resolution, and we aim to demonstrate its potential for p53-focused research.TP53 mutational status. Results: The p53 protein can be detected in cell lines and in primary samples by mass cytometry. By combining antibodies for p53-related signaling proteins with a surface marker panel, we show that mass cytometry can be used to decipher the single cell p53 signaling pathway in heterogeneous patient samples. Conclusion: Single cell profiling by mass cytometry allows the investigation of the p53 functionality through examination of relevant downstream signaling proteins in normal and malignant cells. Our work illustrates a novel approach for single cell profiling of p53.Purpose: The p53 protein and its post-translational modifications are distinctly expressed in various normal cell types and malignant cells and are usually detected by immunohistochemistry and flow cytometry in contemporary diagnostics. Here, we describe an approach for simultaneous multiparameter detection of p53, its post-translational modifications and p53 pathway-related signaling proteins in single cells using mass cytometry. Method: We conjugated p53-specific antibodies to metal tags for detection by mass cytometry, allowing the detection of proteins and their post-translational modifications in single cells. We provide an overview of the antibody validation process using relevant biological controls, including cell lines treated in vitro with a stimulus (irradiation) known to induce changes in the expression level of p53. Finally, we present the potential of the method through investigation of primary samples from leukemia patients with distinct  TP53 is mutated, the expression level of the (mutant) p53 protein is elevated. This is largely due to the absence of activation of a p53 downstream response, including transactivation of Mdm2, which leads to stabilization and accumulation of the mutated p53 protein  (REA529), p53and Ki67 a. To furm AML017 b. By perTP53 mutated patient. We investigated the force directed layout by visualizing the cells originating solely from patient AML017  was characterized by lower p53 expression. By assessing the expression of the p53 downstream target p21(CIP1/WAF1) and the proliferation marker Ki67, we observed a seemingly inverse correlation. Indeed, the Ki67 expression was high, while p21(CIP1/WAF1) expression was low. The reversed was observed in the p53 low population . Ki67 was also expressed in this population.Individual analysis of the different patients using viSNE can be found in In this study, we demonstrate the use of mass cytometry to examine p53 expression in cell lines and patient samples of leukemia. Investigation by mass cytometry demands for single cell suspension. Hematological malignancies are therefore particularly suitable, as the samples naturally are in single cell suspension. Thus, peripheral blood and bone marrow do not require extensive processing by mechanical and/or enzymatical degradation as solid tissues demand for. Peripheral blood and bone marrow samples can be fixed shortly after sampling using commercially available reagents  or proteomic stabilizer (Smart Tube Inc.). This greatly reduces the risk of altering intracellular signaling profiles prior to fixation. Furthermore, due to the possibility to simultaneously perform broad phenotyping and detect expression of multiple intracellular proteins, mass cytometry allows for assessment of protein expression in specific cellular subsets in primary samples. However, limitations of the technology do exist, and these are thoroughly addressed in Spitzer et al. . In shorTP53 mutations (specific amino acid sequences in mutated TP53) could enable mass cytometry to distinguish different mutations. Alternative ways of detecting specific mutations could be to use RNA probes toward specific mutations; a strategy similar to proximity ligation assays for RNA (PLAYR) or metal in situ hybridization (MISH) [In contrast to immunoblot analysis of p53 proteins, single cell detection by mass cytometry does not allow discrimination of molecular weight of the p53 species detected. This is especially a challenge in regard to the detection of p53 isoforms. To our knowledge, no isoform-specific antibodies developed have currently been validated to show compatibility with detection of non-denatured proteins by flow- or mass cytometry. Development of such specific antibodies could allow the assessment of biology and functionality of the p53 isoforms. Future development of antibodies with improved specificity could ease the identification of endogenous p53 isoforms, and as mass cytometry allows for high-dimensional data collection, thorough assessment of the individual isoforms\u2019 properties in a natural system could be achieved. As the method\u2019s span of detection relies solely on appropriate probes , development of specific monoclonal antibodies directed toward specific n (MISH) ,35.Detection of p53 using antibodies is limited by the antibody specificity, sensitivity, and affinity to p53 in the particular sample preparation. The p53 protein is encoded by a gene that may be the origin to more than 13 pre-mRNA splicing isoforms . FurtherTP53 mutations affect the p53 expression level by using different cell lines for validation experiments. As these cell lines express p53 in varying degrees, we show the importance of titrating antibodies on relevant samples. HL60 cells do not express full-length p53 due to deletion in both alleles [TP53 mutation, which results in high protein expression [Careful use of technical and biological validation is essential for establishing mass cytometry detection, e.g., by a standardized set of cell lines. Similarly, post-translational modifications may be validated by in vitro stimulated cell lines. Here, we demonstrate how  alleles , while Npression . An antipression ,42. Of npression . In thisIn general, mass cytometry is regarded as a less sensitive platform than flow cytometry and low-abundant targets may therefore theoretically be more difficult to detect by mass cytometry. However, the low background signal combined with almost negligible signal spillover in mass cytometry partly compensates for this . The detResting healthy cells express only low levels of p53, and thus, to properly detect p53 by mass cytometry, we argue that careful selection of isotope conjugate during antibody panel design is of importance. This was also evident from our observations when conjugating the same p53 antibodies to two different metal isotopes. Therefore, it is important that the antibodies in a panel are tested and titrated on that exact instrument. We encourage users of mass cytometry to carefully assess the signal generated by the different metal channels, and if feasible, conjugate aliquots of the same antibody to different isotopes for comparison of signal intensity. In our case, the two 153Eu antibody conjugates consistently generated stronger signals than the two 175Lu conjugates in all cell samples expressing low levels of p53 protein. In the cell lines expressing high levels of p53 protein, the signal differences were visually indistinguishable.We previously compared two-dimensional gel immunoblot with flow cytometry analysis of intracellular proteins in AML . ImmunobTP53 mutations, and that these mutated subpopulations could indicate higher risk of relapse [TP53 mutations in clinical diagnostics is sequencing, while loss of TP53 can be detected by fluorescent in situ hybridization (FISH). Although TP53 is mutated, the protein can still be translated. The resulting protein product may deviate from normal conformation and thus not retain normal protein function. Downstream targets of p53 might therefore not be affected by p53 activation. Our investigation of patient AML017 exemplifies such an event, where p53 is expressed at high levels in the majority of the malignant population, although the downstream target p21(CIP1/WAF1) remains low. We hypothesize that this pattern is due to the protein product of mutated TP53 in this patient, which is unable to induce p21(CIP1/WAF1) transcription as a consequence of loss-of-function. However, we also observed a second small population, corresponding to approximately 1% of the cells in this patient, where lower levels of p53 were detected. Here, the expression pattern of p21(CIP1/WAF1) and Ki67 was reversed; a pattern indicative of normal p53 function. This could be due to expression of wild type p53 in this small subpopulation of malignant cells. An alternative explanation could be p53-independent activation of p21(CIP1/WAF1) [TP53 wild type patient, we observed the expected expression pattern of p53 and p21(CIP1/WAF1). Yet, despite high p21(CIP1/WAF1) expression, the malignant cell population also exhibited high Ki67 expression, indicative of high mitotic activity. An explanation for this inconsistency could be that this patient harbors other driver mutations causing excessive mitotic activity. Numerous other mutations and aberrant signaling pathways in AML have been described to be associated with non-mutational p53 dysfunction [An increasing number of reports have shown that hematological malignancies may have a minute cell population carrying  relapse ,49. We hP1/WAF1) . Unfortufunction . Howevermut (Y5) antibody unexpectedly yielded signal in both the TP53 wild type and the TP53 mutated cell lines and patient samples. This suggests that the Y5 antibody binds to wild type p53, and illustrates the importance of careful validation of the antibodies. However, a recent report demonstrated the presence of p53 protein with \u201cpseudo-mutant\u201d conformation in pre-leukemic hematopoietic stem/progenitor cells and in leukemic blast cells in TP53 wild type patients [In our experiments, the p53patients . This waThe work presented here is focused on the application of mass cytometry in assessment of hematological malignancies. Although we argue that peripheral blood and bone marrow are especially suitable samples for this application, the method is by no means limited to samples of such origin. Several publications presenting mass cytometry analyses of single cell samples originating from solid tissues have been published ,54,55. WIn summary, mass cytometry has a great potential for p53-centric investigation of primary patient samples. It allows for thorough investigation of surface markers, enabling broad immunophenotyping combined with intracellular signaling proteins, far exceeding current routine methods such as flow cytometry. As precision medicine is emerging and the span of patients eligible for this treatment approach is expanding, increased knowledge of the disease state in every individual patient is of importance and can potentially provide clinical information relevant for future treatment choices. In this work, we demonstrated how currently available mass cytometry technology can be applied to investigate the p53 network in primary clinical samples. Development of p53 isoform-specific antibodies as well as modification- and conformation-specific antibodies compatible with the mass cytometry platform would add increasing value to experiments focusing on p53 and its functions in different malignancies.This study has been performed in accordance with the Declaration of Helsinki and in agreement with approved local protocols. The study was approved by the regional ethics committee; Regionale komiteer for medisinsk og helsefaglig forskningsetikk (REK) Vest, University of Bergen, Bergen, Norway (Project REK2012/2245). Before study initiation, all subjects provided written informed consent.w/w), 20.35% formaldehyde (w/w) and 15.65% diethylene glycol (w/w)) according to manufacturer\u2019s protocol. The samples were stored at \u221280 \u00b0C in NaCl 0.9%. Bone marrow from the HCL patients was aspirated by standard medical procedure into syringes containing heparin, and further sample processing was performed as described for peripheral blood.Blood samples were collected by phlebotomy of a peripheral vein or drawn from a Hickman central venous catheter into EDTA vacutainers . Red blood cells were lysed, and white blood cells were fixed using BD lyse/fix Buffer 5X . OV-90 and Raji cells were purchased from American Type Culture Collection . All cell lines were cultured in RMPI-1640 medium  supplemented with 10% heat inactivated Fetal Bovin Serum , 1% L-arginine (Sigma-Aldrich) and 1% Penicillin-streptomycin (Sigma-Aldrich), in incubators at 37 \u00b0C and 5% COmut (Y5) were purchased from Cell Signaling Technology , Miltenyi Biotec  and Abcam , respectively. These antibodies were ordered as custom formulation suspended in BSA- and azide free buffer. This is important for the mass cytometry technology, as carrier BSA will compete with the antibody for binding to the metal tags during conjugation. The antibodies were conjugated to the metal tags according to Fluidigm\u2019s MaxPar Antibody Labeling Kit protocol . In the conjugation process, the antibodies are covalently bound to a polymer. The polymer includes a chelating group which forms a non-covalent non-reversible interaction with the metal.The details of the mass cytometry antibody panel are described in The p53-specific antibodies were titrated and validated using appropriate cell line and primary cell controls, as described in the results section. Antibody titrations were performed by five 1:1 serial dilution of the antibodies, starting at 1:50 dilution. Five equal barcoded cell aliquots were stained with the resultant titration series. During titration, all channels susceptible for potential spillover were kept empty, in order to evaluate the actual signal spillover the concentrations tested.k-nearest neighbor density estimation to form cell clusters, using a range of different k-values. The optimal k-value (number of clusters) was found using the \u201celbow point/switch point\u201d. To increase biological interpretability, similar clusters were manually merged. We visualized the data by a force directed layout, which separates the clusters according to relative similarity. Unsupervised clustering using x-shift and graphical representation of the data was performed using the VorteX stand-alone software, downloaded from nolanlab/vortex GitHub.All samples included in the validation and titration were barcoded as described in Fluidigm\u2019s MaxPar Cell ID protocol (PRD 023 V1). After barcoding, the cells were stained with extracellular and intracellular antibodies according to the Fluidigm Phospho Protein staining protocol (PN 400278 A4), with some minor adjustments. This includes the addition of two 20 min incubation steps with 200IU heparin  immediately preceding the addition of the extracellular and intracellular antibody cocktails. The final heparin concentration during antibody incubation was 100IU. This was done to block unspecific antibody binding by eosinophils, as previously described . In addiTP53 gene, and the post-translational modification by acetylation at Lysine 382 can be detected by mass cytometry using antibody probes. We further demonstrate the potential of the technology by analyzing patient samples, including assessment of p53 expression in specific cellular subsets. Mass cytometry allows detection of multiple parameters simultaneously, enabling mapping of the p53 network. Thus, these results demonstrate that mass cytometry is a promising tool, which could reveal novel insight into p53 biology in multiple malignancies. Future development of p53 isoform-specific antibodies compatible with this technology could have the potential to further advance the field of p53 research by mass cytometry.In this work, we demonstrate that the p53 protein, the non-functional protein of a mutated"}
{"text": "Interactions between DNA and DNA-binding proteins play an important role in many essential cellular processes. A key function of the DNA-binding protein p53 is to search for and bind to target sites incorporated in genomic DNA, which triggers transcriptional regulation. How do p53 molecules achieve \u201crapid\u201d and \u201caccurate\u201d target search in living cells? The search dynamics of p53 were expected to include 3D diffusion in solution, 1D diffusion along DNA, and intersegmental transfer between two different DNA strands. Single-molecule fluorescence microscopy enabled the tracking of p53 molecules on DNA and the characterization of these dynamics quantitatively. Recent intensive single-molecule studies of p53 succeeded in revealing each of these search dynamics. Here, we review these studies and discuss the target search mechanisms of p53. Genome editing method has been used to investigate and engineer gene functions in life science. The genome encompasses DNA sequences that encode genes, and gene editing is the genetic engineering of a specific DNA sequence, including insertion, deletion, modification, and replacement. The main player in genome editing is a type of protein that can bind to DNA, known as DNA-binding proteins. DNA-binding proteins include enzymes, which can cut DNA or ligate two DNA molecules, and transcription factors, which can activate or deactivate gene expression. These proteins are classified into DNA sequence-specific and nonspecific binders.6 and 109 bp of DNA, respectively. In contrast, the DNA sequence that is bound by the proteins is just typically 5\u201330 bp. For example, assuming that a single protein searches for the target coded in 109 bp of genome by repeatedly associating and dissociating with random sites in the genome (3D diffusion), the search time is estimated to be much longer than the physiological time which is estimated from several minutes to a half of an hour in cells. However, the natural proteins complete the target search exactly within the physiological search time. This gap is called the \u201ctarget search problem.\u201d How do DNA-binding proteins solve this problem and function on the target?The sequence-specific DNA-binding proteins have a common mechanism to search for and bind to their target DNA sites encoded in a genome. Successful target binding by transcription factors can trigger the regulation of the cellular functions, whereas the failure of the target search and binding is known to cause various diseases including cancers. In addition, the target binding is delicately regulated depending on genome functioning. Accordingly, the target search and binding by DNA-binding protein are one of the indispensable reactions in life. The bacterial and human genomes comprise approximately 10To solve this problem, three target search dynamics are currently proposed: 3D diffusion; 1D diffusion, in which a protein moves along DNA; and intersegmental transfer, in which a protein transfers between two different DNA strands without dissociating ,3. TheorThe transcription factor p53 can induce multiple tumor suppression functions, such as cell cycle arrest, DNA repair, and apoptosis. Furthermore, p53 is known as the guardian of the genome, and it contains an N-terminal (NT) domain, core domain, linker, tetramerization (Tet) domain, and C-terminal (CT) domain . The corTo solve the target search problem, it is necessary to differentiate and characterize each search dynamic. Single-molecule fluorescence microscopy can track a single molecule moving along DNA and/or transferring between two DNA molecules ,22,23,24Several elegant methods for producing DNA arrays were developed in order to acquire data on many molecules simultaneously ,27,28. AIn 2008, Tafvizi et al. succeeded in observing the movement of p53 tetramer along a non-target DNA sequence using single-molecule fluorescence microscopy . A thermTafvizi et al. further revealed that 1D diffusion of p53 along the non-target DNA sequence does not depend on a monovalent salt concentration; this suggested \u201c1D sliding,\u201d in which p53 moves along DNA while maintaining contact with the DNA ,29. BaseWe clarified that p53 possesses two 1D diffusion modes with different diffusion coefficients by altering the structure of p53 on DNA ,36 Figu. The fasIf p53 reaches the target DNA sequence using 1D diffusion, it will recognize and bind to the target. Considering the fast target search by DNA-binding proteins, one might predict a high efficiency of target recognition and binding . ContrarIn the cell, many DNA-binding proteins bind to and cover DNA, which could block the 1D diffusion of p53 along DNA and hinder its target search. In the nucleus, genomic DNA is stored at high concentrations. In these conditions, it may be possible that p53 possesses a special mechanism to bypass the obstacles on DNA by transferring from one DNA strand to another a.8 M\u22121s\u22121). This suggests that even if p53 encounters DNA-bound obstacles in the cell, it can bypass these via ultrafast transfer. In contrast, p53 does not transfer from the target DNA sequence. Accordingly, once p53 binds to the target DNA sequence, it is not repelled from the target DNA by nearby DNA in the cell.We conducted a kinetic ensemble measurement to monitor the transfer process of p53 from fluorescent to non-fluorescent DNA . FluoresTo directly examine p53 transfer between DNA segments, we prepared crisscrossing DNA in the flow cell using the modified DNA garden method and tested whether p53 moves between DNA segments at the crisscross points  Figure b. The crConsidering the in vitro data described above, p53 is presumed to solve the target search problem by utilizing 3D diffusion, 1D diffusion along DNA, and intersegmental transfer between two DNAs in the cell. In 3D search, p53 can associate to non-target DNA near the diffusion limit . The assConsidering the biological functions of p53, one may wonder why the target search by p53 is not optimized in terms of facilitation. Under various cellular stresses such as DNA damage and tumorigenesis of the cell, p53 turns the function \u201cON\u201d and induces DNA repair and apoptosis. In contrast, p53 should keep the function \u201cOFF\u201d under normal conditions. It would be dangerous if p53 were to accidentally bind to the target DNA sequences and induce apoptosis in normal conditions. To prevent this malfunction, p53 may keep target recognition low under normal conditions at the expense of target search speed in stress conditions.To regulate multiple functional pathways as well as the discrete ON/OFF switch, p53 has three different strategies. One strategy is to increase the p53 copy number under stressed conditions . The incThe target search dynamics of p53 in vivo have been investigated by single-molecule fluorescence imaging. For example, Mazza et al. succeeded in tracking p53 molecules one by one in live cells using HILO illumination . They shTarget search is not limited to DNA-binding proteins, such as p53. Various processes can be considered as target search problems, including biomolecules searching for binding partner molecules or animals seeking food. Interestingly, a common mechanism known as intermittent search is widely used in biology . IntermiSingle-molecule fluorescence microscopy has clarified that p53 solves the target search problem by utilizing 3D diffusion, 1D diffusion along DNA, and intersegmental transfer between two DNAs. As predicted by theoretical studies, target search by p53 can be facilitated by combining these three strategies. In particular, considering the ultrafast association to DNA, 1D diffusion along DNA, and ultrafast intersegmental transfer, target searching by p53 is somehow optimized toward facilitation. In contrast, the low target recognition by p53 decelerates target searching. Furthermore, p53 may prevent malfunction under normal conditions at the expense of the target search speed in stress conditions. Accordingly, the target search strategy of p53 may be well designed to satisfy biological requirements.Further single-molecule investigations could deepen the understanding of target search by p53. In the cell, histone proteins bind to and condense DNA, which may affect target search by p53 . Single-Since many DNA-binding proteins exist in the cell and have different structures and functions, it is interesting how such DNA-binding proteins solve the target search problem. For example, if certain DNA-binding proteins differ in structure and function, their target search mechanisms may each differ as well ,104,105."}
{"text": "TP53 genomic locus is a target of mutational events in at least half of cancers. Despite several decades of study, a full consensus on the relevance of the acquisition of p53 gain-of-function missense mutants has not been reached. Depending on cancer type, type of mutations and other unidentified factors, the relevance for tumour development and progression of the oncogenic signalling directed by p53 mutants might significantly vary, leading to inconsistent observations that have fuelled a long and fierce debate in the field. Here, we discuss how interaction with the microenvironment and stressors might dictate the gain-of-function effects exerted by individual mutants. We report evidence from the most recent literature in support of the context dependency of p53 mutant biology. This perspective article aims to raise a discussion in the field on the relevance that context might have on p53 gain-of-function mutants, assessing whether this should generally be considered a cell non-autonomous process.The  Mutant p53 GOF effects have been shown to vary in different settings, potentially depending on cancer types, experimental models and conditions.Reciprocal interaction between microenvironment and mutant p53 GOF effects exists.Similarly to wt p53, mutant p53 proteins can contribute to transduction of extrinsic signalling.What is the effective contribution of mutant p53 GOF effects to the cancer progression?How relevant is the microenvironmental context in determining mutant p53 GOF effects?Can microenvironmental factors be targeted to abolish mutant p53 GOF pro-oncogenic functions?5. Originally identified as a major executor of the response to DNA damage, with a more recent revision, p53 is considered a molecular hub for the interactions between stressors  and cellular biological responses12  theory in p53 mutation26. The shift of p53 from the wild-type status to the mutant protein therefore appears to turn p53 into an oncogene. This basic, consistent and generally accepted consideration has, however, not always found solid support in the experimental data. Clear GOF phenotypes have been shown for many hot-spot mutations, such as p53 R175H and R273H. Introduction of p53 R175H and R273H into p53-null cells promotes growth in in vitro soft-agar assays and in injected nude mice. In contrast, genetically engineered mouse models carrying mouse homologous mutations (p53 R172H and R270H) did not show any alteration in survival compared with p53-null mice.An additional critical aspect of the mutations in p53 is the frequently observed protein products. Eighty percent of p53 mutations are missense, leading to generation of neomorphic proteins36. However, a complementary approach based on functional and transcriptional analyses of CRISPR-Cas9-generated isogenic human leukaemia cell lines suggested a selective advantage associated with a dominant negative effect of p53 mutants but no evidence of any GOF. This study also reported no evidence of GOF effects on the clinical outcome of patients with myeloid malignancies37. Whether this is different from solid tumours, where a pro-invasion and pro-metastatic programme might benefit from mutant p53 GOF, remains to be determined. However, formal evidence for the existence of p53 mutant GOF effects could be found in the dependency displayed by cancers on sustained expression of p53 mutant proteins38. Ablation of mutant p53 in allotransplanted, xenotransplanted and autochthonous mouse cancer models impairs tumour growth, promoting cell death and tumour regression or stagnation42. While these results allow pharmacological targeting of the mechanisms leading to p53 mutant stabilisation as an anticancer approach, at the basic molecular level, they formally demonstrate that expression of p53 mutant proteins is not equal to the loss of the functional wild-type version. However, a general consensus has not yet been achieved on the relevance of the contribution of p53 GOF effects to tumorigenesis.Puzzling results have also been shown in myeloid malignancies. GOF p53 R172H was seen to accelerate complex-karyotype acute myeloid leukaemia in mouse models by promoting cell fate plasticity through the pluripotency factor FOXH143. This is reflected in an influence of extrinsic signalling on p53 mutant behaviour and on the ability of the p53 mutant to modulate the response to extrinsic factors. In light of the central role of functional (wt) p53 in the response to cellular stress, a fundamental conserved implication of the mutant proteins in the interaction with microenvironment is not surprising  transcriptionally promotes the expression of Hsps, which in turn interact with p53 mutants, inhibit the E3 ubiquitin ligase activity of MDM2 and CHIP and induce the stability of p53 mutant proteins. The mechanisms leading to the stabilisation of p53 mutants are therefore influenced by extrinsic stressors able to trigger the activation of HSF1. Similarly, mechanical stimuli from the tumour tissues have also been shown to result in p53 mutant stabilisation. Rho-A is a sensor of extracellular matrix (ECM) stiffness. In response to stiff a ECM, Rho-A activation and actin-dependent mechanotransduction induce activation of Hsp90 via the mechanosensitive HDAC6 deacetylase, thus leading to stabilisation of mutant proteins50. Similar to HSF1, Rho-A is also frequently hyperactive in tumour tissues. Notably, Rho-A is regulated by mutant p53 activity itself. Activation of Rho-A is influenced by GEF-H1 and RhoGDI, which are influenced by GOF p53 mutants, feeding a forward loop that results in further stabilisation of the p53 mutant. Disruption of this loop with zoledronic acid (ZA) and geranylgeranyl transferase inhibitors (GGTIs), which inhibit the geranylgeranylation of Rho-A required for its activation on the plasma membrane, impairs p53 mutant stability51  determines the expression of fundamental components of the basal lamina, such as laminin-\u03b32 and collagen type VIIa1, in hypoxic tumours56. Consistently, vessel density correlates with the expression of p53 missense mutations in human breast cancers57.GOF p53 mutants can also facilitate a pro-tumorigenic microenvironment by promoting the secretion of IL-8 and GRO-\u03b1 by cancer cells. Similar to the p53 mutant/HIF-1 molecular model, p53 mutants appear to hijack the E2F2 transcriptional factor. The p53 mutant/E2F2 complex controls the promoter of inhibitor of DNA binding 4 (ID4), which binds and stabilise mRNAs of IL-8 and GRO-\u03b1. Conditioned media from p53 mutant cells promote the in vitro proliferation of endothelial cells, and p53 mutants favour angiogenesis in vivo in xenotransplanted models58. Cooperation between mutant p53 and Sp1 facilitates transcriptional control of ENTPD5, and this appears to mediate p53 mutant-dependent growth, architectural tissue remodelling, migration, invasion and lung colonisation as assessed in tail vein-implantation mouse models58.N-glycosylation of cellular surface proteins, such as receptors and integrins, is also altered by p53 status. Ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) is a mutant p53 target gene involved in promoting the folding of N-glycosylated membrane proteins in the endoplasmic reticulumEvidence exists that p53 mutants can influence the microenvironment. Simplification tends to indicate how \u201cmutants\u201d direct the composition of the extracellular milieu; however, it is reasonable to speculate that different mutations will have different impacts on this process. However, the large variety of mutations makes the resolution of this issue very challenging Fig. .Fig. 1p560, Rb62 or Bcl-267. The p53 mutant can dictate instructions to the microenvironment, which in turn are influenced by additional extrinsic factors that direct signals on the cancer cells that are differentially integrated based on the p53 mutational status (Fig. In this perspective, we suggest that p53 GOF is a non-cell-autonomous phenomenon, which would make this gene very peculiar if compared with other oncogenes/oncosuppressors, such as KRas51. The limited ability of in vitro and xenotransplanted models to recapitulate the natural cancer microenvironment might represent a particular concern in the interpretation of the data on p53 GOF. The addiction that cancer cells develop to p53 mutations is a specific relevant aspect in this context. While strong support for the existence and relevance of p53 GOF comes from evidence that in vivo tumours are dependent on p53 expression, no evidence exists of the reproducibility of this effect in vitro. This questions not only the work done to dissect the mechanisms underlying p53 GOF but also the utility of in vitro models for developing therapeutic approaches to target p53 mutant stability.It is remarkable that a significant fraction of work on p53 GOF is performed in in vitro systems or in xenotransplanted models. Compelling evidence indicates that plastic dishes provide altered extracellular matrix interactions to cancer cells, influencing the stability of p53 mutants and consequentially providing artificial responses68.It appears optimistic to envisage general major mechanisms responsible for p53 GOF, but it is possibly also very optimistic to envisage the existence of p53 mutant of similar mechanisms in different contexts. This raises complexity in the study of p53 GOF, but even more, it presents challenges in identifying strategies to target this gene with general approaches in cancer patients. While a massive effort in drug development against p53 mutant tumours has been invested in the two past decades, the outcome is very soon expected to be delivered. This should also help in understanding how feasible the approach is and direct efforts for future investments"}
{"text": "Forty years of research has proven beyond any doubt that p53 is a key regulator of many aspects of cellular physiology. It is best known for its tumor suppressor function, but it is also a regulator of processes important for maintenance of homeostasis and stress response. Its activity is generally antiproliferative and when the cell is damaged beyond repair or intensely stressed the p53 protein contributes to apoptosis. Given its key role in preventing cancer it is no wonder that it is the most frequently mutated gene in human cancer. Surprisingly, a subset of missense mutations occurring in p53 (gain-of-function) cause it to lose its suppressor activity and acquire new functionalities that turn the tumor suppressor protein into an oncoprotein. A solid body of evidence exists demonstrating increased malignancy of cancers with mutated p53 in all aspects considered \u201challmarks of cancer\u201d. In this review, we summarize current findings concerning the cellular processes altered by gain-of-function mutations in p53 and their influence on cancer invasiveness and metastasis. We also present the variety of molecular mechanisms regulating these processes, including microRNA, direct transcriptional regulation, protein\u2013protein interactions, and more. One of the results of the changes in demographic structure of many societies is an increasing prevalence of cancer. It is an extremely diverse disease, with many types of cancers originating in various organs. With progress in molecular diagnostics, cancers that previously have been thought of as a single disease are now known to have molecular subtypes that are predictive of their disease course and outcome. Being diverse as they are, most\u2014if not all\u2014cancers possess several features defined by Hanahan and Weinberg in 2000 as hallmarks of cancer ,2. TheseTP53 gene encodes p53 protein that, in its wild type form, acts as a tumor suppressor. In an active state, it is a homotetramer that binds to its specific target sites and modifies gene expression. Activity of p53 is crucial for control of cell cycle progression, apoptosis, cellular senescence, DNA repair, and genome maintenance. The crucial role of p53 in preventing cancer formation is underlined by the fact that, based on tens of thousands of records in International Agency for Research on Cancer (http://p53.iarc.fr) [The iarc.fr) , over 50iarc.fr) ,7,8; thiiarc.fr) ; this moiarc.fr) ,11,12,13Generation of p53 NULL mice in the early 1990s demonstrated in vivo that it is indeed a major tumor suppressor ,15. The Trp53-/R248Q and Trp53R248Q/R248Q genotypes when compared with their Trp53\u2212/\u2212 counterparts [Trp53R248W/R248W mice presented more complex tumor spectrum than the Trp53\u2212/\u2212 mice, but no significant change in survival [Trp53+/R172H genotype exhibited marked increase in frequency of metastasis when compared with their Trp53+/\u2212 counterparts, but no decrease in overall survival was noted [Trp53+/R172H genotype exhibited marked increase in frequency of metastasis when compared with their Trp53+/\u2212 counterparts [Trp53+/R172P mice [Phenotypic variations of GOF p53 depend on the residue that is mutated as well as the amino acid that replaces the wild type variant. Although pooled patients with germline p53 missense mutations (Li-Fraumeni syndrome) do not develop the tumors earlier than the patients with NULL genotypes, early onset (7 years difference) is observed when the population of patients bearing R248Q (but not R248W) mutation is compared to NULL population. In addition, the distribution of primary tumor sites and the tumor spectrum in the large IARC cohort differs between patients bearing NULL and several \u201chot spot\u201d mutated alleles ,27. Simiterparts , whereassurvival . In a muas noted . In anotas noted , the micterparts . Interes72P mice . In a bi72P mice . In an i72P mice . Similar72P mice ,29. Thes72P mice . AlthougFor local invasion or distant metastasis to happen, tumor cells that are already immortalized still have to break several barriers. First, the cells have to acquire mobility and capability of extracellular matrix degradation. This, in conjunction with sensitivity to certain chemokines, enables directional migration through extracellular matrix. At this stage the tumor is capable of local invasion. If the cells can penetrate lymphatic or blood vessel wall, then they can travel to the local lymph node or a remote location, respectively. To survive in a blood or lymphatic vessel, the cells need to be resistant to anoikis (lose their anchorage dependence) and also evade immune cells. The organs targeted by metastases are non-random and that points to the fact that cancer cells exhibit a certain degree of tropism. That tropism results from sensitivity to certain chemokines and ability to adhere to the endothelium at specific locations. Such phenotypes are often connected to changes in expression of chemokine receptors and integrins. It is important to understand that a similar p53-induced change in phenotype may be caused by a number of molecular mechanisms and by a direct or indirect activity of mutated p53. These mechanisms include changes in miRNA, lncRNA, and interactions of p53 with transcription factors or chromatin. Conversely, a certain molecular mechanism may be involved in modification of various phenotypes.CD44, SERPINE1 (PAI-1) and CCND1 (Cyclin D1) [In the process of spontaneous acquisition of metastatic properties, cancer cells often undergo epithelial\u2013mesenchymal transition (EMT). This is a broad phenotypic change that allows the cells to gain mesenchymal properties such as ability to migrate or detach from surrounding cells. Although the changes in cells are dramatic, the whole process is controlled by a relatively small number of transcription factors such as SNAI1 (Snail), SNAI2 (Slug), TWIST1, and ZEB1/2 ,32,33. Tclin D1) . A more clin D1) , wherebyclin D1) . Indeed,clin D1) . An examclin D1) . Nearly clin D1) . A mechaclin D1) . In thisclin D1) .Following the reports that wild type and mutated p53 variants regulate several aspects of cancer initiation and progression through miRNA ,43 furthTrp53-/R172H cells derived from murine pancreatic ductal adenocarcinoma [Mutations in p53 may lead to phenotypic changes that do not present full EMT profile or that are of different nature, yet still increase metastatic potential. These include sequestration and inactivation of p63 and p73 that has been observed in mouse models  and demoarcinoma , a depenarcinoma . In a siarcinoma . A novelarcinoma .Another, relatively less known class of RNA molecules is long non-coding RNA (lncRNA). These molecules may serve as scaffolds for assembly of protein complexes. It has been recently discovered that two lncRNAs\u2014lnc273-31 and lnc273-34\u2014were upregulated in colorectal cancer cell culture model following the expression of p53-R273H. They are required for increased cell migration, proliferation and chemoresistance in vitro. In a murine xenotransplant model these lncRNAs dramatically accelerate tumorigenesis. Their expression is also significantly elevated in colorectal cancer patient tissues that bear R273H mutation in p53 .Changes in cellular physiology induced by GOF mutations in p53 that manifest as clinically observed increase in metastasis may also concern changes in cytokine production and cytokine sensitivity as well as integrins and cytoskeleton organization. Although some of them are similar to what is happening during EMT, they may also occur as isolated phenotypes. It was reported that cancer cells expressing any of several mutated p53 variants  released exosomes enriched in miR-1246 that were taken up by tumor-associated macrophages. This reprogrammed the macrophages into anti-inflammatory immunosuppression mode with increased release of TGF-\u03b2, which in turn supported tumor growth . A case A link between inflammation and cancer has been established long ago and indeed inflammation is considered one of the \u201challmarks of cancer\u201d ,2. The tTrp53-/R172H mice elevated expression of PLAC8 (onzin) was found when compared to Trp53\u2212/\u2212 counterparts; this resulted in an increase in metastatic traits in osteosarcoma cells, both in vivo and in vitro. The molecular mechanism involved increased expression of CXCL5 that, in an autocrine manner, enhanced ERK1/2 phosphorylation. Observed phenotypes were reproduced by addition of exogenous CXCL5 and they could be abolished by specific inhibition of ERK1/2. However, some phenotypic changes resulting from p53-R172H expression were not prevented by Cxcl5 gene silencing, indicating that other mechanisms were also involved.A tumor may be likened to a wound that never heals, thus inducing chronic inflammation. There are multiple ways in which tumor cells adapted to sustain and benefit from an ongoing inflammatory process. Due to the limited scope of the article the effects of loss of p53 activity will not be elaborated upon, but rather some examples of GOF p53 mutation-induced changes in the development of inflammation and their consequences for tumor invasion and metastasis will be presented. The key cellular regulator of inflammation is NF-\u03baB and it was demonstrated in a number of in vitro and in vivo models that mutated p53 prolongs its activation in response to TNF-\u03b1 and thus promotes chronic inflammation that eventually leads to tumorigenic transformation . In glioTrp53\u2212/\u2212 or Trp53-/R270H background it was shown that while the Trp53\u2212/\u2212 animals rarely developed invasive tumor phenotype, it was commonly found in Trp53-/R270H mice. The authors conclude that this might have been caused by an observed increase in chromatin accessibility resulting in upregulation of approximately 350 genes, many of which could be classified as proinflammatory [Several mechanisms related to mutated p53-induced changes in epigenetic gene expression regulation were proposed. In a transgenic mouse colon cancer model carrying deletions in Apc in a ammatory . In a reammatory . A mechaammatory . The incammatory . An indiammatory . Interesammatory . Table 2The best characterized metabolic shift in cancer cells is known as Warburg effect . In brieAfter forty years of scientific efforts directed at dissecting the role of p53 in tumor development and evolution an enormous amount of observations was made. An initial view that p53 was an oncoprotein was abandoned in favor of its tumor suppressor role. Several years later it turned out that under certain circumstances p53 may indeed become an oncoprotein. If one looks at the tumor development as a process of evolution and adaptation to hostile environment\u2014patient organism\u2014one cannot miss the high prevalence of certain mutations in p53. These overrepresented gain-of-function mutations were demonstrated to provide the tumor with numerous advantages in its struggle for survival and expansion. However, that also means that tumors rely on and are addicted to mutated p53 which potentially presents clinically actionable opportunities. This brief review presented a number of molecular mechanisms utilized by mutated p53 to exert its activities: epigenetic regulation, direct and indirect transcriptional control, miRNA-mediated control, and mRNA stability modification. Some of these act in a cell-autonomous manner and some in autocrine or paracrine manner. Certainly there are effects and modes of action that are unknown yet. However, due to the high prevalence of these mutations in cancer patients, every effort directed at identifying the molecular mechanisms of clinically relevant phenotypes may result in identification of actionable therapeutic targets."}
{"text": "Due to Here we have organized this Research Topic titled \u201cRole of p53 in cell metabolism, ferroptosis, and stemness\u201d and wish to collect manuscripts about the emerging roles of p53 in mediating cell metabolism, ferroptosis, and stemness, which link p53 with diverse diseases, particularly tumors . We haveReinisch et al. found that under acute fasting, p53 in BAT was activated to induce the expression of miR-92a-1-5p. They also demonstrated a fructose transporter Slc2a5 to be a repressive target of miR-92a-1-5p. The downregulation of Slc2a5 reduced the import of fructose, glycolysis, and finally thermogenesis. This study revealed a novel p53/miR-92a-1-5p/Slc2a5 axis in fasting-regulated thermogenesis.MicroRNA is a type of short non-coding RNA with diverse functions by targeting different genes . p53 is Lei et al. in their comprehensive review focused on the role of p53 in the brain. They firstly introduced a basic background of p53 and its regulation. They then discussed how p53 regulates various diseases in the brain, including glioma, cerebral stroke, and neurodegenerative diseases.Zhou et al. wrote a short review about ferroptosis and lymphoma, highlighting that targeting the p53-mediated ferroptotic pathway may be a promising method to treat lymphoma. pan and Wang contributed a review about p53 and ferroptosis in osteosarcoma. In this review, they classified ferroptosis into typical and atypical types. In both types of ferroptosis, p53 has a vital role. Ferroptosis also contributes to diseases other than cancer, such as in the regulation of diabetes. In the review by Du et al., authors depicted the major mechanism of ferroptosis and its effect in diabetes. Notably, they pointed out the crucial role of p53-regulated ferroptosis in the pathology of diabetes. In an interesting study from Hu et al., authors investigated the function of a bioactive peptide G1dP3 in MH7A cells. They showed that G1dP3 significantly suppressed the viability of MH7A cells by inducing ferroptosis. Mechanistically, they demonstrated that G1dP3 could activate p53 expression, resulting in the suppression of the SLC7A11/GPX4 axis.Ferroptosis is a newly discovered cell death type, which is caused by the iron dependent peroxidation of cell membranes . p53 hasLiu et al. presented an informative review about how p53 mutation or deletion remolds the tumor immune microenvironment. There are at least three ways that mutant p53 or loss of p53 contributes to an immunosuppressive status: 1) induce immunosuppressive cytokines and downregulate proinflammatory factors; 2) augment the expression of immunosuppressive ligands; and 3) facilitate immunosuppressive cells differentiation. Correspondingly, targeting mutant p53 would be a reasonable choice to restore the tumor immune microenvironment. Corazzari and Collavin wrote an attractive review to summarize the ferroptosis-regulatory activity of both wild type and mutant p53. They proposed that the regulated ferroptosis could be a stress response to diverse stimuli in and out of the tumor cells. K120 is a critical site for p53 function. Monti et al. analyzed the transactivation activity of the p53 K120R mutant. The K120R mutation abrogates the acetylation at this site, preventing p53 from promoting apoptosis. However, this mutant could still retain some activity to modulate cellular metabolism. Mutant p53 also influences the efficacy of tumor treatment. Li et al. reported a pulmonary sarcomatoid carcinoma case with p53 T125K mutation. Fortunately, treatment of Tislelizumab in combination with Anlotinib enabled this patient to go into complete remission. We hope that this therapeutic regimen may benefit other similar patients.p53 functions as a checkpoint molecule to suppress tumorigenesis. To overcome this, p53 is often mutated or deleted in tumor cells. Mutant p53 may have different functions compared to wild type p53 . Liu et Wei et al. discussed the crosstalk between p63 and STAT3 in the regulation of cancer stemness.Other members of the p53 family, such as p63 and p73, also have fundamental roles in tumor . Signal To summarize, this Research Topic of papers present novel information about some unconventional functions of p53 (and p63). Elucidating the mechanisms of p53 that underlie its versatile roles will not only benefit the basic research about p53 and tumor biology, but also open promising doors for developing therapeutics targeting p53 and related pathways for the treatment of cancer and other diseases. We hope this Research Topic can bring new knowledge and ideas to those who are interested in the function of p53."}
{"text": "These findings reveal unique aspects of the amount of various cannabis forms smoked/vaped in relation to gender and provides preliminary evidence of cannabis consumption behaviors in relation to changing social and cultural contexts.Limited research examines changes in quantities of various forms of smoked/vaped cannabis among regular consumers, including emerging adults  in Canada. This information is particularly relevant in the current context of emerging cannabis behaviors among EAs related to political amendments , vaping-related lung illnesses (EVALI), and unprecedented pandemics (COVID-19). This study investigated the impact of legalizing recreational cannabis use in Canada, the EVALI epidemic, and the COVID-19 pandemic on the quantity of smoked/vaped forms of cannabis in relation to gender differences. EAs retrospectively self-reported the quantity of herb, hash, concentrates, joint size, and the number of joints and vaping cartridges in relation to three consecutive developments: pre-legalization, post-legalization; pre-EVALI, post-EVALI, pre-COVID-19, and during COVID-19. The quantity of herb use significantly increased among heavy users, and vaping quantity significantly increased among light users. Overall, an increasing incremental trend was observed in the average quantity of cannabis forms used over time. Males consumed higher quantities of all cannabis forms than females. More males than females reported using concentrates ( In Canada, cannabis consumption rates are characterized by marked differences in relation to age and sex ,2. For eContrary to its widespread permissive use and its presumptions as a \u2018benign\u2019 substance among EAs ,6, cannaThe complexity of measuring cannabis-related effects and risks are further complicated by sex-specific neurobiological mechanisms, gender-specific consumption differences, and motives for use. Nascent evidence suggests a higher sensitivity to cannabis among females, and their vulnerability based on the accelerated progression to problematic use , althougEmerging trends of cannabis consumption behaviors and diversified profiles associated with recreational cannabis legalization in Canada since 17 October 2018 warrants research to examine the quantity of cannabis used, as it may vary across products and methods of use ,15. The p < 0.001). This increase was more prominent among students aged 21 and above , rising from 40.4% to 60.9% (p < 0.001). Similarly, according to recent nationwide data from Canada, there was an increase in cannabis use between 2018 and 2019, specifically among individuals aged 25 and older (from 13.1% to 15.5%) and males (from 17.5% to 20.3%) .n = 11), did not smoke or vape cannabis in the last 12 months (n = 13) , or did not consent (n = 7).Participation was limited to the following inclusion criteria: reported smoking and/or vaping cannabis in the last 12 months, currently living in Canada at the time of the study, and 18 to 29 years old. The survey was restricted to the English language, and consent was required to participate. Respondents that met the inclusion criteria and consented were then directed to the survey questions, which was designed to take 20 to 25 min to complete. No compensation was provided. All responses were anonymous. Convenience sampling was used to recruit participants 18 to 29 years old through social media platforms (Facebook and Twitter) across Canada and from Ontario Tech University. Recruitment and data collection occurred from 4 May 2020 to 31 August 2020. Respondents were identified as cannabis users if they reported smoking and/or vaping cannabis in the last 12 months. Results of 343 individuals were reviewed, of which 312 were eligible. Respondents were excluded if they were not 18\u201329 years old  Prior to Legalization (approximately October 2018); (2) since legalization; (3) prior to EVALI (approximately November 2019); (4) since EVALI; (5) prior to imposed COVID-19 preventative measures (approximately March 2020); (6) since imposed COVID-19 preventative measures.International Cannabis Policy Study (ICPS) . Reference images were provided to quantify 1 g of hash and concentrate (derived from ).For vaping cannabis oil/vape juice, participants were asked: \u201cWhat size vaping cartridge do you \u2018primarily\u2019 use?\u201d, and \u201cHow many vape cartridge(s) or refill(s) did you use in a month?\u201d. Vaping quantity was determined based on the number of vape cartridges/refills used in a usual month . Numeric assumptions were inputted for options \u201cless than\u201d or \u201cmore than\u201d the amount indicated. Measuring vaping quantity by the number of cartridges over a longer timeframe than a day is found to be effective in measuring consumption. Therefore, the consumption of vaping was examined by the average of vaping cartridges used in a month. Evidence shows most respondents prefer to report the number of cartridges or tank refills (51%), and fewer users finish 1 cartridge/tank in a day . Data foParticipants reported the cumulative lifetime days of smoking or vaping, using the following questions: \u2018How many days in your lifetime have you smoked cannabis?\u2019; \u2018How many days in your lifetime have you vaped cannabis?\u2019. A binary variable was created to differentiate an occasional user  from an experienced user  based on lifetime days of use. Occasional users included smoking or vaping: 1 to 2; 3 to 10; or 11 to 99 days in their life. Experienced users included those that reported 100 to 999 or more than 1000 days of lifetime smoking or vaping . FrequenDue to the limited outbreak and awareness of EVALI in Canada, participants were asked, \u201cPrior to taking this survey did you know about EVALI?\u201d. The options were dichotomized (\u201cyes\u201d or \u201cno\u201d). Demographic characteristics included sex  and postal region. Participants were asked, \u201cwhat was your sex at birth?\u201d, and provided the first three digits of their postal code . Postal codes were grouped by province.p < 0.05) using the estimation method for Rouder\u2019s mixed design. The Bayes factor test yields a BF quantifying how well the alternative hypothesis (H1) predicts the empirical data relative to the null hypothesis (H0) [Data were analyzed using SPSS v 27 (IBM SPSS Statistics 27 \u00a9). Chi-square tests were used to analyze differences in relation to the form of cannabis used most of the time by sex . A one-wsis (H0) ,59. The sis (H0) . AnalyseThe average quantity of cannabis use per month for each form was calculated by multiplying it by the frequency of smoking/vaping categories to reflect the number of days used per month  in relation to each time period. Smoking frequency for each time period was multiplied by the herb quantity, joint size and joint quantity reported. Vaping frequency for each time period was multiplied by the number of cartridge refills in order to estimate the average quantity of vaping cartridges used in one month.The six time periods were divided into four time periods by calculating the average quantity of use between the periods after October 2018, and before November 2019 to create one category (October 2018 to November 2019) given that the quantity of herb, joints and vaping remained consistent in each period. In addition, the average quantity between the two periods\u2014since EVALI (November 2019) and before COVID-19 (March 2020)\u2014were calculated to show the quantity of use from November 2019 to March 2020.2 (1) = 5.02, p < 0.05). Hash was indicated as the least primary form used by males (3%) and females (5%) . More than three quarters of participants (76%) indicated a postal region from Ontario; few participants were from other Canadian provinces and territories (24%). The most popular form of cannabis used as the primary form was herb for males and females (91%), followed by cannabis vaping oil/vape juice, which was endorsed by 15% of males and 15% of females. More males (15%) than females (7%) reported using cannabis concentrates as the primary form (\uab53les (5%) . Only 24The majority of the sample comprised highly experienced (exclusive) cannabis smokers, with 63% of males and 54% of females reporting 100 or more lifetime days of smoking. The mean (SD) age of smoking cannabis onset was 17 (\u00b12) years, while the age of vaping onset was higher at 21 (\u00b13) years (only 2% of users had exclusively vaped cannabis in the last 12 months).BF (14) = 4.496), p < 0.001, showing strong evidence for an effect on the quantity of herb used over time in the sample population (Bf > 3)  = 0.939, p = 0.41), but a statistically significant difference for females  = 5.585, p = 0.004), showing that herb quantity almost doubled from 0.67 g to 1.2 g from pre-legalization to since the COVID-19 pandemic. Post hoc comparisons revealed that a significant increase was evident from before and after legalization for females (p = 0.005), in which herb quantity increased from an average of 0.67 g to at least 1 g personally used on a typical day.(Bf > 3) . AnalyseBF (14) = 65.776, p < 0.001) (1) showing an effect on joint quantity over time was indicated (Bf > 3). Analyses separated by sex showed that there was a significant increase in joint quantity for males  = 3.05, p = 0.049 and for females  = 4.902, p = 0.006). The average number of joints smoked on a typical day by females was highest during the pandemic period (M = 0.86). In relation to joint size, the overall results indicate no evidence for an effect on joint size in relation to the events (BF (14) = 0.903, p < 0.001). Although analyses stratified by sex showed no difference for males  = 1.83, p = 0.17), a borderline significant difference was found for females  = 2.67, p = 0.049).Joint quantity and joint size significantly differed over time (< 0.001) . Strong BF (14) = 0.195 p < 0.001)  = 2.84, p = 0.40) or females  = 2.49, p = 0.11). Changes in hash quantity also indicate moderate evidence for no effect across the time intervals (BF (14) = 0.066, p < 0.001). Due to the small subsample of participants using hash, we were unable to stratify based on sex.In addition, results indicate no difference in concentrate quantity in relation to the events (< 0.001) . AnalyseBF  = 0.954 p < 0.001), although the Bf < 3  = 2.922, p = 0.048). Although there was no significant difference for males  = 1.296, p = 0.29), an average increase is seen before and after legalization and since the COVID-19 pandemic, but a decrease from before and after EVALI  = 9.25, p < 0.001) for the total sample and for females specifically  = 7.86, p < 0.001). The quantity of use calculated based on the average joint size and number of joints also significantly increased for the total sample  = 6.25, p = 0.003) and for females  = 4.06, p = 0.022). F  = 9.03, p < 0.001). Joint quantity also increased significantly among heavy users  = 9.48, p < 0.001). There was an increase over time in relation to vaping for both heavy and light users; however, this association was only significant among light users F  = 5.89, p = 0.005.This study contributes to the complex and evolving landscape of cannabis consumption by examining changes associated with various quantity measures of cannabis forms consumed through smoking and vaping before and after political amendments , public health concerns (EVALI) and during unprecedented natural societal health threats (COVID-19). The findings from this study provide insight into possible changes in cannabis consumption among EAs as a result of public health events. The current legislation on recreational cannabis consumption puts EAs in a social environment that allows for easy accessibility and experimentation and makes them vulnerable to health risks. Recent evidence has shown an increase in the prevalence of cannabis users since legalization . The curFor a clearer estimation of quantity, the quantity measures of the various cannabis forms in this study can be compared to a recent Canadian study that established an algorithm for measuring the quantity of various cannabis forms in standard joint size equivalents . CallaghOverall, the primary findings of this study suggest that cannabis quantity may change over time and in relation to specific events. This is indicated by incremental increases in the average quantity of cannabis forms including dried herb, concentrates, number of joints, and vaping cartridges, from before legalization of cannabis in Canada (October 2018), to after the declaration of the COVID-19 pandemic (March 2020). Specifically, an increasing trend was found over time in relation to the quantity of dried herb, which was the most common form used in this study. It is also interesting to note that in some cases, the quantity of use is lower at the start of each new event relative to the end of the previous one. Although joint size and the number of joints smoked on a typical day increased after legalization, an average decrease was observed from the time frame following legalization and before EVALI was declared an epidemic. However, while joint size decreased, the number of joints per day increased from before to after the declaration of the pandemic for females. These inconsistent patterns which show an average increase in relation to specific events followed by a decline suggest that cannabis consumption behaviors may be associated with changing social and cultural contexts, events, and life trajectory (negative events) among EAs. Additional longitudinal research is needed to further explore the varying patterns of cannabis use over time.Although THC-containing vaping products have been found to play a key role in the propagation of the EVALI epidemic ,61, our In line with the literature, cannabis vaping may lead to an increase in quantity over time, as vaping becomes a consistent and repetitive behavior, and leads to more puffs being taken during each session, thus limiting the time that one vaping cartridge might last . ConsequThe increase in quantity of cannabis used after legalization found in this study, may have been a result of developed interest for the effects and engagement in the liberty of cannabis laws that allowed accessibility to diverse strains and forms . FollowiDuring the COVID-19 pandemic, studies focused on the quantity of joints used in a day, suggesting boredom, stress, and loneliness as motives of increased substance use ,71. SpecIt is important to note that although research indicates an increase in the prevalence of females using cannabis, more research is required to delineate the effects of specific forms of cannabis used in relation to sex-specific mechanisms . For exaThis study sheds light on cannabis-use trends in relation to single-item indicators by examining specific measures in relation to the quantity of various cannabis forms where limited research exists. These findings reveal unique aspects of the amount consumed of various cannabis forms in relation to sex and may aid in determining adverse or harm-reduction measures of consumption. Continued efforts to examine quantity can have important implications for the prevention and treatment of cannabis-related problems. In addition, to our knowledge, this is the first study to examine how quantity measures of multiple cannabis forms may change in relation to legalization, EVALI, and COVID-19 by sex-specific analyses in Canada. At the same time, this paper contributes to establishing standardized criteria for measuring cannabis, which would promote uniformity in quantifying its consumption, thereby aiding the development of research and clinical assessment standards. This, in turn, would enable clinicians to better identify individuals who are at a higher risk of acute and/or chronic issues resulting from cannabis use by gaining insights into how consumption patterns affect health outcomes. Additionally, considering gender-specific factors is important, as significant epidemiological and clinical distinctions have been observed between males and females in relation to cannabis use.Despite these strengths, there are limitations to consider. First, asking participants to retrospectively report cannabis consumption behaviors in relation to specific measurements of quantity may have introduced recall bias, and the events of legalization, EVALI and COVID-19 were primed in the survey as trigger points for recall during data collection. Second, our study recruited individuals that were 18 years old who were minors during the time of cannabis becoming legalized in Canada (approximately 16 years old); therefore, they may have experienced different changes in cannabis use than respondents of age. In addition, the sample size was relatively small and largely from Ontario.Due to the cross-sectional exploratory design of this study, we cannot make any causal inferences. Therefore, it is unclear if the events had a direct effect on the quantity of consumption. Similarly, any changes in relation to cannabis quantity consumed post-legalization of recreational cannabis use in Canada (17 October 2018) may be attributed to legalization and not necessarily reflect the subsequent public heath events, including the outbreak of EVALI and the COVID-19 pandemic. Differences in length between the time periods is also a limitation, given that the mean consumption in one period may not reflect exactly the same process and has the same meaning in other periods. Nonetheless, there seems to be a slow adoption of incremental increases in consumption post-legalization, which was followed by successive events that are relevant to cannabis use and may have influenced users to adapt their consumption. In addition, experimental use is prone to influence the natural progression of increased consumption, as users may transition to more frequent and daily use over time . FurtherMoreover, infrequent, or varying use, and sharing\u2014which is common among cannabis users\u2014can limit the reported capacity ,54. It iIn addition, more people reported using the herb in its dried form and in the form of joints than any other form. This may have limited the power to examine significant changes in quantity for other forms in this study, including, hash and concentrates. Due to power considerations, we were not able to stratify by sex for changes in relation to hash quantity. Finally, examining vaping quantity is difficult as there are a diverse range of vaping devices, forms of cannabis used in vaping devices, as well as the size of cartridges and tanks used .Given the substantial changes  in relation to cannabis policy, corresponding health risks associated with cannabis products and its derivatives, and emerging respiratory illnesses (EVALI and COVID-19) that make cannabis users that smoke or vape particularly vulnerable, examining changes in consumption can provide timely evidence of health-risk behaviors. Greater access to cannabis may increase the quantity of use over time and subsequently build tolerance, particularly for heavy users. Therefore, the quantity of consumption may be critical in addressing the progression of cannabis use disorder, as well as the relationship between measures of consumption and physiological health risks. Sex-specific differences in cannabis smoking and vaping behaviors underscore the need for gender- and sex-focused research as sub-groups may respond differently to factors that may influence cannabis use. Further research is needed to examine the predictors of changes in cannabis consumption behaviors and how these patterns may be influenced by the complex interactions of biological and psychosocial factors."}
{"text": "For malignant transformation, the expression of p62 in the nucleus (p = 0.03) was significantly lower in malignant transformation cases, whereas the expression of p62 in the cytoplasm (p = 0.03) and the aggregation expression (p < 0.01) were significantly higher. Our results suggest that the function of p62 is altered by its subcellular localization. In addition, defects in selective autophagy occur in cases of malignant transformation, suggesting that p62 is a potential biomarker of the risk of malignant transformation of OPMDs.To determine the intracellular behavior of p62, a marker of selective autophagy, in oral potentially malignant disorders (OPMDs). This retrospective study includes 70 patients who underwent biopsy or surgical resection and were definitively diagnosed with OPMDs. Immunohistochemical staining for p62, XPO1, p53, and ki67 was performed on all samples and positive cell occupancy was calculated. We statistically investigated the correlation between protein expression in OPMDs and the association between malignant transformation, clinicopathological characteristics, and occupancy. ki67 expression was negatively correlated with p62 expression in the nucleus ( Oral potentially malignant disorders (OPMDs) are a newly defined concept by the World Health Organization (WHO), which defined them in 2017 as \u201cclinical conditions that carry risk of cancer development in the oral cavity, regardless of whether they are clinically definite precursor lesion or normal mucosa\u201d [Anil et al. reported the presence and extent of epithelial dysplasia as predictors of the development of oral leukoplakia in cancer . AlthougTreatment options for OPMDs include follow-up, removal of disease triggers such as inappropriate restorations and sharp edges of teeth, medication, and surgical resection ; howeverSelective autophagy is a mechanism that selectively degrades bacteria, specific organelles such as mitochondria, and aggregates of polyubiquitinated proteins . DegradaRecently, autophagy has been linked to various diseases, including Alzheimer\u2019s disease, Parkinson\u2019s disease, and cardiovascular disease . It has Yoshida et al. reported the expression of p62 in oral leukoplakia; however, to the best of our knowledge, there are no studies on the expression of p62 in OPMDs . Lin et Of the 104 patients who visited the Department of Oral and Maxillofacial Surgery at the University of Tsukuba Hospital between 2014 and 2021, underwent biopsy or surgical resection, and received a definitive diagnosis of OPMDs, 70 patients provided their informed consent to participate in the study. The specimens retrieved from the patients were formalin-fixed, and paraffin-embedded blocks were prepared for pathological examination. For patients who underwent both biopsy and surgical resection, the specimen obtained during surgical resection was used based on its condition. Clinical and clinicopathological data were obtained from medical records. All the specimens were diagnosed by at least two pathologists. Regarding malignant transformation of OPMDs, since OPMDs have the propensity to recur after surgical resection and is similar to OSCC, the following definition of local recurrence of OSCC was used as a reference: a local recurrence is defined as less than 2 cm away from or occurs within 3 years of the primary tumor and a second primary tumor as more than 2 cm from or occurs more than 3 years after the primary tumor in OSCC [Clinicopathological characteristics including age, sex, drinking habits, smoking habits, location, disorder, presence of epithelial dysplasia, and development of cancer in the 70 cases included in the study are shown in This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the University of Tsukuba Hospital .All tissue sections had been fixed in formalin, paraffin-embedded, and sectioned to a thickness of 4 \u03bcm. Human liver cancer tissue sections were used as positive controls for p62, p53, and ki67, and human endometrioid adenocarcinoma tissue sections were used for XPO1 as specified in the protocol. Staining without primary antibodies was used as the negative control.After deparaffinization and rinsing, samples were pretreated with a high pH system antigen activation solution  in a microwave oven at 95 \u00b0C for 20 min. Endogenous peroxidase activity was removed by treatment with a mixture of 0.3% hydrogen peroxide in 100% methanol for 10 min. After thorough rinsing, the samples were incubated with primary antibodies against p62 , XPO1 , p53 , and ki67  for 60 min at room temperature, and ki67  primary antibodies at room temperature for 60 min. The samples were washed and treated with a secondary antibody (Histofine Simple Stain MAX-PO(MULTI), Nichirei Bioscience, Tokyo, Japan) for 30 min at room temperature and treated with 3,3\u2032-diaminobenzidine tetrahydrochloride  for 5 min to detect antigen\u2013antibody binding. Finally, hematoxylin was used to counterstain the nuclei.The expression of p62, XPO1, p53, and ki67 in the mucosal epithelium of the stained sections was evaluated. Although both strong and weak expressions were observed, we judged the cells as either positive or negative. A staining intensity equivalent to that of the positive control was considered positive. Images of representative strong and weakly expressing cases of each marker are shown in All immunostained markers were assessed using an optical microscope . Regions showing representative staining in sections were selected at a low-magnification field of view (5\u00d7) and evaluated at a medium-magnification field of view (20\u00d7 and 40\u00d7). Three oral surgeons  determined the number of all epithelial cells and the number of positive cells for each marker at random. The positive cell occupancy rate for each was calculated, considering each receiver operating characteristic.Staining for p62 was evaluated separately for nucleus, cytoplasm, and aggregation. In accordance with our previous research, we defined a cell positive for p62 aggregation staining as one with at least one dot of accumulation in the cytoplasm in 20\u00d7 and 40\u00d7 field of view . RepreseThe correlation of protein expression in all OPMDs cases was evaluated using Spearman\u2019s correlation test and is presented as a scatter plot. In addition, univariate analysis was performed using Fisher\u2019s exact test with clinical characteristics and the Mann\u2013Whitney U test for the association with each protein expression rate in cases of malignant transformation of OPMDs.p values less than 0.05 were considered statistically significant.All SPSS software ver. 28  was used for statistical analysis.p < 0.01). The expression of p62 in the cytoplasm positively correlated with ki67 expression (p < 0.01) and XPO1 expression (p < 0.01). XPO1 expression positively correlated with ki67 expression (p = 0.03) and p53 expression (p = 0.03). The correlations for proteins other than these five were not significantly different.The expression levels of p62, XPO1, p53, and ki67 in OPMDs, which were correlated using the Spearman\u2019s correlation test, are shown in p = 0.03), p62 in cytoplasm (p = 0.03), and aggregation (p < 0.03) were associated with malignant transformation. Actually, the expression of p62 in the nucleus was significantly lower in the malignant transformation cases, whereas p62 expression in the cytoplasm and aggregation was significantly higher. In contrast, the expression levels of XPO1, p53, and ki67 were not associated with malignant transformation.The results of univariate analysis of the association between the malignant transformation of OPMDs and the clinical characteristics or the expression rate of each protein are shown in This study yields two major findings. First, in OPMDs, nuclear p62 expression was negatively correlated with cell proliferation, whereas cytoplasmic expression was positively correlated with cell proliferation. In addition, p62 cytoplasmic expression correlated with XPO1 expression, suggesting that p62 may be transported to the cytoplasm by XPO1. In the cases of OPMDs malignant transformation cases, the p62 expression was altered in the subcellular localization from the nucleus to the cytoplasm, and the p62 aggregation expression was also significantly higher, suggested that selective autophagy is abnormal.Selective autophagy plays a role in maintaining cellular homeostasis but is known to play a dual role in malignant tumors, depending on the type, stage, and genetic context of the cancer. In the early stages of tumorigenesis, it prevents chronic tissue damage by maintaining the genomic stability and by preventing the accumulation of carcinogenic mutations and inhibits the accumulation of cancer inducers. This enabled it to function as a cancer suppressor; however, in the late stages of tumorigenesis, it strengthens cancer cell survival and stress resistance by preventing tumor cell damage and satisfying high metabolic cravings. This eventually promotes tumorigenesis and leads to the accumulation of therapeutic resistance, which contributes to the progression and metastasis of malignant tumors . In othep62 is an adaptor protein for selective autophagy, which is resolved by autophagy along with its target proteins; therefore, the accumulation of p62 is thought to signify autophagy impairment . AlthougOriginally, p62 was a shuttle protein that continuously and rapidly moved between the nucleus and cytoplasm, and this movement was regulated by the phosphorylation and aggregation of p62 . Variouski67 is a protein expressed in all cell cycles except quiescence (G0 phase) and is used as a cell proliferation marker . ki67 isXPO1 binds to the nuclear export signal (NES) of its target protein, forms a complex, and is transported from the nucleus to the cytoplasm via the nuclear membrane . XPO1 paIn addition to autophagy, p62 is involved in the Keap1-Nrf2 pathway . p62 hasp53 acts as a tumor suppressor by inducing genes involved in cell cycle arrest, apoptosis, senescence, and repairing DNA . p53 is Several studies have reported changes in p62 subcellular localization in normal tissues and malignant tumors. Lin et al. reported no difference in p62 nuclear expression but significantly increased the p62 cytoplasmic expression in OSCC compared to normal oral mucosa. The combination of the high p62 cytoplasmic expression and the low p62 nuclear expression in OSCC is also associated with a lower overall survival and disease-specific survival . KitamurThis study has several limitations. First, in this study, 6 cases (8.6%) were malignantly converted, which is similar to the malignant conversion rate reported previously . HoweverIn this study, we found that the properties of p62 in OPMDs with respect to cell proliferative capacity were reversed between the nuclear and cytoplasmic expression. In addition, in cases of malignant transformation of OPMDs, the subcellular localization of p62 changes from nuclear to cytoplasmic, and p62 aggregation increases, suggesting that selective autophagy is impaired. This indicates that changes in the subcellular localization of p62 may be a potential biomarker for assessing the risk of malignant transformation in OPMDs."}
{"text": "Despite the ongoing debate on the risk\u2013benefit ratio of vestibular schwannoma (VS) treatment options, watchful observation and radiation are usually favored in the elderly (>65 years). If surgery is inevitable, a multimodal approach after deliberate subtotal resection has been described as a valid option. The relationship between the extent of resection (EOR) of surgical and functional outcomes and recurrence-free survival (RFS) remains unclear. This present study aims to evaluate the functional outcome and RFS of the elderly in relation to the EOR.This matched cohort study analyzed all consecutive elderly VS patients treated at a tertiary referral center since 2005. A separate cohort (<65 years) served as a matched control group (young). Clinical status was assessed by the Charlson Comorbidity Index (CCI), the Karnofsky Performance (KPS), and the Gardner and Robertson (GR) and House & Brackmann (H&B) scales. RFS was evaluated by Kaplan\u2013Meier analysis using contrast-enhanced magnetic resonance imaging to identify tumor recurrence.surgELDERLY was 67.33 \u00b1 42.02 months and 63.2 \u00b1 70.98 months in the surgCONTROL.Among 2,191 patients, 296 (14%) patients were classified as elderly, of whom 133 (41%) were treated surgically. The elderly were characterized by a higher preoperative morbidity and worse gait uncertainty. Postoperative mortality (0.8% and 1%), morbidity (13% and 14%), and the functional outcome  did not differ between the elderly and the young. There was a significant benefit in regard to the preoperative imbalance. Gross total resection (GTR) was accomplished in 74% of all cases. Lower grades of the EOR  raised the incidence of recurrence significantly. Mean time to recurrence in the Surgical VS treatment aiming for complete tumor resection is feasible and safe, even in advanced age. A higher EOR is not associated with cranial nerve deterioration in the elderly compared to the young. In contrast, the EOR determines RFS and the incidence of recurrence/progression in both study cohorts. If surgery is indicated in the elderly, GTR can be intended safely, and if only subtotal resection is achieved, further adjuvant therapy, e.g., radiotherapy, should be discussed in the elderly, as the incidence of recurrence is not significantly lower compared to the young. Among benign nerve sheath tumors, the vast majority are vestibular schwannomas (VSs) with a reported incidence rate of 1.52 per 100,000 , 4. HistIn contrast to other patient cohorts, there seems to be a general agreement that watchful observation and radiation treatment should be favored in the elderly (>65 years of age) assuming a higher operative morbidity and lower life expectancy , 11. Howelderlies (>65 years of age) is of paramount importance, as an incidence peak is described in this specific patient group  the patterns of VS management in a tertiary neurosurgical center, (2) the onco-functional outcome, and (3) the incidence of recurrence in relation to the EOR in a large cohort of elderly patients compared to a matched-control cohort.nonsurgELDERLY) or surgical treatment (surgELDERLY) depending on tumor size, the presence of hydrocephalus, clinical presentation, and a patient\u2019s individual preference. A separate cohort of patients with <65 years of age, who underwent elective VS surgery, served as a matched control group (surgCONTROL) to specifically compare surgical treatment in the elderly and the young , gender, and tumor size (closest match). All patients were treated by the retrosigmoid approach with intraoperative neurophysiological monitoring. Large VS  were generally treated in the semi-sitting position, while small VS  in the prone position  patients (>65 years) with unilateral VS treated at a German academic, tertiary referral center between April 2005 and October 2020. Patients were referred to non-surgical  were retrospectively analyzed to determine the tumor size according to the Koos grading system in a blinded fashion . The EORt-test was used. Recurrence-free and overall survival were estimated using the Kaplan\u2013Meier method and compared between cases and controls using a log-rank test. The length of follow-up for recurrence-free survival was calculated from the date of surgical intervention to the date of either recurrence or the last clinical visit. Significance was defined as the probability of a two-sided type 1 error being <5% (p < 0.05). Data are presented as mean \u00b1 standard deviation (SD) if not indicated otherwise. Due to the low incidence of complications and perioperative morbidity, for its analysis and comparison dependent on the EOR, DS and STR were grouped together.Statistical analysis was performed in R Studio (Version 1.2) using descriptive statistics. To compare nonnumeric parameters of both groups, the chi-square test and Fisher\u2019s exact test were applied. For numeric parameters, Welch\u2019s two-sample nonsurgELDERLY) by either SRS  or watchful observation . 133/296 (45%) of elderly patients were treated microsurgically (surgELDERLY) via a retrosigmoid approach. A separate cohort of patients younger than 65 years (N = 133) served as a matched control group (surgCONTROL). The mean age of the surgCONTROL cohort was 46.3 \u00b1 11.9 years  were of >65 years of age at date of diagnosis and classified as elderly Figure\u00a01The most common initial symptom in the elderly was functional hearing loss in 172/296 (58%) cases, followed by vertigo in 117/296 (40%) patients. Tinnitus and gait uncertainty were similarly common with 84/296 (28%) cases and 85/296 (29%) cases, respectively. Facial palsy was a leading initial symptom in only 27/296 (9%) cases in the elderly. Trigeminal affection and swallowing deficits were very rare clinical symptoms with 24/296 (8%) and 5/296 (2%) cases, respectively. No patient presented with double vision Table\u00a01.surgELDERLY with the younger surgCONTROL cohort, they presented with a worse initial KPS  poorer preoperative comorbidity status , yielding in a significantly higher CCI in the surgELDERLY compared to the surgCONTROL group  (surgELDERLY had a higher incidence of pre-operative gait uncertainty (p < 0.001), but complained of less tinnitus than their controlled matches (p = 0.016).When comparing < 0.001) 2. HowevsurgELDERLY and nonsurgELDERLY cohort did not differ in age, gender, the clinical status in KPS. The incidence of cystic morphology and MR-graphic progression was higher in the surgically treated but did not reach statistical significance in this cohort. However, nonsurgELDERLY were characterized by a significant smaller tumor size (p < 0.001). The nonsurgELDERLY cohort had less initial severe CN deficits  than surgELDERLY. The CCI was not significantly different in both cohorts . There were no significant group difference in the initial incidence of facial palsy, functional hearing loss, vertigo, trigeminal affection, swallowing deficit, headache, gustatory deficit and hydrocephalus in a matched control comparison (\u201csurgELDERLY\u201d vs. \u201csurgCONTROL\u201d). Remarkably, surgELDERLY had a higher incidence of gait uncertainty but complained of less tinnitus than their controlled matches. Additionally, surgELDERLY had a poorer preoperative comorbidity status , yielding in a significantly higher CCI in the surgELDERLY compared to the surgCONTROL group  (The eristics Table\u00a01.surgELDERLY and 240.0 \u00b1 82.1 minutes in the surgCONTROL (p = 0.398).Surgery was performed by a retrosigmoid craniotomy in all cases using either a semi-sitting  or prone position  in both surgical study groups. Mean operating time (skin to skin) was noted similarly at 248.0 \u00b1 75.2 minutes in the surgELDERLY  and surgCONTROL group  (p=0.858). Larger tumors (Koos \u00b0III and \u00b0IV) more often yielded in postoperative complications . Postoperative hemorrhage, venous thrombosis and symptomatic pneumoncephalon occurred more frequently in the surgELDERLY, but this did not reach any statistical significance. In contrast, younger patients were more prone to postoperative CSF leakage. Overall, the complication rate in the surgELDERLY cohort was not significantly raised. One patient suffered a postoperative hemorrhage and treatment was terminated after the patient\u2019s presumed will, yielding in a mortality rate of N = 1 . There was no significant difference in discharge modality (p = 0.377). Impeccable facial function (H&B = 1) was completely unaffected by the surgery in 38/112 (34%) and 53/123 (43%) in surgELDERLY and surgCONTROL, respectively (p = 0.096). After surgery-related deterioration, facial function recovered to a favorable outcome  in 45/75 (60%) and 46/70 (65%) surgELDERLY and surgCONTROL patients, respectively  than in surgCONTROL at 71%  with p = 0.164. New trigeminal deficit was observed in 2%. Of the 20 patients suffering from preoperative trigeminal affection , 17 patients recovered clinically in the surgELDERLY group (85%); the same was observed in 20/22 patients (90%) in the surgCONTROL group (p = 0.277). New postoperative gait uncertainty was observed in N = 4 in the surgELDERLY but none in the younger surgCONTROL group. Postoperative recovery of symptomatic gait uncertainty was observed in 71% (37/52) and 76% (12/16) of the surgELDERLY and surgCONTROL, respectively (p = 0.382). The recovery of vertigo symptoms was observed in N = 40/55 (71%) of all surgELDERLY presenting with preoperative vertigo; this rate was higher in the surgCONTROL cohort with N = 37/40 (90%) (p = 0.013). The incidence of new postoperative vertigo was N = 0 (0%) in surgELDERLY but N = 8/91 (9%) in its control cohort. Considering new postoperative tinnitus, the incidence was higher in the young (N = 1 in surgELDERLY and N = 9 in surgCONTROL), whereas the postoperative improvement of known tinnitus was similar at 14% and 16%, respectively (surgELDERLY and surgCONTROL) (p = 0.585).Postoperative KPS did not differ between ectively Table\u00a05.surgELDERLY cases . When taking the EOR into account, there was no significantly higher postoperative CN affection  in surgELDERLY associated with GTR and STR/DS (surgELDERLY compared to the surgCONTROL (p = 0.035). Additionally, within the surgCONTROL group, STR/DS was associated with a significantly higher incidence of permanent facial deterioration (p = 0.036) compared to GTR.GTR was achieved in the majority of d STR/DS Table\u00a06.surgELDERLY: p = 0.015; surgCONTROL: p = 0.003). The incidence of recurrence was statistically insignificant in surgELDERLY compared to surgCONTROL, when treated with GTR (p = 0.621). Mean time for surveillance was 38 \u00b1 36 months in surgCONTROL (median: 25 months) and 31 \u00b1 37 months (median: 34 months) in surgELDERLY. The overall incidence for recurrence was 5/133 (4%) and 10/133 (8%) after neurosurgical tumor resection in the surgELDERLY and surgCONTROL, respectively, with no statistical significance (p = 0.143). Mean time to recurrence was 67.33 \u00b1 42.02 months in surgELDERLY and 63.2 \u00b1 70.98 months in surgCONTROL. Kaplan\u2013Meier analysis on RFS depending on the EOR is shown in surgELDERLY and surgCONTROL cohorts (p < 0.001). Mean time to recurrence in surgELDERLY was 67 \u00b1 53 months and 79 \u00b1 71 months in surgCONTROL.STR/DS was significantly associated with the incidence of recurrence compared to GTR in both subgroups  in a tertiary neurosurgical center and to assess the oncological and functional outcome of this particular cohort in comparison to matched young controls and in regard to the EOR. The elderly represented approx. 14% of all VS patients in our cohort. The majority was eligible for non-surgical treatment. Patients were allocated for surgical treatment due to large tumor size affecting the brainstem and worse preoperative clinical symptomatology . In comparison to a matched-control cohort, elderly selected for surgery were characterized by a worse preoperative clinical condition (KPS and CCI). Despite this, postoperative mortality, morbidity and functional outcome did not differ between both surgical groups. These data suggest that the retrosigmoid approach for VS resection is safe in the elderly >65 years of age. Patients significantly benefit in regard to preoperative imbalance. GTR was accomplishable in the majority of patients. Notably, the EOR determined RFS.surgELDERLY), preoperative CN deficits  were distinctively more severe than in the conservatively managed (nonsurgELDERLY). Same was described in a nationwide registry study in Sweden with 58 elderly patients and by a series of surgically managed elderly patients by Samii et\u00a0al. , perioperative complication rates and morbidity are statistically comparable. Perioperative complications in the elderly have been described by current literature from 20% to 57% . The rate of permanent facial deterioration was significantly higher in surgELDERLY compared to surgCONTROL in the GTR group suggesting decreased postoperative rehabilitation potential in facial function with advanced age  comparing the nced age .The explanation for the inversely proportional relationship of permanent facial deterioration and GTR vs. STR/DS in the elderly  and the young  could be attributed to the actual intention-to-treat. In the young, we usually intend GTR unless there is a deterioration of the intraoperative neuromonitoring . This approach explains the GTR rate of 74% in the present study. However, in a larger cohort of the same group, GTR was even higher with approx. 93% . This faLong-term results should be chosen to compare functional outcome between such different treatment modalities as radiosurgery and microsurgery, e.g., in hearing outcome. Watchful observation (\u201cWaS\u201d) abandons CN function  to the natural history of VS and tumor dynamic. Thus, it has been shown that 12% lose functional hearing in the course of the VS natural history . In lineIt is not to be forgotten that functional outcome, as physicians and/or treating surgeons may define it, does not necessarily transfer to quality-of-life in the patients\u2019 eyes in a proportional way. Leaving residual tumor behind or treating the tumor by non-invasive treatments such as SRS can impact mental health or illness perception .Due to the study design (matched by tumor size and the EOR), this analysis does not allow any general statement considering the frequency of the achieved EOR in the elderly compared to the young alone. However, when put into context with another series published and treated by the same group and institution with N = 572 primary VS patients, the rate of GTR was significantly higher among a general VS patient cohort of any age. Therefore, we can assume that the management of elderly patients with VS is carried out more conservatively\u2014even in regard to the surgical EOR .Mean time to recurrence was >5 years in both groups, proposing that long-term follow-up in VS patients should be carried out regardless of age. Still, the overall incidence of and mean time to recurrence were statistically insignificant in the elderly compared to the young concordant to previous matched cohort studies with distinctly smaller patient numbers , 10. In The EOR is significantly associated with RFS in both groups independent of age with noted early tumor recurrence in DS compared to STR and GTR. Pre-existing data show that the volume of residual tumor correlates with the incidence of recurrence , 32. GTRA definite statement on surgical treatment of VS is generally very difficult to acquire due to several reasons: (1) the level of evidence: there are no published randomized controlled clinical trials or even prospective studies on surgical resection in the current literature , (2) hetIt is apparent that the retrospective nature of this study bears its limitations and biases. Firstly, this is a single-center study. Therefore, the generalizability and reproducibility of the results may be limited to specialized centers with a comparative caseload in VS. Furthermore, although the number of patients classified as elderly may be regarded as the largest cohort compared to previously published studies , 10, stiThe evidence level of VS management is remarkably low, and the debate on the risk\u2013benefit ratio of surgical treatment is still ongoing, especially in the elderly. The present matched-cohort study shows that the microsurgical tumor resection of VS is safe and does not bear additional perioperative morbidity or worse functional outcome in the elderly as compared to a young control group. The overall incidence and of recurrence was statistically insignificant in the elderly compared to the young. When treating with surgery alone, the EOR determines RFS and the incidence of recurrence/progression in both study cohorts. For maximal tumor control, GTR should be intended. As incidence of recurrence is not significantly lower compared to the young. Postoperative follow-up should be carried out as mean time to recurrence was > 5 years in both groups.\u2003If leaving relevant tumor residual is inevitable, adjuvant therapy, e.g. SRS, should be evaluated in the Elderly.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the participants was not required to participate in this study in accordance with the national legislation and the institutional requirements.SW contributed to the acquisition, analysis, interpretation of data, conception and design, and writing the first draft. KM and FE contributed to interpretation of the data and critical review of the final manuscript. GN contributed to data analysis, interpretation of the data, and reviewing the final manuscript. MT contributed to the interpretation of the data and critical review of the manuscript. All authors contributed to the article and approved the submitted version."}
{"text": "Neurotensin (NTS) is a peptide discovered in 1973, which has been studied in many fields and mainly in oncology for its action in tumor growth and proliferation. In this review of the literature, we wanted to focus on its involvement in reproductive functions. NTS participates in an autocrine manner in the mechanisms of ovulation via NTS receptor 3 (NTSR3), present in granulosa cells. Spermatozoa express only its receptors, whereas in the female reproductive system , we find both NTS secretion and the expression of its receptors. It consistently enhances the acrosome reaction of spermatozoa in mammals in a paracrine manner via its interaction with NTSR1 and NTSR2. Furthermore, previous results on embryonic quality and development are discordant. NTS appears to be involved in the key stages of fertilization and could improve the results of in vitro fertilization, especially through its effect on the acrosomal reaction. Neurotensin (NTS) is a 13 amino acid peptide initially described in 1973 in the bovine hypothalamus , and itsNTS has a physiological role and is implicated in several functions, mainly in digestive and central nervous systems. In the digestive system, NTS acts on intestinal motility  and faciNTS effects are mediated by three receptors (NTSR): NTSR1 and NTSR2 are G-protein-coupled receptors with seven transmembrane-spanning domains, and NTSR3 or sortilin (SORT1) is a Vps10p receptor family member, which is characterized by the presence of an extracellular region containing a cysteine-rich domain and only one transmembrane domain . NTSR1 aThe NTS/NTSR1 complex is involved in the progression of many cancers, such as colic adenocarcinoma , small-cSeveral publications have reported the expressions of NTS and its receptors in reproductive tissues, related to their roles in reproduction functions, particularly in ovulation, sperm capacitation, fertilization, and embryonic development. It has recently been highlighted that NTS receptors are located on male gametes and that NTS is expressed in the uterus. NTS could play a role in reproduction issues by participating in follicular rupture during ovulation ,27 and iSynthesize knowledge regarding the expression and function of the neurotensinergic complex in the different models in which it has been studied;Propose an integrative mechanistic interpretation;Open perspectives for future research.The aim of this review was to synthetize the worldwide knowledge about NTS and its potential role in reproductive functions in order to achieve the following:On the PubMed database, the keywords \u201cfertility\u201d, \u201cfertilization\u201d, \u201covulation\u201d, \u201coocyte\u201d, \u201cembryo\u201d, \u201cendometrium\u201d, \u201cspermatozoa\u201d, \u201coviduct\u201d, and \u201creproduction\u201d associated with NTS were tested. After abstract studies, we considered 15 publications of interest ,40,41,42NTS was described in the female tract in the oviduct epithelium using Western Blot, immunohistochemistry, and RT-PCR ,29,33,39Some authors have been interested in the presence of NTS/NTSR1 in the tissues of the human myometrium and leiomyoma and, more particularly, in women having hormonal treatment for in vitro fertilization (IVF) . Rodrigucumulus oophorus and corona radiata granulosa cells do not express NTSR1 and NTSR2; NTSR3 was not studied i levels were increased just after the addition. The calcium intensity when 50 \u03bcM of NTS was added was higher than that when 10 \u03bcM was added, providing evidence for dose dependency in the [Ca2+]i response. However, the number of sperm cells that immediately increased their calcium levels were low at 50 \u03bcM  and 10 \u03bcM , whereas almost all the sperm experienced increased calcium mobilization when ionomycin was added  [In human and non-human primates, Campbell et al. studied the NTS effects on the spermatozoa acrosomal reaction using immunofluorescence . They de < 0.05) . They al < 0.05) . Hiradat < 0.05) . When ad < 0.05) . Moreovexamined) .The potential role of NTS in spermatozoa is illustrated in Fertilization was specifically studied in only one publication. The NTS pretreatment of monkey sperm reduced the fertilization rate of monkey MII oocytes in vitro from 72% . Moreovep < 0.05) [p = 0.03). In this treatment group, the hatching rate was 28.1 \u00b1 3.8% versus that of the control group  [Two publications studied the role of NTS in embryo development. The addition of NTS to an IVF medium significantly improved the levels of cleavage in the bovine embryo  . However < 0.05) . The eff < 0.05) . Embryos = 0.08) . No dataThe role of NTS in reproduction was first discussed in 1981 by Clark et al., who analyzed the effect of NTS on blood flow in the uterus of non-pregnant sheep . They coIn 1987, Reinecke et al. showed using an isometric force displacement transducer that NTS enhanced autonomous oviduct contractility up to 55% in vitro and that their amplitude was 9-fold more than that without NTS addition .p < 0.005), while siRNA-NTS induced the apoptosis of EECs (p < 0.005). In addition, NTS may play a role in endometrial receptivity by increasing the levels of certain biochemical markers of the receptive endometrium, such as leukemia inhibitory factor (LIF), COX2, and HOXA10, in EECs in vitro [The effects of NTS on endometrial receptivity appear to be very different depending on the species. In dairy goats, Zhang et al. analyzed the concentrations of NTS mRNA and other molecules in the receptive endometrium and the pre-receptive endometrium . Using iin vitro . Howeverin vitro . Conflicin vitro . The NTS physiological functions in mammalian reproduction are summarized in Data regarding the important role of NTS and its receptors are now well-established. About 15 publications that we reviewed exhaustively show the action of NTS in ovulation and fertilization.The data concerning ovulation are still incomplete. The exponential presence of NTS in granulosa cells, especially after LH, hCG, or eCG triggering, and the co-expression of the specific receptor NTSR3 point us towards an autocrine and/or paracrine mode of action. The limited experimental data support a necessary but not sufficient role in causing ovulation. Mechanistic studies conducting the inhibition testing of signaling pathways confirm a direct action via the MAPK pathway. Likewise, the presence of NTS in the female genital tract, particularly after ovulation, indicates a physiological role. Data concerning the stages of fertilization, as well as the presence of NTSR1 and NTSR2 on the sperm membrane , could suggest a major paracrine effect presiding over capacitation and the acrosomal reaction. However, fertilization rates with NTS-treated sperm were lower than those obtained with untreated sperm. It would be interesting to experiment to determine whether these fertilization rates remain low if NTS is added directly to the fertilization medium rather than to the sperm preparation, as the NTS-induced acrosomal reaction should be performed as close to the oocyte as possible. NTS could potentially play a role in IVF in cases of fertilization failure.The role of NTS in endometrial receptivity is not clear across species, yet implantation failures are a major cause of IVF failure, and its potential mechanisms are still poorly understood. The study of NTS expression at the endometrial level in women could be of interest in patients presenting repeated implantation failures after embryo transfer in this context."}
{"text": "The protein p53 is a well-known tumor suppressor that plays a crucial role in preventing cancer development. In Chinese philosophy, the concept of Yin and Yang is used to describe inseparable opposites, where Yin represents the negative or inhibitory force, and Yang represents the positive or promoting force. In this context, the wild type of p53 can be considered Yin, as it acts as a negative regulator of cancer progression, while most p53 mutations can be considered Yang, as they promote cancer progression.Some p53 mutants, such as p53 S46F and S121F, exhibit higher transactivation activity than wild type, which can be considered as a super Yin of p53 . In contIt has been known that some mutated p53 can appear in the cytosol , and cyt"}
{"text": "In addition to its role in tumors, p53 has a widespread expression in the brain and participates in most cell processes, such as dendrite formation, oxidative stress, apoptosis, autophagy, DNA repair, and cell cycle arrest. Therefore, abnormalities in p53 and its related signaling pathways play an important role in the diagnosis and treatment of central nervous system diseases. This review mainly discusses the latest findings regarding the role of p53 in some central nervous system diseases, such as brain tumors, Alzheimer disease, Parkinson disease, autism, epilepsy, spinocerebellar ataxia, and so on, to provide a comprehensive interpretation of the treatment of neurological diseases from a new perspective.The transcription factor p53, a widely accepted tumor suppressor, regulates the expression of many oncogenes and their downstream signaling pathways, resulting in a series of biological outcomes. Mutations and deletions of the  Thus, abnormal expression of p53 protein can lead to the occurrence and development of neurological diseases. p53 is an important cancer suppressor factor that has a high mutation frequency in human cancers. Through the pathological analysis of a large number of tumor patients, approximately 50% of the tumor samples had p53 deletion or mutation, including brain tumors  is one of the target genes for p53. A mutation in P53V143A can block the transcription of p21 and its function of inducing cell cycle arrest, leading to cell cycle disorder and promoting the occurrence of malignant glioma  often occur . TherefoAD is a common neurodegenerative disease, and its neuropathological features are the accumulation of amyloid-\u03b2 (A\u03b2) and nerve fiber tangles formed by tau hyperphosphorylation, resulting in neuronal dysfunction . A high As mentioned above, p53 is present at a low level in normal cells. In addition to posttranslational modifications, the conformational state of p53 also affects intracellular stability. Unfolded p53 has been detected in early-onset AD patients . UnfoldeTargeting p53 as a therapeutic target to prevent or treat AD remains a great challenge. Although p53 is related to neuronal death and is involved in the pathological processes of AD, only in vitro evidence has been promising, and in vivo studies are needed to support the potential for p53 targeting to be translated into the clinic. As mentioned above, phospho-p53 (Ser15) and phospho-p21 (thr145) are biomarkers for the diagnosis of AD in peripheral blood lymphocytes of patients with AD . In addiDJ-1 is a PD gene that could provide valuable reference ideas for the treatment of PD. In a zebrafish PD model deficient DJ-1, p53 and the proapoptotic factor Bax were overexpressed before toxin exposure, and no significant neuronal cell death was observed, suggesting that subliminal activation of the cell death pathway may have therapeutic effects on PD, such as inhibition of the p53 pathway  is also known as autism. Its clinical manifestations include social interaction difficulties, communication impairment, and stereotyped repetitive behavior patterns, which are the result of a combination of genetics and external environmental factors . HoweverSCA is an autosomal dominant genetic disease that mainly manifests as behavioral movement disorders and speech disorders. There is currently no specific drug treatment, so it is very important to study its pathogenesis and pathways . The patp53 is not only involved in the pathogenesis of brain tumors, AD, and spinocerebellar ataxia, but also in the occurrence and development of other CNS diseases, such as schizophrenia (SCZ), depression, epilepsy, and cerebral ischemia. SCZ and depression are common affective disorders with a high incidence . It has Epilepsy is a common neurological disease with a complex etiology. In addition, p53 is significantly upregulated in both posttraumatic epilepsy and drug-resistant epilepsy rat models, accompanied by increased apoptosis of nerve cells in the mesial temporal lobe and hippocampus . SimilarFigure 2). At present, evidence for the role of p53 in related neurological diseases is still relatively limited and needs more research support. In addition, as people grow older, the risk of co-occurrence tumor and neurodegenerative diseases increases significantly; whether and how p53 is involved in this process should be given more attention. It should be noted that most preclinical models are constructed using adult animals, which ignores the effect of aging and may hinder hypothesis clarification (In this review, we describe the role of the p53 pathway and genetic alterations of p53 in the progression of different CNS diseases (fication . TherefoI/LH have been utilized to reverse the abnormal structure of p53 or downstream signaling pathway changes caused by p53 mutation (Studies have demonstrated a strong correlation between tumors, neurological disorders, and p53. Consequently, antitumor drugs that target p53 may have potential therapeutic benefits for treating AD, PD, autism, and so on. Some of these drugs function by inhibiting the ubiquitination degradation of p53, which is regulated by E3 ligase MDM2 H. . To restmutation . Some coIn addition, when discussing p53, the role and function of its family members p73 and p63 should not be forgotten . Accordi"}
{"text": "The COVID-19 pandemic and associated social restrictions had an extensive effect on peoples\u2019 lives. Increased rates of weight gain were widely reported, as were declines in the general populations\u2019 mental health, including increases in perceived stress. This study investigated whether higher perceived levels of stress during the pandemic were associated with greater levels of weight gain, and whether poor prior levels of mental health were a factor in higher levels of both stress and weight gain during the pandemic. Underlying changes in eating behaviours and dietary consumption were also investigated. During January-February 2021, UK adults (n = 179) completed a self-report online questionnaire to measure perceived levels of stress and changes (current versus pre-COVID-19 restrictions) in weight, eating behaviours, dietary consumption, and physical activity. Participants also reported on how COVID-19 had impacted their lives and their level of mental health prior to the pandemic. Participants with higher levels of stress were significantly more likely to report weight gain and twice as likely to report increased food cravings and comfort food consumption . Participants reporting an increase in food cravings were 6\u201311 times more likely to snack and to have increased consumption of high sugar or processed foods . Females reported a far greater number of COVID-19 enforced lifestyle changes and both being female and having poor mental health prior to the pandemic were significant predictors of higher stress and weight gain during the pandemic. Although COVID-19 and the pandemic restrictions were unprecedented, this study suggests that understanding and addressing the disparity of higher perceived stress in females and individuals\u2019 previous levels of mental health, as well as the key role of food cravings, is key for successfully addressing the continuing societal issue of weight gain and obesity. After the declaration of the SARS-CoV-2 coronavirus COVID-19) pandemic in March 2020 9 pandemi. These iAlthough COVID-19 restrictions were implemented to improve the health outcome of the pandemic, the high level of hospitalisations and deaths in the UK  caused cSince the start of the pandemic there was also an overall reported increase in food intake and weight gain both in the UK and other countries \u201318. StudPrior to COVID-19, obesity was already considered to be a global epidemic \u201326. In aPrior to the pandemic, long-term chronic stress already had a significant positive association with weight gain \u201334. SeveFood cravings are defined an intense desire to consume a particular food or food type, often in the absence of hunger . Higher Some of the main barriers to weight loss and weight maintenance that have been identified in recent studies are mental distress and binge eating (or comfort eating) , negativThe aim of this study was to investigate whether higher levels of stress reported by the general UK population during the pandemic were linked to higher levels of weight gain, and whether these changes were directly associated with increased food cravings and comfort eating. Secondly, we aimed to investigate whether individuals\u2019 level of mental health prior to the pandemic were linked to higher levels of stress and weight gain during the pandemic. In addition, we addressed whether specific lifestyle changes caused by COVID-19 and the UK imposed restrictions were linked to higher levels of stress. We hypothesized that individuals with higher perceived stress would report greater weight gain during the pandemic, with people who had poor mental health before COVID showing the strongest effects. Furthermore, we hypothesized that the weight gain and perceived stress were strongly linked with increased food cravings and comfort food consumption.The study used a convenience sample and the eligibility criteria was that participants were aged 18 or over and had been resident in the UK since the beginning of COVID-19 restrictions in March 2020. Participants were recruited through open adverts on social media and through internal advertising in Bangor University School of Human and Behavioural Sciences. To achieve adequate power for testing a minimum of 92 participants were required, based on a two tailed priori power analysis of 25% effect size, 0.8 power, 0.05 error . Effect size used was based on previously reported increases in food intake and weight gain when under high levels of stress, both prior to and during COVID-19 restrictions , 17. TheData was collected using a bespoke online survey that was administered via Qualtrics . The survey was completed anonymously, with no data collected that could identify participants. The survey was designed to be easy to answer and completed in as short a time as possible, to ensure minimal drop out of participants. To enable this, where appropriate, questions were asked about categorical changes  rather than absolute measures. As well as simplifying the questionnaire, the use of categorical measures, rather than absolute measures, enabled the reporting of overall trends while avoiding inaccuracy problems with self-report measures.At the start of the survey participants were provided with a brief overview of the study aims and the participant inclusion criteria (over 18 and based in the UK since the beginning of COVID-19 restriction). For participants to take part in the study they were required to click on a button of \u201cI meet the inclusion criteria and consent to take part in the study\u201d. After providing informed consent participants reported on the following information:Participants reported on the demographic measures on age, gender, main current occupation, country of the UK based, height and weight . Participants were asked to report how their weight had changed since the beginning of the COVID-19 restrictions within the following categories: lost more than 3kg/6 pounds, lost up to 3kg/6 pounds; weight remained stable, gained up to 3kg/6 pounds, gained between 3kg/6 pounds and 6kg/a stone, gained more than 6kg/a stone.Participants were asked to report whether their general hunger, food cravings, number of meals consumed, snacking in the daytime and snacking in the evening had decreased, increased or stayed the same in the past 3 months, compared to prior to COVID-19 restrictions. Participants also reported if they believed any of the following reasons had increased their dietary consumption: being at home, boredom, stress anxiety or low moods, feeling tired or lacking in energy, The behaviour categories were based on categories reported by other COVID-19 studies , 47.Participants were asked to report if their consumption of any of the following categories had decreased, increased or remained the same in the past 3 months, compared to before the start of the COVID-19 restrictions: fruit and veg; high fibre foods , red or processed meat , dairy products, vegetarian/vegan meat or dairy alternatives, processed foods , home cooked food, tinned or frozen food; high sugar foods , coffee or tea, fruit juices or smoothies, fizzy drinks (including diet drinks), alcohol, water. The range of dietary categories was based on dietary categories reported in other COVID-19 studies \u201317.To ensure that any reported changes in weight were due to changes in dietary consumption rather than changes in physical activity, participants were asked if their level of vigorous/moderate exercise, walking and sedentary time had decreased, increased or stayed the same in the past 3-months compared to before COVID-19 restrictions.As part of the survey participants completed the Perceived Stress Scale PSS-10  to evaluParticipants were asked to report on ways in which ways COVID-19 and the associated imposed restrictions had impacted their lifestyle and food availability. Participants were asked if the restrictions had impacted them in any of the following ways: continued to work outside the home but had to use personal protective equipment (PPE), moved to work from home, experienced job uncertainly, experienced job loss, had confirmed COVID-19 infection, had to self- isolate due to possible COVID-19 infection, had to self-isolate due to underlying health issues, had additional caring responsibilities including school-aged children or other dependents, had less access to fresh or healthy food, had less money to spend on food, had less time to prepare healthy food.Descriptive statistics (mean \u00b1SD) were used to characterise survey respondents and summarise their changes in weight gain, eating behaviours, dietary consumption and physical activity. Eating behaviours and dietary consumption were re-categorised in to an \u201cincreased\u201d or \u201cno increase\u201d categories (which included the \u201cstayed the same\u201d and \u201cdecreased\u201d categories). This was as this study was specifically interested in the likelihood of increased food cravings and comfort foods being linked to stress and weight gain.Although the PSS-10 questionnaire gives a continuous score outcome, to enable us to carry out logistic analysis with the categorical variables reported for changes in weight, dietary consumption and exercise, participants were recategorized into a higher or lower stress group, with categorization based on the mean PSS-10 score of trial participants (19.3\u00b16.9). Participants with a PSS score of 19.3 or lower were allocated to the lower stress category and participants with a PSS score of 19.4 or higher were allocated to the higher stress category.p < 0.05 was applied to all statistical analyses.Ordinal regression analyses were used to identify factors linked to a greater likelihood of reporting weight gain. Pearson\u2019s chi-square analyses were used to investigate a significant difference in likelihood in response between the higher and lower stress groups to: weight gain, increases in eating behaviours, increases in dietary consumption, and changes in physical activity. Binary regression analysis was used to identify factors linked to a greater likelihood of belonging to the higher stress group. Odds ratios were also calculated for all chi-square analyses, to allow direct comparison of results with the binary and ordinal regression analyses. All statistical analyses were carried out using the Statistical Package for Social Sciences . A 95% confidence interval (95% CI) was selected, and a significance level of In total, responses were collected from 220 participants between 6th January until 2nd February 2021. After removing duplicates and incomplete questionnaires, 179 participants\u2019 data sets remained. There was no significant demographic difference between survey completers and non-completers. The full survey dataset is available in 2.Most survey respondents were female (75%) and aged between 18\u201355 (85%). Two thirds of respondents were based in Wales (66%) and a third (34%) in England. The mean BMI of respondents was 26.10(\u00b15.46) kg/mMany respondents reported that their main current occupation involved working from home, with a higher proportion of females being based at home (F = 40%:M = 27%) and more male respondents continuing to work outside the home (F = 15%:M = 27%). As the questionnaire was advertised within the university, unsurprisingly students made up 25% of respondents, although 8% of responders were in the retired category. Notably, only females reported that caring for children or dependents was their main occupation (5%). The overall level of participants unemployed or furloughed was low .When asked about the impact of COVID-19 restrictions on their lifestyle, a comparable number of women and men reported moved to work from home (F = 50%: M = 46%). A higher proportion of women reported worked outside the home using PPE (F = 35%:M = 25%), having to isolate due to potential COVID-19 infection (F = 25%:M = 16%) or having additional caring responsibilities for children or other dependents (F = 39%:M = 9%). Males reported higher levels of job uncertainty (F = 12%:M = 23%), job loss (F = 5%:M = 7%), having had COVID-19 (F = 4%:M = 7%) and self-isolating due to underlying health issues (F = 4%:M = 11%). The difference in the level of impact of COVID-19 restrictions by gender was notable. When comparing the total number of changes that participants were affected by, 53% of females were affected by 2 or more lifestyle changes compared to 39% of males. The impact of COVID-19 restrictions on food availability was also far more likely to impact females. Females were around twice as likely to report less money to spend on food (F = 16%:M = 9%), less access to healthy food (F = 12%:M = 7%) or less time to cook healthy food (F = 11%:M = 5%). Full demographic statistics for survey participants are available in Participants were asked to report their weight change in categories rather than absolute weight changes to avoided inaccuracy problems with self-report measures. There was a notable difference between the genders for reported weight gain, with females almost twice as likely as males to report an overall weight gain since the start of the COVID-19 restrictions (F = 50%: M = 27%). and twice as likely to report the highest level of weight gain of more than 6kg (F = 10%: M = 5%). see 2 = 14.639, p = 0.012, phi = 0.286). This confirmed the study\u2019s main hypothesis that participants reporting a higher level of stress during the pandemic were statistically more likely to report a higher level of weight gain. Although the aim this study was not specifically focus on gender differences, due to the large differences in weight changes reported by males and females a Chi-square tests for independence was carried out, which showed that the association between gender and weight gain was statistically significant, with small to medium effect, \u03c72 = 11.986, p = 0.035, phi = 0.259).Ordinal regression analysis showed that the likelihood of the higher stress group reporting a greater level of weight gain was statistically significant, with a medium effect size, \u03c7In the survey, participants were asked to report on changes in eating behaviours that are often linked to weight gain, including hunger, number of meals, cravings, and snacking. Overall increases were reported for all eating behaviour categories, apart from number of meals see . Althoug2 = 70.877, p<0.001), with increased food cravings , increased number of meals , increased snacking in the day  and eating due to stress, anxiety or low mood  all significant factors linked to weight gain. Although being at home or boredom were given by the participants for the primary reasons for increased food consumption, there was no statistical significance of these factors and actual weight gain. An ordinal logistic analysis was performed to assess whether increases in the 5 eating behaviours and 4 reasons given for increased eating increased the likelihood of respondents reporting weight gain. The full model was statistically significant, \u03c7\u03c72  = 6.402, p = 0.011, phi = 0.202;) OR = 2.3 and snacking in the day \u03c72  = 4.192, p = 0.041, phi = 0.165), OR = 1.9. Participants in the higher stress category were also more likely to report increased eating due to stress, anxiety or low mood \u03c72  = 25.684, p<0.001, phi = 0.390), which is as expected, as both measures are reporting on levels of stress. In comparison, there was no statistical significance for higher stress and increased number of meals. In summary, increased food cravings and snacking in the day were the only behaviours correlated with both higher stress and weight gain.To determine whether these behaviours were more likely in the higher stress group a chi-square test was carried out. This showed that participants who were in the higher stress category were twice as likely to report increased food cravings Like changes in eating behaviours, there was a polarisation of changes dietary consumption. Over half of respondents maintained their pre-COVID-19 consumptions levels of fruit and veg (55%), high fibre (70%), red/processed meats (52%), white meat/fish (60%), dairy products (71%), vegetarian/vegan alternatives (62%) and tinned/frozen foods (68%). A notable change was seen with home cooked food category, where 50% increased their consumption. The other categories which showed the greatest change in consumption were those that can be categorised as comfort foods. There was comparable split between decreased, increased or remained the same for processed foods . Forty percent of participants reported an increase in consumption of high fat/salt foods, and over half (57%) of participants reported an increase in high sugar foods. With the drink categories, the majority respondents reported maintaining their pre-COVID-19 consumption level of fruit juices/smoothies, fizzy drinks and water . In comparison almost half of respondents reported increased consumption of coffee and tea, and alcohol . A full summary of the dietary changes for survey participants is available are available in \u03c72 = 75.291, p<0.001), although only 4 of the 16 categories made a significant contribution. Increased consumption of high sugar and processed foods were linked to a 2.5\u20133.2-times likelihood of an increase in weight gain . However, the inverse effect was seen in the categories of high fibre foods  and water , where increased consumption was linked to a greater likelihood of weight loss. To determine which dietary categories were linked to weight gain an ordinal logistic regression was carried out. The full model, containing all 16 food and drinks categories, was statistically significant, \u03c72 = 4.001, p = 0.045, phi = 0.161), OR = 1.9 and processed foods \u03c72  = 6.310, p = 0.012, phi = 0.201), OR = 2.5. In comparison, there was no statistical significance for changes in the consumption of high fibre food or water and higher levels of stress. In summary, increases in high sugar and processed food consumption were the only dietary categories linked both to a higher level of stress and weight gain.A chi-square analysis was carried out to determine whether the dietary categories linked to weight change were also linked to higher levels of stress. This showed that participants in the higher stress group were twice as likely to have increased their consumption of both high sugar foods \u03c72(1) = 48.008, p<0.001, phi = 0.534; OR = 11.2), and over 6 times more likely to report increased consumption of processed foods, \u03c72 (1) = 22.920, p<0.001, phi = 0.374, OR = 6.3), although there was no significant link with high fat/salt foods. There was also a statistically significant link between increased cravings and increased snacking in the day (but not snacking in the evening), with participants reporting increased food cravings 6 times more likely to report an increase in snacking during the day, \u03c72 (1) = 29.181, p<0.001, phi = 0.419; OR = 6.0).To investigate further the relationship between increased food cravings and increased comfort food consumption , a chi-square analysis was performed. We also investigated if an increase in food cravings was linked to an increase in snacking, as comfort food is often consumed outside mealtimes . Partici\u03c72 = 25.354, p = 0.005, phi = 0.271), but not for changes in level of walking or sedentary time. Chi-squared tests of level of activity versus stress levels were non-significant \u03c72 = 1.40, p = 0.50, phi = 0.088). Consequently, although a reduction of moderate/strenuous activity increased the likelihood of weight gain, it was not a mediator of the effect of stress on weight gain. A full summary of the physical activity changes for survey participants is available in To discount the role of physical activity in the effect of stress on weight gain, participants were also asked to report in their changes in physical activity. Almost half of respondents (47%) reported a reduction in their level of moderate or strenuous activity compared to their pre-COVID-19 levels of activity. In comparison, almost half (43%) of participants reported that they had increased their level of walking compared to pre-COVID-19 level. However, the category with the greatest reported difference, was sedentary behaviour, with 78% or respondents reporting an increase in sedentary compared to their pre-COVID-19 levels. Chi-squared tests for level of activity versus weight change did show a statistical significance for a reduction in moderate/strenuous activity and weight gain, Participants were asked to report both their current levels of perceived stress, using the PSS-10 questionnaire, and their mental health prior to the start of the pandemic. With both categories a higher level of stress was reported by women. The overall PSS-10 score for trial participants was (19.3\u00b16.9). However, the average score for women was 20.4\u00b16.6, compared to an average score of 15.7\u00b16.7 for men. Half of participants (54%) reported that prior to COVID-19 they had generally coped well with stress. 25% reported that they had some level of stress, anxiety or depression prior to COVID-19 restrictions, but had not received a formal diagnosis and 17% reported that they had previously been diagnosed with stress, anxiety or depression. Again, women were far more likely than men to report having been diagnosed with stress, anxiety or depression prior to COVID-19 (F = 19%:M14%) or have had some level of stress anxiety or depression but had not been diagnosed (F = 26%:M = 20%).p = 0.014, OR = 3.6), which was independent of their levels of stress prior to COVID-19. As the pandemic caused so many enforced changes to peoples\u2019 everyday life, one of the aims of this study was to see if any of these changes directly increased participants level of perceived stress. As reported in the descriptive statistics, moving to work from home was the highest reported change and was comparable for women and men. There were also notable differences between the genders in the numbers of changes they were affected by and some key categories such as caring from children and other dependents. A binary regression analysis was carried out which included all lifestyle changes, and gender, age and mental health prior to COVID-19. Surprisingly, the analysis did not show a significant link between any of the lifestyle changes and higher levels of perceived stress. However, gender and mental health prior to COVID-19 were both significantly linked to perceived stress during the pandemic \u03c72 = 58.95, p<0.001). Participants who had a prior diagnosis of stress, anxiety or depression were almost six times more likely to be in the higher stress category , confirming our hypothesis, with participants who had not been officially diagnosed, but reported some levels of stress, anxiety, or depression over three times more likely to be in the higher stress category . Females were around 4 times more likely to be in the higher stress category than males  diagnosis prior to the pandemic were almost six times more likely to be in the higher stress category (OR = 5.7). In addition, participants who had not been officially diagnosed, but reported some prior levels of stress, anxiety, or depression were also three times more likely to be in the higher stress category (OR = 3.6). Other studies have also reported prior mental health, stress and weight gain. Participants with a diagnosis of psychiatric illness or obesity prior to the pandemic were more likely to report declines in weight gain protective behaviours . Having R = 3.6), which was independent of their levels of mental health prior to COVID-19. Other published studies have also highlighted higher stress levels during COVID-19 in women than in men [Whilst this study was not specifically designed to investigate differences by gender, our results reported significant differences between women and men. Women reported higher levels of weight gain (F = 50%: M = 27%) and were twice as likely to have reported the highest weight gain category F = 10%: M = 5%). Women were more likely to have a mental health diagnosis prior to COVID-19  or have low mental health but not diagnosed . In addition, during the study they were also over 3 times more likely to be in the higher stress category than males  being within in the 18\u201355 age range. We believe, however, that the study results are valid, as the survey achieved adequate power . In addition, the results are in line with other similar larger published studies cited in this paper. With respondents being mainly young-middle aged adults, we recognise that this population is not fully representative of the UK adult population. However, as many of the COVID-19 imposed restrictions  were more likely to impact the working age population, we believe that with this study sample enabled a realistic evaluation the impact of the pandemic restrictions on people\u2019s lives.The primary aim of this study was to investigate the relationship between higher stress and weight gain for a wide range of factors. To enable us to do this using logistic analyses we categorised all factors. However, it is accepted that by using categorical rather than continuous measures for perceived stress scores we may have limited the ability to track smaller changes in lifestyle factors on stress levels, particularly as former studies indicate that females routinely report higher levels of stress \u201312. We aPerhaps the most important limitation of this work that it is a correlational study, so although we are proposing that higher stress leads to weight gain, it needs to be considered that weight gain could also be the driver of stress. We acknowledge that there are complex interactions between stress and weight gain, with different responses shown in females and males . HoweverIn summary, the results from this study corroborate previous research, that higher levels of stress are significantly linked to increased weight gain. The study also highlights significant differences in perceived stress and weight gain during the pandemic by gender and the role in mental health prior to the pandemic in weight gain. Further work is necessary to understand how stress reduction strategies could be incorporated into weight loss/maintenance approaches, including a greater awareness of individuals\u2019 stress levels and history of mental health when developing weight loss and maintenance strategies. A more gendered approach to weight loss may also be required, either to take into consideration differences in perceived stress levels between females and males or to determine if the role of stress in weight gain is a gendered factor.The study also reiterates the key role of food cravings in the link between stress and weight gain. Food cravings were linked to a 3 times increased likelihood of reporting weight gain, and to 6\u201311 times increased likelihood of snacking and consumption of high sugar and-processed foods. This strongly suggests that re-focusing weight loss methods on the ability to control cravings, possibly by using stress reduction techniques, will be a more effective approach to than the current focus on reduction of the downstream behaviours of increased snacking, and consumption of processed and high sugar foods.S1 Dataset(ZIP)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file."}
{"text": "Due to physical differences between the genders, it is hard to study whether women also vary from men in their competitive drive in sports. The Olympic committee instituted major rule changes in the sport of shooting in the Tokyo Olympic Games (2021), leveling the playing field. We explored performance in a myriad of competitions including newly established mixed-gender doubles events in this unique sport of very limited physical input. Men performed better than women in events which required \u201cdynamic\u201d qualities (following moving targets) but when the competitions were held most constant (rifle shooting with stationary targets indoors) and regardless of distance or posture, women performed equally to men exhibiting seemingly similar competitive drive. The last finding should propel the Olympic committee to fully integrate rifle events. In the broader sense, we find that when the playing field is even, the genders, at least among highly trained selective competitors compete equally. Sometime in the early 2000\u2019s Uri Gneezy and Aldo Rustichini conducted a very intriguing field study in an elementary school in Israel . The parNotwithstanding experimental concerns in regards to the criterion of who was assigned to the control vs. the experimental condition, time measurement , possiblAbundant of research in this field is still in agreement with the results of the original running study, arguing that when the stakes are especially high males show greater competitive tendencies, which lead them to superior performance over females. Three main categories of research make this claim. Laboratory experiments ,7, archiDue to the limiting circumstances of gender separation, which preclude direct comparisons, researchers need to show ingenuity in exploring gender differences in sport. Also of note is the type of sports studied whereupon some competitions are characterized as closed skill  ,16 such In tennis, an interactive sport, Paserman  studied Shifting to more closed and continuous type of sport, performance in New York City Marathon (2007\u20132014) was studied where female runners start the race 30 minutes earlier than their male runners, which leads some elite women to be eventually passed by the fastest men . It was Other research in gender differences in professional sports focused on financial rewards and their impact on performance \u201323. For Most closely approximating the sport of shooting, Duffy  in quasiSpecifically in rifle shooting, the focus of the current investigation, competitors perform in a highly controlled environment. The sport is characterized as closed in terms of skill execution where the environment is mostly predictable and static , with fiIn the same sport, an important rule change in 2018 was examined when the Olympic shooting protocols increased the number of women\u2019s shots from 40 to 60, making them equal to men . This stIn the current investigation, we endeavored to test this conclusion when the stakes are highest during the Olympics where contestants can win substantial sums of money  and sponIn the way of specific directional hypotheses, we predicted based on past research ,30 that This study was approved by the Ethics Committee of the Polytechnical University of Madrid. The participants of the study were 357 shooters of 101 countries. Of the total participants\u2019 number, two shooters were excluded because one was disqualified, and the other did not start the competition. The final sample was composed of 355 shooters with an average age of 29.9 \u00b17.65 years . The gender distribution was 178 women and 177 men.The qualification system for the mixed team events according to the ISSF rules is as follows: \u201cMixed Team entries may come from athletes entered in Mixed Team quota places obtained at the 2018 WCH Changwon, KOR and from double starters who are also entered in individual events.\u201d . In consequence, those athletes qualified in the mixed events during the world Championship in 2018 were directly classified for the Olympics in the mixed events. In addition, those shooters who qualified in the individual\u2019s events with an opposite gender mate who qualified independently in the same event, were able to join forces in the mixed event. For example, a male air pistol shooter and a female air pistol shooter who has qualified in the individual event could participate in the mixed event together. Lastly, it is important to note that the individual events were finished before the mixed events had commenced as to not undermine performance in the individual modalities. Shooters who qualified for the team competition hence increased their chances of winning a medal without jeopardizing their focal individual pursuit.See The participants took part in a total of 15 shooting modalities, five modalities for each weapon: pistol, rifle, and shotgun. Additionally, six shooting modalities were for men, six for women and three for mixed team modalities. Among the competitors, 95 men and 125 women were double starters, competing in two events. nd to August 2nd. The results were tabulated from the International Shooting Sport Federation (ISSF) webpage once the results became official .The data of the present study were collected during the Olympic Games Tokyo 2021 from July 22According to the new International Olympic Committee competition protocol, the number of shots allotted were matched and to balance the competition by gender. In addition, prone rifle, free pistol and double trap modalities were replaced by three mixed team modalities in the Tokyo 2021 schedule , in which both men and women competed together and simultaneously .As inclusion criteria, only those modalities in which men and women fired the same number of shots and under the same conditions, both individually and in mixed teams, were selected to be analyzed. Consequently, the modalities of rapid-fire pistol (only men) and sport pistol (only women) were excluded from the study.Additionally, the final rounds were excluded from the analysis due to the specificity of these stages\u2019 rules and the low number of participants , as they could entail insufficient statistical power.Regarding the qualification round structure, athletes fired 60 shots in sets of 10 shots for individual events of air pistol and air rifle (both men and women) while mixed events of air pistol and air rifle have a total 60 shots per team in sets of 10 shots, (each gender shot 30 shots). Additionally, 3x40 rifle event consist in 120 shots in sets of 10 shots divided 40 shots by position . On the other hand, clay target individual modalities of trap and skeet consist of 125 targets in sets of 25 shots (both men and women) while, in the mixed trap team event, each gender shot 75 targets for maximum score of 150 hits. See Performance were measured using the total points in the precision events and total number of hits in trap and skeet events according to the ISSF rules and regulations . Furthermore, to make possible the performance comparison between modalities with the same weapon but with different number of shots (for example: air pistol vs. air pistol mixed team and air rifle vs. air rifle mixed team) the average points per shot was calculated .Performance outcomes were measured using the total points (continuous score) in the precision events. More specifically, points with decimals in 10m Air Rifle and 10m Air Rifle Mixed Team and points without decimals in the rest of events. The total number of hits (a fraction score) was computed for the trap and skeet events and trap mixed team events according to the ISSF rules and regulations  see .M) and standard deviation (SD). The normal distribution of the variables was checked using the Kolmogorov\u2013Smirnov and Shapiro\u2013Wilk tests.The data are described by arithmetic mean (T tests used to contrast gender and competition type). In our main analysis, we utilized A two-way mixed ANOVA to analyze the differences between gender , the shooting event , and the interaction between them. Effect size with a 95% interval confidence was calculated using Cohen\u2019s d and partial eta squared. Three benchmark points were defined to indicate the effect size,  and partial \u03b72 = 0.01 small; \u03b72 = 0.06 medium; \u03b72 = 0.14 large. . All statistical analysis was conducted with SPSS version 28.For descriptive purposes, the cells means by gender and type of competition are presented in (t(87) = -3.1; p = 003; d = 0.60, IC95% ); skeet (t(56) = -2.89; p = 005; d = 0.76, IC95% (0.23\u20131.30)); trap (t(38.61) = -5.04; p < 001; d = -1.38, IC95% (0.80\u20131.98)). Contrary, no differences by gender were found in 10m air rifle nor 50m rifle 3 positions (p > .05). In order to increase confidence in the overall reliability of the results we also conducted a repeated bootstrapping procedure. The results remained the same for the t-tests with a simulation of 2000 samples and a 95% confidence interval.The comparisons between gender in the individual competitions showed that men shot better than women in 10m air pistol t(38) = -2.97; p = .005; d = 0.94 IC95% (0.29\u20131.59). No differences by gender were found in trap nor 10m air rifle (p > .05). Also, no differences were found between the individual and mixed competitions in any event by gender (p > 0.05). See In addition, the comparisons between gender in the mixed competitions showed that men perform better than women in10m air pistol  as the repeated measure was carried out. See F = 8.33; p = .007; partial \u03b72 = .19) with men (M = 9.59) performing better than women (M = 9.48). There was no main effect of type of event on shooting performance  = 1.32; p = .260; partial \u03b72 = .04), nor was there an interaction  = .73; p = .400; partial \u03b72 = .02), showing that women did not deteriorate further in the mixed events beyond their individual performance level.For the 10-meters pistol shooting events  gender has a main effect  = 6.29; p = .018; partial \u03b72 = .19) with men (M = 0.96) performing marginally better than women (M = 0.94). There was no main effect of type of event on shooting performance  = .007, p = .940, partial \u03b72 = .00), nor was there an interaction  = .21; p = .65; partial \u03b72 = .008), showing that women did not deteriorate further in the mixed events beyond their individual performance level.In the trap shooting events  a significant gender effect was found  = 1.06; p = .308; partial \u03b72 = .02). There was no main effect of type of event on shooting performance  = 1.36; p = .250; partial \u03b72 = .03), nor was there an interaction  = .17; p = .680; partial \u03b72 = .003), showing that women did not deteriorate in the mixed events in comparison to their individual performance level.Regarding the 10-meters air-rifle shooting events  our results showed no main effect for gender . The latter competitions are unique as they afford the most control (initiation of trigger pull and stationary targets indoors) as well as physical support (the rifle is held close to the trunk of the body and a support vest is sustaining the weight of the rifle). These findings lead us to the conclusion that when movement is kept minimal and regardless of distance or posture, women are not less competitive than men are, and they perform equally. This was true both when they compete in gender segregated events as well as in the mixed-team competition. It is important to note that by no means do we deem rifle events to be \u201ceasier\u201d or less demanding in comparison to other shooting events. Rifle elite shooting requires intense focus and sustained effort  as well However, the results should be also analyzed through a much broader lens extending beyond Olympic shooting competitions. They show that under unique and probably not easy to attain set of circumstances, highly trained experts of both genders compete equally. Still it seems that even minor barriers may cause uneven competitive environment. Coming full circle to Gneezy and Rustichini\u2019s  contemplA psychological analysis contrasting the \u201cmixed-double\u201d events with the individual competitions highlights several important dimensions . In the We choose to pursue our gender exploration based on average performance analysis since this approach was most robust and valid statistically allowing maximum power to test the hypotheses under the current sample size limitations. However, Olympic medal winning competitions (and other sports) are a case of outlier performance . In otheAlthough this is the first study that have analyzed gender performance differences comprehensively in all shooting modalities, general limitations should be pointed out such as the Covid 19 pandemic timing which disrupted practice and competition routines  as well More specifically, it is important to note that the number of shooters who participated in both, the mixed and in the individual competition were smaller than those who competed only in the individual or the mixed competitions, a dimension that could reduce our statistical power. Nevertheless, the results of this study corroborate earlier findings from the world championship event  and collTwo major consequences arise if indeed in a direct competition scenario, the genders perform equally. First, recent years highlighted the controversy around the participation of trans competitors in sports . This coWomen and men shooters performed separately but equally in the 2021 Tokyo Olympics in \u201cstatic\u201d rifle shooting modalities. Men were superior in \u201cdynamic\u201d  shooting events. In the newly formed \u201cmixed\u201d team events  these performance patterns were maintained and the mixed gender competitive environment did not impede women\u2019s performance beyond. Supported by earlier research ,30 we enS1 File(SAV)Click here for additional data file."}
{"text": "O. ficus-indica (prickly pear cactus) is an important forage and food source in arid and semiarid ecosystems and is the most important cactus species in cultivation globally. The high degree of apomixis in the species is a hindrance in plant breeding programs where genetic segregation is sought for the selection of superior genotypes. To understand if in ovulo embryo rescue could increase the proportion of zygotic seedlings, we compared the mature seed-derived seedlings with those regenerated from in vitro embryo rescue at 20, 25, 30, 35, and 40 post-anthesis days (PADs) in four Italian cultivars. The seedlings were classified as apomictic or zygotic based on molecular marker analysis using inter-sequence single repeat (ISSR) primers. Multiple embryos were recovered from all the cultured immature ovules, and plantlets were regenerated and acclimatized to the field post hardening, with success rates ranging from 62% (\u2018Senza spine\u2019) to 83% . The level of polyembryony differed among cultivars and recovery dates, with the highest being \u2018Rossa\u2019, producing 4.8 embryos/ovule at 35 PADs, and \u2018Gialla\u2019, the lowest, with 2.7 at 40 PADs. The maximum number of embryos observed within a single ovule was 14 in \u2018Trunzara bianca\u2019. ISSR analysis revealed that ovule culture at 35 PADs produced the highest percentage of zygotic seedlings in all the cultivars, from 51% (\u2018Rossa\u2019) to 98% , with a high genotype effect as well. Mature seeds produced much fewer seedlings per seed, ranging from 1.2 in \u2018Trunzara bianca\u2019 to 2.0 in \u2018Rossa\u2019 and a lower percentage of zygotic seedlings . Our research opens a pathway to increase the availability of zygotic seedlings in O. ficus-indica breeding programs through in ovulo embryo culture. Opuntia ficus-indica (L.) Mill.), also called cactus pear, Barbary fig, or nopal cactus, is a member of the Cactaceae family; it originated in central Mexico and is considered the most important cactus species in horticulture worldwide, with a global distribution , (AC)8YT (Ta 50.3 \u00b0C), (AG)8YT (Ta 50.3 \u00b0C), (GT)8YG (Ta 52.1 \u00b0C), and (CA)8RG (Ta 51 \u00b0C). To distinguish apomictic from zygotic seedlings, a genetic analysis based on ISSR analysis was performed, as previously described in detail  (145 DAP) [We attempted embryo rescue over five PADs periods from 20 to 40 days in four Italian cultivars with contrasting morphologies, and in all the cultivars, 35 PADs embryo rescue was the most successful in terms of the percentage of responsive ovules (80\u201395%) and the mean number of responsive embryos per ovule (4.4\u20135.3) in all four cultivars. In contrast, mature seeds produced very low numbers of seedlings per seed . Our method of acclimation and hardening of immature embryo-derived seedlings was efficient, and the success rate was from 62% (\u2018Senza spine\u2019) to 83% . The next step in our research was to identify the origins of the seedlings, and we used ISSR markers, which are highly efficient and reliable ,50,51. A145 DAP) .in vitro development of the zygotic and apomictic embryos present in the immature ovules. Interestingly, we observed that in some combinations, PGR stimulated callus formation and the production of adventitious embryos from the different tissues of immature ovules. On the contrary, the PGR-free medium allowed the regular development of the zygotic and apomictic embryos already present in the immature ovules without the production of callus and adventitious embryos. Therefore, the immature ovules used in the present study were incubated on PGR-free medium to facilitate the recovery of zygotic embryos. The in vitro protocol used in our research is simple, as it consists of only MS media supplemented with malt extract and sucrose. For in ovulo embryo rescue of Hylocereus interspecific hybrids, another cactus of horticultural significance, Cisneros and Tel-Zur [in vitro ovule culture, casein hydrolysate seems to be an essential ingredient. Thus, a reduced form of organic nitrogen seems to be essential for embryo growth in vitro, as also suggested by Sahijram et al. [In preliminary experiments (data not presented), different combinations of plant growth regulators (PGR) were added to the culture medium to stimulate the  Tel-Zur  used a c Tel-Zur  and gent Tel-Zur  in vitrom et al. .Opuntia spp., apomixis can occur mainly through sporophytic agamospermy [Opuntia [in vivo during the seed maturation processes is not known, and we are focusing on this aspect in our current research. Our hypothesis is that the numerous embryos of apomictic origin that are contained in the ovule  compete with the zygotic embryo by using the resources necessary for its development, causing its abortion. This hypothesis is substantiated by the increase in the proportion and number of zygotic embryos when the immature ovules are incubated on a culture medium providing sufficient nutrients, which enhances the chances of survival of zygotic embryos.It is known that in mospermy ,22,23, w[Opuntia . Howeverin ovulo embryo culture can increase the number of zygotic seedlings and their ratio to apomictic seedlings; therefore, this can play a significant role in crop improvement programs of apomictic O. ficus-indica involving hybridisation and selection in segregating populations.In conclusion, it can be stated that"}
{"text": "Endometrial cancers with p53 mutations tend to recur and develop metastases more frequently. From this point of view, the detection of new potential therapeutic targets such as HER2 is particularly interesting. In this study, we demonstrated that overexpression of HER2 (++ or +++) was present in 31.4% of p53-mutated endometrial carcinomas and that among these, 36.3% showed amplification of the HER2 gene by CISH (Chromogenic In Situ Hybridization). These results suggest that various anti-HER2 agents could constitute interesting future treatments in this type of cancer.p53-mutated endometrial carcinomas tend to recur and develop distant metastases. Therefore, the detection of new potential therapeutic targets such as HER2 is particularly interesting. In this retrospective study, which considered over 118 endometrial carcinomas, the p53 mutation was detected in 29.6% of cases. In these cases, the HER2 protein profile was studied via immunohistochemistry, and an overexpression of HER2 protein (++ or +++) was noted in 31.4%. The CISH technique was used in these cases to determine if gene amplification was present. In 18% of cases, the technique was not conclusive. Amplification of the HER2 gene was observed in 36.3% of cases and 36.3% of cases showed a polysomal-like aneusomy for centromere 17. Amplification was found in serous carcinomas, clear cell carcinomas and carcinosarcomas, highlighting the future potentiality of HER2-targeted therapies in these variants of aggressive carcinomas. More than 400,000 women worldwide were affected by endometrial cancer in 2020. The mortality rate of endometrial cancer is estimated to increase by 1.9% per year. The morbidity rate is higher in North America and Western Europe, which could be explained by a lifestyle marked by risk factors for endometrial cancer such as obesity [In 2020, endometrial cancer was the fourth most common tumor in Europe with an incidence of 12.9 to 20.2 per 100,000 women. It is the most frequent gynecologic cancer in Europe [It is established that a high body mass index (BMI) is a modifiable risk factor for endometrial cancer, and it is a more important risk factor for endometrial cancer than for any other cancer .Obesity is a risk factor for endometrial cancer because adipocytes convert androgens into estrogens and estrogens stimulate endometrial proliferation and therefore potentially the development of hyperplasia and cancer. In addition, hyperglycemia and insulin resistance, which are associated with obesity, promote IGF-1 signaling abnormalities, leading to increased cell proliferation [Initially, endometrial cancers were classified into two categories: type I, which represents approximately 80% of cases with a hormone-dependent profile, and type II for the remaining 20% of cases presenting a hormone-independent profile and a poor prognosis .The Cancer Genome Atlas of molecular analyses for different histologic types of endometrial cancers reveled four tumoral subtypes, each associated with a different survival profile and of great interest for prognosis, namely: POLE -mutated tumors; MMR (Mismatch Repair)-stable tumors; MMR-instable tumors; and p53-mutated tumors .This new classification, according to the ESGO , ESTRO (European Society for Radiotherapy and Oncology) and ESP (European Society of Pathology) 2020 recommendations, allows some modulations of the therapeutic schemes, adding to its therapeutic prognosis value . TherefoP53-mutated tumors are classically associated with more aggressive tumor histological types and a pejorative prognosis, such as high-grade endometrioid carcinomas, serous carcinomas and carcinosarcomas .The p53 protein is normally involved in the preservation of the cellular genome by blocking the replication of cells containing genomic alterations. It is a beneficial element at the cellular level; therefore, one can understand how important it is for this element to stay active. If a mutation occurs, efficiency decreases or is totally annihilated according to the mutation type. This protein is found to be mutated in 2/3 of cancers, preventing apoptosis when genetic abnormalities are present and thus encouraging cancer survival. On the endometrial cancers\u2019 side, this mutated protein is found in 20 to 40% of cases. One of the characteristics of the mutated p53 protein is that its half-life is increased, and therefore it can be detected by immunohistochemistry .The p53 protein \u201cwild type\u201d has a short half-life and is rapidly degraded after activation; thus, it is kept at low levels in the cell. This mechanism is mainly due to MDM2 and MDM4, which decrease the transcriptional activity of the p53 protein, in particular by making it the target of degradation by proteasomes. When the p53 protein is mutated, this cascade is broken either because the mutated p53 protein does not have the ability to activate MDM2 or because MDM2 becomes a stimulator of p53 protein transcription.In both cases, the p53 protein accumulates in the cell and thus becomes detectable via immunohistochemistry .(a)\u201cNul type\u201d: complete absence of immunohistochemical detection of the protein in tumor cells .(b)\u201cWild type\u201d: nuclear immunohistochemical reactivity of low and/or moderate and/or diffuse intensity detected in less than 80% of tumor cells.(c)\u201cMutant\u201d: strong and diffuse immunohistochemical reactivity detected in 80 to 100% of cells.(d)\u201cCytoplasmic\u201d: significant cytoplasmic reactivity that is associated with variable nuclear reactivity but does not reach the high and diffuse detection threshold.The immunohistochemical profile of the p53 protein in endometrial carcinomas has been classified into four patterns :(a)\u201cNul The histochemistry of the p53-mutated protein is of interest, but we will refrain from going too much in depth on the subject since it is not the aim of this study.As p53 mutations in tumors tend to recur more frequently and develop distant metastases within five years in 50% of cases, it is of great interest to determine the presence of potential targets for future targeted therapies, which be of some benefit in the treatment of these varieties of endometrial cancer, whether or not they are targeted in conventional radio/chemotherapy .In particular, it has recently been suggested that overexpression of the HER2 protein may be observed in approximately 25\u201330% of endometrial serous carcinomas, which are typically p53-mutated and therefore could constitute a potential therapeutic target  ,11.HER2 is a transmembrane receptor involved in cell proliferation. When the HER2 receptor is activated, it stimulates signaling pathways that lead to oncogenic transformation through cell survival, proliferation, angiogenesis and metastasis. HER2 has no known ligand and can therefore be constitutionally activated. In addition, when HER2 is overexpressed, it forms heterodimers with other members of the HER family, inducing particularly important downstream oncogenic signaling. HER2 overexpression is used in breast cancer to select patients who may potentially respond to targeted therapy with an anti-HER2 agent; this concerns 15 to 30% of breast cancers. Targeted anti-HER2 therapies have significantly improved the prognosis of breast cancer patients .The aim of this study was to assess HER2 protein overexpression and the secondary gene\u2019s amplification using CISH (Chromogenic In Situ Hybridization)-improved NGS (Next-Generation Sequencing) p53-mutated endometrial carcinomas to determine if this could be a good approach for future studies on new targeted therapies to add to the therapeutical panel of endometrial cancer.-The MMR and the p53 proteins analyzed by immunohistochemistry: MLH1 (MutL Homolog 1) ; MSH2 (MutS Homolog 2) ; MSH6 (MutS Homolog 6) ; and PMS2 (Postmeiotic Segregation 2)  and p53  .-Immunostaining was performed using a fully automated immunohistochemical system .-\u00ae Fluorometer in combination with the Qubit\u00ae dsDNA HS Assay Kit . NGS was then performed in order to sequence hotspot mutations in the following panel of genes , the database of the Department of Pathology was studied. This database lists every gynecological case analyzed by the Department of Pathology. The cases concerning breast pathologies, ovarian pathologies, vulvar pathologies and cervix pathologies were not retained. Every endometrial case between 2019 and 2022 was analyzed to finally extract endometrial cancers meeting the following criteria:Once this selection was achieved, 118 cases between 2019 and 2022 were included in this series.First, the proportions of histological types in the series were determined according to the WHO 2020 classification: low-grade endometrioid carcinomas, high-grade endometrioid carcinomas, serous carcinomas, clear cell carcinomas and carcinosarcomas; finally, other extremely rare histological types, such as neuroendocrine tumors, were excluded.Next, the molecular profile protocoled via NGS was analyzed. The cases were classified according to the 4 tumoral subtypes identified by The Cancer Genome Atlas: pole-mutated; MMR-stable; MMR-instable; and p53-mutated.The pole-mutated and MMR-instable tumors were excluded. Indeed, 8% of endometrial carcinomas with microsatellite instability and a third of Pole-mutated carcinomas are also carriers of a mutation in the TP53 gene .However, it seems that these mutations are events encouraged by this initially present microsatellite instability and therefore do not affect the molecular profile or phenotype of these tumors because these mutations affect sequences of the TP53 gene that do not impact the function or expression of the p53 protein. These cases of unstable MMR tumors with a mutation of the added TP53 gene also have a better prognosis than tumors with a mutated P53 profile .For the remaining cases with a TP53 mutation detected via NGS, the HER2 protein profile was studied.The slides were again reviewed by two experienced pathologists in order to classify the cases according to their immunohistochemical classification, as seen in  and FiguTo finalize our description, in the cases with an immunohistochemistry \u201c++\u201d or \u201c+++\u201d, to determine if a gene was amplified, CISH (Chromogenic In Situ Hybridization) was performed . The VenA total of 68.64% of cases were low-grade endometrioid cancers; 10.17% were high-grade endometrioid cancers; 214.41% were serous cancers; 4.24% were clear cells cancers; and 2.54% were carcinosarcomas .Concerning the 118 cases included in the study , they weA total of 5.93% of cases had a Pole-mutated profile; 40.68% had an MMR-stable profile; 23.73% had an MMR-instable profile; and 29.66% had a p53-mutated profile .The 35 cases with a p53 protein mutation were classified according to their histological type. Altogether, 22.86% were low-grade endometrioid cancers; 11.43% were high-grade endometrioid cancers; 48.57% were serous cancers; 14.28% were clear cells cancers; 8.57% were carcinosarcomas .Concerning the 81 low-grade endometrioid cancers in our series, 9.88% were p53-mutated; of the 12 high-grade endometrioid carcinomas, 33.33% were p53-mutated; of the 17 serous cancers, 88.24% were mutated; of the 3 clear cell cancers, 66% were mutated; and of the 3 carcinosarcomas, 100% were mutated .A total of 88.24% of the 17 serous endometrial cancers of this series were mutated for the p53 protein; 5.88% were MMR-stable and 5.88% were MMR-instable .Concerning the cases in this study that had a TP53 gene mutation detected using NGS, 1 of them was unavailable and the other 34 were analyzed by two experienced pathologists. Using the criteria previously described, 38.24% had a \u201c0\u201d profile for HER2 protein immunohistochemistry expression; 29.41% had a \u201c+\u201d profile; 23.53% had a \u201c++\u201d profile; and 8.82% had a \u201c+++\u201d profile .For the 11 cases in the series that had an immunohistochemistry profile corresponding to \u201c++\u201d or \u201c+++\u201d for the HER2 protein, 9 were analyzable. Four of them presented an amplification for the HER2 gene; four others presented an aneusomy, which looked like a CEP17 polysomy; and one case was not amplified.With regard to the four cases presenting an amplification of the HER2 gene, 50% were clear cell carcinomas; 25% were carcinosarcomas and 25% were serous cancers . The ampOf the four other cases presenting an aneusomy for CEP17 (polysomy-like), 75% were serous cancers and 25% were carcinosarcomas . The p53We first aimed to answer: Is this series sufficiently representative of the distributions found in other series in order to be able to draw conclusions?Regarding the proportions of histological types and molecular profiles, they followed the trends published in the literature; the endometrioid type affected 80% of patients while the others types of non-endometrioid carcinomas corresponded to 20% of tumors, with this group presenting mainly serous carcinomas . A literThe second question is as follows: Is HER2 amplification worth studying in the future to add some new targeted therapies to the therapeutical panel of the p53-mutated endometrial cancer?A randomized clinical trial that began in 2011 and is currently in phase II (NCT01367002) is investigating the therapeutic response of stage III or IV or recurrent serous carcinomas to carboplatin/paclitaxel compared to carboplatin/paclitaxel to which trastuzumab, an anti-HER2 agent, is added . The firCurrently, it is recommended HER2 gene amplification be tested for only in serous histological types of endometrial cancer, which is considered the most aggressive histological type of endometrial cancer. However, Ross et al., in their retrospective study in 2021, report that in 94% of cases of endometrial cancers with amplification for the HER2 gene, there is a mutation of the TP53 gene as well . This TPMoreover, Ross et al. have observed at least one case of amplification of the HER2 gene in all histological types of endometrial cancer . InsteadRegarding the 21.57% of cases of mutated P53 tumors presenting a polysomal-like aneusomy for centromere 17, this is a phenomenon already described in breast cancers for which the clinical impact and effects on the expression of the HER2 protein are still uncertain .A recent study showed that polysomy of centromere 17, without amplification of the HER2 gene, would be associated with a higher immunohistochemical expression of the HER2 protein, corresponding to \u201c++\u201d/\u201d+++\u201d scores . TherefoAgain, the data reported in the literature diverge. However, Hofmann et al., in 2008, had already described two cases of breast cancers overexpressing the HER2 protein in the presence of polysomy of centromere 17 and which presented a therapeutic response to trastuzumab . MoreoveOf course, all these data concerning HER2 expression and/or amplification in endometrial cancers must be tempered, firstly, because endometrial tumors are often large (>30 mm) and HER2 expression/amplification may be heterogeneous. However, it has been shown that in other cancers , this heterogeneity can affect not only the prognosis of these carcinomas but also their response to the various HER2 therapies. In addition, most of the clinical studies that tested the different anti-HER2 therapies are only based on the immunohistochemical score and do not consider HER2 amplification. Finally, in breast cancer, tumors considered HER2-negative (+ or ++ without HER2 gene amplification) may still respond to new HER2 therapies. Finally, in breast cancer, tumors that are considered HER2-negative may still respond to new HER2 therapies .In this study, we have demonstrated that overexpression of HER 2 (++ or +++) was present in 31.4% of p53-mutated endometrial carcinomas and that among these, 36.3% showed HER2 gene amplification by CISH. This overexpression and gene amplification are not restricted to serous carcinomas but were also found in clear cell carcinomas and carcinosarcomas. These results suggest that various anti-HER2 agents could constitute interesting future treatments in this type of cancer. Naturally, our findings should be confirmed by larger studies in the future. Furthermore, in the era of personalized medicine, we advocate for the continuation of studies on targeted anti-HER2 therapies for cases with HER2 amplification. In addition, their extension to endometrial carcinomas with polysomal aneusomy for centromere 17 could be proposed, following studies already initiated for breast cancers."}
{"text": "ACCs). In addition, the expression of FLNA was associated with a less aggressive tumour behaviour in ACCs. Indeed, the subgroup of ACCs with high FLNA showed a lower ENSAT stage, Weiss score, and S-GRAS score compared to ACCs with low FLNA expression (p < 0.05). Moreover, patients with high FLNA had a longer overall survival than those with low FLNA (p < 0.05). In conclusion, our data suggest that FLNA may represent a \u201cprotective\u201d factor in ACCs, and the integration of FLNA immunohistochemical expression in ACC tissues along with other clinical and molecular markers could be helpful to improve diagnostic accuracy and prognosis prediction in ACCs.The insulin-like growth factor 2 (IGF2) promotes cell growth by overactivating the IGF system in an autocrine loop in adrenocortical carcinomas (ACCs). The cytoskeleton protein filamin A (FLNA) acts as a repressor of IGF2 mitogenic signalling in ACC cells. The aims of this study were to test FLNA expression by immunohistochemistry in 119 ACCs and 26 adrenocortical adenomas (ACAs) and to evaluate its relationship with clinicopathological features and outcome in ACCs. We found that 71.4% of ACCs did not express FLNA, whereas FLNA absence was a rare event in ACAs (15.4%,  Adrenocortical tumours affect 3\u201310% of the population and include a variety of diseases ranging from frequent benign adrenocortical adenomas (ACAs) to rare and generally aggressive adrenocortical carcinomas (ACCs). The differential diagnosis between ACAs and ACCs is based on the Weiss score, which is composed of nine histopathological items  ,3.The prognosis of ACCs is generally poor, and the median overall survival (OS) ranges between 3 and 4 years . HoweverFrom a pathogenic perspective, several mechanisms have been demonstrated to be involved in adrenal tumorigenesis, including altered regulation of the cell cycle and activation of the insulin-like growth factor (IGF) system . Indeed,The cytoskeleton actin binding protein filamin A (FLNA) has recently been demonstrated to play an important role in negatively regulating the IGF system in ACC cells . FLNA isIn ACC cell line H295R, FLNA acts as a specific repressor of the IGF2-induced signalling cascade that promotes cell proliferation through extracellular signal-regulated kinases (ERKs) activation. Indeed, genetic silencing of FLNA induced an increase in the insulin-like growth factor 1 receptor (IGF1R) expression and signalling in H295R and ACC primary cultured cells and potentiated cell responsiveness to the IGF1R-insulin receptor (IR) inhibitor linsitinib . WesternThe present study aimed to test the expression of FLNA in a large, multicentre, and well-characterized cohort of ACC and ACA tissues by immunohistochemistry and to test its relationship with the clinicopathological features and outcome, with the final purpose of assessing its relevance as a biomarker to improve the diagnostic accuracy and/or prognostic classification in ACCs.n = 119, In the ACC group n = 119, , most ofIn total, 85.1% of patients received mitotane postoperatively, and 50% of these were also treated with systemic chemotherapy . Data abn = 26, In the ACA group n = 26, , we obsep < 0.05 and p < 0.001, respectively).Comparing the ACA and ACC groups, we found a higher age at diagnosis and a prevalence of patients with single hormone secretion in ACA vs. ACC . Representative pictures are shown in In tumours with any degree of FLNA expression, positivity was observed mostly in a very heterogeneous fashion. In FLNA-positive tumours, the immunoreactivity score (IRS) was 1 in 31/34 ACCs, while the remaining three ACCs showed a FLNA IRS of 2, 3, and 4, respectively. In total, 18 ACCs showed a percentage of positive cells \u22645%, 13 samples showed a percentage included from 6% to 30%, 2 from 31% to 60%, and 1 from 61% to 100%.p < 0.001 compared to ACC, In contrast, 22 out of 26 of ACAs (84.6%) were positive for FLNA (p = n.s.) . FLNA was localized in the cytoplasm in all positive samples, and in a subgroup of tumours, a plasma membrane localization was also observed  A.p < 0.05 vs. low FLNA) (p < 0.05 vs. low FLNA). In addition, given that the Weiss score is a multiparameter scoring system based on nine histological criteria, we sought to better understand which of them was associated with FLNA expression. We found a reduced mitotic rate in the group with high vs. low FLNA (p < 0.05) (p < 0.05). Accordingly, in the high FLNA group, the number of patients with an S-GRAS score higher than 3 was significantly lower than the low FLNA group (p < 0.05)  from the ACCs with a percentage of positive cells higher than 5% . We found that the ENSAT stage and Weiss score were significantly lower in the high FLNA group (ow FLNA) . Indeed, < 0.05) . Moreove < 0.05) .No association was found between the high or low FLNA group and the other clinicopathological features analysed  .Regarding FLNA intracellular localization, the subgroup of ACCs showing a plasma membrane FLNA immunopositivity did not present any significant difference in the clinicopathological features compared to the subgroup of ACCs with cytoplasmic FLNA.Lastly, no association between FLNA staining and clinical data in the ACA cohort was found.p < 0.05)  (p = 0.08)  . No diff = 0.09)  and adju = 0.08) .No association was found between the intracellular localization of FLNA and the clinical outcome.The present study indicates that FLNA expression is lost in the majority of ACCs but not in ACAs, and high FLNA expression in ACCs is associated with a less aggressive tumour behaviour compared to low FLNA.By analysing FLNA immunoreactivity in 119 ACCs, we found that FLNA was completely absent in 85 samples (71.4%). An opposite situation was found in ACAs, where FLNA was expressed in 22 out of 26 samples. These observations are in line with previously published data analysing FLNA protein expression by Western blot in a small group of ACC patients , breast The observation that FLNA absence is typical of ACCs, while it is a rare event in ACAs, suggests the possible use of FLNA expression as an immunohistochemical marker to discriminate ACCs from ACAs in those situations where histologic differentiation could be challenging ,35,36. MIn ACCs, the sole presence of FLNA was not associated with any clinicopathological features. However, we observed that the group of ACCs considered positive for FLNA expression included tumours with a very low percentage of immunopositive cells. We hypothesized that the effect of FLNA in these tumours could be negligible. We thus divided ACCs based on the percentage of FLNA-positive cells less than or equal to 5%  from ACCs with a percentage of positive cells higher than 5% (\u201chigh FLNA\u201d). We found that the small group of high FLNA ACC displayed less aggressive features, with a lower ENSAT stage, Weiss score, and S-GRAS score, and longer survival than ACCs expressing low FLNA levels. Moreover, we demonstrated that FLNA expression was associated also with the individual Weiss parameter mitotic rate, with a lower number of mitoses in the high FLNA group. This finding agrees with the previously demonstrated role of FLNA in decreasing IGF2 mitogenic effects in ACC cells through a reduction in ERK phosphorylation . To noteInterestingly, we found that high FLNA ACC patients showed a longer OS compared to low FLNA ACCs. However, in the multivariate survival model performed with FLNA adjusted for S-GRAS, we observed only a tendency toward the protective role of FLNA, the lack of significance being probably due to the small sample size. Moreover, it is important to note that after adjustment for the ENSAT tumour stage and Ki67, even if not significant, the FLNA protective hazard ratio did not change (HR = 0.2).In the literature, the role of FLNA expression in the OS of patients varies according to the type of tumours. High levels of FLNA were associated with longer OS in colorectal cancer , while iWe can hypothesize that in the absence of FLNA, IGF2 mitotic effects are potentiated, leading to a final worse outcome in ACCs. Indeed, we previously demonstrated that FLNA is able to bind IGF1R, and FLNA silencing induced a reduction in the IGF1R expression and a simultaneous decrease in ERK phosphorylation, cyclin E1 expression, and cell proliferation in human ACC cell line H295R and in primary cultured ACC cells , supportOur previous and current findings suggest that FLNA may represent an alternative drug target along the IGF2/IGF1R pathway, possibly overcoming the failure of IGF1R inhibitors in clinical trials. Therapeutic approaches aimed to restore FLNA expression could represent a novel strategy in the treatment of ACCs.Admittedly, the evaluation of FLNA expression detected by immunohistochemistry does not take into account posttranslational modifications that are crucial in determining FLNA activity. Since one of the main mechanisms that control FLNA functions and proteolysis is phosphorylation on Ser2152 ,42,43, fIn conclusion, the present study indicates that FLNA is absent in the majority of ACCs but not in ACAs, and high FLNA expression is associated with a less aggressive tumour behaviour compared to low FLNA in ACCs. Overall these data suggest that FLNA may be a cancer suppressor factor in adrenocortical tumours. The integration of FLNA expression assessment with other clinical and molecular markers will be helpful to improve both the diagnostic accuracy and prognosis prediction in ACCs.www.ensat.org). In total, 119 patients, who underwent adrenalectomy because of ACCs, were included in the study, i.e., 74 patients from the University Hospital of Wuerzburg (Germany), 29 from the University of Florence , and 16 from Laiko Hospital Athens (Greece). Moreover, 26 tissue sections derived from patients with histological diagnosis of ACA that had surgery in Foundation IRCCS Ca\u2019 Granda Ospedale Maggiore Policlinico were analysed.This is a retrospective European multicentre study conducted on behalf of the ENSAT  status, tumour-related hormone excess at diagnosis, symptoms, date of last followup, and status of patient  were recorded. The S-GRAS score for the ACC samples was calculated as previously published , resultiMoreover, a database for ACA patients with the following data were created: sex, age, date of diagnosis, histopathological characteristics, tumour-related hormone excess, date of last followup, and status of patient.The study was approved by the local ethics committees, and each patient gave written informed consent to the use of their tumour sample and clinical information.Slides of 4 \u00b5m thickness were obtained from representative formalin-fixed paraffin-embedded (FFPE) blocks of adrenocortical tumours (119 ACC and 26 ACA). Particularly, only primary tumours were investigated.Immunohistochemical staining with anti-FLNA antibody  was performed using an automated stainer , after EDTA buffer retrieval and via diaminobenzidine revelation . The internal positive control for proper staining was represented by stromal cells. The following parameters were assessed by 2 independent investigators: percentage of positive tumoral cells; pattern of staining ; intensity of staining. Discordant results were reviewed and discussed among the 2 investigators until a consensus was reached. Immunoreactivities were graded manually using an optical microscope  according to an immunoreactivity score (IRS) that takes into account both the percentage of positive cells  and the staining intensity , with a final score being obtained by multiplying both parameters together (range 0\u20139). Representative scans were captured via an Aperio AT-2 scanner . Tissues with FLNA staining >5% of positive cells were considered \u201chigh FLNA\u201d, whereas those with FLNA staining \u22645% of cells were considered \u201clow FLNA\u201d.Continuous variables were reported as the mean \u00b1 standard deviation (SD) or median and IQR, according to their distribution, and were compared using parametric or non-parametric tests, respectively. Discrete variables were described as numbers and percentages and compared through the chi-square test or Fisher\u2019s exact test, when appropriate. The Kaplan\u2013Meyer method was used to describe the OS and PFS according to FLNA expression and different patient characteristics. The log-rank test was used to test the difference in survival across groups. The prognostic role of FLNA was also assessed in multivariate analysis according to the Cox regression model. We evaluated the potential prognostic role of FLNA adjusted for the S-GRAS score since it was recently demonstrated to have a superior prognostic performance respect to tumour stage and Ki67 in ACCs . MoreoveStatistical analyses were carried out using the SAS software, version 9.4 , and IBM SPSS statistics, version 28.0.1.1 ."}
{"text": "This study systematically reviewed all human longitudinal exercise interventions that reported changes in the gut microbiota; frequency, intensity, duration and type of exercise were assessed to determine the influence of these variables on changes to the gut microbiota in both healthy individuals and clinical populations (PROPERO registration: CRD42022309854). Using PRISMA guidelines, trials analysing gut microbiota change with exercise interventions were included independent of trial randomisation, population, trial duration or analysis technique. Studies were excluded when microbiota abundance was not reported or when exercise was combined with other interventions. Twenty-eight trials were included, of which twelve involved healthy populations only and sixteen involved mixed or clinical-only populations. The findings show that participation in exercise of moderate to high-intensity for 30\u201390 min \u22653 times per week (or between 150\u2013270 min per week) for \u22658 weeks is likely to produce changes in the gut microbiota. Exercise appears to be effective in modifying the gut microbiota in both clinical and healthy populations. A more robust methodology is needed in future studies to improve the certainty of the evidence. The human gut microbiota has been implicated in numerous aspects of health and wellbeing, including disease risk and healthy aging ,2,3,4. TCross-sectional studies have reported that sedentary and active populations have different gut microbiota characteristics . Both Ban = 25) [n = 7) [n = 38) [The earliest systematic review examining a relationship between exercise and the microbiota that included some human data was published in 2019; only five of the 25 included studies reviewed in this paper included human data . In 2020n = 25) , older a [n = 7)  or exclu[n = 38) . While tThis systematic review was conducted and is reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . The revOn 21 February 2022, five databases were systematically examined by A.N.B: PubMed, Scopus, SportDiscus, CINAHL, and EMBASE. Searches were limited to full-text articles published in the English language in peer-reviewed journals. Key search terms included: \u2018gastrointestinal microbiome\u2019, \u2018microbiome\u2019, \u2018microbiota\u2019, \u2018exercise\u2019, \u2018physical activity\u2019, \u2018adult\u2019, \u2018human\u2019 and \u2018NOT animal\u2019  design: randomised controlled trials, cohort studies and case-control studies; (ii) population: adults aged 18 years and older; (iii) intervention: any frequency, intensity, time or type of physical activity; (iv) control: comparison group receiving a different physical activity prescription, control group not receiving the intervention at any time point during the trial, waitlist control or crossover group or no comparison/control group; and (v) outcome: faecal analysis of microbiota diversity and/or microbiota taxonomy. Any study that also incorporated strategies that may have influenced the outcome , where the effects of physical activity could not be isolated, was excluded.Covidence was used for screening the titles and abstracts of articles identified through the search process. Duplicate removal was automated, and articles were screened by A.N.B. and R.E.W. to exclude those outside of the scope of the review. Following the screening, full-text articles were retrieved and independently assessed by A.N.B. and R.E.W. for eligibility according to the outlined inclusion criteria. Where discrepancies in article eligibility were identified, eligibility was discussed in the research group, with an independent assessment made by a third blinded arbiter if a consensus was not achieved (T.L.S.). Reference lists of eligible articles were examined to locate potential additional studies that met the inclusion criteria.Study details, including the participant characteristics, exercise and control group prescriptions and outcome measures, were extracted independently by two authors (A.N.B. and R.E.W.). Where the assessment of microbiota diversity and/or microbiota taxonomy occurred at more than one time point during an intervention, the pre- and post-intervention outcomes were extracted, and other intra-intervention values were omitted. Participant characteristics and group classification were dependent on only one group; if a study included a group that was of clinical interest, this study was analysed with the perspective of this clinical group compared to the apparently healthy group included.The methodological quality of included articles was independently assessed by A.N.B. and R.E.W. Randomised controlled trials meeting inclusion criteria were assessed for methodological quality using a six-item derivation of the nine-item Delphi list developed by Verhagen et al. Three of the nine Delphi criteria  were deemed not to be appropriate for all types of included interventions. All criteria were equally rated using a \u2018yes\u2019 (1 point), \u2018no\u2019 or \u2018unclear\u2019 (0 points) answer format, with a quality score generated as a percentage of the maximum score for each included study. Non-controlled trials meeting the inclusion criteria were assessed using the Delphi, acknowledging that these would receive a lack of score for randomisation. Any discrepancies in methodological quality ratings were mediated by a third arbiter (T.L.S.). The results of the quality analysis can be found in r = x) and how many studies (including those reporting change) likely were able to observe changes to this level but did not report a change within the bounds of the variable . For instance, the statement \u2018increases seen in Example bacterium \u2019 should be interpreted as, of the studies including this variable, two of the four studies (50%) reporting this level of taxonomy found an increase, and thirteen studies (including the four listed) potentially had the capacity to observe a change, but it was not reported, suggesting no change was reported. This approach was used unless the review is comparing the frequency of reporting a microbe in response to a single variable. In this case, the only number reported (nr) would be compared to the number that likely could assess this change  within this variable, and this would only be reported once in each statement, e.g., Example was more commonly reported in x interventions  compared to y intervention (nr = 1). Taxa that were reported in all studies likely to be capable of assessing this change in relation to the variable investigated were reported simply as (n = x).Results were analysed and reported using a combination of quantitative, descriptive and narrative data synthesis. All reported changes in the gut microbiota of studies included were collated with study and intervention characteristics by reports of increase, decrease or no reported change. Thereafter, summaries of reported changes in the gut microbiota across the included literature were produced . Most clinical populations were characterised by metabolic disorders  [n = 1, 6%) [n = 1, 6%) [n = 1, 6%) [n = 1, 6%) [n = 1, 6%) [Participant characteristics are shown in studies) ,41,43,44= 1, 6%) , autoimm= 1, 6%) , inflamm= 1, 6%) , myalgic= 1, 6%)  and elde= 1, 6%) .n = 19) [Details of the exercise interventions are summarised in n = 19) ,41,42,44n = 19) ,39,43,45n = 19) . Three sn = 19) ,33. Anotn = 19) ,26,36,41n = 19) ,41,45, nn = 19) ,27,31,44n = 19) ,36,42,43n = 19) ,41. Of tn = 26), whole genome shotgun sequencing (n = 5) and one study [n = 9) except for two studies, one of which collected fourteen samples per participant (every two weeks) and the other collected twenty-eight samples per participant (two samples per week)Stool samples were sequenced with 16S rRNA gene amplicon sequencing (ne study  did not n = 6) compared to aerobic-only interventions . Beta diversity was reported to increase in 83% of concurrent trials (n = 6) compared to 42% of aerobic-only interventions (n = 12). Aerobic-only exercise interventions were the only interventions to report Roseburia and Lachnospira, where increases were reported in  and  of studies, respectively. Dorea abundance was reported to increase more often in response to aerobic interventions  compared to concurrent exercise interventions (n = 1). Similarly, aerobic interventions reported a higher abundance of Ruminococcus more commonly than concurrent interventions . Changes in Bifidobacterium, Veillonella and Akkermansia were similar between intervention types .Concurrent exercise interventions  more commonly reported increases in measures of alpha diversity , while 45% of studies examining changes in beta diversity reported a higher diversity (n = 11). Two studies found an increase in Bacteroides, with another reported no change . A total of 50% of studies reporting Faecalibacterium changes used low-to-moderate intensity exercise . At the level of species, Faecalibacterium prausnitzii was not found to change in two studies, though a third did report an increase . This intensity of exercise included 66% of studies reporting an increase in Prevotella copri .Thirteen studies used low-to-moderate or moderate-intensity exercise in at least one study arm ,40,41,45n = 6), while 75% of studies reporting beta diversity found a positive change (n = 4). At the level of phylum, consistent increases in Firmicutes were seen in all studies that reported this level of taxa . Similarly, decreases in Bacteroidetes abundance were found in studies reporting at this level . At the level of family, 67% of studies reporting improvements in Lachnospiraceae and Ruminococceae used moderate-to-high-intensity exercise . At the level of genus, moderate-to-high and high-intensity exercise included, most studies that reported a positive change in Dorea , Bifidobacterium , Ruminococcus  and Akkermansia  and 50% of studies reported improvements in Veillonella . The only two studies reporting an increase in Streptococcus utilised this exercise intensity . Two of three studies reporting Roseburia at this exercise intensity found a decreased relative abundance . Both studies reporting Bacteroides at this intensity reported a decreased abundance of this genus . Conflicting data were found for Lachnospira , Faecalibacterium , Prevotella  and Collinsella . At the level of species, both studies reported an increase in Ruminococcus bicirculans and utilised moderate-to-high intensity exercise .Nine studies used moderate-to-high and high-intensity exercise ,27,31,44n = 4), while three of the four studies reported an increase in beta diversity (n = 4). Three studies reported at the level of genus, and two studies of these reported an increase in Bifidobacterium (n = 2). There were conflicting data for Roseburia; one study reported increases in Dorea, Ruminococcus and Veillonella abundance and a decrease in Prevotella. None of the HIIT studies reported changes at the level of species.Four studies utilised HIIT ,36,42,43n = 2) or beta diversity (n = 1) when using maximal effort or SIT protocols. One study utilising maximal intensity and the study utilising SIT found that Lachnospira abundance increased. Otherwise, no consistent changes were observed. One study that included maximal exercise efforts found increases in Ruminococcus and Romboutsia.Three studies utilised SIT  or maximn = 13), most did not find a change in alpha diversity; only one study reported an increase . A total of 58% of studies reporting beta diversity found an increase (n = 12). At the level of genus, studies that involved two to three training sessions per week included 50% of studies reporting increases in Lachnospira , 60% of studies reporting improvements in Bifidobacterium , 67% of studies reporting improvements in Roseburia  and both studies reporting improvements in Oscillospira .Of those studies that used a training frequency of two to three sessions per week (n = 5) reported an increase in alpha diversity in 33% of studies reporting this metric (n = 3) and 67% for those reporting changes in beta diversity (n = 3). No consistent changes were seen at the level of genus.Studies with a training frequency of four to five sessions per week (n = 4) found an increase in alpha diversity in 67% of studies reporting this change (n = 3). However, there was no change in beta diversity (n = 1). No consistent changes have been reported at the level of genus, though Prevotella copri was found to increase in two of the studies .Studies with a training frequency of greater than five sessions per week (n = 6).Interventions that did not specify exercise frequency or involved less than one training session a week were not included in the frequency analysis (n = 13) reported improvements in beta diversity compared to shorter interventions of 0% (n = 4). At the level of genus, Roseburia decreased more commonly in interventions that lasted less than one week (n = 2), compared to a longer duration intervention (n = 1). All improvements in Roseburia occurred in response to interventions lasting more than four weeks , while Lachnospira was reported to increase in studies lasting eight weeks or less . Dorea was reported to increase in studies ranging from one week to twenty-six weeks , while increases in Ruminococcus were found both in studies lasting less than one week (n = 3) and also in those ranging from eight weeks to twenty-six weeks (n = 3) . Studies with an intervention duration of over nine weeks reported the majority of increases in Veillonella  and Bifidobacterium . Decreases in Clostridium were reported in studies with a duration of five weeks or less . Changes in Akkermansia were inconsistent in relation to duration, with increases seen from one to twenty-six weeks .Studies that included an exercise intervention for 12 weeks or longer represented 50% of all reported improvements in alpha diversity. All those studies that assessed potential changes in beta diversity with a duration of six weeks or more (n = 6).Increases in alpha diversity were only found in studies that included more than 90 min of exercise per session; a total of 67% of these studies found an improvement in alpha diversity (Lachnospira (n = 3), with <30 min reporting an increase (n = 1) and >60 min reporting a decrease (n = 1). Increases in Dorea were only found in studies that included exercise lasting longer than 50 min . Increases in Ruminococcus were also most commonly reported with exercise sessions lasting longer than 50 min . Increases in Streptococcus were only reported in studies involving exercise sessions lasting more than 95 min (n = 2) . Bacteroides increased in exercise sessions ranging between 30 and 60 min (n = 2) and decreased in studies lasting more than 90 min (n = 2) . Increases in Bifidobacterium were most commonly reported in studies that included exercise sessions lasting between 30 and 60 min . Akkermansia was more commonly found to increase in response to interventions that included 90 min exercise sessions .Studies that involved 30 to 60 min of exercise per session most commonly reported improvements in n = 21), 18% of those in clinical populations reported an increase (n = 2), with the remainder finding no change (n = 9). In healthy populations, 40% of studies reported an increase in alpha diversity (n = 4), with the remainder finding no change (n = 6). Beta diversity (n = 17) was increased following exercise for 60% (n = 6) of clinical population studies that reported this metric (n = 10), with 57% (n = 4) reporting this change in healthy populations (n = 7).Of those studies that reported alpha diversity , and n = 4 and n = 0 , respectively). Indeed, healthy populations reported decreases in Roseburia (n = 3). Increases in Dorea, Ruminococcus, Romboutsia and Bifidobacterium more commonly reported in healthy populations following exercise . Healthy populations were more likely to show decreases in Roseburia (n = 3 vs. n = 0), Prevotella (n = 2 vs. n = 0) and Bacteroides (n = 2 vs. n = 0). Both healthy and clinical populations reported decreases in Odoribacter and Clostridium, as well as increases in Veillonella and Akkermansia with similar frequency.Twenty studies reported a change at the genus level and increases in n = 6); two studies found that Ruminococcus bicirculans and Prevotella copri increased in healthy populations, and one study reported an increase in Prevotella copri in clinical populations. No other consistent changes in species were reported.Ten studies reported changes at the species level. These changes were more commonly reported in healthy populations , exercise interventions, sequencing techniques and targets of sequencing. Exercise interventions were assessed by exercise type, intensity, frequency, and time, as well as the duration of the training intervention. Outcomes that were based on the population were also investigated; clinical and apparently healthy populations were compared in a number of studies. Clinical populations included metabolic conditions, neurological disorders, autoimmune disorders, myalgic encephalomyelitis and elderly populations. Healthy populations included apparently healthy, recreational and elite athletes and military populations. The findings suggest that differences in exercise delivery influence changes in the gut microbiota in response to training. While population variations were found to produce a similar response at the level of diversity, genus responses were found to vary.Roseburia, Lachnospira, Dorea and Ruminococcus, in particular). Exercise type was only directly assessed in one randomised controlled trial [Concurrent exercise  appeared to influence alpha and beta diversity more than aerobic-only interventions. Aerobic-only exercise interventions reported more common changes at the level of genera  compared to those that have used two to three sessions a week (n = 13). This may explain why some genera were not consistently reported in higher frequency interventions; six studies did not report exercise frequency, and these were not included in the analysis. It is unclear whether these changes may have been observed in other studies had different analysis techniques been utilised.It appears that while exercising two to three times per week is sufficient to influence beta diversity and abundance at the level of genus, four to five weekly sessions are needed to elicit some change in alpha diversity (33%)\u2014and more than five sessions a week are needed for more consistent increases (67%). Again, the influence of diet on those changes reported in response to exercise interventions is not well understood. As very high levels of physical activity are often accompanied by an increase in energy intake, future research needs to control for diet when assessing potential changes in alpha diversity with exercise training. Additionally, relatively few studies have included more than three and five exercise sessions a week (Roseburia (decrease), Ruminococcus (increase) and Clostridium (decrease). These changes are likely to be compounded by the maximal intensity or very high exercise session durations used in these studies. Of interest, Lachnospira appeared to increase in response to eight weeks of training, after which there was no further change. It is unclear as to whether this suggests that the perturbation of exercise only influences this genus in the short term. Veillonella is one of the most highly discussed genera in the context of exercise and microbiota interactions, with Scheiman et al. (2019) identifying its potential role in endurance exercise as a metaboliser of lactate [Veillonella increases, suggesting that repeated and prolonged exercise behaviour may be linked to this genus [Veillonella following an ultra-marathon. Veillonella was also shown to increase in exercise studies with clinical populations, potentially supporting Scheiman\u2019s suggestion that Veillonela can increase based on exercise demands. However, the same can be said for other genera assessed in this review. Based on the relative interest in Veillonella and the mixed findings, with some studies finding significant changes and others finding no change, it is possible that those individuals showing an increase in Veillonella have a significant presence of this microbe prior to training.Exercise interventions lasting more than four weeks were responsible for most of the reported changes at the genus level. Changes in diversity metrics were found at six weeks (beta diversity) and 12 weeks . The longer duration (greater than eight weeks) exercise interventions were more likely to find changes in the microbiota at the genus level. In noting this, interventions lasting less than four weeks reported changes in  lactate . In agreis genus . It appeLachnospira and Bacteroides; however, longer sessions appeared to blunt this increase or even result in decreases. Similarly, 50 min per session was associated with increases in Dorea, Bifidobacterium and Ruminococcus with decreases in Ruminococcus reported in studies where training sessions lasted longer than 90 min. These longer duration sessions (>95 min) also appear to be related to increases in Streptococcus. While alone this is not concerning, at the level of genus it may be a potential tool to further investigate the environmental changes that may be linked to microbiota change with exercise, authors of both articles reporting an increase in Streptococcus comment that this is a genus that does contain pathogenic species [Akkermansia was also reported to increase in these longer duration sessions; Akkermansia is a commonly reported genus that has a higher abundance in active individuals [The duration of exercise sessions was highly predictive of change in the gut microbiome diversity, with sessions lasting more than 90 min producing the most consistent change in alpha diversity. Thirty minutes appeared sufficient to produce increases in  species ,23. Akkeividuals . This reividuals  and obesividuals . Given tividuals .It appears that aerobic training at higher intensities is most likely to elicit changes to the gut microbiota, particularly changes in alpha diversity. There appears to be a dose\u2013response, with a possible \u2018inverted U\u2019 describing the relationship; low frequency and duration of exercise results in little to no change, 30\u201390-min sessions 3\u20135 times per week produce most of the change, and exercise lasting longer than 90 min and more than five times per week produces some change but a higher likelihood of a potentially negative change to the gut . Indeed,The available evidence suggests that a certain level of disruption or hormesis is necessary to influence changes in the gut microbiota of humans; however, there may be a ceiling effect, as seen with the plateaus in microbiota change in some studies in this review. To this point, an exercise intervention duration of eight weeks or more appears to produce the most consistent change across alpha diversity, beta diversity and most genera. Furthermore, the majority of studies collected samples only before and immediately after the exercise intervention, and the time course of changes is not clear. Of the two studies that sampled faecal matter during the exercise intervention, the limited available data suggest that most of the observed changes in the microbiota may occur within two weeks of training before plateauing . In the Based on the changes observed in this review, the removal of the exercise stimulus reverses changes in as little as three weeks in previously sedentary individuals . This isRoseburia and, to some extent, Lachnospira, did increase with exercise more commonly in clinical populations, with these genera commonly seen to be reduced in clinical populations at baseline compared to healthy populations and are linked with potential benefits to health [Many clinical populations present with gut microbiota dysbiosis or different microbial composition compared to healthy individuals ,63,64. To health ,67. Exero health ,69,70. Ro health .Bacteroides, which is a highly discussed and observed genus in the wider human gut microbiota literature; however, this review found only three studies reported this genus. It is, therefore, likely that the one study reporting no change was supported by an unknown number of other studies. As such, the results of this review should be interpreted as exercise likely having either no effect or a positive effect (in the case of Bacteroides) as per each change reported.The primary limitation of this review is that results were only included as they were reported in each study. Given that the reporting increase, decrease and no change across every level of taxonomy for each study is not feasible, this means that many of these studies likely found no change in microbes with no need to report this, especially those using metagenomic analysis. An example of this is The current review is also limited by the high heterogeneity of studies assessed. This has been the nature of microbiota research as an emerging field in exercise and in humans; however, future research will benefit from standardisation of some of the techniques from stool sample collection to downstream analysis. A recent study highlighted that the most popular and currently feasible way of analysing the gut microbiota  may underestimate the changes made in the gut over time due to the imposed constraints of abundance analysis . An alteControl standards varied considerably between studies, primarily concerning diet analysis, repeated sample collection and frequency of sample collection. Again, there is no consensus as to what ideal standards should be, and as the literature stands, much can be interpreted from the collection of studies presented in this review. However, for the strength of individual studies, understanding the intra-individual variation and influence of diet on stool samples and the interpreted microbiota will strengthen the conclusions drawn from these studies.To support the intraindividual variation and its role in microbiota research in humans, a growing body of literature suggests that case-wise/intrapersonal assessment in a functional capacity may be a positive way to understand microbiota changes  and to aFinally, this review did not assess the data recorded in repositories and, as such, all results were drawn directly from the body of articles or supplementary files where possible. There is a risk of under- or over-reporting some findings in this review due to the analysis techniques used, and perhaps a review investigating repository data, such as the work by Bisanz et al. (2019), in high fat diets in murine models may be warranted . SimilarThis is the first systematic review to assess only longitudinal study designs and varied populations when considering the interaction between exercise and the human gut microbiota. We were able to compare exercise dose through frequency, time, type and intensity of exercise across 28 studies. While single bouts of high-volume exercise have been shown to influence the gut microbiota, it was found that moderate-high, but especially high-intensity interventions, for more than 30 min, three or more times per week and for more than eight weeks resulted in the most consistent changes on the human gut microbiota. When potential differences between clinical and healthy populations were compared, microbiota changes were observed in both populations with exercise; however, specific genera that responded to exercise appeared to differ. Further research is required to verify these findings, and future studies would benefit from waitlist or crossover controls to account for inter and intraindividual variability in the gut microbiota. An improved understanding of how different types of exercise affect the human gut microbiota of different populations will inform the development of the most appropriate interventions to improve health via the gut microbiota and assist in chronic disease prevention and management."}
{"text": "Polyphenols are natural substances with variable phenolic structures and are elevated in vegetables, fruits, grains, bark, roots, tea, and wine. There are over 8000 polyphenolic structures identified in plants, but edible plants contain only several hundred polyphenolic structures. In addition to their well-known antioxidant effects, select polyphenols also have insulin-potentiating, anti-inflammatory, anti-carcinogenic, anti-viral, anti-ulcer, and anti-apoptotic properties. One important consequence of ischemia is neuronal death and oxidative stress plays a key role in neuronal viability. In addition, neuronal death may be initiated by the activation of mitochondria-associated cell death pathways. Another consequence of ischemia that is possibly mediated by oxidative stress and mitochondrial dysfunction is glial swelling, a component of cytotoxic brain edema. The purpose of this article is to review the current literature on the contribution of oxidative stress and mitochondrial dysfunction to neuronal death, cell swelling, and brain edema in ischemia. A review of currently known mechanisms underlying neuronal death and edema/cell swelling will be undertaken and the potential of dietary polyphenols to reduce such neural damage will be critically reviewed. Polyphenols are natural compounds with variable phenolic structures and are rich in vegetables, fruits, grains, bark, roots, tea, and wine ,2. Whileet al. i can induce cell swelling as demonstrated in lactacidosis-induced glial swelling [2+]i. Also, such blockade of the rise in [Ca2+]i by blockers of the L-type calcium channel as well as modulation of [Ca2+]i through BAPTA, a calcium chelator, reduces cell swelling in C6 glial cultures [2+]i following administration of polyphenols. Quercetin attenuated the H2O2-induced calcium dysregulation in PC12 cells [+/Ca2+ exchanger [2+ concentration in RBL-2H3 cells induced by antigen stimulation [2+]i increase by attenuating ionotropic Ca2+ influx in PC12 cells [2+]i was observed after exposure to tannins in HL-60 cells [2+]i in U87 cells by influx of extracellular Ca2+ as well as by releasing calcium from intracellular stores. Whether this difference in modulating [Ca2+]i by polyphenols is due to the difference in cell types or the polyphenols tested or the stressor involved is not clear. It is also possible that the difference arises from assessing [Ca2+]i in control conditions (in the absence of stress) compared to examining the effects of polyphenols in the presence of stress. Nevertheless, these studies indicate that polyphenols have the potential to modulate calcium channels that are involved in cell volume regulation, but their role in attenuating glial swelling/cytotoxic edema in ischemia needs to be further elucidated.The effects of polyphenols in regulating ion channels or ion co-transporters, following cerebral ischemia, are not clear, and are a subject of current investigation. An increase in intracellular calcium is a key feature of ischemic injury ,185. Furswelling ,187 and swelling . Studiescultures . Other s12 cells . Quercetxchanger . Apple cmulation . EGCG re12 cells . In cont60 cells . Red win60 cells . EGCG inm) by immunosuppressants, cyclosproin A (CsA), but not FK506, are consistent with the role of the mPT mediating such events. Similar to CsA, CPE, and green tea polyphenols, also significantly prevented OGD-induced cell swelling and the decline in \u0394\u03a8m in C6 glioma indicating that one mechanism by which CPE and GTE exert their protective effects is possibly by blocking the mPT. Interestingly, quercetin significantly attenuated cell swelling in C6 glial cells following OGD but did not block the dissipation of the \u0394\u03a8m [Mitochondrial dysfunction is an important characteristic of ischemia ,195. The the \u0394\u03a8m  indicatiet al. [et al. [et al. [in vitro [An increase in inflammatory markers has been associated with brain edema  and coulet al.  which ex [et al.  and Lee  [et al.  report a [et al.  but the  [et al. . Curcumi [et al.  but as w [et al.  most lik [et al. . Il-1 re [et al. . Taken tin vitro ,210. A rin vitro  as well in vitro  and TNF-in vitro . In addiin vitro , as wellin vitro  and otheOxidative stress and mitochondrial dysfunction are key features of cerebral ischemia that affect neuronal viability after ischemia. In addition, these factors also affect brain edema which is a major consequence of ischemia and can be fatal if not resolved. Edema can further aggravate neuronal injury by affecting cerebral perfusion. Currently, there are few remedial agents to effectively reduce neuronal death or brain edema not only in ischemia but also in other neural injuries including traumatic brain injury. The potential for the use of polyphenols in the preventing cell loss or damage and edema in cerebral ischemic injury is tremendous. However, the cellular and molecular actions of polyphenols involved in neuroprotection have to be elucidated further. Given the large proportion of the population affected by stroke and traumatic brain injury, and with few strategies to effectively attenuate brain edema and associated neural damage, it is important to determine the potential beneficial effects of dietary polyphenols in the prevention and alleviation of such damaging effects."}
{"text": "The sensitivity of parasite isolates to anti-malaria drugs were also evaluated for a possible association with polymorphisms in these genes.In 2005, Ghana replaced chloroquine with artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. The aim of this work was to determine for the first time, polymorphisms in the putative Plasmodium falciparum genes were assessed from filter-paper blood blot samples by DNA sequencing. In vitro drug sensitivity test was carried out on some of the blood samples from volunteers visiting hospitals/clinics in southern Ghana using a modified version of the standard WHO Mark III micro-test.The prevalence of point mutations in the above 50 value for artesunate was 0.73 nM , amodiaquine 30.69 nM  and chloroquine 58.73 nM . Twenty point mutations were observed in pfATPase6 gene, with no L263E and S769N. All mutations found were low in frequency, except D639G which was observed in about half of the isolates but was not associated with artesunate response (p = 0.42). The pftctp gene is highly conserved as no mutation was observed, while CVIET which is chloroquine-resistant genotype at codon 72-76 of the pfcrt gene was identified in about half of the isolates; this was consistent with chloroquine IC50 values (p = 0.001). Mutations were present in pfmdr1 gene but were not associated with artemisinin response (p = 1.00).All successfully tested parasite isolates were sensitive to artesunate; while 19.4%, 29.0% and 51.6% were resistant to quinine, amodiaquine and chloroquine respectively. The geometric mean of ICpfATPase6 gene is highly polymorphic with D639G appearing to be fixed in Ghanaian isolates. These may just be spontaneous mutations as all parasite isolates that were tested displayed satisfactory in vitro response to artesunate. However, there is no improvement in susceptibility of the parasites to chloroquine five years after its proscription.The  Plasmodium falciparum parasites that are resistant to conventional anti-malaria drugs in almost all endemic countries , while a pair of primer was sufficient for the relatively short pftctp gene [GenBank:DQ141561). Two pairs of primer were employed in sequencing portions of the pfmdr1 gene [GenBank:M29154.1] spanning codons 86, 184, 1034, 1042 and 1246; the first pair of primer targeted the first two codons whereas the second primer set were for the last three codons . This study also received ethical approval from Tokyo Medical and Dental University, Japan (Certificate Number 955).in vitro for their drug sensitivities, out of which 7 (18.4%) failed the test due to poor growth or contamination ; amodiaquine, 30.69 nM ; chloroquine, 58.73 nM ; quinine, 355.37 nM  , that of artesunate and chloroquine was medium, but were all statistically significant  that were used for drug sensitivity test (phenotyping). A total of twenty point mutations were found in pfATPase6 gene (Table p = 0.423)  isolates harboured the N86Y mutation (TAT nucleotides) while 43/60 (71.7%) with Y184F mutation (TTT nucleotides) in the pfmdr1 gene  pressure in Ghana; nonetheless, this study presents a picture in time since there is no report of artemisinin resistance in Ghanaian isolates. This study therefore describes for the first time, polymorphisms in pfATPase6 gene in such isolates. By using PCR sequencing assay, neither L263E nor S769N point mutation was observed; and this is consistent with the in vitro micro-test results. A total of twenty point mutations were observed in this gene, confirming its molecular diversity than previously demonstrated [The limitation to this study was that polymorphisms in the nstrated ,36,37; tnstrated .pftctp gene in artemisinin activity, our present result confirms its extreme conservation, and thus not a strong candidate for resistance to artemisinin and its derivatives due to the fact that it is not under major selective drug pressure [pfmdr1 and pfcrt genes in some isolates conferred chloroquine resistance [pfmdr1 gene and K76T allele in the pfcrt gene; but they had no part in the modulation of artesunate response as the lowest and highest artesunate IC50 values corresponded to parasite isolates with pfcrt K76T. This was corroborated by Fisher's exact test. Afonso et al [Plasmodium chabaudi, can develop stable resistance to artemisinin but lack mutations in candidate genes including atp6, tctp, mdr1 and cg10.Even though studies have shown the involvement of the malaria parasite pressure ,29. Studsistance ,39, but sistance ,18. Our so et al  in theirMore than half of the parasite isolates were resistant to chloroquine after five years of its proscription in Ghana, which is a reflection of our molecular results but at variance to the Malawian situation where sensitive isolates were recovered after stopping the use of chloroquine ,22. The Plasmodium falciparum isolates from southern Ghana showed no improvement in susceptibility to chloroquine, but they exhibited satisfactory in vitro response to artesunate. No L263E and S769N mutations in the SERCA-type PfATPase6 were observed, albeit evidence is clear on the greater molecular diversity of PfATPase6 than previously demonstrated as twenty mutations were detected with D639G appearing to be fixed in this parasite population. Whereas these mutations may be spontaneous and as such without any particular role in the parasites response to artemisinin, continual monitoring of P. falciparum susceptibility to artemisinin is welcome.The authors declare that they have no competing interests.in vitro drug assessment test while BKB and TK did the molecular work. NO, MDW and DAB were involved in all stages. BKB wrote the manuscript, which was read and approved by all authors.BKB and NO conceived the idea, while BKB, IA, TS, JO, WKA and JHNO performed the field work. BKB, IA, TS, JO, JHNO, HA, MFO and NA did the"}
{"text": "In response to extra- or intracellular stresses, the cellular gatekeeper p53 is able to integrate multiple diverse signals to determine the outcome of cell fate, by regulating the expression of numerous responsive genes Salmonella or Helicobacter infections Bacterial and viral infections represent a major type of cellular stress, triggering different biological countermeasures, notably mediated by the p53 pathway. In the process of evolutionary adaptation to the host environment, pathogens have developed diverse strategies to hijack and exploit such host machinery. This is particularly well-illustrated for viruses, with many examples of virally induced deregulation of the p53 pathway  TP53 gene physiologically expresses several protein isoforms, due to the use of alternative promoters, splicing sites, and translational initiation sites , a RNA, and a DNA virus  Although several studies report on the suppressive function of p53 isoforms and related deregulation of their expression in human cancers Within SV40 lytic infection, p53 is targeted and inactivated by T-Ag, and \u0394p53 has been identified as a new player in SV40 replication by Rohaly and colleagues. They revealed that an ATR\u2013DNA damage response pathway mediates the phosphorylation and stabilization of \u0394p53, enhancing its transcriptional activity in a promoter-dependent manner In human lung epithelial cells, we have investigated the roles of both \u0394133p53\u03b1 and p53\u03b2 isoforms in the context of influenza virus infection Helicobacter pylori has been shown to interfere with p53 function via up-regulation of the \u0394133p53 isoform both in vitro  and in vivo (Mongolian gerbil) H. pylori infection (H. pylori pathogenesis and associated tumorigenesis.Recently, nfection . This stH. pylori have an impact on \u0394133p53 to interfere with full-length p53 activity via mechanisms remarkably similar to those previously described in the field of cancer In conclusion, these three studies, based on different pathogen models, have highlighted for the first time the functional role of different p53 isoforms in the context of infection. A preliminary model emerges in which the isoforms act as regulators of the p53-mediated cellular response against pathogens. As an illustration, both influenza viruses and"}
{"text": "Overexpression of the tumor suppressor gene p53 and the marker for cellular proliferation Ki67 in open lung biopsies are indicated as predictor factors of survival of patients with lung cancer. However, the prognostic value of p53 and Ki67 in fiberoptic bronchial biopsies (FBB) has not been fully investigated. We evaluated p53 and Ki67 immunostaining in FBB from 19 with Non Small-Cell Lung Cancer .FBB specimens were fixed in formalin, embedded in paraffin, and immunostained using anti-p53 and anti-Ki67 antibodies. Slides were reviewed by two independent observers and classified as positive (+ve) when the number of cells with stained nuclei exceeded 15% for p53 or when >25% positive cells were observed throughout each section for Ki67.Positive (+ve) immunostaining was found in 9 patients for p53 (47.37%) and 8 patients for Ki67 (42.10%). We examined overall survival curves of the patients with Mantel's logrank test, both p53 -ve and Ki67 -ve patients had significantly higher survival rates than p53\u2009+\u2009ve (p\u2009<\u20090.005) and Ki67\u2009+\u2009ve , respectively.This study suggests that negative immunostaining of fiberoptic bronchial biopsies for p53 and Ki67 could represent a better prognostic factor for patients with NSCLC. Lung cancer accounts for the most cancer related deaths in both men and women. Cigarette smoking is by far the most important risk factor for lung cancer. Risk increases with quantity and duration of cigarette consumption. The 1-year relative survival for lung cancer increased up to 40% in the last ten years, largely due to improvements in surgical techniques and combined therapies. However, the 5-year survival rate for all stages combined is only 15%. Despite, the 5-year survival rate is 50% for cases detected when the disease is still localized, but only 16% when lung cancers are diagnosed out of this early stage, screening for early/localized lung cancer detection has not yet been proven to reduce mortality.s in its wild-type conformation as a transcription factor and DNA binding protein, and this activity results on inhibition of cell proliferation by blocking entry into the S-phase of the cell cycle [Some independent prognostic factors have been suggested for predicting survival and helping in the management of patients with lung cancer. Regarding this issue, during the last few years there has been a growing interest in molecular biology of lung cancer. As the molecular characteristic of cancer have become better understood, prognostic models have been developed for lung cancer that incorporate biological markers, immunohistochemical properties and genetic features in addition to hystologic subtype, age of patients and extent of disease (TNM-stage). Mutation of the p53 tumor suppressor gene, which is localized on human chromosome 17p13, it has been observed in many human cancers and is the most common mutation in lung cancers [ll cycle . Mutant ll cycle , severalll cycle . Actuallll cycle .The antibody Ki67, which recognizes a nuclear antigen in proliferating cells, has been widely used to estimate growth fraction in different cancer lesions ,12. DespAim of the present study is to determine the correlation of p53 protein and Ki67 antigen, immunohistochemically detected in bronchial biopsies, with survival of patients with non small cell lung cancers.We studied samples obtained from 19 consecutive patients that had been undergone to bronchoscopy, with diagnostic intent, in our Unit, between January 2008 to December 2010. We used flexible fiberoptic bronchoscopy  and all samples were collected by the same operator. Only patients with positive histological diagnosis entered into the study. The clinical staging was defined with the usual method . The stuImmunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections of 5 um with monoclonal antibodies to the p53 protein  and Ki67 . Similar procedures were followed for the usual treatment of deparaffination, with xylene, absolute alcohol and distilled water. Ki-67 and p53 sections were heated in a microwave oven 4 times, each of 5 min, in citrate buffer-solution. The slides, after at least 30 min of air-drying, were incubated with the primary antibodies against p53 and Ki67. Dilutions (raging between 1:25 and 1:50) and time of incubation (30 to 60 min) were depending on the batch of antibody. After washing with Phosphate Buffered Saline (PBS) secondary antimouse antibodies were added  both for Ki-67 or P53. Afterwards the slides were incubated with streptavidin-peroxidase reagent  and again the time of incubation was depending by the \u201cdata-sheet\u201d of the antibody. After another PBS wash, the final staining was provided using a 0,05% solution of diaminobenzidinetetrahydrochloride in PBS usually for 10 min. Finally, sections were counterstained with Meyer\u2019s haematoxylin. For p53 a tumor was considered to be immunopositive when the number of cells with nuclei red-stained exceeded 15% .The Kaplan-Meier method was used to estimate the probability of overall survival as a function of time (starting from the date of diagnostic bronchoscopy to that of death from cancer), and difference in the survival of subgroups of patients were compared with Mantel\u2019s log-rank test. Multivariate analyses were performed using the Cox regression model to study factors that independently influenced overall survival. Statistical significance was set a P value <0.005 and all test were two-tailed as were the reported P values.We found p53 reactivity in 9 of the patients (47.37%). The antibody used in this study reacts with wild type and mutant type of the p53 protein and the nuclear reactivity, as expressed before in \u201cMethods\u201d section, was designed to be \u201cpositive\u201d (+ve). For Ki67 immunostaining, reactivity between antibody and antigen localised in nuclei of proliferating cells, designed + ve as above indicated, was found in 8 of the patients (42.10%). By contrast we designed as \u201cnegative\u201d (\u2212ve) reactivity against p53 antibodies or Ki67 under the \u201ccut-off\u201d points of 15% for p53 and 25% for Ki67. Only tumoral section showed immunoreactivity both for p53 or Ki67, indicating that our immunostaining procedures do not generate false positives. In Figures\u2009We compared Kaplan-Meier survival curves for the p53 and Ki67 + ve groups and p53 and Ki67 \u2013ve groups in all 19 patients with NSCLC Figures\u2009 and 2b. First histological diagnosis of lung cancer is often obtained by fiberoptic bronchial biopsies. However the utility of extend histological studies with immunohistochemical methods in lung cancer is still debated. We have shown the feasibility of immunohistochemical detection of p53 and Ki67 in bronchial biopsies, performed with diagnostic intent, in patients with histological diagnosis of NSCLC. In our study, the incidence of p53 positivity in NSCLC was 47.34%, and it was 42.1% for Ki67 positivity. Furthermore, utilizing these small biopsy lung specimens we compared p53 and Ki67 positivity with survival rate. There are few studies describing p53 overexpression in samples obtained in a non surgical setting and it relationship to survival rate ,22-24. MThe use of Ki67 immunostainining in lung cancer is not easy to apply and the feasibility of Ki67 in specimens of small size like the bronchial biopsies is still controversial.Scagliottiet all.  firstly Simultaneous expression of p53 and Ki67 was studied as a prognostic factor in resected lung cancer . SurprisWe have shown the utility of p53 and Ki67 immunostaining of bronchial biopsies in NSCLC. Our data indicate that p53 and Ki67 immunostaining of small size specimens obtained during diagnostic procedures with fiberoptic bronchoscopy may be useful in predicting prognosis in patients with NSCLC. The incidence of immunohistochemical positivity in NSCLC was approximately the same as reported in other studies, and, furthermore, no histologically negative samples was stained, thus indicating the validity of our method. However, these results were obtained in a relatively small number of patients, such as we were not able to predict a relationship between positive immunostaining and resistance to chemotherapy.We believe that a simple, inexpensive method, as that reported in the present study should be useful in predicting prognosis of patients with NSCLC. More studies are needed to understand the possible relationship of positive stain for p53 and Ki67 with the resistance to chemotherapy.The authors declare that they have no competing interest."}
{"text": "FK506 binding protein 51 (FKBP51) is an immunophilin physiologically expressed in lymphocytes. Very recently, aberrant expression of this protein was found in melanoma; FKBP51 expression correlates with melanoma aggressiveness and is maximal in metastatic lesions. FKBP51 promotes NF-\u03baB activation and is involved in the resistance to genotoxic agents, including anthracyclines and ionizing radiation. FKBP51 is a cochaperone with peptidyl-prolyl isomerase activity that regulates several biological processes through protein-protein interaction. There is increasing evidence that FKBP51 hyperexpression is associated with cancer and this protein has a relevant role in sustaining cell growth, malignancy, and resistance to therapy. There is also evidence that FKBP ligands are potent anticancer agents, in addition to their immunosuppressant activity. In particular, rapamycin and its analogs have shown antitumor activity across a variety of human cancers in clinical trials. Although, classically, rapamycin actions are ascribed to inhibition of mTOR, recent studies indicate FKBP51 is also an important molecular determinant of the drug\u2019s anticancer activity. The aim of this article is to review the functions of FKBP51, especially in view of the recent findings that this protein is a potential oncogene when deregulated and a candidate target for signaling therapies against cancer.  The deseceptors . The N-teceptors . The aimFKBP51 is capable of immunosuppression, mediated by calcineurin (CaN) inhibition, when complexed to FK506 . CaN is FKBP51 interacts with glucocorticoid, androgen, estrogen, progestin and mineralocorticoid receptors -20. The FKBP51 protects cells against oxidative stress . Very reIn the nervous system, FKBP51 regulates clearance of microtubule-associated protein tau and stabilizes microtubules . AbnormaThe FKBP51 homolog in Arabidopsis, PAS-1, plays a critical role in the growth and development of this organism . In mammThe concept that FKBP51 is an essential factor for cell proliferation is also supported by studies on myeloproliferative disorders -35. FKBPFKBP51 controls activation of NF-\u03baB transcription factor that is involved in carcinogenesis, metastasis and resistance to anticancer therapy . FKBP51 An additional mechanism that indirectly implicate FKBP51 in the regulation of NF-\u03baB activation is through the inhibition of steroids response. Glucocorticoids are potent inhibitors of NF-\u03baB activation, mediated by enhanced transcription of I\u03baB\u03b1, that traps NF-\u03baB in inactive cytoplasmic complexes . In addiIn the canonical pathway of NF-\u03baB activation, IKK\u03b1 forms with IKK\u03b2 and a regulatory subunit IKK\u03b3 or NEMO the IKK kinase complex, that mediates the phosphorylation of I\u03baB proteins in response to various stimuli . PhosphoThe essential role of FKBP51 in the activation of NF-\u03baB and NF-\u03baB-regulated genes has been demonstrated, both in normal  and tumoet al. low levels of FKBP51 resulted in increased Akt phosphorylation and decreased chemosensitivity [et al. found that insulin increased binding of FKBP51 to pAkt (S473) in non-small cell lung cancer cells, A549, and that, in the presence of atorvastatin, the levels of pAkt bound to FKBP51 decreased in 5 min [et al. are not in concert with the notion that FKBP51 is an immunophilin capable of CaN inhibition [The Phosphatidylinositol 3-kinase (PI 3-kinase)/Protein kinase B (PkB or Akt) is an important pro-survival signaling pathway that is often deregulated in cancer. Recently, it has been found, in tumor cell lines, that FKBP51 acted as a scaffold to facilitate the interaction between Akt and PH domain leucine-rich repeat protein phosphatase (PHLPP), which mediates dephosphorylation of pAkt at S473 . Accordisitivity . On the in 5 min . Althougin 5 min . The finhibition ,53. AccoIperexpression of FKBP51 has been documented in several human cancers. Enhanced FKBP51 expression is associated with apoptosis resistance and enhanced proliferation in gliomas . A studyIncreased levels of FKBP51 are also associated with tumor aggressiveness in melanoma ,49. A stIn prostate cancer, FKBP51 is part of a superchaperone complex that includes androgen receptor (AR) and androgen . Depletiet al. [An immunohistochemistry study of expression of FKBP51 in 50 tumoral samples including breast, lung, pancreas, ovary, and prostate (10 samples for each tumor), and a comparable number of normal tissue samples showed an intense signal in 38 out of 50 tumors analyzed, whereas normal tissues of the same histotypes showed a weak/absent immunohistochemical signal [et al. . All proThe first evidence supporting the involvement of FKBP51 in the resistance to cancer therapies came from mechanistic studies of the antitumor activity of rapamycin ,9. RapamIn a study conducted in melanoma, a tumor in which PTEN is often mutated , rapamycThe role of FKBP51 in the resistance of tumors to cell death has been extensively studied in melanoma ,49. In tFKBP51 is a protein with a progressively emerging role in cancer biology. Although its function is still far from being fully elucidated, there are clear data suggesting this immunophilin plays an active role in cell proliferation in both the physiologic conditions of cell growth and differentiation -31 and iFinally, increased FKBP51 seems to be a common denominator between depression and cancer . In the"}
{"text": "Pyramidal cells are characterized by markedly different sized dendritic trees, branching patterns, and spine density across the cortical mantle. Moreover, pyramidal cells have been shown to differ in structure among homologous cortical areas in different species; however, most of these studies have been conducted in primates. Whilst pyramidal cells have been quantified in a few cortical areas in some other species there are, as yet, no uniform comparative data on pyramidal cell structure in a homologous cortical area among species in different Orders. Here we studied layer III pyramidal cells in V1 of three species of rodents, the greater cane rat, highveld gerbil, and four-striped mouse, by the same methodology used to sample data from layer III pyramidal cells in primates. The data reveal markedly different trends between rodents and primates: there is an appreciable increase in the size, branching complexity, and number of spines in the dendritic trees of pyramidal cells with increasing size of V1 in the brain in rodents, whereas there is relatively little difference in primates. Moreover, pyramidal cells in rodents are larger, more branched and more spinous than those in primates. For example, the dendritic trees of pyramidal cells in V1 of the adult cane rat are nearly three times larger, and have more than 10 times the number of spines in their basal dendritic trees, than those in V1 of the adult macaque , which has a V1 40 times the size that of the cane rat. It remains to be determined to what extent these differences may result from development or reflect evolutionary and/or processing specializations. Studies in primates have revealed marked differences in pyramidal cell structure among different cortical areas , the bushveld gerbil (Tatera branstii), and the four-striped mouse (Rhabdomys pumilio) were included for study. The rodents used in the present study were caught from wild populations in South Africa with permission and supervision from the appropriate wildlife directorates. All animals  were treated and used according to the guidelines of the University of the Witwatersrand Animal Ethics Committee, which parallel those of the NIH for the care and use of animals in scientific experimentation. Although the ages of the animals is not known, the body mass and physical development indicated that they were sexually mature adults. Animals were sedated with an I.M. injection of ketamine hydrochloride (40 mg/kg) and xylazine hydrochloride (4 mg/kg) and overdosed by I.P. injection of sodium pentobarbital (100 mg/kg).Three African rodent species, including the greater cane rat  for approximately 10 min at room temperature. These sections were then mounted into a perspex chamber on a fixed stage fluorescence microscope (Zeiss Axioskop II Plus) and individual cells were injected with Lucifer Yellow .Methodology used in the present investigation was exactly the same as used in our previous cell injection studies. Animals were perfused transcardially with phosphate buffered saline  then paraformaldehyde (4% in PB). The brains were removed and the right hemispheres were flat-mounted  in stock solution  for 5 days at room temperature. 3,3\u2032-diaminobenzidine  was used as the chromogen Figure . MoreovePyramidal cells were drawn with the aid of a Zeiss Axioskop 40 equipped with a camera lucida. Dendritic tree size  and somal size were determined in 2-dimensions with the aid of NIH-Image software   was larger than that in the bushveld gerbil (14.63 mm2), which was larger than that in the four-striped mouse (11.58 mm2).The primary visual area was readily identified in \u201cflat-mounts\u201d prepared from the cortical hemispheres and processed for cytochrome oxidase Figure . The absn = 108, mean \u00b1 SD: 123.15 \u00b1 24.90 \u00d7 103 mm2) were larger than those in the bushveld gerbil  which, in turn, were larger than those in the four-striped mouse Figure n = 62, 38.55 \u00b1 12.80 \u00d7 103 mm2; Figure F(2) = 393.52, p < 0.001]. Post-hoc Scheffe tests revealed significant differences among all three species (p < 0.001).The basal dendritic trees of layer III pyramidal cells in the greater cane rat  = 167.66, p < 0.001]. Post-hoc Scheffe tests revealed all species comparisons to be significantly different.Plots of the results of Sholl analysis in which we counted the number of dendritic intersections in successive concentric circles with radii of 25 \u03bcm increments revealed that the peak branching density in the basal dendritic trees of layer III pyramidal cells in the greater cane rat (mean \u00b1 SE: 21.99 \u00b1 5.77) was greater than that in the bushveld gerbil 17.43 \u00b1 4.12), which, in turn, was greater than that in the four-striped mouse  was less than that in the bushveld gerbil , which was greater than that in the four-striped mouse  = 10.18, p = 0.001]. Post-hoc Scheffe tests revealed all species comparisons to be significantly different.A total of 6484 spines were drawn and tallied. As reported previously  was greater than that in the bushveld gerbil (4316), which, in turn, was greater than that in the four-striped mouse (2141).SD: n = 108, 170.71 \u00b1 19.84 mm2) were larger than those in the bushveld gerbil at , which, in turn, were larger than those in the four-striped mouse Figure n = 62, 107.07 \u00b1 15.86 mm2; Figure F(2) = 239.75, p < 0.001]. Post-hoc Scheffe tests revealed significant differences among all three species (p < 0.001).The cell bodies of layer III pyramidal cells in the greater cane rat  the absolute size of the cerebral cortex occupied by V1 differs up to 4-fold among the three species; (2) there are differences in the size, branching structure, spine density, and total number of spines in the basal dendritic trees of layer III pyramidal cells in V1; (3) the spine density may vary independently of the size and branching structure of the dendritic trees among species; and (4) there is a trend between the size and number of spines in the dendritic trees of pyramidal cells and the size of V1 such that the larger V1 the larger and more spinous are the cells.Comparison of these data obtained from African rodents with those sampled by the same methodology from V1 of other species such as the South American rodent cutia  and approximately twice as spinous (7903 spines) as the largest, most spiny cells observed in the macaque during development (53.93 \u00d7 103 \u03bcm2 and 3900 spines, Elston et al., Developmental studies in the macaque monkey reveal that the dendritic trees of layer III pyramidal cells at birth are larger than those observed in the adult four-striped mouse and bushveld gerbil, but then become smaller during maturation into adulthood (Elston et al., The difference in the relationship in rodents and primates between the size of pyramidal cells and the size of V1 could hardly be more dramatic Figure . StatistPreviously it has been suggested that the dendritic trees of pyramidal cells may be larger and more branched, may be smaller and less branched, or be the same, in species with increasingly larger cerebral cortex (see Figure 37 of Elston, One possibility is that the data reflect fundamentally different evolutionary principles in rodents and primates (see Gould, As each spine receives at least one excitatory input (DeFelipe et al., The observed differences in pyramidal cell structure detailed here between primates and rodents may also reflect a difference in the processing of visual information in V1 of the different species. For example, the tangential area occupied by the \u201caverage\u201d dendritic tree of layer III pyramidal cells in V1 of the adult cane rat is nearly three times the size of that in the adult macaque monkey Figure . DiffereFurthermore, in primates it is well known that several, 35 or more, topographically organized visual cortex areas are present (Felleman and Van Essen, The present physiological data suggests that single unit responses to visual stimuli in rodents are not that different to those in primates (Tiao and Blakemore, Pyramidal cell structure in rodent V1 differs considerably to that in primates. In rodents, pyramidal cells are progressively larger, more branched and more spinous in species with progressively larger brains. In primates, there is relatively little difference in the size, branching structure, and number of spines in pyramidal cells in species with progressively larger brains. The two distinct trends observed in rodents and primates cannot reasonably be attributed a single scaling rule or allometric equation, but appear to reflect fundamentally different modes of cortical organization. As more data becomes available it is becoming increasingly clearer that not only should principles of cortical microcircuitry not be generalized across cortical areas within a given species, but they should not be generalized across species for a given cortical area.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."}
{"text": "As a transcription factor, p53 responds to a variety of stresses to either induce apoptosis (cell death) or cell cycle arrest (cell preservation) to suppress tumor development. Yet, the effect p53 has on the non-cancer aspects of aging is complicated and not well understood. On one side, p53 could induce cellular senescence or apoptosis to suppress cancer but as an unintended consequence enhance the aging process especially if these responses diminish stem and progenitor cell populations. But on the flip side, p53 could reduce growth and growth-related stress to enable cell survival and ultimately delay the aging process. A better understanding of diverse functions of p53 is essential to elucidate its influences on the aging process and the possibility of targeting p53 or p53 transcriptional targets to treat cancer and ameliorate general aging. Caenorhabditiselegans p53 gene (Cep-1) because it increases lifespan ; heterozygous p53+/\u2212 mice also succumb to cancer before control mice but at a later age than p53\u2212/\u2212 mice ; they are produced by different promoter usage, alternative splicing, and alternative translation initiation sites ,40. Thespromoter . Such dipromoter ,44. ThusIsoforms of p53 with N-terminal truncations could conceivably enhance aging since their overexpression reduced cancer but accelerated aging in mice ,46. ThesMutations in p53 are found in half of all human cancers, and other parts of the p53 pathway are altered in many others. These mutations confer a selective advantage on the tumor cells, allowing them to evade cell cycle checkpoints, avoid apoptosis and senescence, and proliferate under conditions where normal cells cannot. Most tumor-associated p53 mutations are within the DNA-binding domain. These affect the ability of p53 to bind to target genes to varying degrees, and hence may alter the overall protein conformation. Most can still tetramerize with wild-type p53 and exert a dominant negative effect. Many engineered mutations in p53 that affect its regulation and function have been tested in cell culture and in mice. Although studies of function must generally start by utilizing tissue culture systems, the use of mouse models is important to interrogate the complex processes of tumorigenesis.It would be difficult to identify analogous mutations that affect aging, because they would presumably not be selected in the same manner. However, a polymorphism recently identified in human populations suggests that subtle changes to the p53 protein can also affect longevity and aging. A simple substitution of a proline for an arginine at codon 72 hinders the ability of p53 to induce apoptosis and leads to an increased cancer risk. However, this polymorphism was enriched in the older population and was associated with longevity, even though some had died from cancer \u201315. We pA number of studies have shown some benefit in targeting endogenous wild-type p53 or mutant p53 in order to enhance its function and slow tumor growth. The most promising approach involves drugs able to reactivate unfolded or mutant p53 (as well as the related p63 and p73 proteins in some cases). Because of its diverse activities and profound biological outcomes, p53 is tightly regulated by multiple mechanisms that control activity, stability, and localization . As a coMDM2 (murine double minute 2) and MDM4 (a.k.a. MDMX) regulate p53 and their overexpression effectively negates functional p53 to enhance cancer risk. MDM2 is a ubiquitin ligase that leads to degradation of p53. When MDM2 levels are high, p53 becomes polyubiquitinated and targeted for degradation by the proteasome. MDM2 also directly represses p53 activity. The MDM2 gene is also a target for p53, setting up a negative feedback loop. When p53 is activated, MDM2 levels increase, which then turns down p53. MDM4 is not a ubiquitin ligase but enhances p53 ubiquitination in a heterocomplex with MDM2. MDM4 also regulates p53-mediated transcription. MDM2 and MDM4 are not redundant. Deletion of either protein causes embryonic lethality in mice, and p53 deletion rescues either mutation \u201334. Apopcis-imidazoline analogue Nutlin-3 disrupts the p53\u2013MDM2 interaction to enhance p53 function and promises to be an alternative to chemotherapy. Nutlin-3 also shows a synergistic effect in combination with certain therapeutics, such as TRAIL or bortozemib  showed enhanced p53\u0394p-mediated suppression of oncogene-induced tumors after MDM4 loss, supporting MDM4 as an anti-cancer target  situation; p53 activity is likely to be regulated in a graded manner, and different activities of p53 are undoubtedly regulated differentially by different modifications. These modifications can profoundly affect the p53 activity in tumor suppression, and are likely to affect the effects of p53 on aging as well.The p53 protein has multiple Ser/Thr residues that serve as phosphorylation sites for a number of protein kinases. The phosphorylation state of many of these sites changes upon stress signaling. Many important regulatory sites are concentrated in the transactivation domain at the N-terminus, with some also present in the C-terminal regulatory domain. Some sites can be phosphorylated by multiple kinases and some kinases can phosphorylate more than one site. Two important sites are Ser 15 (Ser 18 in mouse p53) and Ser 20 (Ser 23 in mouse). These sites become phosphorylated by ATM and other kinases in response to DNA damage. When these serines are phosphorylated, the interaction with the negative regulator MDM2 is weakened, stabilizing p53 and allowing it to interact with coactivators and activate transcription. Mutant mice with a Ser to Ala mutation at these sites (cannot be phosphorylated) show increased p53 stability and transactivation. However, the phenotype is tissue specific and not as severe as would be predicted from the effects in tissue culture experiments, suggesting that phosphorylation is not the only means of controlling p53. Interestingly, mice generated by a knock-in of the Ser 18 (Ser to Ala) mutation show accelerated aging, suggesting that active p53 in response to DNA damage protects against aging . A knockAcetylation is a major mechanism for regulating p53 activity. Multiple lysine residues in p53 are acetylated; many of these same lysines are also targets for ubiquitination by MDM2. Therefore, acetylation of these lysine residues prevents their ubiquitination, stabilizing p53. In addition, acetylation can inhibit the interaction of MDM2 with p53. Finally, acetylation recruits cofactors allowing p53 to activate its transcriptional targets. Acetylases that are responsible for modification of p53 include p300, CBP, PCAF, TIP60, and hMOF . In tissRecent work showed that manipulation of p53 acetylation in mice could separate the classic p53 functions of cell cycle arrest, induction of apoptosis, and senescence from the anti-cancer activity of p53 . In thisThere are other ways p53 can be modified. p53 can be methylated on lysines and arginines by several different methylase enzymes, which can either activate or repress its activity depending on which site is methylated and the number of methyl groups added . p53 canIt is clear that p53 protein activity can be modulated by several types of posttranslational modification, and that the overall activity of a given p53 protein molecule will be determined in a complex manner by the balance between different amounts and combinations of modifications at any given time. Some of the modifications are likely to affect other modifications, particularly those that target the same p53 amino acid residues. Affecting p53 activity by altering amounts, timing, and specificity of protein modification represents a promising avenue to affect the aging process.Ribosome biogenesis refers to the production and processing of ribosomal RNA. It is a complex process that occurs in the nucleolus of the cell. RNA polymerase I (Pol I) transcribes tandem arrays of ribosomal DNA (rDNA) (about 400 repeats in humans) in the nucleolar organizer region (NOR) to delineate a nucleolus . This prInterestingly, ribosome biogenesis serves as a sensor of cellular stress and defects in ribosome biogenesis activate p53 . FurtherInhibitors of ribosome biogenesis (genotoxic or non-genotoxic agents) that induce a p53 response could potentially function as potential anti-cancer agents . DNA dam1-arrested cells to transit from quiescence (where they still retain the ability to proliferate) to senescence. However, p53 might not induce cellular senescence beyond G1 arrest, so its role in senescence induction is unclear -related family \u201392 and iRapamycin has been shown to increase lifespan for heterogeneous outbred mice ,98 and fGlucose is the major source of energy for ATP generation and glycolysis is an ancient metabolic pathway that converts glucose to pyruvate to produce ATP and NADH. Pyruvate then enters the tricarboxylic acid (TCA) cycle to generate ATP through OXPHOS. Contrary to normal cells, cancer cells primarily generate energy (ATP) through aerobic glycolysis to increase anabolism and perpetuate tumor growth, known as the Warburg effect . In addic oxidase 2 (SCO2)  ,113 and 2 (SCO2) ,115 thatScutellaria baicalensis Georgi that induces apoptosis in HeLa cells  induce AMPK and as a result induce p53. Metformin also inhibits mTORC1 via AMPK . Metform0 or G1. Thus, Ap53\u2212/\u2212 mice is increased by this intervention  is a well-characterized intervention to extend lifespan and ameliorate physiological aging across species, including mammals . CR causrvention . However+/\u2212 mice . It rema1 arrest in response to damage or cellular stress to protect the cell, but does not induce cellular senescence directly. Instead pro-growth pathways may enable an arrested cell to enter a senescent state. Recent data that show p53 allows cells to survive serine depletion highlight a protective role (p53 connects cell metabolism and DNA integrity to various cellular outcomes including cell cycle arrest, cellular senescence, and cell death. Although the role of p53 in tumor suppression has been well studied, recent evidence suggests that p53 also affects aging. There are multiple ways to target p53 as an anti-cancer therapeutic . Howeverive role . These o"}
{"text": "PADI4 gene to form a susceptibility haplotype for rheumatoid arthritis (RA); nevertheless, results in association studies appear contradictory in different populations. The aim of the study was to analyze if the presence of three SNPs in PADI4 gene susceptibility haplotype (GTG) is associated with ACPA positivity in patients with RA. This was a cross-sectional study that included 86 RA patients and 98 healthy controls. Polymorphisms PADI4_89, PADI4_90, and PADI4_92 in the PADI4 gene were genotyped. The susceptibility haplotype (GTG) was more frequent in RA patients; interestingly, we found a new haplotype associated with RA with a higher frequency (GTC). There were no associations between polymorphisms and high scores in Spanish HAQ-DI and DAS-28, but we did find an association between RARBIS index and PADI4_89, PADI4_90 polymorphisms. We could not confirm an association between susceptibility haplotype presence and ACPA positivity. Further evidence about proteomic expression of this gene will determine its participation in antigenic generation and autoimmunity.Peptidyl arginine deiminase IV (PAD 4) is the responsible enzyme for a posttranslational modification called citrullination, originating the antigenic determinant recognized by anti-cyclic citrullinated peptide antibodies (ACPA). Four SNPs (single nucleotide polymorphisms) have been described in  Rheumatoid arthritis (RA) is an autoimmune disease, characterized by articular inflammation which can lead to joint destruction. RA prevalence is 1% worldwide with considerable variation between ethnic groups, with a higher prevalence in Caucasians compared with Asiatic populations . This diloci for RA located within several chromosomes, one consistently implicated is the HLA-DRB1 gene , 28PADI4The present study represents the first one in Mexican population which is the result of genetic admixture of Spanish, Indian, and Black populations, and it should be emphasized that the genetic background introduced from Spaniards includes genes from Romans, Greeks, Visigods, Arabs, and Jews . We shouPADI4 polymorphisms are associated with RA susceptibility, regardless of ACPA titers. This is consistent with results published by Hoppe and Kang et al. in German [PADI4 gene polymorphisms could have a higher susceptibility role in Asiatic populations compared to Caucasians, but soon after that, an association between PADI4 gene and RA was confirmed in a French population [We found that n German  and Koren German  populatin German , since Pn German  concludepulation . The lacP = 0.0002), but more interestingly, we found a new haplotype (GTC) which was both significantly associated (P = 0.006) and more frequent than previous susceptibility haplotype. We believe that since this second haplotype was present in a higher frequency among RA patients, it should be looked forward.When we analyzed haplotype frequencies, we found that the susceptibility haplotype (GTG) was significantly associated with RA  positivity was present in 74%, similar to previous reports , 49, 50.i et al. . We did i et al.  and seemi et al. . For thii et al. , but thei et al. . As a coPADI4 translation, as the increase in mRNA stability has been demonstrated, generating an increase in protein levels and as a consequence a higher occurrence of citrullinated proteins [Supporting the first theory is the fact that susceptibility haplotype presence can affect proteins . CitrullPADI4 gene polymorphisms and clinical variables, except for high punctuation in RARBIS index and presence of susceptibility allele (G) in PADI4_89 or susceptibility homozygous (T/T) in PADI4_90 of PADI4 gene.Finally, we did not find associations between Limitations in this study include sample size since only one gender is represented in the present investigation; it is well known that RA is a condition that affects mainly women, but it would be desirable to increase sample size in order to be able to include male patients and to verify if association with the susceptibility haplotype remains constant, since in our study we were able to find only one HC with the susceptibility haplotype.In conclusion, we confirm the association between the susceptibility haplotype (GTG) with RA patients, observing also that a new haplotype (GTC) could be associated with RA in Mexican mestizo patients. Further studies including other regions in Mexico and increasing the sample size are required in order to confirm our findings; furthermore, the biggest challenge for association studies is the identification of genetic variants that, combined in haplotypes, can be described as causal effects to RA."}
{"text": "The function of p53 is best understood in response to genotoxic stress, but increasing evidence suggests that p53 also plays a key role in the regulation of metabolic homeostasis. p53 and its family members directly influence various metabolic pathways, enabling cells to respond to metabolic stress. These functions are likely to be important for restraining the development of cancer but could also have a profound effect on the development of metabolic diseases, including diabetes. A better understanding of the metabolic functions of p53 family members may aid in the identification of therapeutic targets and reveal novel uses for p53-modulating drugs. TP53 allele display an enormously increased cancer risk, a condition known as Li-Fraumeni syndrome  progress to irreversible senescence, a process named geroconversion, whereas cells in which mTOR is inhibited  ultimately achieve a reversible quiescent state . However, the majority of cancer cells display alterations in glucose metabolism  cells, pyruvate is subsequently fed into the mitochondrial tricarboxylic acid (TCA) cycle in order to generate NADH and FADHtabolism . Often, tabolism . Intermetabolism  but alsotabolism . Furthertabolism ; therefotabolism . Increastabolism . FurtherTP53-induced glycolysis and apoptosis regulator (TIGAR), which acts as a phosphatase that degrades F2,6BP and thereby decreases the activity of PFK1. Hence, p53, via TIGAR, lowers the glycolytic rate and would be predicted to promote the diversion of glycolytic intermediates into the PPP  takes place in the mitochondria and breaks down fatty acids into two-carbon units in order to yield acetyl-CoA, NADH, and FADHduce ATP . Fatty aduce ATP . Multiplduce ATP . Additioduce ATP , are knomulation .p53 plays a crucial role in lipid metabolism, participating in both normal and pathological conditions. True to its role as a tumor suppressor, p53 generally functions as a negative regulator of lipid synthesis by activating fatty acid oxidation and inhibiting fatty acid synthesis . Mouse eThe regulation by p53 of carbohydrate and lipid metabolism is tightly linked to another important function of p53: the regulation of ROS. In its role as a \u201cguardian of the cell,\u201d p53 can eliminate the deleterious effects of oxidative insult either by limiting ROS damage in cells that can be salvaged or by using ROS to eliminate cells damaged beyond repair. Hence, if the insult is transient and\u00a0repairable, then p53 can activate a suite of antioxidant responses, many of which are linked to carbohydrate or lipid metabolism. For example, the activation of the PPP via TIGAR produces NADPH that can reduce glutathione, an important cellular antioxidant. Interestingly, a recent study showed that p53 can also directly promote GSH synthesis at the expense of nucleotide synthesis after serine deprivation, and, thereby, it actively controls ROS levels under conditions of metabolic stress . MitochoAutophagy (here referring to macroautophagy), which literally translates as \u201cself-eating,\u201d is an important cellular catabolic process carried out in the cytoplasm. Autophagy is characterized by the formation of double-membraned autophagosomes around cytoplasmic components that have been targeted for degradation. Once enclosed around their cargo, autophagosomes fuse with lysosomes, forming autolysosomes in which the delivered contents are then catabolized . AutophaThe relationship between p53 and autophagy is complex, given that p53 can both promote and inhibit autophagy in a context-dependent manner . This abDRAM-1 is activated by p53 and encodes multiple isoforms that regulate autophagy , thereby promoting cell-cycle arrest and survival in response to nutrient starvation (The activation of autophagy by (nuclear) p53 is well established and can occur through a variety of mechanisms. The mTORC1 pathway is a major inhibitory regulator of autophagy. Hence, by negatively regulating mTORC1 , p53 actutophagy . The proutophagy . Recentlutophagy . The tumutophagy . Recentlnditions , suggestnditions , ATG7 prarvation .\u2212/\u2212 HCT116 cells also inhibited autophagy, the mutants showing the highest cytoplasmic localization having the greatest effect (The role of cytoplasmic p53 in the regulation of autophagy was established by suppressing p53 expression in colon cancer (HCT116) cells. Surprisingly, this resulted in the upregulation of autophagy, whereas the reintroduction of p53 repressed autophagy . In simit effect . It seemt effect . Under nt effect .Clearly, the identification of an ever-increasing number of functions for p53 provokes the important question of how the final response of the cell is determined. Many of the examples discussed above indicate that the particular outcome of the activation of p53 depends on the level of the insult. This may influence the activity of p53 itself as well as that of other proteins or transcriptional factors that ultimately determine the fate of the cell. p53 has clear relationships with other transcription factors that are known to be critical in the response to metabolic stress, most notably PGC, SREBP, and Nrf2. PGC-1\u03b1 is a critical regulator of glucose, lipid, and energy metabolism, whose functions include the regulation of mitochondrial biogenesis and the expression of antioxidants. Under conditions of mild metabolic stress, p53 has been shown to increase PGC-1\u03b1 expression, resulting in the induction of an antioxidant response . MoreoveTp63 and Tp73 genes are transcribed from two distinct promoters, resulting in either full-length proteins that retain a full transactivation (TA) domain (TAp63 and TAp73) or N-terminally truncated isoforms (\u0394Np63 and \u0394Np73) that lack part of this domain but still retain some ability to activate transcription. In addition, alternative RNA splicing results in different C termini for both p63 and p73 isoforms, termed \u03b1-\u03b5 and \u03b1-\u03b7, respectively. TAp63 and TAp73 can supplement p53 function by transactivating p53 target genes, resulting in cell-cycle arrest and apoptotic cell death, although p63 and p73 also have distinct transcriptional targets. In contrast, \u0394Np63 and \u0394Np73 generally have antiapoptotic functions and have been shown to act as dominant negatives for inhibiting the function of p53 family members. However, \u0394N isoforms also activate specific sets of target genes and function independently of other p53 family isoforms. Despite these similarities and overlap in activity, p63 and p73 each have functions that are strikingly different from those of p53, and both proteins play critical roles in development . Unlike p63, p73 is essential for proper neural development, and p73 knockout mice display developmental defects in the CNS. Analysis of selective TAp73 or \u0394Np73 knockdown suggested that \u0394Np73 isoforms are necessary for neuronal survival, whereas TAp73 is required for the long-term maintenance and differentiation of neuronal stem cells. Both p63 and p73 are important for germ cell maintenance. p63 controls the quality of the female germline by eliminating damaged oocytes, whereas p73 maintains genomic stability of the oocyte pool.p53 belongs to a family of transcription factors that also includes p63 and p73, functional homologs of p53 that show high sequential and structural similarity. Both the Sirt1, AMPK\u03b12, and LKB1 and thereby coordinates fat and glucose metabolism. Loss of TAp63 results in defects in lipid utilization, fatty acid synthesis, and FAO, and mice lacking TAp63 consequently display insulin resistance as well as symptoms of obesity, type 2 diabetes, and premature aging  had a shortened lifespan  and increased levels of ROS . Furtherem cells . Another+/m mice . HoweverThe metabolic functions of p53 are emerging as critical not only for tumor suppression but also for maintaining normal cellular homeostasis. Nevertheless, many questions remain. Clearly, different stress signals can activate different p53 responses, as measured by transcriptional profiles, and several proteins that contribute to the ability of p53 to promote adaptation to metabolic stress are preferentially activated by p53 in response to nutrient deprivation . However"}
{"text": "In this study we aimed at characterizing the regulation of hepatic metabolic pathways by the p53 transcription factor.Analysis of gene expression following alteration of p53 status in several human- and mouse-derived cells using microarray analysis, quantitative real-time PCR, chromation immunoprecipitation and reporter gene assays. A functional assay was performed to determine lipid transfer activity.plfp, abca12 and cel) was further characterized. In addition to HepG2, the genes were induced following activation of p53 in human primary hepatic cells isolated from liver donors, p53-dependent regulation of these genes was evident in other cell types namely Hep3B cells and mouse hepatocytes. Furthermore, p53 was found to bind to the genes\u2019 promoters in designated p53 responsive elements and thereby increase its transcription. Importantly, p53 augmented the activity of secreted PLTP, which plays a major role in lipoprotein biology and atherosclerosis pathology.We identified a novel role for the p53 protein in regulating lipid and lipoprotein metabolism, a process not yet conceived as related to p53, which is known mainly in its tumor suppressive functions. We revealed a group of 341 genes whose expression was induced by p53 in the liver-derived cell line HepG2. Twenty of these genes encode proteins involved in many aspects of lipid homeostasis. The mode of regulation of three representative genes (These findings expose another facet of p53 functions unrelated to tumor suppression and render it a novel regulator of hepatic lipid metabolism and consequently of systemic lipid homeostasis and atherosclerosis development."}
{"text": "The human waking EEG spectrum shows high heritability and stability and, despite maturational cortical changes, high test-retest reliability in children and teens. These phenomena have also been shown to be region specific. We examined the stability of the morphology of the wake EEG spectrum in children aged 11 to 13 years recorded over weekly intervals and assessed whether the waking EEG spectrum in children may also be trait-like. Three minutes of eyes open and three minutes of eyes closed waking EEG was recorded in 22 healthy children once a week for three consecutive weeks. Eyes open and closed EEG power density spectra were calculated for two central (C3LM and C4LM) and two occipital (O1LM and O2LM) derivations. A hierarchical cluster analysis was performed to determine whether the morphology of the waking EEG spectrum between 1 and 20 Hz is trait-like. We also examined the stability of the alpha peak using an ANOVA.The morphology of the EEG spectrum recorded from central derivations was highly stable and unique to an individual (correctly classified in 85% of participants), while the EEG recorded from occipital derivations, while stable, was much less unique across individuals (correctly classified in 42% of participants). Furthermore, our analysis revealed an increase in alpha peak height concurrent with a decline in the frequency of the alpha peak across weeks for occipital derivations. No changes in either measure were observed in the central derivations.Our results indicate that across weekly recordings, power spectra at central derivations exhibit more \u201ctrait-like\u201d characteristics than occipital derivations. These results may be relevant for future studies searching for links between phenotypes, such as psychiatric diagnoses, and the underlying genes  by suggesting that such studies should make use of more anterior rather than posterior EEG derivations. The EEG can be recorded non-invasively and inexpensively, with accurate estimates of cortical oscillations made with just a few minutes of recording. EEG recordings are of great utility not only for learning about the healthy brain, but also reflect changes in brain structure/function associated with psychiatric disorder. The heritability and stability of the EEG add to its utility. For example, studies of twins have found that the human waking EEG is one of the most heritable traits with heritability estimates ranging between 0.55 and 0.9,2, depenIn addition to being highly heritable, the waking EEG spectrum is stable across time. A number of studies in adults, adolescents, and children have shown high test-retest reliability between sessions  for both absolute and relative spectra,9-11. Mientire waking EEG spectrum in early adolescents (ages 11 to 13 years) over weekly intervals and examine the extent to which the waking EEG spectrum is a biological trait in this age range. We used a cluster analysis based on distance between the three weekly-recorded power density spectra to assess whether the EEG spectrum is trait-like. Compared to ICC analysis, in cluster analysis the entire EEG spectrum is taken into account, making it more representative of an individual, thus enhancing its utility and validity. Unlike ICC, cluster analysis does not rely on a priori knowledge of repeated measures within a given individual and thus does not make use of intra and inter-individual variability. Rather, in cluster analysis, spectra are grouped according to their similarity. Furthermore, we examined whether trait-like characteristics, as determined by the cluster analysis, vary in central and occipital EEG derivations. In order to compare our results with previous stability studies, we also examined the stability of the alpha peak across recordings using an ANOVA.The aim of the current study was to examine the stability of the Twenty-two healthy right-handed early adolescents, aged 11 to 13 years  participated in this study. All participants were pre/early pubertal (n = 13) or mid-pubertal (n = 8) with the exception of one female who was late pubertal.Participants were recruited using television and newspaper advertisements, as well as at schools, and at a meeting of the association for parents with gifted children. The Zurich cantonal ethical committee (KEK) for research on human participants approved the protocol and the participants\u2019 legal guardian gave written informed consent. This study conforms with the Code of Ethics of the World Medical Association (Declaration of Helsinki), printed in the British Medical Journal (18 July 1964). Participants were compensated for their participation by cinema, book or CD vouchers and a T-shirt.Participants were healthy, had no history of neurologic and psychiatric disease, and were medication free. For three days before each experimental session participants were asked to abstain from caffeine and medication and to adhere to a regular sleep\u2013wake schedule . Wrist-worn actimeters and sleep logs verified compliance. The data used in the current study is the control data from another study.Participants came to the laboratory on three different occasions in weekly intervals, always at the same time of the day. At each session, a baseline waking EEG was recorded to assess the stability of waking EEG in early adolescents over weekly intervals.EEG , electrooculogram (EOG), and electrocardiogram (ECG) were recorded with a polygraphic amplifier Artisan . The analog signals were high-pass  and low-pass filtered (\u22123 dB at 67.2 Hz), sampled at 256 Hz, and recorded using Rembrandt DataLab .Waking EEG was recorded continuously for 6 min . Participants sat on a chair, rested their head on a chin rest, and were instructed to avoid movement. Vigilance was ensured by continuous online visual inspection of the recordings and alerting the subjects via intercom when signs of drowsiness were present.Artifacts were visually marked and artifact-free segments were subjected to spectral analysis  using MATLAB . All participants had at least thirty 2-s epochs of artifact free data in each condition  which were used to calculate the power density spectra. Frequencies between 1 and 20 Hz were analyzed.2 test to assess whether the rate of correct clustering was significantly different between derivations or \u2018eyes open/closed\u2019 conditions.Hierarchical cluster analysis based on Euclidean distance was used to examine whether the log-transformed EEG spectra from 1 to 20 Hz) were trait-like using MATLAB functions PDIST and LINKAGE (average aggregation strategy). Cluster analysis was performed in several steps. First, a power spectrum was obtained for each recording/participant and represented as a vector consisting of power density at all frequency bins . Next, the distance between all vectors  was calculated and vectors with small distances between them were clustered together, while vectors that were far apart were clustered separately. Finally, the distance between vectors is represented visually as a dendrogram, which consists of upside-down U-shape lines where the height of the U represents the distance between the connected objects  of the alpha peak. The alpha peak was determined on an individual basis because significant interindividual variability in the frequency of the alpha peak has been shown[To be able to compare our results with previous studies and investigate whether the characteristics of the alpha rhythm vary across sessions and derivations, we determined the frequency and height  visual analog scale similar to Aitken, was admIn order to compare the morphology of the entire spectrum we performed a hierarchical cluster analysis (see Methods). The number of participants that clustered successfully over all three recordings was dependent on EEG derivation Figure\u00a0. For the2(1) = 13.54, p = 0.0002; Eyes open: \u03c72(1) = 9.40, p = 0.002). To demonstrate this difference, four participants whose spectra clustered in the eyes closed condition for derivation C3LM but not O1LM are shown in Figure\u00a02(1) = 0.23, p = 0.63, Eyes open: \u03c72(1) = 0, p = 1) or left and right occipital (Eyes closed: \u03c72(1) = 2.28, p = 0.13, Eyes open: \u03c72(1) = 0, p = 1) derivations  and frequency (Hz) of the alpha peak in the waking EEG spectrum using an ANOVA with factor \u2018week\u2019 . Average power spectra are shown in Figure\u00a0We examined the stability of the peak height  = 7.86; p = 0.001; O2LM: F = 7.134, p = 0.006). Peak height increased between weeks one and two (O1LM: t(21) = \u22122.68; p = 0.014; O2LM: t(21) = \u22122.78; p = 0.011) and between weeks one and three (O1LM: t(21) = \u22124.12; p <0.001; O2LM: t(21) = \u22122.9; p = 0.009). Means and standard deviations for peak height and frequency are shown in Table\u00a0A change in mental tension across weeks was observed (mean (SD): week 1 = 30.3 (16.8); week 2 = 29.7 (24.2); week 3 = 27.6 (21.1); F = 4.248; p = 0.036). A post-hoc paired t-test revealed a decline in mental tension between weeks one and three (t(21) = 2.37; p = 0.028) but not between weeks one and two (t(21) = 1.88; p = 0.075). No other variables showed a significant change across weeks.We included mood as a covariate in the ANOVA analysis in order to examine whether the change in mental tension had an impact on the observed effects in the occipital derivations. Examining the data in this way, there was no longer an effect of week for O1LM peak frequency in the eyes closed condition and O2LM peak height in the eyes open condition.The current study used two different measures to examine the stability of the EEG spectrum in early adolescents: a cluster analysis approach and characterization of the alpha peak. Overall, the results showed that the waking EEG is highly stable across recordings and unique to an individual. In addition, we were able to demonstrate that the degree to which the EEG spectrum is trait-like is dependent on the brain region.entire spectrum rather than limited to the alpha band. Although the alpha oscillation comprises an important cortical rhythm, functional neuroanatomy is more accurately reflected in the entire EEG spectrum.The current study used cluster analysis to examine whether the waking EEG in early adolescents represents a trait. Thus, the unique contribution of our analysis is not solely the examination of stability, but also the quantification of interindividual variability. Another advantage of the current analysis is that clustering is based on the interindividual variability rather than low intraindividual stability may account for the lower rate of clustering in occipital versus central derivations. For example, greater variability across individuals was observed in the frequency and shape of the waking EEG spectrum in central compared to occipital derivations , we would not expect successful clustering for any participant by chance. We do not expect that the difference between central and occipital derivations is due to less stability of the alpha peak in the occipital regions because our ANOVA analysis revealed a change in alpha peak characteristics in only the eyes open condition, whereas the difference between occipital and central derivations was present in both eyes open and closed conditions. The lack of In a previous study of trait-like characteristics of the sleep EEG in a different sample of adolescents, Tarokh et al. found thThe alpha frequency band is an important rhythm to consider when examining the heritability and stability of the waking EEG. Several studies have shown that alpha peak power is a highly stable EEG parameter and thatSeveral limitations of this study are important to note. With respect to our analysis of the stability of the alpha frequency peak, our frequency resolution was 0.5 Hz, which limits our ability to detect more subtle changes in frequency across recording session. Furthermore, this analysis was restricted to four derivations, which limits our ability to examine regional differences in further detail. We examined a narrow age range and the degree to which the regional differences we observe are specific to this developmental stage is unknown. In fact, there are large differences in the maturational trajectory of different cortical regions. Future We used a novel method to show not only that the waking EEG spectrum is trait-like, but that the degree to which the EEG is trait-like depends on brain region. This finding has implications for resting state waking EEG studies in search of biological markers of cognitive capabilities and psychiatric disorders suggesting that such studies should use central rather than occipital derivations.The authors have no competing interests to declare.SPL and PA designed the study. LT, SPL and DB performed data analysis. DB and SPL participated in data collection. LT, SPL, DB and PA wrote the manuscript. All authors read and approved the final manuscript.Peter Achermann, Sarah P Loughran Shared senior authorship.Dominik C Benz, Leila Tarokh Shared first authorship."}
{"text": "Posttraumatic stress disorder (PTSD) and major depressive disorder (MDD) frequently co-occur after traumatic experiences and share neurocognitive disturbances in verbal memory and executive functioning. However, few attempts have been made to systematically assess the role of a comorbid MDD diagnosis in neuropsychological studies in PTSD.The purpose of the current study is to investigate neurocognitive deficits in PTSD patients with and without MDD. We hypothesized that PTSD patients with comorbid MDD (PTSD+MDD) would have significantly lower performance on measures of verbal memory and executive functioning than PTSD patients without MDD (PTSD\u2013MDD).n=84) and patients with PTSD\u2013MDD (n=56).Participants included in this study were 140 treatment-seeking outpatients who had a diagnosis of PTSD after various single traumatic events and participated in a randomized controlled trial comparing different treatment types. Baseline neuropsychological data were compared between patients with PTSD+MDD (The PTSD+MDD patients had more severe verbal memory deficits in learning and retrieving words than patients with PTSD alone. There were no differences between the groups in recall of a coherent paragraph, recognition, shifting of attention, and cognitive interference.The results of this study suggest that a more impaired neurocognitive profile may be associated with the presence of comorbid MDD, with medium-sized group differences for verbal memory but not for executive functioning. From a clinical standpoint, being aware that certain verbal memory functions are more restricted in patients with comorbid PTSD and MDD may be relevant for treatment outcome of trauma-focused psychotherapy. Posttraumatic stress disorder (PTSD) and major depressive disorder (MDD) are common outcomes after experiencing a traumatic event , as well as consistent deficits in verbal memory  a PTSD diagnosis according to DSM-IV; 2) a single traumatic event that led to the development of PTSD and had stopped at the time of inclusion; 3) age between 18 and 65 years; 4) mastery of the Dutch language. Exclusion criteria were: 1) acute suicidality; 2) current severe MDD or current severe alcohol or substance dependence according to DSM-IV; 3) a lifetime psychotic disorder according to DSM-IV; and 4) a severe personality disorder according to the SCID-II screener , 1 (= rarely), 3 (= sometimes), and 5 (= often) and the total PTSD score (range 0\u2013110) consists of the sum of the scores. The depression scale of the Hospital Anxiety and Depression Scale (HADS) was used to measure the severity of the depressive symptoms by self-report  is a test to measure shift of attention, planning and cognitive flexibility Reitan, . PatientThe Stroop Color Word Test is thought to measure selective attention and cognitive flexibility . If this overall test was significant, we examined group differences on the separate test variables within the univariate general linear model. Two-tailed tests were applied throughout and level of significance was set at \u03b1=0.05 for the multivariate tests. Level of significance for the univariate analyses was set at \u03b1=0.01 to correct for multiple testing. Partial-eta squared was calculated as an effect size for significant differences.Analyses were conducted using SPSS version 19.0 . Chi-square tests and independent Demographic and clinical characteristics of the two groups are displayed in F-test revealed a significant difference between the groups for the CVLT, F=2.89, p=0.011. Univariate analyses revealed that the PTSD+MDD group scored significantly lower on CVLT sum of trials 1\u20135, F=8.63, p=0.004, \u03b72=0.058; CVLT short-term cued recall, F=8.15, p=0.005, \u03b72=0.057; CVLT long-term free recall, F=10.98, p=0.001, \u03b72=0.069; and CVLT long-term cued recall, F=7.77, p=0.006, \u03b72=0.053, than the PTSD\u2013MDD group. Multivariate F-tests did not reveal significant differences between the groups for the RBMT, F=2.90, p=0.059; TMT, F=1.10, p=0.337; or Stroop, F=1.88, p=0.137.Mean scores and standard deviations of the two groups on the neurocognitive tests are displayed in The main finding of the current study is that treatment-seeking patients with PTSD and comorbid MDD have more pronounced verbal memory deficits than patients with PTSD alone, with medium-sized differences between the groups. These verbal memory deficits in the group with PTSD and MDD seem to be restricted to the encoding, short-term retrieval and long-term retrieval of separate words, since there were no significant differences in retrieval of a coherent paragraph or in recognition between the two groups. Various aspects of executive functioning in the present study were similar for both groups as well, as expressed by a lack of differences on measures of mental speed, shift of attention, selective attention, and cognitive interference. In sum, encoding and retrieval of separate words was a bigger challenge for PTSD patients who had comorbid MDD diagnoses, than for those who had PTSD without MDD. Results of this study thus suggest a somewhat more impaired neurocognitive profile for PTSD patients with comorbid MDD.Our results confirm and extend the studies of Sachinvala et al.  and JohnWe note that the group with PTSD and comorbid MDD in our study also had more severe PTSD symptoms than the group without comorbid MDD. Therefore, it is possible that the differences we found between the groups are partly attributable to greater PTSD symptom severity. Moreover, it could be the case that PTSD symptom severity contributes to the other neuropsychological processes  for which we were not able to confirm statistically significant group differences. However, more severe PTSD symptoms and a comorbid MDD diagnosis are such intertwined constructs that it is very difficult, and may not even be clinically meaningful, to statistically separate the influence of these two variables (Miller & Chapman, Limitations of this study include that we did not administer tests for all types of attention and that we had to exclude patients with severe MDD, as this was an exclusion criterion for the treatment trial. It would be interesting to examine especially sustained attention in samples with PTSD+MDD versus PTSD alone, as depressive symptoms were also found to play a role in sustained attention in PTSD (Meewisse et al., In conclusion, patients with PTSD and comorbid MDD seem to have more difficulty in learning separate units of verbal information and retrieving this information in the short and long term than PTSD patients without comorbid MDD, even when the severity of the MDD diagnosis is only mild to moderate. PTSD symptom severity may contribute to these difficulties. From a clinical standpoint, it is good to be aware that these aspects of verbal memory are more restricted in patients with comorbid PTSD and MDD, as verbal memory performance can influence the treatment outcome of trauma-focused psychotherapy (Wild & Gur,"}
{"text": "This tumour suppressor protein has three main domains; the N-terminal transactivation domain, the CTD  and the core domain (p53C) that constitutes the sequence-specific DBD (DNA-binding region). Most p53 mutations related to cancer development are found in the DBD. Aggregation of p53 into amyloid oligomers and fibrils has been shown. Moreover, amyloid aggregates of both the mutant and WT (wild-type) forms of p53 were detected in tumour tissues. We propose that if p53 aggregation occurred, it would be a crucial aspect of cancer development, as p53 would lose its WT functions in an aggregated state. Mutant p53 can also exert a dominant-negative regulatory effect on WT p53. Herein, we discuss the dominant-negative effect in light of p53 aggregation and the fact that amyloid-like mutant p53 can convert WT p53 into more aggregated species, leading into gain of function in addition to the loss of tumour suppressor function. In summary, the results obtained in the last decade indicate that cancer may have characteristics in common with amyloidogenic and prion diseases. Disruption of the p53 network usually has severe consequences that favour cell survival and tumour progression \u20133. p53 iThe human p53 protein comprises 393 amino acid residues and three main functional regions: the N-terminal activation domain, which is able to interact with a variety of proteins; the CTD, responsible for tetramerization; and the core domain (p53C) comprising residues 94\u2013312 that constitute the sequence-specific DBD (DNA-binding region) of the protein  Figure . More thTP53) are frequently associated with increased susceptibility to cancer development. Inactivation of p53-regulated pathways has been described in over 50% of all human cancers, making them interesting targets for cancer therapies  is located at 17p13.1 and is composed of 11 exons  breast cancer  reduction assay  are found in most proteins. These small APRs are normally sheltered from aggregation due to intra-protein interactions or because they are hidden in the hydrophobic protein core. However, when the protein structure is disturbed by mutations or environmental changes, these sequences can be exposed, thus favouring protein aggregation . One of lymphoma . This coThe formation and kinetics of WT and mutant p53 amyloid oligomers and fibrils has been widely discussed ,79\u201382. pThe DNA-binding properties of p53 are frequently lost upon p53 aggregation; however, the small cognate double-stranded DNA has been shown to stabilize both the p53C domain and full-length p53 and to have the potential to rescue aggregated and misfolded species  Figure . TherefoThe presence of intermediate states that participate in the p53 aggregation route has been highlighted in different studies ,72,86. MTP53 gene proposes that the presence of even a single mutant protein in the formation of the p53 tetramer would lead to loss-of-function of p53 [The dominant-negative hypothesis for WT p53 inactivation in cells carrying a mutation in one of the alleles of the n of p53 ,88. Our n of p53 ,16. In on of p53  , conjugated ubiquitin and several cell cycle-regulating cyclins, among other molecules [In addition to self-aggregation and combined mutant-WT aggregation, p53 aggregation has also been described where other proteins function as a platform. This is the case for the above mentioned p53\u2013Hsp70\u2013Mdm2 interaction  and alsoolecules . p53 wasolecules .We conclude that aggregation of p53 into a mixture of oligomers and fibrils sequesters the native protein into an inactive conformation that is typical of a prionoid. Our findings that amyloid aggregates are present in biopsies of breast cancer tissues, especially in the aggressive tumours, show the relevance of this prion behaviour in cancer pathogenesis ,76. In fThe prion-like behaviour of oncogenic p53 mutants, as represented in"}
{"text": "The tumor suppressor gene p53 is involved in a variety of cellular activities such as cellular stress responses, cell cycle regulation and differentiation. In our previous studies we have shown p53\u2019s transcription activating role to be important in osteoblast differentiation. There is still a debate in the literature as to whether p53 inhibits or promotes differentiation. We have found p53 heterozygous mice to show a p53 dependency on some bone marker gene expression that is absent in knockout mice. Mice heterozygous for p53 also show a higher incidence of osteosarcomas than p53 knockout mice. This suggests that p53 is able to modify the environment within osteoblasts. In this study we compare changes in gene expression resulting after either a transient or stable reduction in p53. Accordingly we reduced p53 levels transiently and stably in C2C12 cells, which are capable of both myoblast and osteoblast differentiation, and compared the changes in gene expression of candidate genes regulated by the p53 pathway. Using a PCR array to assay for p53 target genes, we have found different expression profiles when comparing stable versus transient knockdown of p53. As expected, several genes with profound changes after transient p53 loss were related to apoptosis and cell cycle regulation. In contrast, stable p53 loss produced a greater change in MyoD and other transcription factors with tissue specific roles, suggesting that long term loss of p53 affects tissue homeostasis to a greater degree than changes resulting from acute loss of p53. These differences in gene expression were validated by measuring promoter activity of different pathway specific genes involved in differentiation. These studies suggest that an important role for p53 is context dependent, with a stable reduction in p53 expression affecting normal tissue physiology more than acute loss of p53.  In adgulation ,7. In ougulation . Other sgulation ,10. Whilgulation . In embrgulation . So whilThe importance of tissue specificity with regard to p53 function is further supported by studies carried out in p53 transgenic mice ,14. In aSince p53 responds to multiple stimuli and can affect multiple, different cellular processes, changes in p53 expression will affect cells differently depending on whether the change is transient or stable. In this study we test this by using shRNA interference to reduce p53 levels in a bipotential cell line, C2C12, transiently and compared the changes in gene expression of genes involved in p53 pathways with a stable reduction of p53. We found greater changes in expression of MyoD and of other transcription factors with tissue specific roles in the stable p53 knockdown cells, which suggests that long term partial loss of p53 expression affects tissue homeostasis to a greater degree than changes resulting from transient loss of p53. Additionally, we show differences in the promoter activities of genes involved in differentiation between stable and transient loss of p53. Together this points toward the role of p53 as being context dependent, with a stable loss affecting cellular differentiation more than a transient loss.2/95% air.  In order to study the role of p53 in the differentiation of mesenchymal stem cells, the bi-potential cell line C2C12  was selected because it can differentiate into myotubes and can be induced to osteoblasts, depending on the culture media . These cThe p53 HuSH-29 shRNA plasmids , were used to create transient and stable knockdown lines of p53 within the C2C12 cells. We used three different shRNA sequences to knockdown p53. Control cells received in a similar sized non effective scrambled sequence (TR20003). SuperFect  was used for both transient and stable transfections according to manufacturer\u2019s protocol. Additionally, for obtaining stable clones, cells were selected post transfection using puromycin. Single cell clones were expanded, passaged and characterized before use. To determine the changes at the transcriptional level, RNA was isolated either using TRI Reagent  or the QIAGEN RNeasy Mini Kit (cat. no. 74104) prep system depending on the assay used. Genomic contamination of the RNA was removed using the Ambion DNA Free kit . DNA free RNA was quantified using the Nanodrop2000 .Semiquantitative RT-PCR was used to measure the relative expression of p53 after shRNA introduction as well as, the various bone and muscle markers.. RNA was reverse-transcribed using the QIAGEN OneStep RT-PCR kit (cat. no. 210210) using equivalent amounts of RNA. The following primers were ordered from QIAGEN, and used according to their protocol: Gapdh (cat. no. QT01658692) and p53 (cat. no. QT00101906). To measure the intensity of the resulting PCR, the samples were run on a 1% agarose gel containing ethidium bromide and the image captured on a Kodak system. The image was then quantified using the UnscanIt\u2122 software and the relative quantification was calculated using Gapdh to normalize the expression.2 First Strand Synthesis Kit  according to the standard protocol. A master mix using the RT2 SYBR Green/ROX kit  was made for each sample and then the array was then loaded and ran on an ABI Prism 7300 Real Time PCR System, using the settings as defined by SABiosciences for their arrays. Once run, the data was normalized using the included genes on the array, then fold changes were calculated by using the standard 2-\u0394\u0394Ct method, using the array of the control cells as the second \u0394.In addition to looking at the various differentiation markers, a more in depth analysis of pathway perturbations was also assayed using the QIAGEN/SABiosciences p53 Signaling Pathway PCR Array . Genes included on the array are direct targets of p53, regulators of p53, or genes in a p53 pathway that are downstream of p53, and cover many of the different pathways p53 is found to affect, such as apoptosis, differentiation, and cell cycle regulation, as well as control genes for normalization, genomic DNA contaminating and RT efficiency. The RNA obtained for a stable line of shRNA plasmid 34 (S34), 36 (S36), as well as, a transient transfection created from plasmid 36 (T36) and a control line of C2C12 cells was made using QIAGEN RNeasy Mini Kit (cat. no. 74104) prep was first quantified using the Nanodrop 2000, then 1.5 \u00b5g of total RNA was reverse-transcribed using the RT5\u2019-GTTCTTCACGCCCAAAAGATG-3\u2019, reverse 5\u2019-GGACAGTTGGGAAGAGTGTCATT-3\u2019), Pten , p300 , Rb1 , Bag1 , Casp2 , Casp9 , Mcl1, , Numb , and Prkca , with 18S  used as a normalizer. These primers were used in an assay using SYBR Green QPCR on duplicate test samples. Samples of RNA free of DNA contamination were reverse transcribed using the Ambion High Capacity cDNA kit . A master mix containing the Applied Biosystems PowerSYBR\u00ae Green 2x Master Mix , an equal amount of the cDNA reaction, and water to make 25 \u00b5l volume samples were created for each gene. The mix was then aliquoted into a 96 well plate, primer added, and ran on an Agilent 7300 Real Time PCR System. Fold changes were calculated using the 2-\u0394\u0394Ct method using 18S as the first \u0394 with the control samples as the second \u0394.In order to validate results of the e PCR arrays, several genes that underwent changes in expression were selected, and quantitative real-time PCR (QPCR) was performed (see Results section) on triplicate samples of S36, T36, and control C2C12 cells. Specific primers for Realtime analyses are as follows. p53 , MyoD (Integrated DNA Technologies (IDT), Coralville, IA, forward C2C12 clones with the different p53 shRNAs were grown to confluency for stable lines or two days post transfection for transient transfected lines. Total protein was isolated from each plate using M-PER mammalian protein extraction reagent  with supplemented Roche Complete ULTRA Tablets protease inhibitor . Protein concentration was measured using Bradford assay . Following isolation and quantification, the proteins were subjected to western blot analyses. A 25 or 50 \u03bcg fraction of each protein extract was separated on a SDS PAGE gel, then electro transferred onto presoaked PVDF paper . The membranes were then blocked with 5% milk protein in PBS/T, and the primary antibody for either p53  or \u03b2-actin  was added and allowed to incubate overnight at 4\u00b0C. The membrane was then washed with PBS/T three times, and HRP conjugated secondary antibodies to mouse or rabbit IgGs  was added, washed again, and exposed using the ECL Western blotting reagents . The image was then captured on a Kodak system and UnscanIt\u2122 was used to obtain quantification.Several gene specific reporter assays were carried out to examine the effect of transient and stable p53 knockdown. Our reporter constructs consisted of mainly luciferase reporters but some of them were Chloramphenicol Acetyl Transferase (CAT) based reporters. Specific details for both assays are provided below. When stable p53 knockdown lines were used, cells received just the reporter construct. When the assay was carried out in cells with transient knockdown of p53, we either used a specific shRNA construct or a mixture of three p53 shRNA constructs. Specific details are provided in figure legends. Control cells received a scrambled sequence of a similar length in the same plasmid backbone (for knockdown of p53) and/or the empty vector (reporter assays). Forty-eight hours after transfection, they were trypsinized, pelleted, and then resuspended in serum free media. An aliquot was used to count cells for normalization, the rest was assayed using Promega\u2019s Bright GloTM Luciferase Assay Kit . Equal volumes of the resuspended cells and Bright-GloTM were mixed and incubated for 2 minutes. A Turner Designs TD20/20 Luminometer was used to record the luciferase activity for each sample. Samples were read for 10 seconds, each after a two second delay. Luciferase readings for each of the samples were then normalized according to the number of cells/ml. All measurements were carried out on triplicate samples and experiments were repeated at least thrice.In order to evaluate the functional activity of p53 we transfected cells with, pG13-luc , a plasm14C Chloramphenicol. The product was extracted with xylene and radioactivity measured using a liquid scintillation counter. All measurements were carried out on triplicate samples and experiments were repeated at least thrice.Forty-eight hours after transfection the cells were lysed and equal amounts of protein were used to measure chloramphenicol acetyltransferase (CAT) activity using n-Butyryl CoA and Because p53 is involved in the development and differentiation of C2C12 cells into later, more committed cell types, the promoters of certain genes representing various pathways were assayed for activity in the stable and transient p53 knockdowns to test to see if transient or stable loss of p53 had different effects upon the expression of bone or muscle markers. The notch pathway was assayed using the Hey1-luc plasmid . In addi< 0.05 for significance. To visualized the difference between how T36 and S36 differ when compared to control cells, clustering was done using Cluster3 [All statistics were done in GraphPad Prism 5 (Microsoft), using either unpaired t-tests or one-way ANOVA with Tuckey correction for multiple testing and post hoc tests, and requiring a p Cluster3  by usingCluster3 , which wIn order to create a stable knockdown of p53, we isolated puromycin resistant single cell clones after transfecting various p53 shRNA constructs in a plasmid conferring puromycin resistance into C2C12 cells. Individual clones were expanded and characterized. The same constructs were also used for transient reduction of p53, but no selection was performed and cells were harvested after 48 hours for RNA and protein. Our intention was to generate cells with a similar knockdown in p53 using the same shRNA construct. In order to rule out possible dosage dependent effects between and among the different constructs and type of tranfections, several methods were used to assess the knockdown Therefore in addition to determining extent of p53 knockdown at the transcriptional and translational level, we tested the level of p53 functional activity using a construct containing 13 copies of a p53 binding sequence . This coIn order to look for more global differences between acute loss, from the transient knockdown, and a long term loss of p53 the SABioscience Q-PCR array was used to assay the changes in genes related to p53. We chose to compare RNA from stable and transient knockdown created with the shRNA (S36 vs. T36). RNA from both these cells was first compared to control cells receiving a scrambled shRNA sequence. As expected, many of the targets showed changes with loss of p53 expression. Hierarchical clustering of the expression changes between the two lines are shown as compared to control cells. Two types of change were observed one where genes are up-regulated in the acute loss compared to the long term loss of p53, and a second cluster with genes up-regulated in long term p53 loss when compared to the acute loss. . As expeSome of our previous studies demonstrated that the amount of p53 within osteoblasts might affect cell proliferation and differentiation variably -28. SincSeveral genes belonging to apoptosis and differentiation related pathways showing alterations in the array were chosen for validation using realtime PCR. For this analysis we created new stable lines with p53 knockdown using the same shRNA. Transient knockdown of p53 was also carried out in triplicate. RNA from these newly generated cell lines were compared to results obtained from the gene array. As shown in < 0.001; In addition to the expression data, we wanted to determine if specific pathways related to differentiation are activated differently dependent on whether p53 loss was stable or transient. The notch pathway is known to be important for tissue differentiation and has been shown to be important for both myoblast and osteoblast differentiation ,30. When< 0.001; < 0.001; < 0.01; Since the C2C12 cells can differentiate into either myocytes or osteoblasts, we tested the promoter activity of the genes Muscle Creatine Kinase (MCK), a late marker of myogenic differentiation, and osteocalcin, a late marker of osteoblast differentiation. When p53 is stably reduced the loss results in a significant decrease in activity of the osteocalcin promoter (p p53 is a transcription factor that regulates a number of different genes affecting cellular conditions ranging from proliferation, apoptosis, and DNA repair to cellular metabolism and senescence. While a role for p53 in cellular differentiation is well accepted, there is still a debate on whether p53 facilitates or opposes differentiation. This stems from the fact that loss of p53 during development is associated with little or no effect on tissue differentiation . In the In an attempt to separate the different roles of p53 we studied the short and long term effect of p53 loss on its target genes. In this study we utilized C2C12, a bipotential cell line capable of differentiation towards myoblast and osteoblast, to determine the effect of transient and stable knockdown of p53 using RNA interference. An acute loss of p53 lowered the mRNA expression for a number of genes related to apoptosis. This is expected, as one of the major roles of p53 is in regulating apoptosis, a function that represents an immediate response to prevent damaged DNA and or cells from survival. However, these same genes show a small increase in expression after long term loss of p53; perhaps as an adaptation to reduced p53 levels. Specifically, members of the caspase family of genes, Casp2 and Casp9, as well as the Casp2 assembly protein Cradd, are all down-regulated with an acute loss of p53, whereas they appear to be up-regulated when p53 is reduced for the long term. It is also possible that the change seen in caspases with stable p53 loss may reflect other functions unrelated to apoptosis as has been seen for some cell types -40. SimiThe apoptotic pathway is not the only pathway that appears to differ according to the length of p53 knockdown. Genes involved in p53 related differentiation pathways varied in expression based on the type of p53 loss. MyoD and Numb are well known differentiation and cell fate related genes ,43. ValiWe studied lineage specific, as well as, lineage independent signaling pathways that might be affected by the knockdown of p53 in C2C12 cells. We investigated activity of the Notch pathway and did not find differences between stable and transient knockdown as in both cases this pathway was depressed with loss of p53. In a genome wide study carried out to determine p53 regulators, the notch pathway, especially Hey1, was found to be members of an evolutionarily conserved network governing p53 function . It was These studies indicate that while the role of p53 is important to protect cells from damage by regulating key apoptotic genes, p53 has an additional role in maintaining tissue homeostasis. This may be especially important in osteoblasts since bone remodels throughout life. The presence of p53 may be critical to allow for orderly progression of the different steps of the differentiation process. p53 is a direct regulator of matrix proteins osteopontin and osteocalcin ,50. Oste"}
{"text": "AbstractHemiptera: Coccoidea) known from Hungary has increased in the last 10 years by 39 (16.6 %), to a total of 274 species belonging to 112 genera in10 families. The family Pseudococcidae is the most species rich, with 101 species in 34 genera; Diaspididae contains 59 species in 27 genera; Coccidae contains 54 species in 27 genera; and the Eriococcidae contains 33 species in 8 genera. The other 6 coccoid families each contain only a few species: Asterolecaniidae (7 species in 3 genera); Ortheziidae (7 species in 4 genera); Margarodidaesensu lato (5 species in 5 genera); Cryptococcidae (3 species in 2 genera); Kermesidae (4 species in 1genus); and Cerococcidae (1 species). Of the species in the check list, 224 were found in outdoor conditions, while 50 species occurred only in indoor conditions. This paper contains 22 species recorded for the first time in the Hungarian fauna.The number of scale insect species ( Hemiptera: Coccoidea) live on a wide variety of plant species and many of them are important agricultural pests. Publication of new knowledge of this insect group is therefore very important from a practical viewpoint. The distribution data of different species may serve also as a reliable biodiversity indicator in different terPageBreakritories, such as nature reserves and agricultural or urban landscapes. The distribution data may also reflect the progress of climatic changes , Coccidae (54 species) and Eriococcidae with 33 species. The new species to the Hungarian fauna recorded here belong to the Pseudococcidae, Diaspididae and Eriococcidae. Most of the species in the checklist  are native and live outdoors. The check list contains 50 introduced  species, mainly occurring indoors in Hungary on ornamental plants in greenhouses and buildings. Of these indoor species, 33 occurred only in greenhouses or buildings  and 7 were found exclusively on imported tropical/subtropical fruits for consumption. Four of the species living in greenhouses sometimes also occur outdoors. Four other species, which are typically found on imported fruit, also appear in greenhouses from time to time. Two of the newly recorded species were found on imported nursery plant material. In the present list, 22 species are new to the Hungarian fauna. According to these data, Hungary is the most scale-insect-species-rich country in in Central Europe  in the last ten years, and currently totals 274 species in ten families  and 2. Tl Europe .PageBreakPageBreakPageBreakPageBreakPageBreakPageBreakPageBreakPageBreak106 (38.69%) species are considered as widely distributed Pan-Palaearctic species, 75 (27.37%) are widely distributed Euro-Siberian species, 91 (33.21%) are cosmopolitan, and only two species are known to originate from the Mediterranean subregion.No species should be considered as truly endemic only on the basis of its presence in a checklist, because the lack of a species in the surrounding countries is most likely due to inadequate exploration of those areas . Out of PageBreakperate climate, but with several submediterranean, xerophilous habitats. In addition, the Great Hungarian Plain belongs to the steppic province of the Palaearctic Region, which ranges from Hungary to China and the Far East , Aspidiotus destructor Signoret, 1869, Aulacaspis yatsumatsui Takagi, 1977). The following species have become significant pests in Hungary in recent years: Aspidiotus nerii Bouch\u00e9, 1833; Coccus hesperidum Linnaeus, 1758; Planococcus citri ; Pseudococcus longispinus ; Pseudococcus viburni  and Saissetia coffeae   .Pseudococcus elisae Borchsenius, 1957). The number of species in this category is surprisingly low, compared to the number of pests living on various fruits exported from the different regions of production. The low species number reflects the efforts made by exporting countries to prevent the spread of invasive pests. It should be noted that most of these species were unable to establish in Hungary even indoors in greenhouses or on ornamental plants in buildings, despite repeated introductions over more than one hundred years. On the other hand, some of them have become regular pests in Hungary, which has lead to some overlap with the category in Aspidiotus nerii, Planococcus citri and Pseudococcus viburni occur in greenhouses and buildings, while Pseudaulacaspis pentagona and Carulaspis caruelii are found outdoors.A detailed study of the scale insects introduced into Hungary on tropical and subtropical fruits was published by PageBreakPageBreakPageBreak"}
{"text": "As a hallmark of tumor cells, metabolic alterations play a critical role in tumor development and could be targeted for tumor therapy. Tumor suppressor p53 plays a central role in tumor prevention. As a transcription factor, p53 mainly exerts its function in tumor suppression through its transcriptional regulation of its target genes to initiate various cellular responses. Cell cycle arrest, apoptosis and senescence are most well-understood functions of p53, and are traditionally accepted as the major mechanisms for p53 in tumor suppression. Recent studies have revealed a novel function of p53 in regulation of cellular metabolism. p53 regulates mitochondrial oxidative phosphorylation, glycolysis, glutamine metabolism, lipid metabolism, and antioxidant defense. Through the regulation of these metabolic processes, p53 maintains the homeostasis of cellular metabolism and redox balance in cells, which contributes significantly to the role of p53 as a tumor suppressor. Further understanding of the role and molecular mechanism of p53 in cellular metabolism could lead to the identification of novel targets and development of novel strategies for tumor therapy. Tumor suppressor p53 plays a central role in tumor prevention -4. p53 iAs a transcription factor, p53 mainly exerts its function in tumor suppression through transcriptional regulation of its target genes -4. In re18flurodeoxyglucose than normal tissues. Remarkably, recent research has begun to answer these questions. These studies have led to the concept that the metabolic alterations are a hallmark of tumor cells . Th. Th63]. p53 can further regulate glycolysis through its regulation of PI3K/AKT and mTOR signaling pathways Figure . The abeIn addition to glucose metabolism, p53 regulates many other aspects of cellular metabolism, including glutaminolysis and fatty acid oxidation Figure . GLS2 acEmerging evidence also shows that p53 is involved in lipid metabolism. In response to nutritional starvation, p53 induces fatty acid oxidation to drive the TCA cycle to provide energy for cells Figure . Guanidi+\u2009dependent enzyme in mitochondrial matrix, which catalyzes proline degradation in mitochondria and thereby reduces intracellular ROS levels [Oxidative stress and increased levels of ROS in cells play an important role in tumorigenesis. Recent studies have shown that reducing the ROS levels and enhancing antioxidant defense is an important mechanism of p53 in tumor suppression. Mitochondrial oxidative phosphorylation is a main source of endogenous ROS in cells. Whereas p53 promotes mitochondrial oxidative phosphorylation to maintain the homeostasis of cellular energy metabolism, p53 also plays a fundamental role in reducing the intracellular ROS levels mainly resulted from mitochondrial oxidative phosphorylation and maintaining the redox balance. To exert its antioxidant function, p53 induces a group of antioxidant genes, including sestrins 1/2, TIGAR, GPX1, ALDH4, GLS2, and Parkin, especially under conditions of nonstress or low stress, to lower ROS levels and prevent DNA damage (Figure S levels . By incrS levels -53. In aS levels . As a trS levels ,19.Interestingly, in addition to the function of antioxidant defense, p53 can exert prooxidant function through transcriptional up-regulation of a group of prooxidant genes depending on the levels of oxidative stress that cells are facing (Figure Here we reviewed our current understanding of the role and mechanisms of p53 in maintaining the homeostasis of cellular energy metabolism and the redox balance in cells. Whereas cell-cycle arrest, apoptosis, and senescence are traditionally accepted as the major mechanisms by which p53 exerts its tumor suppressive function, these findings provide strong evidence that the functions of p53 in maintaining the homeostasis of cellular energy metabolism and the redox balance in cells contribute significantly to the role of p53 as a tumor suppressor.Metabolic alterations are a hallmark of cancer cells. Ample evidence has shown that the metabolic alterations are critical for the growth, proliferation and survival of tumor cells. Therapeutic strategies targeting cancer metabolism are being developed and tested for cancer treatment. For instance, Metformin, an anti-diabetic drug, which regulates cell metabolism and the AMPK/mTOR signaling, has been already selected as a candidate for tumor therapy ,86. ReceThe authors declare that they have no competing interests.YL and JL prepared the initial draft of the paper. ZF modified and finalized the paper. All authors read and approved the final manuscript."}
{"text": "MYL3, encoding the essential light chain of myosin, are rare and have been associated with sudden death. Both recessive and dominant patterns of inheritance have been suggested. We studied a large family with a 38-year-old asymptomatic HCM-affected male referred because of a murmur. The patient had HCM with left ventricular hypertrophy (max WT\u200921\u2009mm), a resting left ventricular outflow gradient of 36\u2009mm\u2009Hg, and left atrial dilation (54\u2009mm). Genotyping revealed heterozygosity for a novel missense mutation, p.V79I, in MYL3. The mutation was not found in 300 controls, and the patient had no mutations in 10 sarcomere genes. Cascade screening revealed a further nine heterozygote mutation carriers, three of whom had ECG and/or echocardiographic abnormalities but did not fulfil diagnostic criteria for HCM. The penetrance, if we consider this borderline HCM the phenotype of the p.V79I mutation, was 40%, but the mean age of the nonpenetrant mutation carriers is 15, while the mean age of the penetrant mutation carriers is 47. The mutation affects a conserved valine replacing it with a larger isoleucine residue in the region of contact between the light chain and the myosin lever arm. In conclusion, MYL3 mutations can present with low expressivity and late onset.Hypertrophic cardiomyopathy (HCM) is caused by mutations in genes encoding sarcomere proteins. Mutations in  MYL3 which encodes the myosin essential light chain (ELC) of the sarcomere . It. ItMYL3 MYL3 mutation p.V79I. The mutation segregates with disease and is absent in 600 control alleles in a family in which no other mutation was identified in known HCM-associated genes. The phenotypic presentation of the p.V79I mutation is that of classical HCM with asymmetric septal hypertrophy, just as previously described for the p.A57G mutation in two unrelated Korean families [We have described a patient with mild HCM associated with a novel, dominantly inherited, families , 17 but families , p.M149Vfamilies . ExpressThe part of beta myosin containing the IQ1 motif that interacts with the N-C-loop of ELC harbours a number of HCM-associated mutations, that are, p.S782D , p.S782NHomology modelling suggests that the p.V79I mutation may interfere with the interaction between ELC and myosin heavy chain, a mechanism which is believed to be the cause of disease for a number of reported mutations. Based on this evidence we find it likely that the p.V79I mutation is disease causing. However, it can not be ruled out that the mutation may only be associated with hypertrophy when other triggering conditions are present. In this case, the proband is very obese; obesity has recently been associated with a cardiac hypertrophic response in mice fed a high-fat diet through inactivation of the Foxo3a transcription factor via the Akt pathway . As the MYL3 have been associated with sudden death [MYL3-based genetic predisposition, the hypertrophic phenotype and obesity in the proband should also strengthen the recommendation to the proband to lose weight.The finding of a clinically silent mutation with low expressivity and late onset raises the question of whether it should entail a detailed followup of mutation carriers. However, as most mutations in en death , it woul"}
{"text": "A significant amount of evidence suggests that the p38-mitogen-activated protein kinase (MAPK) signalling cascade plays a crucial role in synaptic plasticity and in neurodegenerative diseases. In this review we will discuss the cellular localisation and activation of p38 MAPK and the recent advances on the molecular and cellular mechanisms of its substrates: MAPKAPK 2 (MK2) and tau protein. In particular we will focus our attention on the understanding of the p38 MAPK-MK2 and p38 MAPK-tau activation axis in controlling neuroinflammation, actin remodelling and tau hyperphosphorylation, processes that are thought to be involved in normal ageing as well as in neurodegenerative diseases. We will also give some insight into how elucidating the precise role of p38 MAPK-MK2 and p38 MAPK-tau signalling cascades may help to identify novel therapeutic targets to slow down the symptoms observed in neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The MAPKs are a specific class of serine/threonine kinases which respond to extracellular signals such as growth factors, mitogens, and cellular stress and mediate proliferation, differentiation, and cell survival in mammalian cells. There are 4 distinct groups of MAPKs within mammalian cells: the extracellular signal-related kinases (ERKs), the c-jun N-terminal kinases (JNKs), the atypical MAPKs , and the p38 MAPKs . The p38in vivo. In this paper we will especially focus our attention on the role of two p38 MAPK substrates in neurons: MAPK-activated protein kinase 2  and tau protein. The involvement of the p38 MAPK-MK2 and p38 MAPK-tau signalling cascades in neuroinflammation, actin remodeling, and tau hyperphosphorylation in neurodegenerative diseases will also be discussed. Highlighting the functional role of specific p38 MAPK substrates in neurodegenerative disease will be of particular importance as these could be potential signalling targets which could be exploited therapeutically to slow cognitive decline occurring in normal ageing and in neurodegenerative disease.In the present paper we will give an overview of p38 MAPK localisation, activation, and the functional role of this signalling cascade in the mammalian brain, especially the activation of the p38 MAPK cascade during synaptic plasticity in the hippocampus. Although p38 MAPK isoforms have been shown to be highly expressed in the brain, only a handful of brain-specific substrates for p38 MAPK have been characterised in vitro experiments in 293T HEK cells showed a similar distribution profile for both the endogenous and exogenous MKK3 and MKK6 proteins in that they are both nuclear and cytoplasmically localised within the cell . It. It\u03b1/\u03b2 Mammation , 62. In ytokines . This si\u03b2 plaques or NFTs formed from free aggregated neuronal tau within the brain. However the relationship between these structures and the symptoms of cognitive impairment and memory loss that is associated with the disease remains uncertain. Increasing evidence has shown that the stability of dendritic spines and actin remodelling may participate in the pathology of the disease. The loss of synapses is a common occurrence within postmortem tissue of AD patients , 83\u03b2 is  neurons , contribal cells . In AD, deposits  through  in vivo . Upon thin vitro. It has been demonstrated that release of proinflammatory cytokine IL-1 from activated microglia increased the levels of tau phosphorylation in neurons. These changes are partly mediated through activation of p38 MAPK as a significant increase in the levels of phospho-p38 MAPK was observed upon application of IL-1\u03b2 in cultures of neocortical neurons and microglia [in vitro that IL-1\u03b2-induced tau phosphorylation was considerably decreased in neuronal culture [Glial-neuron interactions and the effect these interactions have on tau phosphorylation have been analysed icroglia . Additio culture , again h\u03b1 has been seen to localise to areas where NFTs, amyloid plaques, and glial cells are present both within human AD brain and transgenic mouse models [\u03b2. This complex, which forms through A\u03b2-peptide-induced dimerisation, suggests a connection between the aberrant processing of APP and the ASK-1-MKK6-p38 MAPK cascade which is involved in inflammation and abnormal tau phosphorylation [In vitro activation of MKK6-p38 MAPK has led to tau phosphorylation at specific sites, the most efficient being Ser-396, which has been suggested to have a functional role in microtubule binding. Abnormal phosphorylation at Ser-396 is observed in AD brain but not in normal functioning adult brain [\u03b2 peptide, can activate the upstream MKKK ASK-1 and MKK6, consequently activating p38 MAPK, which can directly phosphorylate tau protein hence linking deposition of A\u03b2 plaques to downstream tau phosphorylation through the activation of the p38 MAPK signalling cascade under pathophysiological conditions. Under pathophysiological conditions, activated p38e models , 89, 90.e models  and to be models , the prerylation . In vitrlt brain . Furtherlt brain . This suPick's disease is another severe neurodegenerative disorder, which involves progressive dementia and aphasia through the development of Pick bodies, which are comprised of neurofibrils formed of aggregated phosphorylated tau. It is known that oxidative stress is involved in instigating Pick's disease, and since it has been highlighted that the p38 MAPK cascade is activated upon such stimuli, it may play an important role in this disease as well. It has been observed in post-mortem brain tissue that phosphorylated p38 MAPK localises to the Pick bodies which contain highly phosphorylated tau protein, and since p38 MAPK is capable of phosphorylating tau, as described above, it emphasises the importance of p38 MAPK in this disease as well as AD and other related tauopathies .\u03b1-synuclein (SNCA), which is a protein present within the LBs of PD patients and known to play a pivotal role in the development of the disease. \u03b1-Synuclein is highly expressed in neuronal tissue and is subject to many post-translational modifications such as phosphorylation and ubiquitination, although its physiological function is poorly characterised. It has been suggested, however, that these post-translational modifications participate in neurotoxicity [\u03b1-synuclein is the predominant fibrillar component of the proteinaceous Lewy bodies seen in PD. Additionally, it has been observed that amplified \u03b1-synuclein levels are linked to an increase in neuroinflammation as it has been shown that \u03b1-synuclein activates p38 MAPK and other MAPKs in glial cells, a finding that is supported by the fact that extracellular \u03b1-synuclein released from damaged neurons interacts with microglia [\u03b1, which can induce and promote neuroinflammation as a physiological neuroprotective mechanism. If the glial cells become overactivated, however, as may occur through elevated levels of extracellular \u03b1-synuclein released from damaged Lewy body containing neurons, then chronic inflammation could be established and lead to neuronal cell death. Parkinson's disease is the second most prevalent neurodegenerative disease and around 127,000 people in the UK are currently living with the disease, which has been estimated to rise by 28% by the year 2020 . PD invotoxicity  as aggreicroglia . As desc\u03b1, thus causing neuroinflammation and potentially neuronal cell death. An investigation by Culbert et al. [MAPKAP kinase-2, one of p38 MAPK more prevalent substrates has also been implicated within PD, where it has been shown that MK2-deficient mice show decreased levels of neuroinflammation and loss of dopaminergic neurons within the substantia nigra after treatment with the Parkinson's inducing neurotoxin MPTP compared to MK2 wild-type mice . MK2, aft et al.  demonstrConsiderable progress has been made in the understanding of the functional role of the p38 MAPK signalling cascade in synaptic plasticity in the hippocampus and its potential role in neurodegenerative diseases such as AD. However, less is known regarding the role of the direct targets of p38 MAPK, such as MK2 and tau, in regulating neuroinflammation and the actin cytoskeleton in dendritic spines of neuronal cells. Growing evidence suggests that remodelling of actin at dendritic spines plays a crucial role in synaptic plasticity and therefore in cognitive processes such as learning and memory. Furthermore, recent findings in animal models have linked early symptoms of AD with loss of cognitive functions, combined with a reduced number of dendritic spines in the hippocampus. Abnormal dendritic spine morphology has also been observed in brain tissue from patients suffering from AD. Therefore, with an ageing population continuing to grow and the consequent rise in AD, elucidating the precise role of p38 MAPK-MK2 and p38 MAPK-tau signalling cascades in controlling actin remodelling becomes very important as it may identify novel targets to slow down the cognitive decline observed in normal ageing and in the early stages of neurodegenerative diseases."}
{"text": "The transcription factor p73 belongs to the p53 family of tumour suppressors and similar to other family members, transcribed as different isoforms with opposing pro- and anti-apoptotic functions. Unlike p53, p73 mutations are extremely rare in cancers. Instead, the pro-apoptotic activities of transcriptionally active p73 isoforms are commonly inhibited by over-expression of the dominant negative p73 isoforms. Therefore the relative ratio of different p73 isoforms is critical for the cellular response to a chemotherapeutic agent. Here, we analysed the expression of N-terminal p73 isoforms in cell lines and mouse tissues. Our data showed that the transcriptionally competent TAp73 isoform is abundantly expressed in cancer cell lines compared to the dominant negative \u0394Np73 isoform. Interestingly, we detected higher levels of \u0394Np73 in some mouse tissues, suggesting that \u0394Np73 may have a physiological role in these tissues. The Trp73 gene belongs to the p53 family of transcription factors and, like the other members, is transcribed into different isoforms -4. TP73"}
{"text": "TP53, which occur in 96% of serous ovarian tumors, alter the core molecular pathways involved in drug response. One subtype of TP53 mutations, widely termed gain-of-function (GOF) mutations, surprisingly converts this protein from a tumor suppressor to an oncogene. We term the resulting change an oncomorphism. In this review, we discuss particular TP53 mutations, including known oncomorphic properties of the resulting mutant p53 proteins. For example, several different oncomorphic mutations have been reported, but each mutation acts in a distinct manner and has a different effect on tumor progression and chemoresistance. An understanding of the pathological pathways altered by each mutation is necessary in order to design appropriate drug interventions for patients suffering from this deadly disease.Ovarian cancer is the most lethal gynecological malignancy, with an alarmingly poor prognosis attributed to late detection and chemoresistance. Initially, most tumors respond to chemotherapy but eventually relapse due to the development of drug resistance. Currently, there are no biological markers that can be used to predict patient response to chemotherapy. However, it is clear that mutations in the tumor suppressor gene  TP53, in 96% of all serous ovarian tumors. TP53 encodes p53, a tumor suppressor that acts as a major control hub for the cellular response to various stresses, including DNA damaging chemotherapy. Once activated, p53 protects against cancer by functioning as a sequence-specific transcription factor or through protein: protein interactions, activating cell cycle arrest, apoptosis, and DNA damage repair.One of the main goals of clinical research is to establish better drug regimens for patients suffering from specific diseases. In the field of ovarian cancer research, the current standard therapy of platinum and paclitaxel has been in place without change for almost twenty years . The lacRB1 or APC that are largely inactivated by mutations that result in deletion or truncation .,50.49,50R273 are the most common TP53 alterations in ovarian carcinomas, with the most frequent mutations resulting in an amino acid change to histidine or cysteine. Mutation of R273 significantly alters the target DNA sequence without causing structural distortions, thus maintaining the ability to bind DNA [In vivo studies in mice with an engineered R273H mutation in the endogenous locus (p53R273H/\u2212) demonstrate no difference in survival as compared to p53\u2212/\u2212 mice [p53R273H/\u2212 mice develop more carcinomas, including high numbers of lung adenocarcinomas and squamous cell carcinomas. Additionally, cells cultured from these tumors demonstrate an increased capacity to proliferate [TP53 null background results in resistance to cisplatin [Mutations in codon bind DNA ,76. In v\u2212/\u2212 mice . Howeverliferate . In studisplatin ,41. It risplatin .et al. showed that after DNA damage, R273H/C p53 complexes with the transcription factor NF-Y on the promoters of the cell cycle genes cyclin A, cyclin B, cdk1, and cdc25c and activates their expression [The mechanisms underlying increased resistance to some chemotherapeutic agents likely involve new protein interactions as well as new transcriptional targets ,61,77,78pression ,79. Genepression . These ipression . Whetheri.e., NF-Y and SP-1), although the interaction has the opposite effect compared to oncomorphic p53 [In addition to mediating chemoresistance, interactions between R273H/C mutant p53 and other proteins may contribute to oncogenesis or tumor progression. The best characterized R273H p53 interaction is with its family member p63. Specifically, p63 activity is inhibited by R273H p53 binding, which in turn promotes TGF-\u03b2-induced metastasis . The losphic p53 ,69,83. TR248 is the second most commonly altered amino acid in ovarian carcinomas, occurring at a frequency of 6.02% [R248W or R248Q). This alteration does not significantly affect the overall conformation of the p53 protein but changes the DNA binding response element, thereby altering interactions with DNA [248 can increase oncogenicity through increased invasion and chemoresistance to particular drugs. In lung cancer cells, although there are no differences in the proliferation rate of clones stably expressing this mutant, cells with this mutation do display significantly higher migration [P-glycoprotein) [R248W TP53 knock in mice (p53R248W/\u2212), expression of R248W p53 markedly accelerates the development of lymphomas and sarcomas [TP53 R248Q mutant was created, and these mice display accelerated tumor onset as well as a shortened survival [The codon of 6.02% ,34. The with DNA . Cellulaigration . The mecprotein) . As withprotein) . The difsarcomas . In addisurvival .R248 mutations in chemoresistance, disruption of this complex sensitizes cells to adriamycin and cisplatin and induces apoptosis. These data also highlight that, while mutation at R248 is widely considered to predict for resistance to therapy, patients must also be screened for the SNP at codon 72 to achieve an accurate prediction for response.The p53 mutant R248 can activate the transcription of several genes associated with chemoresistance, namely c-Myc, CXCL1, PCNA, ABCB1 (MDR1), and IGF1R ,77,86,87TP53 mutation in all cancers and is the best characterized mutation. Many different types of cancer cell lines that harbor this mutation as well as murine cancer models have been used to study the oncogenic properties of R175H, and these studies have suggested a role in chemoresistance [R273H: greater tumorigenic potential with enhanced proliferative capacity [p53\u2212/\u2212 mice [R175H TP53 transgenic mice display increased genomic instability. In experiments in lung cancer cells devoid of p53, overexpression of the oncomorph R175H p53 enhances resistance to both etoposide and cisplatin [The p53 oncomorph R175H is the third most common sistance . Transgecapacity . Moreove\u2212/\u2212 mice ,90. In aisplatin . Furtherisplatin .Similar to the R273 and R248 p53 oncomorphs, the R175H mutated protein can interact with proteins NF-Y, p63, p73, and SP-1 to increase cell cycle progression, survival, metastasis, and chemoresistance ,79,81,82Y220C mutation occurs in 3.74% of ovarian carcinomas, though it has not been as extensively studied as the other TP53 mutations. Though it is not defined by others as an oncomorphic mutation, one might speculate it has oncomorphic activity given its reported interaction with p63 and p73 and its transcriptional activation of stathmin, which, as a microtubule destabilizer, is implicated in chemoresistance in many solid tumors [The d tumors ,96.Y220C mutation. Specifically, the mutated codon is not in direct contact with DNA but rather creates a binding pocket in the core domain of the protein [in vivo cell or mouse models of cancer. Thus, the binding pocket presents an opportunity for a novel strategy of cancer treatment based on the idea of restoring WT p53 function and thereby sensitivity to DNA damaging chemotherapy.In contrast to the lack of understanding of its functional properties, several structural studies have provided novel insight into the  protein  that is  protein . ImportaTP53 mutation has context-specific effects. Additionally, each oncomorphic mutation may have differing actions in response to different cancer therapeutics used. Experiments should be performed to compare the various oncomorphic mutants in the same parental cancer cell line in order to reduce variability. Two main strategies should be utilized for these studies: over-expression of oncomorphic mutants in p53-null cells, as well as knockdown of WT p53 concomitant with re-expression of the oncomorphic p53 variants  since clearly all TP53 mutants are not equal. Ovarian cancer is an ideal model to study p53 mutant biology because mutations in this gene are so common. Based on our calculations, oncomorphic mutations may comprise up to 20% of all mutations in ovarian tumors. However, a single therapy may not be adequate for all tumors with oncomorphic TP53 mutations. Extensive research in vitro as well as in vivo in mouse models is necessary to examine the biology of each mutation, particularly those that occur with high frequency. The studies described above will pave the way for appropriate comparisons of specific mutants, and possibly identify common signaling pathways that can be targeted for anti-cancer drugs. For example, exciting new methodologies may help progress the field of mutant p53 research by attempting to find oncomorphic p53 binding consensus sequences, as well as transcriptome sequencing to identify oncomorphic p53-associated biomarkers that can be used as predictive tools for resistance to chemotherapy. It will be further necessary to complement in vitro studies with animal models of ovarian cancer to understand the function of these oncomorphs and identify drug strategies that will be most useful.The process of carcinogenesis involves gaining oncogenic activities as well as losing tumor suppressive activities. Tumors that acquire an oncomorphic TP53 oncomorphic mutations can lead to identifying better therapies for patients with oncomorphic mutations in their tumors, and potentially avoid the high incidence of chemoresistance that is currently one of the barriers to improving survival for this deadly disease.As new technologies helps advance methodologies used to study mutant p53 biology, the most useful information will be discovering commonalities between the mutants. Understanding the basic science of in vitro and in vivo studies as well as several clinical trials showing that restoration of WT p53 function causes rapid tumor regression in mice and prolonged survival in humans [TP53 gene into tumors using an adenovirus. Excitingly, 50% (8 of 16) women with recurrent ovarian, peritoneal, or fallopian tube cancer that were treated with the replication-deficient adenovirus encoding human recombinant WT TP53 (SCH 58500) showed a decrease in serum CA125 levels, indicative of clinical response, with minimal side effects [TP53 gene therapy with chemotherapeutic drugs such as cisplatin synergized to enhance clinical efficacy [TP53 gene therapy in ovarian cancer was closed after the first interim analysis because adequate therapeutic benefit was not achieved [One alternative approach is the idea of restoring WT p53 function. The concept of restoring WT p53 activity is strongly supported by n humans \u2013105. Var effects . Moreoveefficacy . UnfortuOther approaches such as targeting cellular proteins responsible for stabilizing mutant p53 are another route that may bring success. A recent development involves inhibiting the heat-shock protein HSP90, which chaperones many mutant p53 proteins ,107 and TP53 mutations.In summary, the information gained from studying the mutant p53 transcriptome and interactome described in this review has solidified the foundation for the development of strategies that can one day be used to treat the large number of cancer patients that harbor"}
{"text": "Fourth, there was a significant gliosis mainly affecting the lower cortex of both disorders. The data suggest that the pattern of cortical degeneration is different in sCJD and vCJD which may reflect differences in aetiology and the subsequent spread of prion pathology within the brain.The laminar distributions of the pathological changes in the cerebral cortex were compared in the prion diseases sporadic Creutzfeldt-Jakob disease (sCJD) and variant CJD (vCJD). First, in some cortical regions, the vacuolation (\u201cspongiform change\u201d) was more generally distributed across the cortex in sCJD. Second, there was greater neuronal loss in the upper cortex in vCJD and in the lower cortex in sCJD. Third, the \u201cdiffuse\u201d and \u201cflorid\u201d prion protein (PrP PrP) gene [sc) is acquired from growth hormone, dura mater grafts, or corneal transplantation [PrP gene [Four subtypes of the prion disease Creutzfeldt-Jakob disease (CJD) have been described to date including familial CJD (fCJD), linked to germline mutations of the prion protein (rP) gene , and iatantation . By contPrP gene , 4. In aPrP gene . VariantPrP gene . VariantPrP gene .sc in the form of discrete deposits or plaques [sc deposit in CJD. Hence, \u201cflorid\u201d deposits are composed of a central \u201ccore\u201d surrounded by a rim of small vacuoles and are especially characteristic of vCJD. Broad bundles of amyloid are present within the core, and the deposits resemble more closely the \u201cclassic\u201d deposits typical of Alzheimer's disease (AD) [sc occurs in the form of \u201csynaptic-type\u201d deposits, and there are relatively few florid deposits or kuru plaques [Neuropathologically, CJD is characterized by the presence in the brain of vacuolation \u201cspongiform change\u201d, neuronal loss, a reactive astrocytosis, and the deposition of PrP plaques , 7. Therase (AD)  rather tase (AD) . In addiase (AD)  which arase (AD) . By cont plaques , 13. sc spreads into the brain in CJD, for example, direct neural transmission from the site of infection, replication of PrPsc in the spleen followed by neural entry through the spinal cord [Differences in pathology between subtypes of CJD could reflect variations in aetiology and the subsequent spread of prion pathology within the brain. Various hypotheses have been proposed to explain how PrPnal cord , infectinal cord , 16, andnal cord . In neurnal cord \u201320. Neocnal cord . Hence, PrP gene or family history of prion disease, and there was no evidence of the known types of iatrogenic aetiology. In the vCJD cases, the pattern of PrPsc deposition typical of these cases was observed with florid-type PrPsc deposits in the cerebral cortex, cerebellum, basal ganglia, thalamus, and brain stem [PrP gene and the presence of Type 1 or Type 2 isoforms of PrPsc [sc (M/M1). All vCJD cases studied were methionine/methionine (M/M) homozygotes at codon 129. In addition, the PrPsc characteristic of vCJD had a uniform glycotype (PrPsc Type 4) [Eleven cases each of sCJD and vCJD  .\u03bcm sections were stained with haematoxylin and eosin (H/E), cresyl violet, or immunostained against PrP using the monoclonal antibody 12F10 (dilution 1\u2009:\u2009250) which binds to a region of human PrP downstream of the neurotoxic domain adjacent to helix region 2: residues 142\u2013160 [Blocks of the frontal cortex (B8) at the level of the genu of the corpus callosum, parietal cortex (B7) at the level of the splenium of the corpus callosum, occipital cortex including the calcarine sulcus, inferior temporal gyrus (B22), and parahippocampal gyrus (B28) were taken from each case. Tissue was fixed in 10% phosphate-buffered formal saline and embedded in paraffin wax. Sequential, coronal 7\u2009sc deposits across the cortical lamainae was studied using methods similar to those of Duyckaerts et al. [sc deposits in sCJD, all pathological changes were counted in 50 \u00d7 250\u2009\u03bcm sample fields, the larger dimension of the field being located parallel with the surface of the pia mater. An eyepiece micrometer was used as the sample field and was moved down each traverse one step at a time from the pia mater to white matter. Histological features of the section were used to correctly position the field. Counts from the five traverses were added together to study the distribution of the pathology within each cortical region. Because of the diffuse nature of the synaptic deposits in sCJD, these lesions were quantified by \u201clattice sampling\u201d, that is, by counting the number of times the intersections of the grid lines encountered a PrPsc deposit [The distribution of the vacuolation, surviving neurons, glial cell nuclei, and PrPs et al. . Five tr deposit . r\u201d) and a significant reduction in the residual sums of squares compared with the preceding curves [\u03c72) contingency table tests.No attempt was made to locate precisely the boundaries between individual cortical laminae. First, the degree of pathological change and cell losses in the CJD cases made laminar identification difficult. Second, identification is especially difficult in the frontal cortex because it exhibits a heterotypical structure, that is, six laminae cannot be clearly identified even when the cortex is fully developed. Instead, variations in lesion density with distance below the pia mater were analysed using a curve-fitting procedure  , 29. Heng curves . The dissc and reveals the strongly immunolabeled florid PrPsc deposits which are composed of a condensed core of PrPsc and the more lightly immunolabeled and irregularly shaped diffuse deposits.sc deposits in the CJD cases studied are shown in sc deposits was fitted by a first-order polynomial  suggesting a linear decline in abundance with distance below the pia mater. By contrast, in sCJD , the distribution of the synaptic PrPsc deposits was fitted by a second-order (quadratic) polynomial  suggesting a peak of abundance in the lower cortical laminae. Typical examples of the laminar distribution of the PrPsc deposits in sCJD and vCJD. The diffuse and florid PrPsc deposits were more frequently distributed in the upper cortex in vCJD while the synaptic deposits were predominantly distributed in the lower cortex in sCJD. There were no essential differences in the laminar distribution of histological features in allocortical areas, for example, PHG and isocortical areas in either sCJD or vCJD.A summary of the laminar distributions of the pathological changes in all brain areas is shown in There were similarities and differences in the laminar distributions of the histological and pathological changes in sCJD and vCJD. First, there were similarities in the distribution of the vacuoles in the two disorders, but in sCJD, there were more brain regions in which the vacuoles were distributed across all laminae while in vCJD, there were more regions in which the vacuolation spared the superficial laminae and the region immediately adjacent to the white matter. Hence, the vacuolation may be more extensively distributed across the laminae in more regions in sCJD than in vCJD.Second, there was a marked difference in the distribution of the surviving neurons in sCJD and vCJD. In normal control brain, cortical neurons are often bimodally distributed with peaks of density in the upper and lower cortex, the two peaks being asymmetric and the upper density peak being significantly larger than the lower density peak \u201320. In s\u03c72 contingency tables): (a) All categories including totals for the bimodal distributions: Vacuoles \u03c72 = 11.87 ; Neurons \u03c72 = 13.27 , Glial cells \u03c72 = 4.43 (P > .05); Diffuse PrPsc versus Synaptic PrPsc\u03c72 = 15.72 , Florid PrPsc versus Synaptic PrPsc\u03c72 = 17.94 ; (b) comparison of unimodal distributions only: Vacuoles \u03c72 = 0.48 ; Neurons \u03c72 = 3.12 , Glial cells \u03c72 = 3.23 ; Diffuse PrPsc versus Synaptic PrPsc\u03c72 = 12.55 , Florid PrPsc versus Synaptic PrPsc\u03c72 = 13.37 ; (c) comparison of bimodal distributions only: Vacuoles \u03c72 = 4.21 , Neurons \u03c72 = 7.56 , Glia cell nuclei \u03c72 = 0.67 , Nonflorid PrPsc\u2009\u2009\u03c72 versus Synaptic PrPsc\u03c72 = 2.76 , Florid PrPsc versus Synaptic PrPsc\u03c72 = 2.76 .Comparisons between sCJD and vCJD (sc deposition, this is unlikely to be the case in vCJD where neuronal losses appear to be greater in the upper cortical laminae. It is possible that in sCJD, the glial cell reaction directly reflects pathological changes in the lower laminae but in vCJD reflects greater degeneration of the afferent and efferent subcortical pathways which have their cells of origin or axon terminals in the lower cortex.Third, there are similarities in the distribution of glial cell nuclei in sCJD and vCJD which showed a marked preference for the lower cortical laminae, a distribution also reported in previous studies . Althougsc deposits were observed in the two disorders. In vCJD, the diffuse and florid PrPsc deposits showed a marked preference for the upper cortical laminae while the synaptic deposits of sCJD were more frequently distributed in the lower cortex. These distributions could be related to the degeneration of different anatomical pathways in the neocortex. Hence, the distribution of the pathological changes in vCJD suggests degeneration of the feed-forward corticocortical projections while degeneration of the feed-back corticocortical pathways and/or the feedback pathways may be present in sCJD [Fourth, significant differences in the laminar distribution of PrP in sCJD , 31. PrP gene [in situ, and the distribution of the pathology will reflect the site of origin and the pathways by which prion pathology spreads from these sites. Hence, if there are multiple sites of origin of the pathology, there may be a less marked topographic pattern to the pathology in sCJD than in vCJD.Differences in the distribution of the pathology in sCJD and vCJD could reflect the differences in the aetiology, and subsequent spread of prion pathology. Hence in vCJD, prions are believed to enter the nervous system by absorption through the gut following consumption of infected food, replication in the spleen, and neural entry via the spinal cord  whereas PrP gene , 4. The PrP gene . SubsequPrP gene . By contThe data are consistent with the hypothesis that there are different patterns of cortical degeneration in sCJD and vCJD. All laminae of the neocortex may be affected in both sCJD and vCJD, but, there is a greater development of the pathology in the lower laminae in sCJD and in the upper laminae in vCJD. Differences in cortical degeneration may be directly related to the different aetiologies of the two disorders and differences in the pattern of spread of the pathology through the brain."}
{"text": "It is a multidisciplinary protein, which is secreted in various body fluids. The ZAG plays roles in lipolysis, regulation of metabolism, cell proliferation and differentiation, regulation of melanin synthesis, cell adhesion, immunoregulation, and so forth. Vitiligo is the most common depigmenting skin disorder, characterized by acquired, progressive, and circumscribed amelanosis of the skin and hair. It commonly begins in childhood or young adulthood. The pathogenesis of this disorder is uncertain, but it appears to be dependent on the interaction of genetic, immunological, and neurological factors. For the first time, we pointed the probable association between ZAG and vitiligo. Herein, I have described this association in different views. By confirming this association, a surprising progression will occur in the treatment of this prevalent debilitating disease.Zinc- For thedipokine  was repodipokine . It was dipokine \u201310.\u03b11, \u03b12, and \u03b13 [\u03b11 and \u03b12 domains are highly conserved [\u03b13 domain is less conserved, indicative of species specificity and evolutionary constrains [ The gene of ZAG, assigned to the chromosome 7q22.1, comprises four exons and three introns . The molonserved , indicatnstrains . The sernstrains . There is high degree of similarity between ZAG and antigen-presenting molecules such as class I MHC molecules both at sequence and structural levels. The ZAG is considered as a member of immunoglobulin gene superfamily  based on The ZAG is a multidisciplinary protein, which is secreted in various body fluids  and is f It appears that renal filtration is an important route for eliminating ZAG; hence, markers of renal function should be considered in studies on ZAG physiology . The gene expression of ZAG is predominately regulated by glucocorticoids , 20, and\u03b23-adrenoreceptor in adipocytes by which it amplifies the induction of ZAG expression [\u03b23-adrenoreceptor leads to an increase in ZAG expression is not known [ Glucocorticoids are able to enhance ZAG expression , 22, 23.pression . Furtherot known , 21.\u03b23-adrenoreceptor agonist BRL37344, suggesting that the sympathetic system can play a role in the regulation of ZAG expression [\u03b23-adrenoreceptor [ It has been shown that the expression of ZAG is increased by the selective pression  and thatreceptor , 20.\u03b3 (IFN-\u03b3) is a potential mediator of the increased ZAG expression in cachexia. It has been shown that IFN-\u03b3 can strongly upregulate expression of ZAG in human epithelial cell lines [ Interferon-ll lines , 24.\u03b1 (TNF-\u03b1), a major product of macrophages [\u03b1 and the PPAR-gamma nuclear receptor [\u03b1 can decrease ZAG expression and secretion [\u03b1 on ZAG is time-dependent; in other words, this effect appears chronic rather than acute [ Studies showed that tumor necrosis factor-rophages , can modrophages , 25. In receptor . TNF-\u03b1 cecretion , 25, whiecretion . It appean acute . Studies The ZAG is a natural adipocyte factor found in both white and brown adipose tissue . Althoug The ZAG is closely related to obesity , 27\u201329. \u03b23-adrenoreceptor [ The ZAG is known to promote lipolysis through stimulation of adenylate cyclase in a GTP-dependent process via binding through receptor , 21. Rusreceptor . Moreovereceptor . Glucocorticoid-mediated upregulation of ZAG expression in white and brown adipose tissues results in lipolysis , 23; henThe ZAG is responsible for cachexia or lipid catabolism seen in cancer patients , 32. It \u03b1 and ACC, thereby activating a pathway central to the regulation of energy metabolism [ The ZAG can stimulate energy utilization in adipose tissue and skeletal muscle by upregulation of UCP isoforms and GLUT4. Findings have shown that ZAG can lead to phosphorylation of AMP kinase-tabolism . Russelltabolism , 35. Thetabolism . The circulating ZAG is negatively correlated with fasting insulin, homeostasis model assessment of insulin resistance , 19, C r The ZAG plays a role in lipid metabolism . It also In detecting of doping with recombinant erythropoietins by isoelectric focusing and western double blotting, ZAG interacts nonspecifically .\u03b11-antitrypsin, related to the forward motility of spermatozoa in human seminal plasma, played important roles during maturation of spermatozoa, from the epididymis through fertilization in the female reproductive tract [ The presence of ZAG in human seminal fluid is 6 times more than that in the human serum, which suggests its role in fertilization . The lipve tract . The role of ZAG in production of melanin is different. In vitro, increased ZAG can decrease melanin production by decreasing levels of tyrosinase protein and its activity, which are the key step for melanin synthesis. Furthermore, ZAG decreases melanin production in vivo, suggesting that ZAG has a different mechanism in vivo and in vitro. Collectively, it appears that the minimal level of ZAG is required for melanin production . It has also been shown that ZAG is produced by normal epidermal keratinocytes, where its expression improves with cellular differentiation , 42. It \u03b1 (TNF-\u03b1) also inhibits melanin synthesis by human primary melanoma, but it appears that ZAG inhibits melanin production via a TNF-independent mechanism [ Tumor necrosis factor-echanism .\u03b22-microglobulin for folding [ Due to high degree of structural similarities to MHC-I, ZAG is considered as a member of immunoglobulin superfamily . The mem folding , 46. The folding . Althoug folding , 18.\u03b13 domain [2+ and Mn2+ for mediating cell adhesion, and surprisingly, the presence of Mg2+ and Mn2+ is also needed for cell adhesion by ZAG. This finding describes the role of ZAG in the integrin-mediated adhesion. Other mechanisms for ZAG-mediated cell adhesion have been described [ ZAG is effective in attaining the properties of cell adhesion between cells and extracellular matrices . This fu3 domain , 47. Int It has been shown that the RNase activity of ZAG has similarities to other RNases such as human pancreatic RNase , 48. The The crystallographic studies of ZAG have shown that its groove can bind small hydrophobic ligands , 12. It  The role of ZAG in malignancies is different. In one hand, ZAG indirectly plays a role in hindering tumor progression via downregulating cyclin-dependent kinase, which regulates G2-M transition, a rate limiting step in the cell cycle. Moreover, ZAG is actively involved in inhibition of tumor growth and proliferation via its linking with immune system . The cap In Jiaxian red cattle, polymorphism of four loci at the coding region of the ZAG gene has been shown including in Z1, Z2, Z3, and Z4. In one study, Guo et al. showed that Z4 locus can be one of the markers of growth traits in these animals . The concentration of ZAG in serum has been shown to increase significantly in carcinomas . Hence,  Some studies have demonstrated that low levels of ZAG expression correlate with metastatic potential and a higher risk of recurrence in prostate cancer, suggesting that ZAG can act as an inhibitor of proliferation , 16, 52. Moreover, there is a higher level of ZAG in the well-differentiated tumors than that in the moderately or poorly differentiated tumors , 7. It w Cancer cachexia is characterized by progressive loss of adipose tissue due to increased lipolysis, resulting in the depletion of 85% of the adipose tissue mass and 30% of weight. A potential mediator of this increased lipolysis is a lipid-mobilising factor (LMF) which has been shown to be identical with the plasma ZAG . In cach\u03b23-adrenoreceptor [\u03b23-adrenoreceptor antagonist. Their study showed that ZAG can directly influence UCP expression, which is responsible for lipid utilization during cancer cachexia [ The role of ZAG in cancer cachexia was described by Bing et al. . ZAG canreceptor . Fastingreceptor , 33. Thireceptor . Furthercachexia . The ZAG is involved in the pathogenesis of proliferative diabetic retinopathy , 54. It \u03b22-microglobulin has significantly higher concentration in nonresponders to thalidomide-based induction therapy compared to responders, while haptoglobin has a lower concentration [ The ZAG is a marker for response to thalidomide-based therapy in patients with multiple myeloma. Rajpal et al. showed that ZAG along with vitamin D-binding protein, serum amyloid-A protein, and ntration . The ZAG is one of the biomarkers in multiple sclerosis. Tumani et al. have shown that the level of ZAG in cerebrospinal fluid is downregulated in the episode of multiple sclerosis . Findings have showed that ZAG can be as a biomarker for human metabolic alterations . The ZAG Vitiligo is the most common depigmenting skin disorder , charact The pathogenesis of this disorder is uncertain, but it appears to be dependent on the interaction of genetic, immunological, and neurological factors . The mel\u03b1 in vitiligo skin, compared with normal skin, reveals an imbalance of epidermal cytokines at sites of lesions [ Many patients with generalized vitiligo have serum autoantibodies and circulating autoreactive T cells against melanocytes and melanocyte components . Moreove lesions , 69. It appears that there may be a relationship between stress and the development of vitiligo, because vitiligo patients tend to have higher scores for anxiety, depression, adjustment disorders, obsessive symptoms, and hypochondria , 70. A new theory is emphasizing that depigmentation in vitiligo patches results from a chronic detachment of melanocytes that is proposed to be designated as melanocytorrhagy, which is possibly related to increased susceptibility to mechanical and other types of stresses , 71, 72. It has been demonstrated that repigmentation occurs in 10\u201320% of patients , 73, 74. Studies have shown that topical steroids and narrowband UVB monotherapy are the most effective and safest forms of treatment for localized and generalized vitiligo, respectively , 77. Som For the first time, we pointed the probable association which might be present between ZAG and vitiligo , 64. It Studies have revealed that ZAG as a keratinocyte-derived factor influences melanocyte proliferation and dendricity , 43, 44.Some patients with vitiligo have serum autoantibodies and circulating autoreactive T cells against melanocytes and melanocyte components . StudiesIt has been revealed that ZAG is effective in attaining the properties of cell adhesion between cells and extracellular matrices . We know\u03b1 is high [\u03b1 can decrease ZAG expression and secretion [In whitish patches of vitiligo, the expression of TNF- is high , 69. Somecretion , 25; henTopical steroids are the most effective and safest forms of treatment for vitiligo, especially for the localized one , 77. StuAs a hypothesis, Bagherani et al. suggested that zinc might be effective in the treatment of vitiligo . Some stThe linkage signals on chromosome 7 in patients with generalized vitiligo and associated autoimmune diseases have been reported , 67. SurSome findings have shown that the minimal level of ZAG is required for melanin production . Hence,  Regarding mentioned subjects, the association between vitiligo and ZAG must be confirmed in one robust study in vitro and in vivo. If we can confirm this association, this discovery will result in a big progression in the field of treatment of this debilitating disease."}
{"text": "Defined by a persistent fear of embarrassment or negative evaluation while engaged in social interaction or public performance, social anxiety disorder (SAD) is one of the most common psychiatric syndromes. Previous research has made a considerable effort to better understand and assess this mental disorder. However, little attention has been paid to social motor behavior of patients with SAD despite its crucial importance in daily social interactions. Previous research has shown that the coordination of arm, head or postural movements of interacting people can reflect their mental states or feelings such as social connectedness and social motives, suggesting that interpersonal movement coordination may be impaired in patients suffering from SAD. The current study was specifically aimed at determining whether SAD affects the dynamics of social motor coordination. We compared the unintentional and intentional rhythmic coordination of a SAD group (19 patients paired with control participants) with the rhythmic coordination of a control group (19 control pairs) in an interpersonal pendulum coordination task. The results demonstrated that unintentional social motor coordination was preserved with SAD while intentional coordination was impaired. More specifically, intentional coordination became impaired when patients with SAD had to lead the coordination as indicated by poorer  coordination. These differences between intentional and unintentional coordination as well as between follower and leader roles reveal an impaired coordination dynamics that is specific to SAD, and thus, opens promising research directions to better understand, assess and treat this mental disorder. Social anxiety disorder (SAD) is one of the most prevalent mental disorders, with estimates of its lifetime prevalence at about 7\u201313% Furmark, . SAD is However, very little attention has been paid to nonverbal behaviors of patients with SAD when interacting with other people. This is surprising given that several studies have demonstrated close relationships between the bodily behavior of interacting people and their mental states during social interactions  underlying the coordination. It has been demonstrated that a stronger coupling, and thus more stable coordination, occurs when the movements of the other person are (a) perceived using central vision compared to peripheral vision   with the coordination of control participants using a task that involved the swinging hand-held pendulums  . Controls (Matched control participants and synchronization partners 1 and 2) were excluded if they had: (1) a history of social phobia or other anxiety disorder, bipolar or psychotic disorder, recurrent depression, substance dependence, or of any substance abuse in the last 6 months based on the SCID or (2) a current major depressive episode.Nineteen patients diagnosed with SAD participated in the study. Diagnoses were made according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria . Patients and synchronization partners 1 and patients for the SAD group and synchronization partners 2 and matched controlparticipants for the control group. Each trial consisted of three 30 s segments that were delimited by an auditory stimulus. Participants were instructed to oscillate their pendulum at the same self-selected tempo whether they were looking  or not looking  at the swinging of the other participant . Finally, in order to further explore the effect of the severity of SAD, we also performed correlational analyses to examine the relationship between the different motor coordination variables and the LSAS Total score obtained by SAD patients and matched control participants (N = 38). Those correlational analyses were performed on the whole sample because beyond the usual approach of considering SAD as a discrete category that is qualitatively distinct from normal social anxiety, we also considered SAD as part of the upper end of a continuum that differs only quantitatively  from normal social anxiety. Indeed, there is now growing evidence that social anxiety refers to a dimensional  rather than a categorical (representing a distinct entity) construct  with the factors Group , Pendulum Combination , and Segment  to examine circular variance of the relative phase during unintentional coordination. We performed 2 \u00d7 3 \u00d7 2 repeated-measures analyses of variance (ANOVAs) with the factors Group , Pendulum Combination , and Pattern  to examine the phase shift and standard deviation of relative phase during intentional coordination. F = 5.20, p = 0.008, F = 8.42, p < 0.001, p = 0.002) and Segment 3 = \u201cNo vision\u201d (p = 0.08)) and when participants had preferred movement frequencies closer to each other ). However, this ANOVA did not reveal any effect of Group indicating that the SAD group synchronized as well as the control group when the coordination was unintentional. Moreover, no significant correlations were found between the LSAS Total score and movement synchronization during the visual interaction (Segment 2) , also suggesting that unintentional social motor coordination was not disrupted in SAD.The ANOVA performed on the circular variance values in the unintentional coordination task yielded significant main effects of Segment  = 24.62, p < 0.001, t-tests indicated phase shifts were significantly negative in the P2-P1 (Follower) condition (t(37) = \u22124.15, p < 0.001), not different from zero in the P1-P1  condition (t(37) = \u22120.54, p = 0.59), and significantly positive in the P1-P2 (Leader) condition (t(37) = 3.02, p = 0.005)  = 0.2, p = 0.88, p-values > 0.10), which confirms this last result and that social anxiety did not modulate the phase shifts from the intended coordination.The ANOVA performed on the phase shift values yielded a significant main effect of Pendulum Combination  = 19.37, p < 0.001, F = 6.29, p = 0.003, p-values < 0.005). Interestingly, the analysis also demonstrated a significant interaction between Group and Pendulum Combination  = 3.70, p = 0.03, p = 0.049)  = 0.36; p = 0.03) and the P1-P2 (Leader) pendulum condition (r(34) = 0.35; p = 0.03)  = 0.19; p = 0.27).The correlational analyses between the standard deviation of relative phase values (average of in-phase and anti-phase coordination) and LSAS Total scores for the three different pendulum combination conditions supported this last result. After removing an outliner in the SAD group (>3 standard deviations above the mean), the results yielded significant positive relationships between these two variables for the P1-P1  pendulum condition  with the rhythmic coordination of control participants. We also examined the relationships between the severity of SAD as assessed by the LSAS and measures of social motor coordination. The results demonstrated that the coordination dynamics were disrupted in the SAD group when the coordination was intended and the SAD participant had to lead the coordination. Specifically, the rhythmic coordination was more variable for the SAD group than for the control group in this condition. We discuss this result in the next section as well as its implications for better understanding the origins of this mental disorder and improving its assessment and treatment.It is important to observe that the disruption of social motor coordination occurred when the coordination was intentional but not when the coordination was unintentional. Such intended-unintended dissociation is in line with previous research that found a similar dissociation in social motor coordination in schizophrenia  and a potential confound did not result in any significant effects. However, the lack of a gender effect should be confirmed in future investigations with larger groups of participants. Such an effect is of particular interest in view of previous research that has shown differences in the social phobia of men and women, with greater fear of scrutiny for women than men  as well as measuring their anxiety in these conditions would be interesting to confirm the current results. It was also initially hypothesized that changes in the coordination of patients could originate from a motion perception deficit or irregular gaze behaviors. It would thus be interesting to examine the gaze behaviors of patients using eye-tracking systems (Roerdink et al., It is important to note that the behavioral pattern observed in SAD patients in the current study appears to be unique to this population. None of the previous studies that investigated social motor coordination in healthy subjects observed such modification of movement dynamics (Schmidt and Richardson, An additional confounding factor that should be investigated in future research is that the coordination exhibited in the current experiment also depended on the properties of the control partner, on his/her visual and attentional processes, his/her motivation and/or his/her reaction to their partner with social phobia. To evaluate such an issue, testing the coordination of patients with a virtual partner instead of a real partner would allow systematic control of the virual partner\u2019s movement variance (Klinger et al., The disruption of the dynamics of social motor coordination demonstrated in the current study with SAD also raises important questions about the origins of these disorders. Do people with SAD have abnormal coordination dynamics because they have anxiety or do they have anxiety because they have abnormal coordination dynamics? Although the causal relation between bodily movement and mental states during social interactions is often unclear, there is growing evidence showing that bodily movement might be first in such a relation (Bernieri et al., Finally, if impaired social motor coordination of patients with SAD contributes to their mental illness and inevitably affects the success of their social exchanges, interventions aimed at improving the social motor coordination might lead to effective treatments. It is possible to develop rehabilitation protocols that can help patients with SAD to more successfully coordinate with others. Such rehabilitation may even be accelerated by using biofeedback and giving real-time information about their coordination with the others, which has been demonstrated in a variety of rehabilitation protocols (Kovacs et al., Manuel Varlet, Ludovic Marin, Delphine Capdevielle, R. C. Schmidt, Robin N. Salesse, Beno\u00eet G. Bardy and St\u00e9phane Raffard conceived and designed the experiment. Manuel Varlet, Delphine Capdevielle, Jonathan Del-Monte, Jean-Philippe Boulenger, and St\u00e9phane Raffard performed the experiment. Manuel Varlet, Ludovic Marin, R. C. Schmidt, Robin N. Salesse, Beno\u00eet G. Bardy and St\u00e9phane Raffard analyzed the data. All authors contributed to and have approved the final 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."}
{"text": "The p53 protein was discovered many years ago as a tumour suppressor gene and despite the wealth of information that has accumulated, a complete understanding of how p53 functions remains still elusive.p53 is a key regulator of the checkpoint response to DNA damage in mammal cells and is affected by loss-of-function mutations in the majority of human cancer. In various normal adult tissues, basal levels of p53 protein are low, but specific stress signals induce post-translation modifications and its stabilization, leading to activation of distinct transcriptional programmers. In response to stress signals p53 mainly acts to repair DNA damage and depending on the severity of the damage, the cell microenvironment and the cell type, p53 can orchestrate different cellular outcomes such as apoptosis and senescence.In addition to its nuclear activity, p53 also possesses biological activities that are transcription-independent, indeed p53 functions also in the mitochondria and cytoplasm, where, depending on the kind of stress either triggers apoptosis or inhibits autophagy , 2.Then, the DeltaNp53 isoform, that lacks the transactivation domain, contributes to regulate p53 functions . Cells fIndeed, emerging evidence suggested that p53 and DNA damage checkpoints have also a prominent but still not well characterized role in the regulation of aging in worm, mice and human . HoweverAs consequence, the existence of two different and not redundant p53 pathways explains why the abrogation of the p53 aging function, as it occurs in p66Shc null mice, does not increase tumour formation."}
{"text": "The viability and subtle developmental defects of p53 knockout mice suggest that p53 does not play major role in development. However, contradictory evidence also exists. This discrepancy mainly results from the lack of molecular and cellular mechanisms and the general fact that p53 activation requires stresses. Recent studies of p53 in mouse and human ES cells and induced pluripotent stem (iPS) cells shed new light on the mechanisms of the developmental roles of p53. This review summarizes these new studies that support the developmental roles of p53, highlights the possible underlying molecular mechanisms, and discusses the potential relationship between the developmental roles and the tumor suppressive function of p53. In summary, the molecular mechanisms underlying the developmental roles of p53 are emerging, and the developmental roles and tumor suppressive function of p53 may be closely related. The history of the p53 field has made several turns. When it was first discovered as a SV40 binding protein, it was believed to be an oncoprotein because it is highly expressed in many types of tumors and the p53 cDNA from these tumors can transform normal cells together with H-Ras -6. LaterGiven that p53 is important and highly expressed during development, it was surprising to observe that p53 knockout mice developed normally, while the adult mice quickly developed tumors . Since tAnother organ that has obvious defects in p53 knockout mice is the testis . The tesThe most direct evidence showing that p53 is able to affect development is from the study of mdm2 knockout mice. Mdm2 knockout mice die between implantation and E5.5, around the stage of blastocysts from which embryonic stem cells are derived ,22. In ap53 also can influence mouse embryo implantation by regulating the maternal Lif levels  Table\u00a011. Lif isAn interesting study showed that p53 dependent apoptosis may be involved in X irradiation-induced teratogenesis . In thisThe potential developmental roles of p53 have been extensively covered in another review article ,39. HoweThe activity and levels of p53 in early embryos are higher than those in late embryos -46. In vWithout stresses, the high levels of p53 do not lead to the apoptosis or differentiation of ES cells because its activity is inhibited by Mdm2 and Mdm4 . RemovalDelta40-p53 is a naturally occurring splicing variant of p53, with an amino terminal deletion of 40 amino acids . Delta40p53 can be activated in ES cell stage under certain conditions, such as Brca1 deletion. Brca1 is a protein that is involved in repairing DNA double strand breaks through homologous recombination. Its deletion in ES cells activates p53, causing the differentiation and/or apoptosis of ES cells. Therefore, ES cells containing Brca1 deletion are not viable ,51  in response to DNA damage ,53, it iDespite the controversy of the apoptotic function of p53, the differentiation regulation is a well-established function of p53 in mES cells. Upon DNA damage, p53 induces the differentiation of mES cells by repressing the transcription of Nanog Figure\u00a0A 56]. A. A56]. AIt is emerging that p53 is fully functional in mES cells in response to DNA damage stress. In mES cells, p53 seems to play an important function by regulating the development-associated genes because these genes are more enriched in p53-regulated genes than apoptosis- and cell cycle-associated genes ,40. ThisOne of the surprising findings about the molecular mechanisms of p53 during development is that the p53 stress signaling and the developmental signaling merge at their downstream gene level . Upon DNIn human ES (hES) cells, p53 plays important roles in regulating their differentiation and apoptosis Figure\u00a0B 53,67]67]. p53 The tumor suppressor p53 elicits many cellular functions after various stresses, such as DNA damage, hypoxia, virus infection, and unfolded protein shock. However, the connection between the cellular function and the tumor suppressive function is still unclear despite more than 30\u00a0years of study. Cell cycle arrest (reversible), senescence (irreversible cell cycle arrest), and apoptosis all have been shown to participate in tumorigenesis, suggesting that they may contribute to the tumor suppressive function of p53 . Cdkn1a \u039411/\u039411) is not compatible with life. Crossing Brca1\u039411/\u039411 mice with p53 heterozygous or null mice completely rescue the embryonic lethality of Brca1\u039411/\u039411 . This example shows that the failure of p53 signaling during development could also lead to tumorigenesis in the adulthood. As described above, p53 loss partially rescued the dys-regulation of other genes, such as Brca2 and Rad51, suggesting that the complete loss of these genes also activates p53-independent pathways to cause embryonic lethality. Further studies are required to determine whether the same scenario exists for other genes or in human development.One typical example demonstrating the relationship between the developmental role and tumor suppressive function of p53 is the functional interaction between Brca1 and p53 (Figure\u00a0There are at least two non-exclusive models for p53 in development (Figure\u00a0ES cells, as an excellent model for early development, have provided many novel insights into the developmental roles of p53. However, ES cells only represent a snapshot of development. p53 regulates very different sets of genes in different cell types . CautionThe authors declare that they have no competing interests.MHS, YH, and JH wrote the review. All authors read and approved the final manuscript."}
{"text": "The characterization and cellular localization of transcription factors like NF-\u03baB requires the use of antibodies for western blots and immunohistochemistry. However, if target protein levels are low and the antibodies not well characterized, false positive data can result. In studies of NF-\u03baB activity in the CNS, antibodies detecting NF-\u03baB proteins have been used to support the finding that NF-\u03baB is constitutively active in neurons, and activity levels are further increased by neurotoxic treatments, glutamate stimulation, or elevated synaptic activity. The specificity of the antibodies used was analyzed in this study.Selectivity and nonselectivity of commonly used commercial and non-commercial p50 and p65 antibodies were demonstrated in western blot assays conducted in tissues from mutant gene knockout mice lacking the target proteins.A few antibodies for p50 and p65 each mark a single band at the appropriate molecular weight in gels containing proteins from wildtype tissue, and this band is absent in proteins from knockout tissues. Several antibodies mark proteins that are present in knockout tissues, indicating that they are nonspecific. These include antibodies raised against the peptide sequence containing the nuclear localization signals of p65  and p50 . Some antibodies that recognize target proteins at the correct molecular weight still fail in western blot analysis because they also mark additional proteins and inconsistently so. We show that the criterion for validation by use of blocking peptides can still fail the test of specificity, as demonstrated for several antibodies raised against p65 phosphorylated at serine 276. Finally, even antibodies that show specificity in western blots produce nonspecific neuronal staining by immunohistochemistry.We note that many of the findings in the literature about neuronal NF-\u03baB are based on data garnered with antibodies that are not selective for the NF-\u03baB subunit proteins p65 and p50. The data urge caution in interpreting studies of neuronal NF-\u03baB activity in the brain. NF-\u03baB is a transcription factor that is ubiquitously present in all cells of the body. It exists as a homo- or hetero-dimer comprising typically p50 and p65 (RelA) subunits, but also combinations of these subunits with other members of the Rel family, such as p52, c-Rel and RelB . ActivatNF-\u03baB function is most studied in the immune system , but it All of the techniques that rely on antibodies require antibody specificity to ensure that the assay is truly tracking NF-\u03baB proteins. An antibody is specific if it recognizes and binds to the epitope in the target protein and to no other molecular or nonspecific entities. Validation of antibody specificity for IHC is typically done by a set of control experiments that involve omission of the primary antibody and co-incubation of the preparation with an excess amount of the peptide used for immunization. Another important test of specificity is the demonstration in western blot that the antibody binds to a single protein that runs in the gel at a molecular weight that is expected of the target molecule. The most stringent of control tests is the demonstration that the binding or staining of the antibody is absent in tissues or cells from a genetic mutant animal that has been engineered to lack that protein, i.e., a knockout (KO) mouse. Surprisingly, there are ample cases of antibodies that \"pass\" all the control tests except the last one. Thus, this study used the resource of tissue from p50 KO and p65 KO mice to test a number of widely used NF-\u03baB antibodies. Only a few antibodies passed all of the tests, and several widely used antibodies failed. The most significant failures were antibodies raised against the region containing the NLS of both p65 and p50. A review of the literature on NF-\u03baB in brain reveals that most of the claims of constitutive or induced NF-\u03baB activity in neurons are based on results obtained with antibodies that failed the rigorous specificity test.rela-/-/tnfr1-/- (p65 KO) mice were a gift of Mollie Meffert [nfkb1-/- (p50 KO) mice were a gift of Christopher Hunter [nfkb1 gene product and precursor to p50, but for clarity in the text, only the p50 KO is noted in the designation. The p65 KO mouse has additional deletion of the Type 1 TNF receptor, which permits survival of the mouse into adolescence [C57BL/6 mice were obtained from The Jackson Laboratories. The  Meffert ,5, and nlescence , and forhttp://web.ncifcrf.gov/research/brb/reagents/AntiseraReagent.aspx). The list of antibodies tested for the study is provided in Table Antibodies directed against p65 and p50 were obtained from commercial sources , Billerica, MA; Calbiochem, St. Louis MO; Cell Signaling, Beverly, MA; Abcam, Cambridge, MA; Epitomics, Burlingame, CA) or from the NIH, including a p65 antibody that was a gift of Ulrich Siebenlist, National Institute of Allergy and Infectious Diseases, NIH, and antibodies deposited by Dr. Nancy Rice, National Cancer Institute, NIH, into the repository of the Biological Resources Branch, NCI, Frederick, MD  (10 mM) and 2-deoxycytidine (100 mM) (Sigma) to inhibit astrocyte growth. After 10 days in culture, the cells were subjected to the various experimental conditions and processed for either immunostaining or immunoblotting.Mouse neurons were cultured from 16-day embryonic C57BL/6 mouse brains as described previously . HippocaMouse brain cells comprising astrocytes, microglia, fibroblasts, and some neurons were cultured from 16-day embryos. Subcortical pieces were dissected out in cold HBSS, homogenized, pelleted, resuspended, and plated for 10 days as above except the medium contained only DMEM with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml).Mice were maintained in the animal colony under standard housing and ad lib feeding conditions. Kainic acid  was dissolved in sterile water immediately before use. From a stock solution of 0.3 mg/ml, 1.0 \u03bcl was slowly infused with a Hamilton syringe stereotaxically lowered to the coordinates A-P +1.5, M-L -2.4, D-V 3+3.8 with respect to the interaural line for intrahippocampal injections. Injections were made i.p. at 30 mg/kg.Samples from whole brain, liver, or spleen or from cultured cells were homogenized on ice (with sonication for tissues) in T-PER reagent . Protein concentration of these whole cell extracts was determined using the BioRad Protein Assay Reagent  according to the manufacturer's instructions. Cytosolic and nuclear extracts of cortical neurons were prepared using the NE-PER kit (Pierce) according to the manufacturer's instructions. Both the T-PER reagent and the buffers in the NE-PER kit were supplemented with both a protease inhibitor cocktail  and a phosphatase inhibitor cocktail .Protein samples of either tissue extracts or individual cytosolic or nuclear extracts were loaded in equal amounts into individual wells of a 10% polyacrylamide-SDS gel and the proteins resolved using MOPS-SDS buffer. Proteins were transferred to a PVDF membrane  for 2 h at room temperature, and then the nonspecific sites were blocked with 5% Dried milk in Tris-Buffered Saline containing 0.05% Tween-20 (TBST buffer) with gentle washing for 2 h at room temperature. The membranes were incubated overnight at 4\u00b0C in a fresh solution of 5% dried milk-TBST containing antiserum for p50 or p65 at optimized dilutions for each antibody. After the overnight incubation, membranes were washed three times with TBST (10 min each) and re-incubated with a goat anti-rabbit IgG-HRP secondary antiserum  diluted in 5% dried milk-TBST for 4 h at room temperature. The membranes were washed three times with TBST (10 min each), reacted with the West Duro Chemiluminescent substrate (Pierce) and exposed to X-ray film to visualize protein present in the samples. Approximate molecular weights of the target proteins were determined by comparison to a BenchMark prestained protein ladder (Invitrogen).Neurons were fixed on coverslips in 4% PBS-paraformaldehyde for 30 min at room temperature, washed with PBS and then permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 15 min at room temperature. The cells were blocked with PBS-0.1% Triton containing 5% Normal Goat Serum (NGS) for one hour at room temperature and incubated overnight at 4\u00b0C with the appropriate primary antiserum diluted in PBS-0.1% Triton-5% NGS. Antiserum tested was sc-372G (Santa Cruz) at 1:700 dilution. After washing with PBS-0.1% Triton, the cells were incubated for 2 h at room temperature with the anti-goat biotinylated secondary antibody followed by avidin, biotinylated enzyme, and diaminobenzidine using the Vectastain Elite ABC kit  for brown color reaction. Coverslips were inverted and fixed to slides with Permount for later examination under a microscope (Leica DMR) and CCD camera .Mice were deeply anesthetized  and transcardially perfused with saline followed by freshly prepared phosphate buffered 4% paraformaldehyde. Brains were removed and stored in the same fixative followed by transfer to a 20% sucrose solution. Microtome-cut, 40-\u03bcm-thick, free-floating sections were processed as above. For IHC, prior to adding the primary antibody, control sections were incubated for 15 min in 3% hydrogen peroxide in PBS to quench endogenous peroxidases and then washed. Quenching had no effect on the staining outcome, so it was eliminated in remaining experiments. The step was omitted in the IC procedure because control experiments (incubating fixed cells in the Vectastain color reaction reagents) indicated that quenching did not affect the color reaction.There were several p65 antibodies that marked a single protein migrating in the gel at the correct molecular weight, and this marked band was absent in lanes containing protein from p65 KO tissue. These were Santa Cruz sc-372, NIH p65, and Invitrogen 2A12A7 monoclonal Figure , 1b, 1d.The failure of the MAB3026 antibody was quite surprising, given that in early publications, then named \u03b1-p65M, it passed stringent tests for validity in cells transfected with a construct overexpressing p65 . Therefonfkb1 gene. Other p50 antibodies labeled multiple bands including a band at 50 kDa MW that was absent in p50 KO tissue. These included Abcam 7971  tissue, in brain, liver, and spleen samples. A similar pattern was found with the NIH p65 antibody. The four antibodies that met this rigorous test of specificity were all raised against a portion of the C-terminus of p65, which includes its transactivation domain (TAD). In contrast, antibodies raised against the N-terminus or against the NLS region did not meet the criteria for specificity. Thus, it is apparent that the C-terminus contains antigenic peptide sequences that are unique to p65, whereas other regions of the molecule are not so selectively and strongly antigenic. The NLS region should be notably difficult to raise selective antibodies against because the aa sequences are conserved for a number of proteins that have NLSs. The N-terminal Rel homology domain (RHD) is conserved across other proteins, so this too may not be a suitable region for antibodies. Finally, not surprisingly, the rabbit monoclonal antibodies gave excellent results. They are new and have not been used in many publications to date.The sc-372 antibody has been used in several publications to characterize NF-\u03baB activity in the brain. A study by  examinedIn human brain infarctions, a selective staining of glial cells was seen by Terai  using thIn a study of status epilepticus seizures, which are associated with neuronal loss, p65 staining with sc-372 was induced in astrocytes and blood vessels mainly in the hippocampus and entorhinal cortex . ConstitFollowing immune challenge by i.p. IL-1\u03b2 injection, Nadjar et al. used the sc-372 antibody for IHC in rats and mice to examine the cellular CNS response . BackgroOther IHC publications are more difficult to interpret. Quite a few studies do not provide sufficient information in the Method section to allow determination of which specific antibody was used. A study by Gabriel  does notIn summary, studies employing the antibody we have characterized here as the best one for localizing authentic p65 in brain have fairly consistently shown that p65 staining is not appreciably present in neurons and is not induced in neurons after various brain insults. Rather, p65 immunostaining is increased in endothelia, astrocytes, and microglia in these studies.KRKRT (NLS underlined). The authenticity of the monoclonal antibody was determined by western blot [In contrast to the above summary, another group of studies shows predominantly neuronal p65 staining, both constitutively and after pathological insults. The most widely used antibody for IHC to depict this pattern of NF-\u03baB activity in the brain is the MAB3026 antibody against \"activated\" p65. The original p65 antibody raised against the NLS region was a rabbit polyclonal made by P. A. Baeuerle  and nameern blot . InteresIn the early years, the MAB3026 antibody was used to support the argument that there was constitutive and induced NF-\u03baB activity in neurons ,31. VeryFollowing peripheral lipopolysaccharide (LPS) administration eliciting an inflammatory response in the brain, ISHH showed strongly induced I\u03baB\u03b1 mRNA expression in non-neuronal cells of the meninges, blood vessels, brain parenchyma, and circumventricular organs ,21. In sOf the tested p50 antibodies, the Epitomics 1559 rabbit monoclonal antibody raised against a peptide sequence located between aas 340 and 370 of p50 was the best antibody in the study. It is new and has not been used in published studies of brain localization or activity. In contrast, the Santa Cruz sc-114 antibody, raised against the NLS region at aas 357-365 to generate an \"activated p50\" antibody, was clearly not directed against p50 in our gels. This fact has already been published -41. ThesA study has already been published showing the lack of specificity of this and other phospho-ser276-p65 antibodies in a number of cell lines . Our fin++ and enzymes like PKC and PKA to phosphorylate nuclear proteins. In one study, glutamate stimulated the production of PKC-phosphorylated proteins at 48, 87, and 120 KDa molecular weight in hippocampal neurons [The amino acids surrounding ser276 of p65 contain sequences that are also found in proteins that are phosphorylated by a number of enzymes, so it is possible that the antibody marked unknown phosphorylated proteins of large molecular weight. Indeed, these proteins were very responsive to glutamate treatment as indicated in western blots by intensified bands of nuclear but not cytoplasmic proteins appearing within minutes of glutamate stimulation, in immunohistochemically stained hippocampal neurons following in vivo KA treatments Figure , and in  neurons . We founWe show here that even antibodies that pass the test of specificity on western blots still label cells nonspecifically when used for IHC. The probable reason for this is that the levels of p50 and p65 in neurons are so low that they cannot be detected by this method. Raising the concentration of the primary antibody to the point that cellular staining becomes visible amounts to nonspecific staining, which we see even at 1:500 dilution of authenticated antibodies. Antibodies that mark additional proteins in the western blots are even more difficult to interpret when used for IHC, especially in reports of changes in staining levels induced by various manipulations. It is unlikely that the changes measured are due to alterations in levels of p65 or p50. Any of the other proteins recognized by these antibodies might contribute to the immunohistochemical outcome. We  and others  have fouThe close functional association of p50 with p65 should result in similar immunohistochemical staining patterns in tissues following manipulations. A few studies have used p50 antibodies other than sc-114 for IHC in brain. Some used antibodies distributed by Warner Greene (Gladstone Institute). A p50 antibody named Ab 392 raised against the N-terminus was used in several IHC publications following its characterization in COS cells . A westeA p50 antibody made by Mariagrazia Grilli  but not commercially available passed the test of marking single bands at 50 and 105 kDa in a western blot that were absent in p50 KO tissue, and it was used to show staining of radial glial cells in the subventricular zone and in the rostral migratory stream . The autWidely used antibodies raised against the NF-\u03baB subunits p65 and p50 were subjected to the test of specificity in WT and KO tissues, and several of the antibodies that failed the test of specificity are the ones that have been used in published studies to argue for neuronal activation of NF-\u03baB in the brain following neurotoxic or other challenges. Antibodies that rigorously passed the specificity test show little specific neuronal immunostaining either in cell culture or brain, whereas they do mark non-neuronal cells in brains subjected to toxic or excitatory challenges. The data help to explain confusion in the literature about the role of NF-\u03baB in neuronal function and injury. The lack of neuronal NF-\u03baB response observed with the authentic p65 antibodies is a finding consistent with studies employing in situ hybridization histochemistry showing non-neuronal localization of induced I\u03baB\u03b1 mRNA expression in injury, infection, and pain models ,54-56 anThe authors declare that they have no competing interests.MH conceived of the project, advised on experiments, prepared the figures, and wrote the manuscript. PR performed in vitro experiments, western blots, and immunohistochemistry of neuronal culture. PB performed animal surgeries and immunohistochemistry of brain sections. SL performed western blots and immunofluorescence. All authors read and approved the final manuscript."}
{"text": "Miceknocked out forp66shchave a lifespan~30% longeranddemonstrate an enhanced resistance to oxidative stress and age-related pathologies such as hypercholesterolemia, ischemia, and hyperglycemia. In this respect, p66shc is a promising pharmacological target for the treatment of age-related diseases. In this review, an attempt has been made to survey and put to a critical analysis data concerning the involvement of p66sh\u0441 in the different signaling pathways that regulate oxidative stress and apoptosis. It is important to note that miceknocked out for p66shc , in comparison with other mouse models with a prolonged lifespan , are fertile and exhibit a normal phenotype [The identification of the mutations that lead to the prolongation of the lifespan of various model organisms shows that aging can be considered as a genetic program . One of henotype . These mhenotype , AGE  , and by four loci in mammals \u2013 Shc ( ShcA ), Sli ( ShcB ), Rai ( ShcC ) [RalP [ShcA locus are designated according to their molecular weights as p46shc, p52shc, and p66shc, respectively. These proteins participate in the regulation of proliferation (p46shc and p52shc) and apoptosis (p66shc) [P66shc is an adaptor protein which is coded for by a single locus in ( ShcC ) , and Ral ) [RalP . The fou(p66shc) .Saccharomyces ), nematodes ( Caenorhabditis ), and insects ( Drosophila ) but appears in amphibians ( Xenopus ), fishes ( Fugu rubripes ), and mammals [P66shc is considered to be a relatively \u201cyoung\u201d protein since it is not found in yeast  and SOS (nucleotide exchange factor), which leads to RAS activation and the induction of the mitogene-activated protein kinases (MAPK) pathway. Although p66shc as p46shc/p52shc is phosphorylated by tyrosine-kinase receptors and interact with the GRb2/SOS complex, it, apparently, does not activate MAPK . The comp66shc  gene revealed decreased levels of intracellular ROS, as determined by means of ROS-sensitive probes, as well as reduced levels of oxidative damages to DNA and proteins, estimated by measuring 8-oxo-deoxiguanosine and nitrotyrosines [p66shc -deficient cell lines  derived from mice, rats, and a human showed that p66shc plays an important role in apoptosis induced by various agents Fig. 2).  p66shc and Regulation of Expression of Antioxidant Enzymes Previous research has shown that p66shc reduces the expression of such antioxidant enzymes and regulatory factors as glutathione peroxidase-1 , MnSOD , 20, 28,It should be mentioned that these data are in disagreement with a number of publications which argue that p66shc does not affect the levels of antioxidant enzymes , 15-17. p66shc and Mitochondria-mediated Apoptosis p66shc gene are resistant to various inductors of mitochondria-mediated apoptosis implies a direct interaction between p66 and mitochondria.The fact that cells carrying deletion of the Mitochondrial localization of p66shc and its transport to mitochondria.  Studies on the cellular localization of p66shc showed that 32% of this protein is localized in the cytoplasm; 24% is in the endoplasmic reticulum; and 44%, in mitochondria [chondria . Inside chondria . Accordi2 O 2  [The stimuli that promote p66shc translocation into mitochondria are usually pro-apoptotic factors, such as UV radiation and treatment with H 2 O 2  , 35. How2 O 2  . However2 O 2  . It was 2 O 2  .2 O 2  treatment includes the activation of PKC\u03b2, which phosphorylates p66 at Ser36. Phosphorylated p66 becomes a target of prolylpolymerase Pin-1 that recognizes a proline residue, which follows phosphorylated serine. After isomerization, p66shc is dephosphorylated by PP2A phosphatase and transported into mitochondria. The latter event is confirmed by the fact that the pool of mitochondrial p66shc is not phosphorylated [According to data presented in , the sigorylated  Fig. 4)2 O 2  trorylated  that theRedox proapoptotic activity of p66shc and the production of ROS.  Current views hold that, in the course of mitochondrial-dependent apoptosis, various signals  promote the formation of a multi-subunit protein complex, which eventually forms a pore in the inner mitochondrial membrane. The permeabilization of both mitochondrial membranes results in the release of Cyt C and other proteins in the cytoplasm, apoptosome formation, and caspase activation [tivation .et al . have suggested a mechanism explaining the role of p66shc in mitochondrium-dependent apoptosis [In vitro showed that the addition of recombinant p66shc to isolated mitochondria with a permeabilized outer membrane results in mitochondria swelling as a result of permeability transition pore formation. This effect was also inhibited by cyclosporine A, catalase, antioxidant dimethylthiourea, and the inhibitor of complex III of the respiratory chain antimycin A, as well as being dependent on respiratory substrates. Therefore, the obtained data is evidence that permeability-transition pore formation is a key step in p66-mediated apoptosis and that ROS and respiration are also necessary in this process.Pelicci poptosis . It turnpoptosis . In vitrThe application of electrochemical analysis and fluorescent redox-sensitive probes revealed that p66 acts as a redox enzyme and transfers electrons from reduced Cyt C to oxygen. The interaction of p66 with Cyt C is mediated by the CB-domain, which was confirmed by ELISA and site-directed mutagenesis . IncomplTherefore, the Pelicci group suggested the following mechanism of pro-apoptotic action of p66shc : under nin vitro . In order to validate this hypothesis, one needs to carry out additional experiments based on fluorescent probes for measuring ROS levels in the mitochondria of cells with an inactive respiratory chain (Rho-0 cells), as well as in cells devoid of Cyt C .It is important to realize that the suggested mechanism of p66shc redox activity, which includes Cyt C as an electron donor, is only a reality It should be noted that the described model has many weak points. For example, p66shc does not contain any well-known redox domains or metal-binding domains. Therefore, its ability to generate ROS only in the presence of copper ions can be an artifact, since copper ions are known to be able to generate ROS during Fenton\u2019s reaction, which could affect the results obtained by means of redox-sensitive fluorescent probes. It is also known that Cyt C, upon oligomerization, can produce ROS as a result of auto-oxidation -52. TakiStudies on an isolated CH2-CB-domain . The mechanism of action of p66shc was also studied in in vitro experiments with an isolated CH2-CB-domain. It turns out that the recombinant CH2-CB-domain can exist in two forms: reduced, as a dimer, and oxidized, as a tetramer (or a dimer of dimers) which contains disulfide bridges between residues Cys59. The addition of both the CH2-CB-domain and the full-length protein to mitochondria resulted in the formation of a permeability transition pore and swelling; however, only the tetramer exhibited such a pro-apoptotic activity. In contradiction to the data presented in [ented in , which s Apart from that, the CH2-CB-domain was shown to trigger ROS production in the presence of dithionite (as an electron donor) and copper ions. However, this effect was not reproduced when dithionite was replaced with Cyt C as an electron donor. Apparently, the other domains of p66shc are necessary either for interaction with Cyt C or for maintenance of the CH2-CB-domain in proper conformation.  As a result of the above-described findings, a refined model of p66-mediated apoptosis was suggested. The model assumes that under normal conditions the tetrameric form of p66 is reduced by mitochondrial antioxidant systems. Under stress conditions, however, antioxidant systems are not able to retain p66shc in their reduced dimeric state, so the oxidized tetrameric forms generate ROS locally, which triggers permeability-transition pore formation and apoptosis.  It is important to note that a direct connection between the ROS-generating and pro-apoptotic functions of p66shc does not necessarily exist. For example, a confirmation of this fact can be the lower level of ROS production in the case of pro-apoptotic tetrameric forms of p66shc in comparison to the dimer, which does not induce apoptosis. p66 as a redox sensor . Extensive experiments on the isolated CH2-CB-domain revealed a new interaction partner of p66shc \u2013 peroxiredoxine 1 (Prx1). Prx1 is a member of the peroxidase family. These enzymes regulate the redox balance in the cell. Prx1 can exist in the form of a dimer, a decamer (5 dimers) or a multimer. Prx1 is basically localized in cytosol, though recent proteomic studies have revealed its presence in the perimitochondrial compartment. Under normal conditions, Prx1 exists predominantly in dimeric form, which functions as peroxidase. When the cell is under stress, Prx1 undergoes oxidation and forms decamers that possess lower peroxidase activity. However, upon these transformations Prx1 acquires a chaperone function. Severe oxidative stress leads to the formation of the multimer, which is also devoid of peroxidase activity but can function as a chaperone [haperone -58.Interactions between the CH2-CB-domain of p66shc and Prx1 result in the destabilization of the decameric form of Prx1 and favor dimeric form transition. In turn, Prx1, in complex with p66shc, retains p66shc in dimeric form by means of a disulfide exchange between the two proteins. The resulting hybrid complex consists of dimeric p66shc, which generates ROS but has low pro-apoptotic activity, and dimeric Prx1, which functions as a peroxidase. Thus, under normal conditions dimeric Prx1 stabilizes the inactive state of p66shc and degrades ROS which are produced by p66shc. Under stress conditions, cystein residues of proteins are oxidized, resulting in disassembly of the complex: so, the uncomplexed p66shc can now participate in apoptosis induction . Therefo According to current views, p66shc is a pro-apoptotic protein which regulates oxidative stress and induces the mitochondrial apoptosis pathway by means of redox activity. Although the physiological role of p66shc has been extensively studied, little is known about the precise mechanism of action underlying its redox functions and participation in apoptosis induction. Further studies will endeavor to elucidate the precise mechanism of p66shc translocation into mitochondria and the localization of p66shc-dependent ROS production in the cell. Although p66shc phosphorylation at Ser36 is one of the indispensible steps of apoptosis, it was shown that the mitochondrial pool of p66 is not phosphorylated. This indicates that p66shc phosphorylated at Ser36 probably participates in pro-apoptotic events outside mitochondria.  P66shc is undoubtedly interesting in terms of the investigation of oxidative stress, apoptosis, and the aging related to it. It is important to note that, to estimate the involvement of p66shc in age-related disorders, it is necessary to better understand its role in cancer. A deeper understanding of the regulatory pathways and structural and mechanistic bases of p66shc redox activity can be important for developing pharmacological approaches to the treatment of age-related disorders."}
{"text": "Squamous cell carcinoma (SCC) of the oral cavity and pharynx represents the sixth most common form of malignancy worldwide. A significant proportion of these cases are related to human papillomavirus (HPV) infection. In general, HPV-associated SCC is more commonly nonkeratinizing and poorly differentiated, whereas non-HPV-associated SCC is typically keratinizing and moderately differentiated. Nevertheless, significant overlap in morphology is seen between these two forms of SCC. The purpose of this paper is to highlight the utility of ancillary studies in the establishment of HPV status of oropharyngeal SCC, including p16 immunohistochemistry, high-risk HPV in situ hybridization, polymerase chain reaction, and newer HPV detection modalities. Malignancy of the oral cavity and pharynx constitutes the sixth most common form of malignancy worldwide . In the More recently, human papilloma virus (HPV) infection has been implicated as a major etiologic agent for SCC development \u201312. HPV Evidence suggests that there is a causal association between HPV infection and SCC of the oropharynx, with molecular characteristics that distinguish it from non-HPV-related SCC, including alterations of p16 and c-myc expression \u201315. The Distinction between HPV- and non-HPV-related SCC is important in relation to clinical outcome. A study by Ang et al. found three-year survival rate of 82.4% for HPV positive tumors versus 57.1% for HPV negative tumors . A numbeDespite the importance of establishing the HPV status of SCC, no consensus has been reached on the optimal way to identify HPV-associated SCC . The focIHC staining for p16 is frequently used as a surrogate marker of HPV infection. It has the advantage of being easy to perform on formalin-fixed, paraffin-embedded (FFPE) tissue, and monoclonal antibodies against p16 are commercially available. HPV protein E7 binds to Rb, a negative regulator of p16 expression. Thus, HPV infection leads to increased nuclear p16 expression. As a result, IHC staining for p16 has a sensitivity approaching 100% for detecting HPV-associated SCC Figures  13, 15], 1513, 1However, p16 is overexpressed in a subset of tumors apparently lacking evidence for the presence of HPV DNA , 22, 23.It is possible, then, that there is a subset of non-HPV-associated tumors with histologic phenotype, molecular characteristics, and prognosis similar to HPV-associated SCC. The percentage of these p16 positive, HPV negative tumors varies significantly between the Lewis and Thavaraj studies. Whether this represents differences in sensitivities of the HPV tests used or true differences in HPV prevalence in different populations is not definitely clear.In any event, p16 positivity is a sensitive marker for nonkeratinizing, poorly differentiated yet prognostically favorable SCCs. While p16 may not be a specific marker of HPV infection, it can provide important prognostic information, and future therapies aimed at targeting this pathway of HPV tumorigenesis may well be effective in treating p16 positive, HPV negative SCC.ISH testing for HPV has the benefit of being the only molecular method allowing for direct identification of HPV in topographical relation to the pathologic lesion in tissue Figures  26]. Un. Un26]. Numerous technically validated HPV ISH assays are commercially available, most containing a cocktail of probes targeting multiple types of HPV. Though probes for individual types can be used if subtyping is clinically relevant, HPV subtype 16 is by far the most commonly found in oropharyngeal SCC , 10, 26.The most common technical difficulties experienced with ISH are background and an absence of signal . BackgroThe sensitivity of the assay is increased by signal enhancement techniques. One such technique is tyramide signal amplification, which has been shown to have a 10 to 100-fold increase on sensitivity . In thisNevertheless, the sensitivity of ISH appears to be less than that seen in PCR analysis, as a metaanalysis by Termine et al. found HPV in 39.9% of cases by PCR compared to 29.8% by ISH . HoweverPCR is a process in which a signal sequence of DNA or mRNA is amplified several orders of magnitude through several rounds of denaturing at high temperature (~95\u00b0C), annealing of complimentary oligonucleotide primers at a lower temperature (~55\u00b0C), and DNA replication at an intermediate temperature (~72\u00b0C) by a heat-resistant DNA polymerase. In theory, it can be used to detect as few as one copy of a DNA sequence, making it a highly sensitive detection assay .Material for PCR can be obtained from FFPE tissue by scraping tissue from a tissue block, digesting, centrifuging, and using the resultant supernatant for PCR studies . In addiPCR has the advantages of being highly sensitive for HPV detection, widely available, and cost effective. However, standard PCR techniques have a number of drawbacks. PCR has lower specificity than ISH and is technically cumbersome to perform . In contSeveral PCR amplification techniques are commercially available. These PCR screening assays commonly have primers designed to amplify a region of DNA that is present in multiple HPV types (most commonly within the highly conserved L1 gene) , 33. SinThe use of real-time PCR has also been assessed in HPV detection. Real-time PCR allows for quantification of target DNA via colorimetric markers that accumulate during PCR amplification, allowing a mechanism of identification of HPV DNA as well as an estimation of viral load , 23. ThiRecent studies have looked into the ability of PCR to distinguish between episomal and integrated HPV DNA. The HPV gene for E2 protein is a common break site prior to viral integration into the host genome . E2 protFinally, there are a number of commercially available assays for the detection of HPV by PCR by reverse transcriptase PCR. These kits target mRNA of the oncogenic E6 and E7 proteins. Thus, they have the advantage of detecting transcriptionally active HPV , 26. It Overall, PCR is a reliable, sensitive marker of HPV DNA. Nonetheless, ISH still has a number of advantages over PCR, including higher specificity, the ability to reliably distinguish episomal from integrated HPV DNA, and the ability to localize HPV to the area of neoplasia.Over the past two decades, a technique has been developed for combining PCR and ISH, referred to as PCR in situ hybridization (PISH) , 39, 40.Another hybridization technique, coined hybrid capture II (HC-II) has been developed and utilized in the detection of HPV. This is an FDA-approved method for HPV detection in cervical pap smears, and studies have demonstrated its utility in demonstrating the presence of HPV in lesions of the cervix and oropharynx , 43, 44.While IHC staining against p16 is frequently used as a surrogate marker of HPV, to date, IHC staining against specific HPV proteins has generally not been performed. Nevertheless, the development of IHC stains against the oncogenic E6 and E7 proteins would have a number of potential advantages over other HPV detection methods. It would have the ability to prove that HPV DNA is being expressed and directly demonstrate that important HPV oncogene proteins are present . DevelopA number of tests for the detection of HPV in oropharyngeal SCC are available, each possessing its own strengths and weaknesses. At the present time, IHC staining for p16 and PCR for HPV appear to be the most sensitive markers of HPV, while ISH confers the greatest specificity. For most clinical laboratories, the combination of a sensitive test  and a specific test  allows for the best potential to accurately establish the presence or absence of HPV in a given case of SCC. The study by Thavaraj et al. utilizes an algorithm originally developed by Weinberger et al. , 45 in wGiven the relative frequency of discordant p16 and HPV results  when using p16 in conjunction with a single HPV test, Thavaraj et al. suggest a three-tiered algorithm. Tumors are still categorized as p16 positive or negative by IHC. Those that are negative are then assessed for HPV by ISH and ultimately categorized accordingly as Class I (p16\u2212/HPV\u2212) or Class II (p16\u2212/HPV+). Likewise, tumors that are p16 positive are initially assessed for HPV by ISH, and if ISH is positive, the tumor is categorized as Class III (p16+/HPV+). However, if the tumor is negative by ISH, a confirmatory PCR test is performed. If the tumor is still negative by PCR, it is classified as Class IV (p16+/HPV\u2212). On the other hand, if the tumor is positive for HPV by PCR, the tumor is considered Class III (p16+/HPV+) despite the negative ISH result.In their study, this three-tiered approach resulted in just a small minority of cases falling under one of the discordant categories , with concordance of p16 and HPV results in 97% . This study suggests that with the three most commonly utilized tests, HPV status can be confidently determined in the vast majority of cases of oropharyngeal SCC.In the future, more recently applied molecular technologies, such as PISH and HC-II, may offer even more accurate diagnosis of HPV in the clinical laboratory, and development of IHC against important viral proteins may ultimately provide the optimal test for active HPV genomic transcription and translation in SCC. Nonetheless, using current ancillary tests in combination with clinical clues and morphology, such as the presence of nonkeratinizing, poorly differentiated lesions, HPV status can be accurately assessed in the vast majority of cases of oropharyngeal SCC."}
{"text": "Elastofibroma dorsi (ED) is a soft tissue tumor found in the subscapular region. The pathogenesis of ED is unclear, but may involve a regenerative or reactive hyperproliferation due to mechanical microtrauma. Magnetic resonance imaging (MRI) is preferred to diagnose ED and complete excision is curative. When bilateral, subscapular masses are identified in the elderly patient and MRI characteristics are typical, biopsy and excision can be avoided. Symptomatic EDs should be excised, and recurrence is rare. Three hundred and thirty cases of ED have been reported since 1980. Fourteen case series and 43 isolated case reports involved 263 women and 67 men (F:M ratio = 3.9:1), with a mean age of 62 years (range 6\u201394 years). Bilateral ED was present in 164 patients and unilateral ED in 157. The reported prevalence in the elderly population ranges from a minimum of 2% to a maximum of 24%. Elastofibroma dorsi (ED) is a benign, slow-growing fibroelastic tumor that was first described by Jarvi and Saxen in 1961. EDs typi3Three hundred and thirty cases of symptomatic ED have been reported in the last 30 years. A comprehensive review of all isolated reports and case series, published since 1980, affirms that EDs are diagnosed almost exclusively in persons over the age of 40, with sporadic reports of ED arising in children and adolescents as young as 6 years. and as hWe report our experience treating an illustrative case of ED and provide a discussion of the significance of physical exam and imaging in the diagnosis of these tumors in order to avoid surgery.A 55-year-old male machinist with no significant medical or surgical history presented with a right subscapular mass causing mild discomfort. He denied debilitating pain, recent trauma, or weight loss. Physical examination revealed a small, 4 cm mass, inferior to the right scapular spine. Laboratory findings were unremarkable. Computed tomography (CT) imaging of the chest with contrast revealed an 8\u00d72.1 cm soft tissue mass, similar in density to muscle, that was located superficial to the ribs and deep to the serratus anterior and lower subscapularis muscles. The lesion was slightly inhomogenous, non-enhancing and contained strands of fatty tissue. A much smaller (1\u00d71.5 cm) contralateral lesion was also noted at that time. Histopathology of samples obtained by core biopsy and a subsequent incisional biopsy revealed normal muscle and adipose tissue and no further intervention was performed.The patient returned 2 years later with increasing discomfort and limited range of motion on the right side. MRI revealed a mass below the body of the scapula, which was isointense to muscle and contained interspersed signals similar to fat, with moderate diffuse enhancement seen on post-gadolinium imaging . ED was The resected specimen consisted of a single, irregular, pink-red fragment of soft tissue that measured 10\u00d76\u00d72.5 cm and weighed 81 g. The cut surface varied from pink to white in color and was soft to rubbery in consistency with focal cystic degeneration . HistopaFirst described by Jarvi and Saxen in 1961, ED is a benign soft tissue tumor located in the subscapular and infrascapular region between the thoracic wall, serratus anterior, and lattisimus dorsi muscle.[et al. speculated that ED may be the result of a physiologic aging process, rather than abnormal elastogenesis or degeneration, based on the discovery of \u201cpre-elastofibroma changes\u201d in their autopsy series. These changes are defined by a weakly elastinophilic material that does not exhibit definite elastic tissue formation.[et al. have reported a familial predisposition for ED, noting that 32% of their cases occurred within a single family.[2The etiology of ED is not fully understood, but most believe that ED results from degeneration of collagen due to repeated mechanical friction between the chest wall and scapular tip, while others have suggested that it may be the cause of a reactive hyperproliferation of fibroblastic tissue rather than a degenerative process.7 Geibel ormation. Additionormation. Nagaminee family. To date, family.210et al. performed a CT study of 258 asymptomatic elderly patients and identified ED in 2% of the patients.[et al. (n = 170),[et al. found ED in 24% of females and 11% of males in people over 55 years of age in a large autopsy series involving 235 cases.[et al. reported that ED occurred in 13% and pre-elastofibroma changes in 81% among autopsies performed on 100 patients.[et al. reported incidental findings of ED in 12 out of 15 patients, nine of whom were diagnosed by CT/MR imaging for symptoms of ED on the contralateral side, and three were diagnosed during surgery for an unrelated thoracic disease.[Historically, ED has been regarded as rare tumors occurring predominantly in the elderly. Brandser n = 170), the liteAn understanding of the classic imaging features of ED is vital to make a presumptive diagnosis and eliminate the need for biopsy or unnecessary surgical resection. MR is the imaging modality of choice in diagnosing ED and typically demonstrates a well-defined, moderately inhomogenous mass with no associated soft tissue edema. On T1-weighted MRI, EDs are isointense with skeletal muscle, which explains how these tumors are often diagnostically overlooked. T1- and T2-weighted images both show interspersed linear and curvilinear, hyperintense areas representing fat.411 Fat s43473Macroscopically, elastofibromas appear as an irregular, poorly defined, unencapsulated, fibroelastic mass with firm, rubbery consistency. Cut surface reveals strands of white and yellow tissue representing adipose tissue intermingled with fibroelastic tissue in a \u201ccheckerboard\u201d pattern. Histolog34611et al. suggested an algorithm for the diagnosis and treatment of a subscapular elastofibromal mass,[Excision may be offered to symptomatic patients, with curative marginal resection proving to be sufficient and is preferred over wide or radical resection. Because mal mass, which comal mass,. In the All patients who underwent a marginal R0 resection (negative macroscopic and microscopic margins) were free of disease at follow-up and experienced no loss in range of motion.671020 Th67104et al.[In conclusion, our case report initially confirmed how CT imaging and general lack of awareness of this mass can lead to a missed diagnosis despite biopsy. Fortunately, on second evaluation by following the protocol set forth by Muratori et al. in which"}
{"text": "The relationship between these BSs and functional p53 response elements (REs) remains unknown, however. We sought to understand whether the p53 REs also reside in transposable elements and particularly in the most-abundant Alu repeats.The p53 tumor suppressor protein is involved in a complicated regulatory network, mediating expression of ~1000 human genes. Recent studies have shown that many p53 We have analyzed ~160 functional p53 REs identified so far and found that 24 of them occur in repeats. More than half of these repeat-associated REs reside in Alu elements. In addition, using a position weight matrix approach, we found ~400,000 potential p53 BSs in Alu elements genome-wide. Importantly, these putative BSs are located in the same regions of Alu repeats as the functional p53 REs - namely, in the vicinity of Boxes A/A' and B of the internal RNA polymerase III promoter. Earlier nucleosome-mapping experiments showed that the Boxes A/A' and B have a different chromatin environment, which is critical for the binding of p53 to DNA. Here, we compare the Alu-residing p53 sites with the corresponding Alu consensus sequences and conclude that the p53 sites likely evolved through two different mechanisms - the sites overlapping with the Boxes A/A' were generated by CG \u2192 TG mutations; the other sites apparently pre-existed in the progenitors of several Alu subfamilies, such as AluJo and AluSq. The binding affinity of p53 to the Alu-residing sites generally correlates with the age of Alu subfamilies, so that the strongest sites are embedded in the 'relatively young' Alu repeats.The primate-specific Alu repeats play an important role in shaping the p53 regulatory network in the context of chromatin. One of the selective factors responsible for the frequent occurrence of Alu repeats in introns may be related to the p53-mediated regulation of Alu transcription, which, in turn, influences expression of the host genes.This paper was reviewed by Igor B. Rogozin (nominated by Pavel A. Pevzner), Sandor Pongor, and I. King Jordan. In renctional . As a cop21 5'-site, most of them remain uncharacterized [Experimentally, the p53 BSs have been identified by two approaches: one is a traditional, single-gene approach, and the other is a genome-wide approach. The first approach  focuses cterized . One criWhile the p53-induced activation of transcription has been studied the most extensively, more than 50% of p53-modulated genes are repressed by p53, and this fraction may actually be as high as 80% . Howeverin silico the putative p53 BSs in the promoters of genes regulated by p53 [MAP4 and survivin, whose p53 REs contain a 3-bp spacer. Furthermore, deletion of this spacer  converts the survivin p53 site into a trans-activating element [To test the hypothesis that the spacer S between two p53 half-sites is related to the p53-mediated trans-activation and repression ,11, we ad by p53 . The sted by p53   This mome-wide , who argTo clarify these issues, we analyzed the two 'extreme' datasets, functional p53 REs and putative p53 BSs. The first set contains a relatively small number ~160) of rigorously defined p53 REs 0 of rigo,36 may bFinally, we examined the three 'hot spots' in the context of nucleosome positions in Alu repeats ,38 and fin vivo [in vivo [Analysis of the known p53 sites and their flanking genome fragments  revealed 24 functional sites that are located in repeats  are associated with the Alu family  have spacer S = 0, whereas for position 85 (Box B), the fraction of p53 sites with spacer S = 0 is less than 30% Figure , Table 1In summary, the functional p53 REs mapped to Alu elements can be separated into two groups, characterized by distinctive locations in Alu repeats, the density and mutation rate of CG dimers in the corresponding Alu regions and the spacer length S. This assessment based on a limited set of p53 REs is corroborated by a genome-wide analysis of putative p53 BSs in Alu elements described in the following sections.First, we searched for putative p53 BSs in masked and unmasked human genome (NCBI Build 36) to understand how these sites are distributed between the repeat and non-repeat regions. A scan of the unmasked genome found ~2 million p53 sites with the PWM-20 scores of 70% or higher (see Methods). Remarkably, distribution of the spacer lengths in these sites is highly non-uniform, with the peaks at S = 0, 3, 8 and 14 bp  of p53 BSs with different spacers Figure , while fNote that in those subfamilies where the predominant spacer is S = 3 or 8, the p53 sites are mostly located near position 85  are located in the inter-nucleosome linker regions, while the sites with spacer S = 0 (position 150) are exposed on nucleosomes. In both cases, the p53 sites are expected to be accessible for p53 binding.The number of p53 binding sites in human genome has been estimated based on experimental data. For example, 37 sites were identified in 1% of human genome (ENCODE) using ChIP assays, which can be extrapolated to ~3,700 sites genome-wide . AnalysiThe studies using various bioinformatics approaches found the larger numbers of p53 sites. In particular, the algorithm based on the p53 affinity for DNA binding sites predicted over 10,000 high-affinity sites and over 200,000 weaker ones with spacer S = 0-1 . A genomA significant number of the p53 BSs detected in genome-wide ChIP experiments  have beeBy analyzing the LTR consensus sequences of the six ERV subfamilies containing p53 sites and comparing them with the subfamilies without p53 sites, Haussler and his colleagues  concludeOur analysis of the functional p53 REs showed that in two out of three locations where the p53 REs are mapped on Alu repeats , the CG dimer occurs frequently in the Alu regions corresponding to the p53 core motif, implying that CG \u2192 TG mutations could give birth to p53 sites, as proposed earlier . By contin vivo; as a consequence, the N-terminal region of p53 would directly interact with TFIIIB, thereby precluding effective assembly of the pol III transcription machinery. Therefore, mutations in p53 leading to defects in DNA binding would result in pol III hyperactivity that is characteristic of many tumors [Previous studies showed that p53 can represses pol III transcription through interaction between the p53 N-terminal domain and TFIIIB, a factor containing TATA-binding protein -53. In ay tumors ,55. In py tumors .Moreover, we hypothesize that the p53 binding to Alu elements and repression of their transcription can change the expression of the host genes. This potential effect is related to the steric interaction between the pol II and pol III machineries, which, in turn, depends on the direction of Alu transcription (pol III) relative to the host gene transcription (pol II). If the two polymerases are moving in the opposite directions, one would expect that the head-on collision would slow down both of them, by analogy with the collision between the DNA replication apparatus and RNA polymerases .  . Accordiet al. , modern By analogy, here we speculate that evolution of Alu repeats  might be influenced by interactions with p53. We suggest that under selective pressure, the recent Alu elements mutated in such a way as to strengthen p53 binding and increase the level of the p53-induced repression of Alu transcription (see above). Keeping Alu expression below a certain level is critical both for the host and for transposable repeats, because excessive Alu transcription would be too exhaustive for the host cell .Following this scenario, invasion of a 'young' Alu element (with a low level of pol III transcription) in the vicinity of a gene promoter, would not disturb pol II transcription of the gene itself. In this way, the p53 BS with a short spacer (S = 0 or 3 bp) and relatively strong p53 binding, would be easily transposed close to the transcription start site, thereby creating (or modifying) the network of p53-dependent regulation of this gene.Finally, we wish to emphasize that the two groups of p53 REs and BSs (located close to the A/A' or B boxes in Alu repeats) are likely to have different chromatin environments, which are critical for p53 binding ,39,46. Tet al. [et al. [The largest collection of p53 REs published recently by Riley et al.  compriseet al. , whereas [et al. . Therefohttp://www.repeatmasker.org) and CENSOR [The selected p53 REs were extended on both sides by 250 bp flanking sequences extracted from human genome (NCBI Build 36). The REs and their flanks were aligned with repeats using RepeatMasker  at each position, see Table S2 in Additional File in vivo REs used for PWM-20 compared to 37 in vitro BSs used by Hoh et al. [Our PWM-20 differs from the published PWM-based approaches used for p53 BS prediction ,64 in seetc.) based on the notations provided by the database.Alu elements  were downloaded from AluGene Database . The eleThe authors declare that they have no competing interests.VBZ initiated and designed the study. FC and MVS created software and performed computations. VBZ and FC wrote the paper. All authors read and approved the final manuscript.The authors suggested that the primate-specific Alu repeats play an important role in shaping the p53 regulatory network in the context of chromatin. This conclusion is based on analysis of experimentally confirmed and computationally predicted p53 binding sites. The authors analyzed 160 functional p53 response elements and found that 24 of them occur in repeats. More than half of these repeat-associated response elements reside in Alu elements. This means that ~10% of experimentally verified p53 response elements are located in Alu elements. This number perfectly coincides with the fraction of Alu elements in the human genome which is ~11%. Thus there is no elevated frequency of p53 response elements in Alu elements. This may be due to a small number of experimentally confirmed p53 response elements. Unfortunately, the authors did not try to analyze recent genome-wide ChIP experiments ,8.Authors' response: Yes, there is no elevated frequency of p53 response elements (RE) in Alu elements, but the number of well established functional p53 REs, 160, is probably too small to make final conclusions. On the other hand, the number of predicted (putative) p53 sites has a higher density than the rest of genome (see below our response to the second fragment of I.B. Rogozin's review).As to the ChIP experiments, its analysis would not improve the above statistics, because the ChIP data provide information about the p53 binding sites (BS), both functional and non-functional.There are several reasons why we did not analyze the ChIP DNA fragments. First, it has been done by the Haussler and Vingron groups ,36. SecoIn addition, there is a principal limitation of the ChIP technology with regard to detecting all functional p53 sites in the repeating elements such as Alu. The ChIP-seq method is based on sequencing the short 'reads' at the ends of the p53-bound DNA fragments  with subsequent mapping of these 'reads' to human genome. If the unique mapping is impossible, the p53-bound DNA fragment is typically ignored. In the case of Alu repeats having a high degree of homology, the probability of occurrence of numerous identical 'reads' is rather high. As a consequence, many 'real' p53 BSs belonging to Alu repeats are missed. As far as the repeating elements are concerned, the p53 ChIP datasets will remain incomplete unless the ChIP-seq methodology is improved . Therefore, in our opinion, the detailed theoretical analysis of the p53 ChIP data would be much more productive one or two years from now.Using a position weight matrix approach, the authors found ~400,000 potential p53 binding sites in Alu elements genome-wide. Using the previous estimate (~10% of p53 RE are in Alu) we could easily calculate that the total number of potential binding sites in the human genome is ~ 4 million sites. I think that this is too generous estimate. Therefore, the vast majority of these potential binding sites are likely to be an over-prediction which is expected taking into account that the p53 consensus sequence is highly degenerate (see the Introduction). In addition, the cumulative work obtained from different laboratories suggests that p53 is not a stand-alone protein; rather, it participates in a complex network of proteins working in concert -68. TherAuthors' response: We agree that the above estimate of ~4 million p53 sites is too generous. In fact, we calculated the number of p53 BSs directly: \"A scan of the unmasked genome found ~2 million p53 sites with the PWM-20 scores of 70% or higher\" . This means that the p53 BSs are over-represented in Alu repeats . See also on page 8: \"the Alu elements have a (somewhat) higher 'density' of putative p53 sites compared to other repeats.\"In any case, there is no doubt that some of these potential elements are functional and they might influence the transcription of neighboring genes immediately after insertion of a new copy of Alu. Although we do not know the number of such case, this possibility poses an interesting question about how primates tolerated a load of p53 binding sites in their genomes. I think that some insertions are deleterious (likely to be removed by purifying selection) and some insertions of Alu together with p53 binding sites may be utilized. This point reverberates with the classical paper of Wojciech Makalowski  I will u\"Interspersed repetitive sequences are major components of eukaryotic genomes. ... they are often quoted as a selfish or junk DNA. Our view of the entire phenomenon of repetitive elements has to now be revised in the light of data on their biology and evolution.... I would like to argue that even if we cannot define the specific function of these elements, we still can show that they are not useless pieces of the genomes. The repetitive elements interact with the surrounding sequences and nearby genes. They may ... acquire specific cellular functions such as RNA transcription control or even become part of protein coding regions. Finally, they provide very efficient mechanism for genomic shuffling. As such, repetitive elements should be called genomic scrap yard rather than junk DNA.\"The p53 binding sites nicely fit this concept taking into account that the rate of turnover of these elements is known to be high . In geneTumor suppressor p53 is unique in the sense that it regulates over a thousand human genes. p53 is known to bind to a cognate site consisting of two decameric half sites separated by a spacer. Recently it has been found that many of the sites that bind p53 in vivo are within transposable elements. One should note that in vivo binding sites (BSs) are not necessarily functional p53 response elements (REs). The interesting study of Cui and associates asks the question whether or not REs also reside in transposable elements, particularly Alu repeats. The authors find that BSs are located in the same regions of Alu repeats as are REs, namely in the vicinity of A/A' and B boxes of the internal RNA polymerase III promoter. The authors conclude that Alu residing p53 sites may have evolved through a mechanism that is different from that of the corresponding Alu consensus sequences and suggest that the strongest binding sites are embedded in the young Alu repeats. My questions are:a) Can one estimate the background probability of occurrence of the motifs tested? In other words, can one assign a statistical significance to the findings?Authors' response: To evaluate the significance of our findings, compare the peaks in Figures http://studentsttest.com, we have p < 10-6 for both cases.For Figure b) At the first glance the periodicity of occurrence vs. spacer length on Figure Authors' response: The peaks of occurrence corresponding to spacer S = 0, 3, 8 and 14 bp are statistically significant (see above). The differences between these S values are 3, 5 and 6 bp. Indeed, 5 and 6 are close to 10.5/2. How can one interpret this?The Alu elements originated from the 7SL RNA, which is a part of the Signal Recognition Particle (SRP) . Therefoet al. report on an analysis of Alu element derived p53 binding sites in the human genome. Transposable element (TE) derived p53 binding sites, and Alu-derived sites in particular, have been treated at some length previously both by the group of Victor Zhurkin, who originally discovered that numerous p53 binding sites are found in TE sequences, and others such as the Haussler and Vingron groups [Feng Cui n groups ,36. So pAuthors' response: First of all, we are sincerely grateful to I. K. Jordan for the detailed and thoughtful review. The reasons why we did not analyze the ChIP DNA fragments are given in our response to I.B. Rogozin's review (see above).One of the most interesting findings in this study is based on results showing that there are two classes of p53 binding sites in Alus: a class that appears to be derived from methylation and deamination of CpG sites as previously reported by Vingron and colleagues  and a clAuthors' response: I.K. Jordan made an interesting point by linking our observation of the progressive decrease in the length spacer (in the p53 BSs) with the existence of two different mechanisms involved in generation of p53 sites in Alu elements.We disagree, however, with the reviewer' assessment: \"The sites derived from methylation and deamination map to the region of the element that is enriched for zero length spacers\" . Below, we show that:(1) not all sites with S = 0 originated through CpG mutations (in the Boxes A/A');(2) not all CpG mutations resulted in formation of the p53 sites with S = 0.(1) To compare different Alu subfamilies in terms of their enrichment for various length spacers, in addition to the absolute numbers presented in Table 2) Furthermore, mutations CG:CG-to-CA:TG are required not only for creation of the p53 sites with S = 0, but also for the sites with S = 8 and S = 14  of the Alu elements themselves.Authors' response: Thank you for the suggestion - we included more biologically-related speculations in the last section of Discussion.The authors propose a selective model whereby young Alus transpose near the promoter regions of host genes bearing p53 binding sites that disrupt the expression of the element, thereby mitigating some of its potential deleterious effects, and also can effect the regulation of the host gene. It is difficult to reconcile this model with the observation that younger Alus are enriched for the short spacer binding sites in the A-box region that appear to have evolved via CpG methylation and deamination. Thus, the intitial effect of many of these insertions would not be to bring that p53 binding site and so selection could not act at that point. This model is also inconsistent with the observation that young Alu elements are depleted near genes relative to older Alu elements.Authors' response: In our opinion, not only the young Alus bear p53 binding sites that could potentially disrupt the expression of the Alu element , but the old Alu elements may also play a similar role. As shown in Figure With respect to the analyses reported here, there are a few rather qualitative conclusions drawn from data that could be quantitatively and statistically analyzed in such a way as to provide more definitive results. The trends that the authors point to do seem to be there, but a more quantitative analysis could provide additional support for their conclusions. I provide a few suggestions to this end below.First, the method for comparing the p53 binding site spacer length distributions for repetitive and nonrepetitive DNA seems indirect. It appears as if the authors compared, 1) the entire genome including repeats and non-repeats . However, to comply with the Reviewer's objection, we substituted \"remarkably\" by \"importantly.\"On page 6, the authors state that \"promoter regions are enriched with Alu elements\" to underscore the potential of Alu-derived p53 binding sites to influence host gene regulation and cite Polak and Domany  in suppoAuthors' response: Thank you for the correction - indeed, these are the upstream regions of the TSS  that are enriched with Alu elements, not the promoters themselves, as we wrote. We changed the end of the Background accordingly.Supplementary Tables S1-S3.Click here for fileSupplementary Figure S1: Alignment of p53 functional REs with the consensus sequences of human repeats.Click here for fileSupplementary Figure S2: Alignment of the p53 functional REs and their flanks with the consensus sequences of Alu repeats.Click here for fileSupplementary Figure S4: Occurrence of putative p53 sites in the subfamilies FLAM-A (A), FLAM-C (B), AluJb (C), AluSq (D), AluSg (E), AluSp (F), AluSc (G), AluSg1 (H) and AluY (I).Click here for filein vivo binding sitesSupplementary Figure S3: PWM-20 score distributions for p53 REs and .Click here for file"}
{"text": "Gallbladder cancer is an infrequent neoplasia with noticeable geographical variations in its incidence around the world. In Chile, it is the main cause of death owing to cancer in women over 40\u2009years old, with mortality rates up to 16.5 per 100,000 cases. The prognosis is poor with few therapeutic options; in advanced cases there is only a 10% survival at 5\u2009years.Several studies mention the possible role of DNA methylation in gallbladder carcinogenesis. This epigenetic modification affects tumor suppressor genes involved in regulation pathways, cell cycle control, cell adhesion and extracellular matrix degradation, in a sequential and cumulative way. Determining DNA methylation patterns would allow them to be used as biomarkers for the early detection, diagnosis, prognosis and/or therapeutic selection in gallbladder cancer. Gallbladder cancer (GBC) is the most frequent malignant tumor of the biliary tract and the fifth most common cancer of the digestive tract. The presenting symptoms are vague, so diagnosis commonly occurs at an advanced stage. This late diagnosis combined with the fact that the gallbladder lacks a serosa result in a rather dismal prognosis -3. The hSeveral factors have been associated with the risk of developing GBC. Lithiasis is one of the main risk factors, presenting in 65% to 90% of cases of GBC ,3,6,7; tin situ and subsequently becomes invasive  through the covalent bond of a methyl group ,19. Non-Genes that show oncogenic properties, that is, that exhibit a methylated promoter region in normal cells, can become reactivated in cancer cells by the loss of this methylation, resulting in hypomethylation . In geneet al..-38.31-38et al. reported), RUNX3 %, APC , CDH13 %, P15INKpatterns . We alsoPSCA is downregulated in non-neoplastic gallbladder lesions such as cholesterolosis, cholecystolithiasis and cholecystitis . However, it was disclosed that the expression was decreased in more than 90% of cancers (40 out of 44) [PSCA enhancer [PSCA was recently reported to be upregulated [Recently it has been reported that egulated , which sAlterations in DNA methylation patterns are commonly found in all cancers, often with concomitant changes in gene expression. In GBC, molecular information is reduced; however, a high rate of methylation of some genes in GBC has been reported and associated with carcinogenesis of other tissues of the human digestive tract.CDH1), located on chromosome 16q22.1, is one of the most important tumor suppressor genes [CDH1 encodes for a transmembrane glycoprotein of 120\u2009kDa that intervenes in cell adhesion mediated by calcium. It is a component of the E-cadherin/beta-catenin complex, which is important for cellular polarity, normal tissue morphology and cellular differentiation [CDH1 belongs to a family of genes directly related to the processes of tumor invasion and cytoskeleton destabilization. CDH1 expression, it has been reported in less differentiated tumors, that generally have an unfavorable prognosis [CDH1 (77.8%) [CDH1 has been described to be hypermethylated in GBC  . CDH1 haCDH1 presented a high methylation frequency in poorly differentiated tumors and in cases with three or more positive nodes [CDH1 expression has been recorded as the lesion progresses, which may be due to methylation [In relation to the degree of tumor differentiation, it was observed that ve nodes . A signihylation . As far hylation ,39.CDH1 and the metastatic phenotype in GBC [Therefore, there is a significant correlation between the methylation of e in GBC , which he in GBC .CDH1 has been reported in two cell lines of biliary tract cancer (SNU-478 and SNU-1079), with silenced mRNA expression. After treatment with a demethylating agent (5-aza-2\u2032-deoxycytidine), CDH1 was successfully re-expressed [Hypermethylation of xpressed .CDKN2A-p16 is a tumor suppressor gene that encodes the protein p16, which is an inhibitor of cyclin-dependent kinase, involved in cell cycle regulation at Checkpoint G1. The loss of p16 expression is usually connected to homozygote deletion, loss of heterozygosity, mutations and methylation. A lower expression of p16 has been reported to increase the activity of D-type cyclin-dependent kinase activity, which translates into aberrant phosphorylation of the retinoblastoma gene, producing uncontrolled cell proliferation [p16 is frequently inactivated by methylation of the promoter and rarely by deletion or mutation [. This is a tendency that is also observed in GBC with a loss of expression of up to 62.5% [et al.[feration ,46. Lossferation . In hepamutation . Inactivmutation -69. Thisto 62.5% ,50. Taka% [et al. reported% [et al..et al. published the methylation status of five genes in advanced carcinoma of the gallbladder and their correlation with the immunohistochemical expression, demonstrating that CDH1, APC, FHIT and CDKN2A might be important in the carcinogenesis of gallbladder [CDKN2A is also one of the main mechanisms that induce the loss of p16 expression [et al. studied different alterations of p16 in 68 tumors in Chinese patients, finding that only 14.8% presented aberrant hypermethylation [In 2006, Roa lbladder . Hypermepression . Furtherhylation . Other ihylation . When thhylation .p16 and p14, it was noted that they presented a high methylation rate (between 40% and 60%) for carcinoma of the biliary duct, GBC and for primary sclerosing cholangitis. In normal tissue and choledocolithiasis, either no or an extremely low methylation rate was observed in the two genes [In the analysis of wo genes .p16  in cases of GBC means a better overall survival rate, and it is thus considered a significant prognostic factor [p16 was related to poorer survival [The absence of alterations in c factor . A similsurvival . This wasurvival .p53, which is commonly altered in numerous human cancers. Reprimo is a highly glycosylated protein located in the cytoplasm that induces cell cycle arrest at the G2 phase, inhibiting the activity of Cdc2 and cyclin B1 [Reprimo by methylation was initially confirmed together with other genes in pancreatic cancer [This is a candidate tumor suppressor gene regulating yclin B1 ,72. The c cancer . As a rec cancer .Hypermethylation of this gene is infrequent in normal tissue; however, methylation percentages of 32% and 27% have been described in the gastric epithelium and colorectal polyps, respectively . A low mUCHL1  is the only gene with a potential oncogenic role that has found to be hypomethylated in the promoter region in GBC [. The product of the gene is a peptide responsible for eliminating Ub from proteins that have it, and to thereby avoid its degradation by the proteasome. Proteins degraded by this mechanism actively participate in cell cycle control, for example, p53 and a variety of cyclins [In vivo analysis of UCHL1-deficient mice suggested that it functions as a regulator of apoptosis in neurons [n in GBC . Is locan in GBC . The pro cyclins . Ub, a p cyclins . In vivo neurons  and also neurons .PGP9.5 promoter is a reliable marker in GBC and that DNA hypomethylation might play a significant role in the re-expression of the gene in GBC [UCHL1 had previously been found in its promoter region in colorectal cancer, and lymph node metastasis was significantly associated with a lower frequency of methylation [The accumulation of Ub has been documented in several types of primary cancers . During e in GBC . In addihylation , poorer hylation .p16CDH1REPRIMODAPK-1 and SEMA3B, is high in GBC [DNA methylation is the epigenetic alteration most studied in the cancer cell. The number of genes with aberrant methylation in the human cancer cell is not known, but it is estimated that around 1% or 250 genes) of the human genome can be aberrantly methylated in a tumor cell . The useh in GBC ,50,51,88 genes ofp16 is frequently inactivated in a wide variety of human cancers by at least three distinct mechanisms: point mutation, small deletions of both p16 alleles, and methylation of CpG islands [p16 promoter in two of eight (25%) cholangiocarcinomas, and four of seven (57%) tumors analyzed by immunohistochemistry, demonstrated an absence of p16 nuclear staining in primary sclerosing cholangitis-associated cholangiocarcinoma [CDH1 are rare events [et al.[et al.[RUNX3 was more frequent in elderly patients [Helicobacter pylori infection [E-cadherin methylation is an early event in gastric carcinogenesis, and is initiated by H. pylori infection [et al.[et al.[et al.[The tumor suppressor gene  islands . Other sarcinoma . Also, aarcinoma . The exparcinoma  and genoarcinoma . In chole events . Downrege events ,92,93, we events ,43. Thiss [et al. found a l.[et al. a frequepatients  and Helinfection  can accenfection . Anothern [et al. and Kogal.[et al. utilizedl.[et al.. All resl.[et al., who useMSP in its classical format is non-quantitative and cannot distinguish between low and high levels of a methylated target sequence. By contrast, combining real-time PCR probes with MSP, as in MethyLight assay, can achieve a quantitative assessment of the level of DNA methylation of a targeted sequence . The effUCHL1 has been reported to be repressed by methylation in other cancers, such as primary head and neck squamous cell carcinoma [UCHL1 is a controversial molecule from an oncologic point of view. Hypomethylation of its promoter has been identified in a subset of human cancers, including GBC, presumably due to its intrinsic oncogenic properties or as a result of transformation. However, arcinoma  and coloarcinoma  and pancarcinoma . Particuarcinoma  and it iChile is considered a very high-risk area for GBC, and its mortality rate reached 16.5 out 100,000 women in 2007 . It is iTherefore, the study of gene methylation has at least five potential clinical applications: reactivating genes inactivated by methylation using demethylating drugs; identifying tumor cells in biological samples, allowing an early diagnosis since the change in methylation frequently precedes the appearance of advanced tumors; determining the methylation of individual genes or methylation profiles for groups of specific genes; being used as response markers to chemo- or hormone therapy; and allowing the transition between neoplastic and normal tissue to be determined in the surgical section margins according to the gene methylation profile ,100,101.Finally, the reversible nature of the epigenetic changes that occur in cancer must be mentioned as these have made epigenetic therapy possible as a treatment option. This therapy is based on reversing the epigenetic modifications that occur in tumor cells and re-establishing a normal epigenome. The possibility of reversing DNA methylation and reactivating the affected genes is an attractive option for a new therapeutic target in the treatment of cancer or preneoplastic lesions. However, the main problem with demethylating drugs is their poor specificity and they therefore cannot be used in the treatment of previously selected genes . NeverthThe authors declare that they have no competing of interests.PL designed the article and participated in writing all sections of the manuscript. PB revised the manuscript and contributed to content relating to methylation studies on the gallbladder cancer. OT contributed to content relating to conclusions and provided guidance for the structure. JCR provided guidance for the overall structure and content of the manuscript. All authors read and approved the final manuscript."}
{"text": "The recently identified class of microRNAs (miRs) provided a new insight into cancer research, since abnormalities of members of microRNAs family have been found in various types of cancer. However, the relationship between five miRNAs  and colorectal cancer remains unclear. In the present study, we examined expression of these miRNAs in 25 pair-matched colon cancer tissues and normal colon mucosa. The expression levels of miR146a, miR155, miR21, miR135a, and miR147b were quantified by real-time PCR. We found that miR21, miR146a, and miR135a were all expressed at higher levels in colon tumors. On the other hand, miR146a and miR147b expressions are significantly higher in left colon compared to right colon. These two miRs, especially miR146a, seemed to be markers for the left colon tumors. Moreover, significant proportional and inverse correlations were found between miR expressions in tumor and healthy tissue, and the correlations profiles were different depending on cancer localization. Taken together, these results lead us to suggest the presence of different mechanisms regulating miRs expression and consequently their target genes in left and right colon. So the pathway of colorectal carcinogenesis would be different according to the site of the tumor. Colorectal cancer (CRC) is the third most common malignancy in the world, accounting for more than 1.2 million cases and 608,700 deaths per year  such as A large number of recent studies have shown the utility of microRNAs as cancer-related biomarkers, supported by the finding that some microRNAs display altered expression profiles in cancers compared to normal tissues .MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, small, and noncoding RNAs of 19\u201325 nucleotides in length that repress protein translation through binding to target messenger RNAs . SubsequSeveral miRNAs have been identified in humans, but much remains to be understood about their precise cellular function and role in the development of diseases . MicroRNMicroRNA155 (miR155) gene located on chromosome 21q21 is involved in the process of carcinogenesis . Indeed MicroRNA21 (miR21) gene located on chromosome 17q23.2 is involved in inflammation, at least in part, by modulating cytokines responses . miR21 iMicroRNA146a (miR146a) gene is located on chromosome 5q34. A recent study provided evidence that miR146a acts as a modulator of the innate immune response and of the adaptive immune response as well. Additional studies showed that miR146a expression is upregulated in thyroid cancer and breast cancer in contrast to pancreatic cancer and gastric cancer in which the expression of this miR is downregulated . It is i stabilin 1 (STAB1) gene on 3p21 and in intron 5 of rhabdomyosarcoma 2-associated transcript (RMST) gene on 12q23. Some studies demonstrated elevated levels of miR135a in colorectal tumor cells [ adenomatous polyposis coli (APC) with a potent effect on Wnt pathway activity. These results suggest that upregulation of miR135a might be involved in CRC pathogenesis [MicroRNA135a (miR135a) is encoded in the human genome by two copies, which are located in the first intron of theor cells  and otheMicroRNA147b gene, which is located on chromosome 15q21.1, is involved in posttranscriptional regulation of gene expression by affecting both stability and translation of target mRNAs. Studies demonstrated the participation of mir147 in a negative feedback loop that is able to inhibit the proinflammatory response of macrophages to multiple TLR ligands . Few stuIn the current study, we aimed to investigate and compare the differential expression of the five miRNAs  in colon cancer (CC) tissue and normal colon mucosa and their relationships to the clinicopathological factors in Tunisian patients and this is in order to assess their involvement in colon carcinogenesis.Paired samples of colon cancer and adjacent normal mucosa in paraffin-embedded blocks came from 25 patients recruited from the Charles Nicolle Hospital of Tunis. They were classified on the bases of their histopathological profiles.Total RNA was extracted from tumor and corresponding normal tissue in paraffin-embedded blocks using the miRNeasy FFPE tissue kit (Qiagen) following the manufacturer's instructions. Total RNA was eluted in RNase-free water and stored at \u221220\u00b0C. The concentration and the quality of RNA were detected by spectrophotometer and by bioanalyser Agilent.\u2212\u0394\u0394CT method.Reverse transcription was performed using the miScript II reverse transcription Kit (Qiagen). Expression of miRNAs was measured using the miScript SYBR Green PCR Kit and miScript Primer Assays (Qiagen) according to the manufacturer's protocol. RNU6B-6 miScript Primer Assay was used as an endogenous control. For each sample, the standard control used is RNU6B-6 as endogenous control. RNU6B-6 corresponds to a small nucleolar RNA that was selected by a previous study from the best performing controls for miR quantification assays. Indeed, based upon its expression level, its relative abundance, and its stability, as determined by several tissues and cell lines analysis, RNU6B-6 is used as endogenous control snRNA chosen as candidate reference in several papers published in the literature on miR quantification , 24. miRt-test and by explorer test. All P values lower than 0.05 were considered statistically significant. Coefficient correlations were calculated using Spearman's rho test. Changes in miR expression between patient subgroups were determined using one-way ANOVA. Cases values with P < 0.05 were considered significantly different. Receiver operating characteristics (ROC) analysis was generated to tag expression of miR146a and 147b in colon tissue. The sensitivity and specificity of the optimum cut-off point were defined as those values that maximized the area under the ROC curve (AUC). All statistical tests were analyzed using SPSS software (version 20.0).For miR quantification performed by real-time PCR, we express our results relatively to endogenous RNA RNU6B-6; thus, it is possible to compare between samples. From this, we have compared the relative expression of each miR between groups that have been stratified according to epidemiological or clinical features. The different tests used for comparison are standard tests displayed by SPSS software and significance of the results is given by this software. The correlation between miR expression and colon cancer was assessed by paired samples The expression levels of five different miRNAs that were known to be deregulated in various cancers were quantified by real-time PCR analysis. We further established the CT values for these genes of interest, in colon tumor and normal mucosa.R which is presented as the n-fold change in gene expression allowed comparing the overall level of each miRNA. The value of R > 1 represented miRNAs overexpression in colon cancer relative to paired normal mucosa. The result reveals that miR21, miR135a, and miR146a are upregulated in colon cancer tissue compared to normal tissue (P = 0.011) and miR135a shows 8-fold overexpression in tumor tissue as compared to control tissue (P = 0.002), while miR146a overexpression is less high (4.02-fold) (P = 0.032). The relative expression ratio for miR155 suggests that it is also overexpressed in tumor tissue (2.84-fold) but statistical analysis did not carry out a significant difference in its expression between compared tissues (P = 0.087). The level of miR147b in colon cancer tissue compared to the healthy tissue tends towards downregulation (0.87-fold) (P = 0.793).In a first step, we analyzed the expression levels of miRNAs in tumor compared to normal mucosa . The rell tissue . Indeed,87-fold) . HoweverTo investigate the clinical significance of miR21, miR146a, miR135a, miR147b, and miR155 expression, the relationships between this expression and clinicopathological parameters of colon cancer patients were assessed. As shown in P = 0.012)  .Considering these results we have then evaluated the correlation between the expression of these miRs according to the tumor localization . As showIn these profiles the correlation between miR21 and miR155 is a constant feature. One has to notice that in healthy tissue correlation profiles in right and left colon are different indicating that regulation of gene expression is normally different according to left or right localization.In both healthy tissues, miR147b does not correlate with the other miRs studied.P = 0.008) and the dissociation of miR146a expression from the other miRs in left colon   .P = 0.003), miR155 (P = 0.04), miR135a (P = 0.02), and miR21 (P = 0.02) in normal tissue, indicating a significative change of miR146a expression in right colon tumor  and seemed to be in part determined in left tumor by that of miR135a in normal left colon (P = 0.03)  .According to this work, we considered miR135a, miR146a, miR21, and miR147b as potential markers in colon cancer and performed ROC curves in order to evaluate the pertinence of their use. We have scored the expression of miR135a, miR146a, and miR21 by curve ROC to confirm their association with colorectal cancer by measuring their degree of sensibility and specificity with respect to this type of cancer . MiR135aMoreover, it appears that miR146a could be considered as a marker of the left colon from a cut-off value of 14.76 with a sensitivity of 78.6% and a specificity of 73% and an AUC value of 0.79 .P = 0.04)   from a c = 0.04) .\u03b2-catenin pathways that are involved in pathogenesis and signaling pathway of CRC. Hence, miRs expression profile analysis enables us to predict prognosis, support diagnosis, and improve therapy responses in cancer [MicroRNAs are considered as potential molecular biomarkers for human malignancies. Indeed, they have an important role in differentiation, proliferation, and apoptosis. Both up- and downregulation of specific miRs have been described in CRC carcinogenesis . Connectn cancer , 25. Indn cancer . TherefoP = 0.011; P = 0.032; and P = 0.002, resp.). This result is in agreement with previous studies that have shown that miR21 is an oncogenic microRNA implicated as an inflammatory mediator and may promote inflammation-associated colon carcinogenesis [P > 0.05). Contrary to our results, miR155 is overexpressed in colorectal cancer and in various tumors, including breast, lung, and pancreatic cancers [In our study, we investigated expression profiles of five microRNAs  in tumor colon tissue compared to healthy adjacent tissue. We showed that miR21, miR146a, and miR135a are overexpressed in tumor tissue comparatively to control tissue  andinterferon regulatory factor 3 (IRF3). Induction of miR147 expression by TLR is capable of downregulating excessive inflammatory responses [On the other hand, we showed that miR147b is not significantly downregulated in tumor colon tissue compared to healthy tissue. On the contrary, some studies have reported significant downregulation of miR147b , 20. Othesponses .Taken together these data indicate that miR135a, miR146a, and miR21 could be considered as a marker of CRC tumor with good specificity and sensitivity.Further, we explored the relationship between expression of these five miRs and colon cancer stratified by tumor aspect, tumor differentiation, tumor histology, TNM stage, lymph node, and metastasis. We found no significant association or any significant differential expression of each miR according to clinical data. In fact, contrarily to our finding, other studies considered miR21 as biomarker for advanced stage of CRC, since its expression level was significantly higher in the late stages of the disease . In addiThe differences observed between this study and previous works could be related to environmental conditions as well as to factors involved in miRs expression and their dysregulation in carcinogenesis and CRC progression. These factors, such as way of life and intestine microorganisms, dramatically vary among populations.P = 0.012 and P = 0.04, resp.). This suggests that miR146a and at a lesser extent miR147b may be biomarkers for a subtype of colon cancer tissue. The other miRs are expressed in the same way in the right or left colon (P > 0.05). In fact, we showed a great difference between profiles of miRs expression correlation. The difference between expression levels of each miR and the correlation coefficient between miR expressions could be due to the mechanisms of regulation related either to genetic factors or to epigenetic factors or both. One has to notice that in normal right and left colon, miRs expression profile is different leading to the suggestion that cells at the two locations do not have the same gene expression profile. Moreover, in the past two decades, a growing amount of data has been reported suggesting that carcinomas of the right and left colon should be considered as different tumor entities. In fact, right- and left-sided lesions exhibit different genetic, biological, and demographical characteristics and risk factors, suggesting that the carcinogenetic mechanism and progression of colon cancer lesions may differ with tumor location [According to tumor location, we found that miR146a and miR147b are both more expressed in left colon tumors than in right colon tumors (location , 38. Thelocation \u201341. On tlocation . A high location . In the location . At the location . Hence, location , 50. All\u03b21/Smad signal transduction pathway [On the other hand, from miRs dysregulation, it can be predicted which targets genes are involved in the carcinogenesis pathway. Few studies have focused on correlation between microRNA and targets. Zhong et al. confirmed gene Smad4 as predictive target of miR146a and showed downregulation of miR146a associated with upregulation of Smad4 at protein level. These data suggested that miR146a might influence proliferation through TGF- pathway . Shibuya pathway .Our study shows upregulation of miR21, miR135a, and miR146a in colon cancer tissue compared to healthy colon tissue. We showed also differences between correlations of miR expression in right- and left-sided colon cancers suggesting that they are differently regulated and that there are different mechanisms of carcinogenesis between the two sites of the colon. Particularly interesting is the miR146a; its expression and role are unclear in several cancers and especially in colon cancer. In addition, overexpression of this miR appears to be specific to the side of colon tissue.Further studies will be necessary to understand the biological role of these miRNAs in colon cancer in order to use them as potential targets for cancer prognosis and therapy."}
{"text": "We report a study that examined the existence of a cognitive developmentalparadox in the counterfactual evaluation of decision-making outcomes. Accordingto this paradox adolescents and young adults could be able to applycounterfactual reasoning and, yet, their counterfactual evaluation of outcomescould be biased in a salient socio-emotional context. To this aim, we analyzedthe impact of health and social feedback on the counterfactual evaluation ofoutcomes in a laboratory decision-making task involving short narratives withthe presence of peers. Forty risky , fortyneutral decisions , and emotions feltfollowing positive or negative outcomes were examined in 256 early, mid- andlate adolescents, and young adults, evenly distributed. Results showed thatemotional ratings to negative outcomes (regret and disappointment) but not topositive outcomes (relief and elation) were attenuated when feedback wasprovided. Evidence of development of cognitive decision-making capacities didalso exist, as the capacity to perform faster emotional ratings and todifferentially allocate more resources to the elaboration of emotional ratingswhen no feedback information was available increased with age. Overall, weinterpret these findings as challenging the traditional cognitive developmentalassumption that development necessarily proceeds from lesser to greatercapacities, reflecting the impact of socio-emotional processes that could biasthe counterfactual evaluation of social decision-making outcomes.  There is now a broad acknowledgement that counterfactual evaluation of outcomes playsa role in everyday decision making. Upon making the decision and observing theoutcomes, people are able to process not only what actually occurred but also analternative course of events that might have occurred if a different option had beenchosen. This complex evaluation requires the cognitive capacity to engage incounterfactual thinking, which is usually accompanied by emotions . Some stOther studies have also characterized regret and disappointment by differentialagency attribution: personal/controlled agency for regret and relief,external/uncontrolled agency for disappointment and elation . AccordiThe present study takes this second approach to the study of counterfactual emotionsin risk decision-making situations. We examined the adolescents\u2019 and youngadults\u2019 decisions involving controlled and uncontrolled events and theircounterfactual evaluation of their negative and positive outcomes to induce therespective emotions of regret, relief, disappointment, and elation. Adolescence is aperiod of increasing risk-taking behavior, including practicing dangerous sports,drinking alcohol, engaging in unsafe experimentation with addictive substances,among others . HoweverThe findings of this study may help to demonstrate the possible existence of acognitive developmental paradox not only in the decision-making process but also inthe realm of the counterfactual evaluation of outcomes. Traditional developmentaltheory presupposes that with age cognitive development proceeds from lesser togreater sophistication, and that increased cognitive skill should decrease thelikelihood of participation in risks . The cogWhat happens with regard to the counterfactual evaluation of outcomes in riskdecisions? Is there a cognitive developmental paradox, too? The studies on thedevelopmental progression of counterfactual reasoning from childhood to adulthoodhave shown that although five- to seven-year-old children are able to experienceregret and relief , 2012, tThere are reasons to suspect, however, that a cognitive developmental paradox couldalso exist in the counterfactual evaluation of outcomes. According to thedual-processes account , adolescThe existence of a cognitive developmental paradox and its possible explanationaccording to the dual-process proposal has not been tested in the realm ofcounterfactual evaluations of decision-making outcomes in social situations. Theexception is one recent study examining the ability to experience regret and reliefin children, adolescents, and young adults who performed a gambling task in asocio-emotional context of competition, in which they were informed that theiroutcome would be compared with that of a competitor . ResultsThe present study uses analogues to real-life social decision-making by means of thesocial context decision-making task SCDT, . This taBoth the risky and the neutral trials involved the same sequence of eventspresented on the screen as illustrated in 1) A second-person scenario describing \u201cyou\u201d as accompanied by aclose friend;2) the two alternative options for the decision-making task in thatscenario;3) the outcome of the choice selected, either positive or negative;extremely bad) to +5 (extremely good), placedat the bottom of the screen; and4) the emotional rating scale, where participants had to indicate \u201chow doyou feel about what just happened?\u201d using a linear scale from -5(5) the feedback information , to inform participants about thegains and losses in health and peer popularity depending on the choice made and thenegative or positive consequence received.The outcomes presented were pre-set by the experimenter following a table ofcontingencies . First, Our main goal was to investigate whether providing feedback on health status and peerpopularity modulates participants\u2019 performance on the counterfactualevaluation of the outcomes . We expected lower emotional ratings when this feedback isprovided, compared to the no feedback condition. When presenting feedback, thenegative peers\u2019 reactions (decrease in popularity) were highlighted, whichmade more salient the socio-emotional context leading to bias in the evaluation ofoutcomes. Specifically, the feeling of regret resulting from risky choices andnegative outcomes would be lessened in the feedback condition. The reason is that inour task risky choices with negative outcomes, though involving health dangers, wereassociated to gains in peer popularity. This would not be the case for the feelingof relief resulting from risky choices with positive outcomes both in health andpopularity. To support our expectation, the feeling of regret was also attenuated ina gambling task by providing a socio-emotional context of competition with peers. We alsoPrevious studies on gender effects reported that adolescent women are more prone thanmen to perceive situations as risky . In factAltogether, the results of the present study could help to demonstrate the existenceof a cognitive developmental paradox in the counterfactual evaluation ofdecision-making outcomes to the extent that the presence of feedback with health andsocially relevant information could affect this evaluation process, as well as thepossible impact of age, and gender effects.MAge = 13.5 years, SD = 0.5; 64mid-adolescents (MA), aged 15-16, 32 female and 32 male,MAge = 15.6 years, SD = 0.5; 64late adolescents (LA), aged 17-18 years, 32 female and 32 male,MAge = 17.50 years, SD = .50, fromone public high school and 64 young adults (YA), aged 19-20, 32 female and 32 male,MAge = 19.5 years, SD = 0.5, from apublic university and one public technical school. After explaining the aim of thestudy to the teaching staff of each academic center and receiving the permissionfrom the officials, students volunteered to participate. Written parental consentwas obtained for children and adolescents prior to the assessment session. Writtenconsent was also obtained for adult participants, who also volunteered afterreceiving information about the research. The procedure was approved by theCommittee for Research Ethics and Animal Welfare at the University of La Laguna.A total of 256 volunteers participated in the study, belonging to four age groups: 64early adolescents (EA), aged 13-14, 32 female and 32 male,very negative)to +5 (very positive). The length of the sentences in the scenarioswas matched in the number of words and unfamiliar words were avoided in all thescenarios.The study used a social context decision-making task (SCDT) involving two types ofverbal materials: forty risk situations and 40 neutral situations . Pilot sExample of a risk situation:\u201cYou are in a disco with your close friend. In the toilet you and your friendmeet a guy who offers you cocaine\u201d.Decision: 1) \u201cYou buy it\u201d (risky choice), 2) \u201cYou tell him thatyou are not interested\u201d (safe choice).Outcomes (risky choice): 1) Negative: \u201cYou got very sick and had to go tothe hospital\u201d, or 2) Positive: \u201cYou had a big \u2019high\u2019 andfelt great\u201dOutcomes (safe choice): Positive: \u201cYou enjoyed dancing with yourfriends\u201d.Example of a neutral situation:\u201cYou are in a restaurant with your friend checking the menu forlunch\u201d.Decision: 1) \u201cYou decided to get a hamburger\u201d, 2) \u201cYou decidedto get a hotdog\u201d.Outcomes: 1) Negative: \u201cThe mayonnaise was spoiled and you got sick and had togo to the hospital\u201d; 2) Positive: \u201cYou enjoyed the meal as it was verygood\u201d.Participants received the scenarios of the risk and neutral situations auditorily andthe choices and outcomes in written format. The presentation of each piece ofinformation was self-paced, allowing for the recording of chronometric data inaddition to the behavioral data. The 80 trials were separated by an inter-trial interval of 5 s, and preceded by a 5-trial practicephase. The stimulus presentation was controlled by means of Cogent 2000, a MATLABToolbox for presenting stimuli and recording responses with precise timing.The task was administered individually to the participants in a quiet room at theirSecondary School. They were asked to imagine themselves (\u201cimagingyou\u201d) as vividly as possible in each situation accompanied with a closefriend and choose between the two alternative actions. They were informed that theirdecisions would have positive or negative outcomes with more or less impact on theirhealth status and their popularity among friends. Half of the participants, randomlyselected from the total sample, were submitted to the feedback condition beinginformed at the end of every trial (by means of bars diagrams) of the specific gainsand losses obtained in peer popularity and health see . Every 1With respect to the procedure, once participants entered a quiet room at the schoolhalf of them completed the battery of self-report assessment measures first  and then, individually, the SCDT inanother room; the other half followed a reversed order (first SCDT and thenquestionnaires).p2 )were calculated . T-test and post hocBonferroni corrected comparisons were used when necessary. Data points lying > 3SD from the grand mean of the dependent variables involvingtime measures in each analysis were considered outliers and were excluded from thatanalysis (2% of the data as average).A mixed factorial design was used with age (four groups) or gender (two groups) andfeedback (present/absent) as between-participant factors, and type of choices and outcome valence (negative and positive) as within-participantfactors. The dependent variables were the emotional rating, rating time, andobservation time of feedback (just in the feedback condition). Rating time is anindex of the cognitive cost allocated to the performing of the emotional rating.Observation time is the time spent watching the feedback information, being anindication of the cognitive effort required to process this information. Fouremotional conditions were analyzed resulting from the combination of type of choicesand valence of outcomes: (a) risky choice and negative outcomes (regret); (b) riskychoice and positive outcomes (relief); (c) neutral choice and negative outcomes(disappointment); (d) neutral choice and positive outcomes (elation). The emotionalcondition resulting from choosing the safe option was not included in the analysesas it always involved positive consequences and could not be crossed factoriallywith the other emotional conditions. In all the cases analyses of variance (ANOVAs)were used and effect sizes , which would suggest that participants experienced regret and disappointmentfor negative emotion scores and relief or elation for positive emotion scores.Means and standard deviations of emotional ratings after reading the consequences areshown in F = 40.4, p = .001,\u03b7p2 = .164 (on average 2.0 and 2.9 respectively).There was a main effect of type of choice, F = 69.85,p = .001, \u03b7p2 = .255, showing thatthe emotional ratings were higher in neutral choices than in risky choices. Therewas also a main effect of outcome valence on emotional ratings,F = 1191, p = .001,\u03b7p2 = .854), showing higher emotional ratings fornegative outcomes (regret and disappointment) than for positive outcomes (relief andelation). However, there was a significant interaction of feedback by outcomevalence, F = 45.88, p = .001,\u03b7p2 =.187  and no feedback conditions . By contrast, emotional ratings for negative outcomes (regret anddisappointment) were significantly lower under feedback conditions than under no feedbackconditions . Significant ageeffects were not observed.Overall, emotional ratings were lower in the feedback condition than in the nofeedback condition, p2 =.187 . Thus, eF = 5.26, p = .023,\u03b7p2 = .025. Women reported higher emotional ratingsthan men when they were told the negative outcome of their decision (regret anddisappointment), whereas gender differences were not significant for positiveoutcomes (relief and elation).There was a significant gender effect according to the outcome valence on emotionalratings, F = 7.49, p = .050,\u03b7p2 = .020. There was a main effect of outcomevalence, F = 19.1, p = .001,\u03b7p2 = .087, indicating that emotional ratings fornegative outcomes took more time than those for positive outcomes. No interactioneffects with feedback were observed.Means and standard deviations of emotional rating times after reading theconsequences are shown in F = 3.52, p = .016,\u03b7p2 = .087 , with only the difference between earlyadolescents and young adults being reliable, p < .05, all othercomparisons p > .10 . However, sensitivity ofrating times to feedback conditions also changed with age, F = 3.27, p = .032, \u03b7p2 = .097,(p < .001) and young adults (p <.05).Age and gender differences were observed in rating times according to feedbackconditions. Overall, rating times significantly decreased with age, = .097,. OverallF = 4.37, p = .038,\u03b7p2 = .021. Simple effects showed thatwomen\u2019s rating times in risky choices (regret and relief) were significantlyshorter in the feedback condition than in the no feedback conditions(p < .01). Men\u2019s rating times in neutral choices(disappointment and elation) were significantly shorter in the feedback conditionthan in the no feedback condition (p < .05).There was an interaction of gender by feedback and type of choice,F = 81.67, p = .001,\u03b7p2 = .415. and after receiving negative outcomes as compared to positive outcomes , F =4.97, p = .029, \u03b7p2 = .028. Botheffects were qualified by a type of choice \u00d7 outcome valance interaction,F = 13.24, p =.001,\u03b7p2 = .103, showing that the above difference wassignificant in the risky choices  butnot in the neutral choices .The last set of analyses was performed in the feedback condition only, since thedependent variable was the observation time of feedback information see . ParticiF = 3.09,p = .030, \u03b7p2 = .075, with thedifference being reliable between early adolescence and mid-adolescence(p = .024), late adolescence (p = .007) andyoung adults (p = .024), .Overall, the observation times decreased with age, This study examined the existence of a cognitive paradox in the counterfactualevaluation of risk decision making by means of exploring the impact of gains andlosses in health status and peer popularity, as well as age and gender effects. Asexpected, the presence of feedback in risk situations determines an attenuation ofthe participants\u2019 emotional experience derived from outcomes as compared tothe no feedback conditions, as shown by the lower emotional ratings, and shorterrating times in the former condition. No effect was obtained from the manipulationof feedback in the neutral situations, as expected. Moreover, what seems to bespecifically affected in risk situations are the emotions of regret anddisappointment which were attenuated in the feedback condition as compared to the nofeedback condition, whereas emotions linked to positive outcomes (relief andelation) were not affected by the presence or absence of feedback. This isremarkable, since overall the processing of negative outcomes demands more effortand provokes higher emotional arousal than the processing of positive outcomes, assuggested by higher emotion ratings, and longer rating times. The impact of feedbackon risky choices is not likely to be due to the instructions received at thebeginning of the task, since in both the feedback and no feedback versionsparticipants were informed that their decisions would have positive or negativeconsequences with more or less impact on their health status and their popularityamong friends. Also it is not likely to be due to a game-like effect according towhich participants tend to disregard the task information waiting for the finalfeedback, since the emotional attenuation is confined to the negative consequencesbut not to the positive ones.As expected, participants evaluating the outcomes in the feedback condition were lesssensitive to regret feelings as compared to relief feelings , both deUnexpectedly, participants were also less sensitive to disappointment feelings underfeedback conditions, as compared to elation, which suggests that the surprise effectprovoked by non-controlled negative circumstances  is also Emotional ratings to outcome information did not vary across age groups, in line withprevious results indicating that the children\u2019s ability to reasoncounterfactually is already in place after 12 years of age . In factGender effects on emotional ratings showed that women experienced more emotionalintensity than men when confronted with negative outcomes (regret anddisappointment) but not when facing positive outcomes, with no feedback effects.That means that overall women become more emotionally activated than men not onlywhen faced with the prospect of receiving negative results but also when actuallyexperiencing harmful consequences . HoweverIn keeping with the ecological validity of the task we are aware of two possiblelimitations of the study. First, feedback information differs under risk  and neutral  situations.Introduction of an arbitrary weight towards gains and losses in peer popularitydepending on the neutral choices, instead of a \u201czero\u201d score, couldhave affected results in unknown ways. However, we managed to perform the analysesand to draw the main conclusions from the comparisons performed within each type ofchoice . Second, it would be interesting to have included a measureof executive functioning to support our interpretation of decreasing times with agein the counterfactual evaluation of outcomes. Third, arguably using real-lifescenarios would have entailed some misunderstandings in the interpretation of riskand neutral situations. To keep this possibility at minimum, we have performed pilotstudies to elaborate verbal material that could be comparable across ages andgenders. We think that simulation of real-life situations is worthwhile to increasethe participants\u2019 chances of visualizing the course of actions and foreseeingtheir consequences, and to facilitate the participants\u2019 actual engagement incounterfactual-related emotional states.In conclusion, this study demonstrated that the cognitive developmental paradox alsoexisted when considering the counterfactual evaluation of outcomes in a salientsocio-emotional context. Evidence of development of cognitive decision-makingcapacities does exist, as young adults were more capable of performing fasteremotional ratings than early adolescents. Moreover, late adolescents and youngadults were able to differentially allocate more cognitive resources to thecounterfactual evaluation than younger participants in situations when no feedbackinformation is available. However, the paradox exists since across the age groupsenhancing the health and the socially relevant consequences of the choices by meansof feedback negatively affected the counterfactual evaluation in risk situations byattenuating the emotional sensitivity to the outcomes of the choices, especially thenegative ones. This could be potentially damaging since there would be less chancesto anticipate regret or to avoid the previously chosen option in the future, twocognitive capacities that are already present in older children . Altoget"}
{"text": "The p53-related gene p63 is required for epithelial cell establishment and its expression is often altered in tumor cells. Great strides have been made in understanding the pathways and mechanisms that regulate p63 levels, such as the Wnt, Hedgehog, Notch, and EGFR pathways. We discuss here the multiple signaling pathways that control p63 expression as well as transcription factors and post-transcriptional mechanisms that regulate p63 levels. While a unified picture has not emerged, it is clear that the fine-tuning of p63 has evolved to carefully control epithelial cell differentiation and fate. At first glance, the tumor suppressor p53 and its family member p63 seem quite similar in function and exhibit a high degree of evolutionary conservation. In particular, the DNA-binding domains are about 60% identical at the amino acid level; however, the adjacent domains and C-termini diverge drastically . While ip63 is also unlike its family member p53 in that it is rarely mutated in human cancers. Instead, mutations in p63 lead to disorders with ectodermal dysplasia such as ankyloblepharon-ectodermal dysplasia-clefting (AEC)/Hay\u2013Wells syndrome, which can include symptoms like cleft lip/palate and skin erosions \u20139.Waf1/Cip1 signaling, indicating that these domains can modulate target gene specificity , and the other set lacking the N-terminal transactivation domain (\u0394N). While the \u0394N form can be dominant negative to the TA isoforms , the \u0394Npcificity , 12.\u2212/\u2212 mice were found to have severe limb and epithelial defects, including partial or missing epithelial stratification, and truncated forelimbs  , 31. IndSo how does p63 impact cancer formation? The last decade has seen a preponderance of direct targets unearthed, including adhesion-related \u03b24 integrin, the tissue integrity factor Perp, the Notch ligands Jagged1 and Jagged2, keratins 5 and 14, and EGF receptor , 37\u201341. p63 is also known to regulate a diverse set of microRNAs. A prominent target is miR-205, a repressor of epithelial\u2013mesenchymal transition (EMT) and metastasis in bladder and pancreatic cancers , 47, 48.A data mining approach also identified p63 and the p53-related p73 gene as key regulators of microRNAs differentially expressed in ovarian carcinomas, including miR-200a, miR-200b, and miR-429 . For morTaken together, p63, like p53 and p73, can regulate a host of processes, some of which are known regulators for or against tumor growth. As suggested by the opposite expression of p63 in different tumor types, the context of the cell type appears to be critical to which p63 targets have the dominant effects in each cell. Whether targets are differentially expressed or have different activities in different cell types needs to be investigated further.As \u0394Np63 is required for the formation of stratified epithelial layers and is the primary isoform expressed in the basal layer of epithelial tissues, it is subject to multiple modes of tissue-specific regulation , 14. As activation of p63 in fibroblasts  are associated with craniofacial abnormalities like cleft lip and/or palate . Both IRR  can also lead to craniofacial disorders such as cleft lip and Crouzon\u2019s syndrome , 88. TheInterestingly, FGFR2 can induce expression of the epithelial-specific transcription factor Elf5, and deletion of Elf5 causes altered expression of \u0394Np63 in the luminal compartment of mouse mammary tissue \u201394. ThisThe tyrosine kinase receptor EGFR has also been found to induce \u0394Np63 expression. In one case, this was through phosphatidylinositol-3-kinase (PI3K) signaling in keratinocytes , while iEpithelial cells can undergo an EMT during development and during carcinogenesis, progressing to a more invasive and metastatic phenotype. This differentiation is thought to allow the cancerous cells greater motility and increased metastatic potential [see reviews by Thiery  and KangOther transcription factors involved in control of EMT are Ovol1 and Ovol2 , 105. ThWhile we have mentioned a number of transcription factors as regulators of \u0394Np63, a clear picture has yet to emerge on which factors are critical direct regulators of \u0394Np63 and through which sequence elements they act near the \u0394Np63 gene.It is possible that multiple pathways regulate p63 through the C/EBP family of transcription factors, as they have been repeatedly found to regulate p63. C/EBP\u03b4 was found to bind to multiple regions of the \u0394Np63 gene in human keratinocytes , 107. AnOther transcription factors have also been found to regulate the p63 gene. An OCT4 binding site within the TAp63 promoter activates its expression, suggesting its involvement in stem cell regulation . Anotherp63 positively activates its own expression through binding to the C38 and C40 intronic enhancers as well as to its own proximal promoter , 113. OvInitially, p63 expression was found to be suppressed by stresses, such as UV irradiation, that stimulate p53 expression \u2013116. Binp63 levels are also regulated by miRNA, ubiquitin-dependent proteasomal degradation, and protein phosphorylation. Notably, miR-203 can repress p63 expression in supra-basal epithelial cells, contributing to definition of the border between progenitor and differentiated epithelial cells , 119. InOther miRNAs have also been found to regulate p63 expression. miR-92 targets \u0394Np63\u03b1 and \u03b2 in the HaCaT keratinocyte cell line and in myeloid cells, respectively, and miR-302 suppressed p63 expression in germ cells , 122. Thp63 protein stability is also regulated by the ubiquitin\u2013proteasome system, adding another layer of regulation . One exaWhile p53 is stabilized by DNA damaging agents, such as UV irradiation, \u0394Np63 is degraded , 132. TwNF-\u03baB can also activate the TAp63 promoter, suggesting that a shift to the TAp63 form could also underlie the DNA damage response . Again, An alternative mechanism for \u0394Np63 degradation as part of the DNA damage response is phosphorylation on threonine 397 by the protein kinase HIPK2 . HIPK2 hp63 has been termed as a master regulator of epithelial cells, and it is often suppressed in order for these cells to differentiate , 142. WeIt will also be important to understand how modulation of p63 levels affects cancer formation. The combination of heterozygous p63 and p53 genotypes in mice yielded conflicting results, giving either greater or reduced tumor burdens , 144. AdOther open questions concern p63 promoter usage and splicing \u2013 what factors determine the balance of usage of the TA and \u0394N promoters, and what governs the presence of different 3\u2032 splicing isoforms? How does the balance of these 3\u2032 isoforms lead to differences in development or oncogenesis? Finally, can the signaling pathways that control p63 levels be controlled to provide a therapeutic benefit in specific cancers? We can hope that the following years will bring a greater understanding of this master regulator of epithelial biology.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. The Guest Associate Editor Wen Zhou declares that, despite being affiliated to the same institution as authors Ron Prywes and Kathryn Yoh, the review process was handled objectively and no conflict of interest exists."}
{"text": "Spontaneous neural activity in the auditory nerve fibers and in auditory cortex in healthy animals is discussed with respect to the question: Is spontaneous activity noise or information carrier? The studies reviewed suggest strongly that spontaneous activity is a carrier of information. Subsequently, I review the numerous findings in the impaired auditory system, particularly with reference to noise trauma and tinnitus. Here the common assumption is that tinnitus reflects increased noise in the auditory system that among others affects temporal processing and interferes with the gap-startle reflex, which is frequently used as a behavioral assay for tinnitus. It is, however, more likely that the increased spontaneous activity in tinnitus, firing rate as well as neural synchrony, carries information that shapes the activity of downstream structures, including non-auditory ones, and leading to the tinnitus percept. The main drivers of that process are bursting and synchronous firing, which facilitates transfer of activity across synapses, and allows formation of auditory objects, such as tinnitus. Spontaneous neural activity is very often considered as neural noise that sets limits on sensory performance. This neural noise idea has been the basis for the optimal processor model in psychoacoustics that typically worked on activity in auditory nerve fibers Green,  and trieThe neural noise hypothesis for spontaneous activity in auditory nerve fibers was apparently boosted by Kiang et al.\u2019s  extensivA decade later, Liberman  raised cJavel et al.  showed fn = 41) having SFR <1 sp/s but in a few others (n = 2) >35 sp/s, the remaining 60 cells had SFRs between 1\u201335 sp/s. Eggermont and colleagues performed studies on spontaneous activity in AI of ketamine-anesthetized cats. In a series of studies  or AI . Huang et al. .Anesthesia will affect SFRs in a way that depends on depth and type of anesthesia. Barbiturates and urethane have a strong effect, ketamine very little. In contrast to studies that measure anesthesia effects on stimulus-evoked activity, there are only a few studies that compared SFRs under anesthesia and awake. Torterolo et al.  reportedg et al.  comparedAnesthesia also changes the prevalence and properties of burst firing in neocortex. Ketamine anesthesia results in spindle-like LFP activity in the 5\u201315 Hz range in cat auditory cortex, depending on the depth of the anesthesia  on SFRs in auditory nerve fibers. Liberman and Kiang  \u201cexposedSuperficial multi-unit recordings, likely from fusiform cells, in the dorsal cochlear nucleus (DCN) of hamsters with 10 kHz, 125 dB SPL, 4 h induced noise trauma  was significantly elevated (about a factor two) compared to sham controls (N = 143). This was more evident in primary-like and onset types of neurons. In addition, mechanical damage to the high frequency region of the cochlea (N = 258) showed similar results as noise exposure, suggesting that it is the hearing loss and not the induction method that causes the SFR changes.Vogler et al.  exposed Ma et al.  exposed Vogler et al. , using tAs in the DCN, acoustic trauma (10 kHz tone at 124 dB SPL for 1 h) in guinea pigs did not immediately resut in SFR changes in the ICC. Two weeks recovery after acoustic trauma resulted in more neurons with high SFR compared to control animals, and a significant increase in the average SFR from control (mean = 1.2 sp/s) values  were further assessed by recording, with two 8-microelectrode arrays, from the same sorted-unit clusters before and after the trauma  and Ab1 (P < 0.001) groups. However, when the three frequency bands were combined, unpaired t-tests revealed a significant increase in the percentage of time of burst-firing (P < 0.0001), number of spikes per burst (P < 0.0001), mean burst duration (P < 0.0001) and mean ISI within a burst (P \u2264 0.001), immediately after the trauma (\u201cAfter1\u201d condition). The burst properties returned to normal a few hours after the trauma (\u201cAfter2\u201d condition). However, as we have seen SFRs increased significantly  and 1.4 for Ab2-Ab2 groups (P < 0.0001). For the After2 condition (well after the trauma), one observes that \u03c1 is significantly increased for the Be-Ab2 (P = 0.001), Ab1-Ab2 (P = 0.0004) and Ab2-Ab2 groups (P < 0.0001). Again, SFRs increased significantly in the After2 condition over a wider frequency range compared to the changes in \u03c1.Spontaneous neural synchrony between spike firing of two neurons is also affected by noise exposure  structure, whereas in auditory cortex there is a hyperexponential distribution  to in particular the ICC and MGB and then increases somewhat in some auditory cortex recordings, likely because they reflect multi-unit activity in contrast to single-unit activity at more peripheral levels Figure . Could oAfter noise trauma, it is often observed that the increase in SFR Figure , and alsThese two distinct views of spontaneous activity, either as unwanted noise or as information carrier, may thus determine how one views the neural mechanisms of tinnitus. If one considers spontaneous activity in the auditory system as unwanted noise, the favored concept about tinnitus is likely that it results from too much noise. The suggested neural substrate will then be increased SFR in the auditory system. On the other hand, if one considers spontaneous activity as the information carrier of the brain, sound modulates this firing rate and reorganizes it. In this model external sound can suppress tinnitus. Tinnitus in this model results from increased neural synchrony, i.e., the pathology also reorganizes the spontaneous firing times either in the form of serial correlations, i.e., burst firing, or as parallel correlations, i.e., as synchronous firing among neurons (Eggermont and Tass, Both serial and parallel synchrony will enable efficient synaptic transmission and may amplify each other. Eggermont et al.  phrased The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
{"text": "Tumor suppressor molecules play a pivotal role in regulating DNA repair, cell proliferation, and cell death, which are also important processes in the pathogenesis of Alzheimer\u2019s disease. Alzheimer\u2019s disease is the most common neurodegenerative disorder, however, the precise molecular events that control the death of neuronal cells are unclear. Recently, a fundamental role for tumor suppressor molecules in regulating neurons in Alzheimer\u2019s disease was highlighted. Generally, onset of neurodegenerative diseases including Alzheimer\u2019s disease may be delayed with use of dietary neuro-protective agents against oxidative stresses. Studies suggest that dietary antioxidants are also beneficial for brain health in reducing disease-risk and in slowing down disease-progression. We summarize research advances in dietary regulation for the treatment of Alzheimer\u2019s disease with a focus on its modulatory roles in BRCA1 and p53 tumor suppressor expression, in support of further therapeutic research in this field. BRCA1 and p53 tumor suppressor genes comprise a variety of cancers. In general, tumor suppressor molecules play a pivotal role in regulating both cell proliferation and cell death in many cell types. Those may also play an important role in progression of the AD lesion coinciding with changes in the cellular composition. For example, alteration of the tumor suppressor gene p53 function, essential in DNA repair and cell apoptosis, often exacerbates cognate behaviors [Alzheimer\u2019s disease (AD) is a progressive degenerative disorder that commonly affects emotional stability and memory domains, and is the most predominant reason of dementia ,2. Pathoehaviors . Other tehaviors ,14 as weehaviors ,16. Suchehaviors ,18. Stanehaviors . The DNAehaviors ,21. ReceThe tumor suppressor BRCA1 implicated in breast and ovarian cancers exerts various properties on the DNA repair system ,22. ActuBRCA1 gene is generally expressed in dividing neuronal cells during development [The elopment , and encelopment   extract up-regulates expression of p21 WAF1 and p27 KIP1 [M. officinalis effectively prevents memory impairment in a transgenic mouse model of AD [M. officinalis, on beta-amyloid-induced neuronal toxicity have been reported [Ginsenoside, one of components in American ginseng herb, activates p53 , which cp27 KIP1 . In addiel of AD . Moreovereported . Baicalireported . The effreported .Gleditsia sinensis, have been shown to increase cell cycle arrest during the G2/M phase, and is associated with increased p53 protein level [Gleditsia sinensis thorns is also associated with up-regulation of p21 WAF1 [Saussurea involucrate induces p21 WAF1 and p27 KIP1 expression, independent of the p53 pathway [Several other promising herbs or life-style conditions involved in p53 activity are as follows. Treatment with thorns from a medicinal herb, in level . Treatmep21 WAF1 . In addip21 WAF1 . On the  pathway . Zinc is pathway . Signali pathway . Zinc th pathway ,85. Furt pathway . Hypoxia pathway . The DNATo maintain normal cellular function, neurons can struggle to avoid oxidative damage throughout aging processes. If the cells cannot keep the balance, the excess toxic stresses may cause neuronal cell death. Because AD is a disease that could last more than ten years, neurons may attempt to survive by attempting to control the damaged or deregulated cells rather than succumbing to apoptosis immediately. These speculations raise the possibility that BRCA1 and p53 accumulate in neurons early stages of AD. In addition, the association of BRCA1 and p53 with neurodegenerative pathology in AD may implicate a neuro-protective function in healthy neurons in AD. Genomic instability as a feature in the pathogenesis of neurodegeneration in AD may as well be a feature of the DNA repair activity in survival neurons. Alteration of BRCA1 and p53 simultaneously and complexly indicate oncogenic and/or neurodegenerative stimuli which are found in cancer, AD, and other neuropathology. There are several examples showing cognitive improvements in dementia patients undergoing chemotherapy against cancer. Notably, tumor suppressors are crucial molecules at this point. Neuroprotective factors have been suggested as a possible target for drug design efforts with the goal of delaying neurodegeneration from neuronal apoptosis. This may represent the rational basis for the development of dietary treatment of AD. Further mechanistic studies are required to understand the precise molecular mechanisms, and to determine whether an appropriate dietary intake is related to the improved brain function and the preservation of brain health. Long-term clinical studies are also necessary to clarify effects of treatment in the management of AD.The functions of BRCA1 and p53 are involved in the pathogenesis of AD as well as carcinogenesis. Some diets may contribute to neuro-protection, disease prevention, and possible better prognosis in AD via regulation of the expression of tumor suppressors."}
{"text": "AMTN was characterized in rodents only. In this study, we applied various approaches, including in silico screening of databases, PCRs and transcriptome sequencing to characterize AMTN sequences in sauropsids and amphibians, and compared them to available mammalian and coelacanth sequences.Amelotin (AMTN) is an ameloblast-secreted protein that belongs to the secretory calcium-binding phosphoprotein (SCPP) family, which originated in early vertebrates. In rodents, AMTN is expressed during the maturation stage of amelogenesis only. This expression pattern strongly differs from the spatiotemporal expression of other ameloblast-secreted SCPPs, such as the enamel matrix proteins (EMPs). Furthermore, AMTN structure changed during tetrapod evolution. To infer AMTN function, we studied spatiotemporal expression of AMTN during amelogenesis in a salamander and a lizard, and compared the results with available expression data from mouse. We found that AMTN is expressed throughout amelogenesis in non-mammalian tetrapods, in contrast to its expression limited to enamel maturation in rodents.We showed that (i) AMTN is tooth (enamel) specific and underwent pseudogenization in toothless turtles and birds, and (ii) the Taken together our findings suggest that AMTN was primarily an EMP. Its functions were conserved in amphibians and sauropsids while a change occurred early in the mammalian lineage, modifying its expression pattern during amelogenesis and its gene structure. These changes likely led to a partial loss of AMTN function and could have a link with the emergence of prismatic enamel in mammals.The online version of this article (doi:10.1186/s12862-015-0329-x) contains supplementary material, which is available to authorized users. AMTN, AMEL, ENAM and AMBN) in the coelacanth genome  was commissioned to GATC Biotech.Pleurodeles waltl , and two sauropsids, Caiman crocodilus  and Tarentola mauritanica  were assembled at ISEM-Montpellier 2 (France) and screened for AMTN transcripts with BLAST using the Montpellier Bioinformatics Biodiversity (MBB) platform [The sequenced jaw transcriptomes (see below \u201cJaw transcriptome sequencing\u201d) of the caudate amphibian, platform .AMTN were translated into amino acid sequences and aligned to mammalian AMTN using Se-Al v2.0a11 [The protein-coding regions of  v2.0a11 . The put v2.0a11 ,39 and p v2.0a11 ,41.P. waltl, and several young adults A. carolinensis (3\u00a0month- to 2\u00a0year-old) were used. Immediately after dissection, the lower jaw quadrants were fixed in a mixture of glutaraldehyde (1.5%) and paraformaldehyde (1.5%) in PBS for 2\u00a0h, at room temperature. They were demineralized for three weeks, under gentle agitation at 4\u00b0C, in the same fixative to which 5% EDTA was added. The solution was changed every two days. After washing overnight in PBS, tissues were post-fixed for 2\u00a0h in 1% osmium tetroxide, rinsed in PBS, dehydrated through a graded series of ethanol then immersed in propylene oxide prior embedding in Epon 812 (EMS). 2\u00a0\u03bcm-thick sections were obtained using a Reichert OMU-3 ultramicrotome, deposited on a glass slide, stained with toluidine blue, and photographed with an Olympus BX61 microscope equipped with a QImaging camera using Image Pro Plus software .One adult AMTN transcript fragments were amplified from A. carolinensis  and P. waltl (703\u00a0bp) cDNA, using primers designed from the gDNA and transcriptome sequences, respectively , and transformed into competent E. coli TOP10F\u2019 bacteria. Colonies containing the vector and the insert were selected for plasmid purification . Purified plasmids were linearized by PCR using M13 forward and reverse primers and the sense and antisense RNA probes were synthesized using SP6 and T7 RNA polymerases  in the presence of digoxigenin-UTP  and purified . waltl 70\u00a0bp cDNA,The jaws were dissected in quadrants and fixed overnight, at 4\u00b0C, in Formoy  solution. Tissues were demineralized in 10% acetic acid for 20 to 30\u00a0days at room temperature and under gentle agitation. The solution was changed every two days. Samples were then dehydrated through an increasing series of ethanol, shortly immersed in toluene and embedded in Paraplast . The sections (8\u00a0\u03bcm-thick) were obtained with a Leica RM2245 microtome and deposited on Superfrost PLUS slides .2). The digoxigenin-labeled probes were revealed at 37\u00b0C using NBT/BCIP (nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indodyl phosphate). The slides were mounted in Aquatex mounting medium , and photographed (Olympus BX61 microscope).The sections were dewaxed in toluene, rehydrated through a decreasing series of ethanol, then in PBS, treated with proteinase K (0.6\u00a0\u03bcg/ml) for 5\u00a0min at 37\u00b0C, rinsed in PBS, post-fixed for 30\u00a0min in 4% paraformaldehyde, rinsed again in PBS and then in 2\u00d7 SSC. The slides were incubated overnight, at 65\u00b0C, with the digoxigenin-labeled antisense probe (0.25\u00a0ng/\u03bcl) in the hybridization buffer . The following day, the slides were washed three times, at 65\u00b0C, in the washing buffer , and rinsed, at room temperature, in the Maleic Acid Buffer Tween (MABT), pH\u00a07.5. Non-specific binding sites were blocked for 2\u00a0h in a blocking solution . Then, the slides were incubated overnight with the anti-digoxigenin antibody coupled to alkaline phosphatase  in the blocking solution. After four baths of MABT, the slides were rinsed in NTM pH\u00a09.5  and the French Rural Code .AMTN sequences of sarcopterygian species were obtained using in silico screening of public databases, RT-PCR on jaw cDNA, and screening of assembled jaw transcriptomes: six species representative of mammalian lineages , eight sauropsids , two amphibians , and one coelacanth . Genbank accession numbers of AMTN sequences obtained from cDNA are indicated in Additional file AMTN was detected in the PCR products obtained from the cDNA of all non-mammalian species used in this study. In the gDNA sequences analyzed in GenBank, AMTN was always found upstream AMBN, from 5\u00a0kb in X. tropicalis to 73\u00a0kb in B. taurus.A total of 17 AMTN sequences were inferred either from the comparison of cDNA and gDNA sequences obtained for the same species  or from sequence comparison of related species . The AMTN sequences in P. molurus and P. regius, on the one hand, and in A. mississippiensis and C. crocodilus, on the other hand, were nearly identical . For H. sapiens, M. musculus, B. taurus, L. africana, M. domestica and O. anatinus the exons were previously identified [P. waltl AMTN (cDNA data only) the exon-intron boundaries were estimated from the alignment of all AMTN sequences. However, the limit between exon 4 and exon 5 could not be clearly defined because of the high variability of this region when compared to the homologous region in other AMTNs. For L. chalumnae (gDNA data only), the exons were identified with UniDPlot then aligned to other AMTN sequences.The exon-intron boundaries of nine non-mammalian entified . For P. AMTN structure resulting from these comparisons is represented in Figure\u00a0AMTN (exons 1\u20139), and three newly identified exons, i.e. two exons located between exons 2 and 3, and one exon inserted between exons 3 and 4. In order to conserve the current nomenclature of AMTN exons, we named 2b, 2c and 3b these three new exons, the former exons 2 and 3 becoming exons 2a and 3a , and the smallest was seven in X. tropicalis . It is worth noting that the opossum AMTN (gDNA data only) may have either the same structure as in other mammals, i.e. the short exon 8 plus the exon 9 as suggested by Gasse et al. [AMTN, i.e. only a large exon 8 as proposed by Kawasaki and Amemiya [AMTN sequences could be present in opossum as splice variants , most in exon 1 plus 15\u00a0nt located at the beginning of exon 2a. In non-mammals, the sequence ends with exon 8 that is larger than in placental mammals and comprises the stop codon followed by the 3\u2032UTR. The latter is also composed of a variable number of nucleotides. In placental mammals, the sequence ends with exon 9 that includes the termination codon and the 3\u2032UTR. In non-placental mammals, platypus and opossum, both structures could occur (see below).The 5\u2032 UTR of X. tropicalis) amino acids (aa), and the first two residues of the mature protein are encoded by the last six nucleotides of exon 2a. Given the different AMTN structure in the various lineages, the length of the encoded protein varies from 252 aa in X. tropicalis to 355 aa in P. waltl.A single signal peptide (SP) encoded by exon 2a was detected in the N-terminal region of all AMTN obtained in this study  and a SxE (SeE: aa 82\u201384) phosphorylation motifs adjacent one another in the region encoded by exon 7. A conserved Arg-Gly-Asp (RGD) motif (putative integrin-binding site) was found in the C-ter region encoded by exon 8 in non-mammals but it is missing in mammals, with the possible exception of marsupials  previously identified in mammals in the region encoded by exon 7 is conserved in sarcopterygian AMTN (SseE)  while a conserved motif \u201cPQQL\u201d is encoded by exon 5. Also, exon 6 encodes 12 amino acids that are well-conserved in all species studied. No particular function is known for these conserved motifs.In addition to these features, in sauropsid AMTN exon 8 is in average 975\u00a0bp-long and encodes a variable region of the protein, the C-terminal region, which houses a well-conserved RGD motif . A ninth exon possessing eight nucleotides followed by a stop codon and the 3\u2032UTR was then recruited. In platypus and opossum, however, screening of the gDNA sequence provided two putative sequences depending on whether the intraexonic splicing occurs in exon 8 or not  could be identified within the 15\u00a0kb genomic region upstream \u03c6 AMBN. They display many nucleotide substitutions and indels  were identified using UniDPlot within the 10\u00a0kb upstream \u03c6 AMBN. They display less variations compared to A. platyrhynchos AMTN  and of a monotreme (platypus: Ornithorhynchus anatinus) (not shown). This exon 2c (57\u00a0bp) possessed high nucleotide identity with sauropsidian exon 2c and displayed correct putative splice sites, which suggests that exon 2c is functional in marsupial and monotreme AMTN. Exon 2c was not found in placental mammals but weak nucleotide similarity in the homologous genomic region of various mammalian species (not shown) suggests that exon 2c was present in an ancestral placental AMTN, then invalidated early, before the diversification of current placental lineages. In contrast, neither functional nor pseudoexons 2b and 3b were found in any mammalian genomes studied to date, which suggests that these two exons were either not present in the AMTN sequence of the last common ancestral mammal or invalidated early in the mammalian lineage, and no longer recognizable in gDNA.In mammals, many onfirmed , but pos Figures\u00a0 and 2, tAMTN is composed of 11 exons in squamates (no exon 9) and of 10 exons in crocodiles (lack of exons 3b and 9)  of sauropsids  using mammalian exon 9 sequence did not provide valuable hit. Exon 3b was identified in squamates only. We checked whether this exon was missed in A. mississipiensis gDNA, but no hit (even as pseudoexon) was obtained.In sauropsids,  Figures\u00a0. The exoAMTN was found to be composed either of 10 exons (absence of exons 3b and 9) in P. waltl (caudates) or of seven exons  in X. tropicalis (anurans)  of X. tropicalis did not provide valuable hit. Similarly, exons 2b, 3b, 4 and 5 were not identified in the target genomic regions (introns 2 and 3) of X. tropicalis and no pseudoexons were found.In amphibians, AMTN .In the late cytodifferentiation stage, ameloblasts have differentiated from the inner dental epithelium of the enamel organ and they are facing mesenchymal cells of the dental papilla differentiated into odontoblasts. As soon as a thin layer of predentin was deposited between the ameloblasts and odontoblats, h Figure\u00a0A, B. AMTd Figure\u00a0C-F. In ts Figure\u00a0G, H. AMTe Figure\u00a0I, J. AftAMTN expression was monitored in larvae (from stage 34 onwards) and in adult specimens .s Figure\u00a0. In larvx Figure\u00a0A. In conx Figure\u00a0F, G. Dure Figure\u00a0B, H. Onct Figure\u00a0C, I. Befl Figure\u00a0D, J. AMTIn this study, we combined RNA-seq data (transcriptomics), genomic data screening, isolation, characterization and expression of the AMTN gene to investigate the evolution of this SCPP in tetrapods. Our results suggest that AMTN was a component of the enamel matrix in the last common ancestor of tetrapods and that its structure changed and the function most likely changed in mammals, probably as a consequence of drastic events that occurred in the coding and/or regulatory region of the gene in the last common ancestor of mammals.AMTN is located close to AMBN and ENAM in the same DNA region, and always upstream AMBN in the genome of sarcopterygian species annotated to date [AMTN and AMBN could be closely related SCPP genes, an hypothesis which is supported by the presence of remarkable motifs in the two proteins, in particular a SxxE motif encoded by a small exon in the middle region of the protein  [AMTN and AMBN (but not that of AMEL or ENAM) is modified by the FAM20C kinase in mice [e.g. polypterids and lepisosteids, and of chondrichthyans to better understand the evolutionary history of these EMPs in gnatosthomes.According to its genomic position, AMTN could be related to ENAM and AMBN because  to date ,18. This in mice . NeverthAMTN structure in sarcopterygian lineages suggests that the ancestral sequence was composed of either nine or 10 exons, depending on whether exon 5 was present in the ancestor and then lost in the coelacanth lineage (or not found in gDNA) or acquired in the lineage leading to tetrapods , nor in Dipnoi (lungfishes), nor in representatives of the basal actinopterygian lineages . Furthermore, when only gDNA is available coding exons could be missed, as illustrated by the discovery of the putative exon 2c in monotreme and marsupial AMTN, after this exon was identified in sauropsids. Indeed, this exon was not detected in opossum AMTN when using comparison with mammalian sequences that do not possess exon 2c [The comparison of s Figure\u00a0. To date exon 2c  but was  exon 2c  when usiAMTN and in most of the tetrapods analyzed here derive from those of an ancestral SCPP, from which AMTN and EMPs originated after duplication. Indeed, AMTN exons 2a and 2b are homologous to exons 2 and 3 (exon 4 and 5 in ENAM) of all SCPPs, respectively. In addition, AMTN shares several other characteristics with all EMPs, i.e. the signal peptide encoded by exon 2a, the conserved Ser-X-Glu motif (putative phosphorylation site) encoded by exon 2b, and the presence of a variable region encoded by a large exon, as previously observed by Kawasaki and Weiss [Our results suggest that some coding exons present in the coelacanth nd Weiss .AMTN exon 2b in salamander cDNA and its absence in clawed frog, for which gDNA and cDNA sequences are available, indicates that this exon has been lost either early in the anuran lineage (no AMTN sequences available in other frog lineages), or later in the lineage leading to Pipidae  encoded by exon 2b, the presence of which characterizes most SCPPs, is not required for the correct AMTN function during amelogenesis in clawed frog and mammals. This leads to the question of whether or not the phosphorylation site (SxxE motif) encoded by exon 7 compensates, in mammals, for the lack of that encoded by exon 2b.The presence of e Figure\u00a0. Exon 2bAMTN and in non-placental mammals. The presence of this exon in representative species of the four sarcopterygian lineages  indicates that its origin is to be found before the divergence of these lineages. On the other hand, its absence in placental mammals, suggests that exon 2c was probably lost in the common ancestor of placentals.Exon 2c was found in non-mammalian sarcopterygian AMTN only, a finding which suggests that this exon was acquired either in the lepidosaurian lineage after its divergence from other sauropsid lineages or soon after Squamata diverged from the sphenodontian lineage , a finding which indicates that, as proposed for exon 2b this exon was lost either early in the anuran lineage or later, in the lineage leading to Pipidae. We did not find this exon in platypus AMTN, indicating that exon 4 was secondarily lost in this lineage. However, in absence of AMTN cDNA in this lineage, the lack of exon 4 could also result from a genome assembly artefact.Exon 4 is absent in the clawed frog AMTN cDNA) and in platypus, although the lack in the latter could result from a badly assembled genomic sequence.Exon 5 has not been found in coelacanth, clawed frog and platypus genomes, and is not present in clawed frog cDNA either. As discussed above we cannot know whether this exon was present in the sarcopterygian ancestor or was recruited later in a tetrapod ancestor. Parsimonious analysis suggests that exon 5 was secondarily lost in clawed frog  the motif is functional (conserved during more than 430\u00a0Ma), and (iii) AMTN exon 7 is homologous to AMBN exon 12 and to ENAM exon 8, all of them preceding the large exons 8, 13 and 10, respectively. In clawed frog AMTN, the exon 7 not only encodes a SxxE/D motif, but also codes for a second phosphorylation site. Because the SxE motif encoded by exon 2b was lost in the pipid (or anuran) lineage, we suggest that the second SxE motif, encoded by exon 7, could compensate for the loss of the former. The presence of two phosphorylation sites, that could be located in different regions of the protein, was probably essential for AMTN to fulfill its function in non-mammalian sarcopterygians.Exon 7 encodes an SxxE motif (putative phosphorylation site) that is found in all AMTN sequences analyzed so far. When phosphorylated, this motif promotes hydroxyapatite precipitation in vitro . Such moin vitro . These fAMTN exon 8 is shorter than in the other lineages and has lost a large part of the 3\u2032 region, including the region encoding the RGD motif in non-mammals [AMTN possesses an exon 9, which encodes the three last amino acids of the protein followed by the stop codon and the 3\u2032UTR. Sequence comparison showed that a mutation in exon 8 generated an intraexonic splice site in an ancestral placentalia AMTN. This intraexonic splicing occurred within a codon, leading to the recruitment of exon 9 encoding an appropriate stop codon. It appears that encoding the stop codon could be the only role of exon 9 as several mammalian species lack this last exon because a mutation generated a stop codon at the extremity of exon 8 [Exon 8 experienced two different evolutionary histories in sarcopterygians. In non-mammals, exon 8 is large and encodes a variable region of the protein, the C-terminal region of which includes a well-conserved Arg-Gly-Asp (RGD) integrin-binding motif that likely plays a crucial role for the protein function. RGD motifs are recognized by integrins and mediate the attachment of the cell to the extracellular matrix . As for -mammals , and hen-mammals . In addif exon 8 .AMTNs displaying a similar environment favorable for intraexonic splicing as observed in the homologous region of placental mammals, and (ii) exon 9 sequence, similar to that in placental mammals, present in both gDNA. The second possibility, suggested by Kawasaki and Amemiya [AMTN exon 8 did not occur in an ancestral marsupial but took place later in an ancestral placental. This finding is supported by the conservation of an RGD motif in the extremity of opossum AMTN, as in non-mammalian species. Such a long lasting conservation of RGD motif, although the rest of the sequence is less conserved, could indicate that this region is functional. A third interpretation could be that both AMTN variants are present in marsupials, the one with a short exon 8 and exon 9 (no RGD encoded), the other with the large exon 8 only (encoding an RGD). In platypus, the interpretation could be the same. However, if the intraexonic splicing does not occur and a large exon 8 transcribed, the latter does not encode an RGD. This suggests a different evolution of AMTN in this lineage. We previously showed in mammals that AMTN is enamel specific [AMTN sequence, including mutations in the sequence encoding the RGD motif.In monotremes and marsupials, the question of the efficient presence of the intraexonic splicing is posed. Indeed, the unavailability of cDNA data in both lineages has led to various interpretations. The first possibility we already proposed  is that  Amemiya , is thatspecific . In platspecific . This coA RGD motif is present in all members of the acidic SCPPs (dentin and bone SCPPs) and is also found in the ENAM sequence of several mammalian species . This sui.e. from the maturation phase onwards, previously reported during murine amelogenesis [AMTN expression in two selected tetrapods has answered partially this question.Taken together our results suggest that in non-mammalian sarcopterygians AMTN may play an important role in ameloblast adhesion to the enamel matrix. This role in ameloblast adhesion was then probably lost, either in the last common mammalian ancestor or later in the last common ancestor of placentals, as a consequence of a mutation generating an intraexonic splice site in exon 8, suppressing the encoded RGD motif. Could the absence of this RGD motif explain the late expression of AMTN, ogenesis ,3,24? The.g. opossum [AMEL expression during amelogenesis was similar in all tetrapods [AMBN, ENAM, or MMP20 in non-mammalian tetrapods.In lizard and salamander, tooth formation is roughly similar to that described in other vertebrates, but unlike mammals, ameloblasts do not exhibit Tomes\u2019 processes, rod and interrod enamel structures are absent, and hence enamel is not prismatic -53. Ther opossum . These cetrapods ,55. No dAMTN appear when comparing lizard or salamander amelogenesis to the one of rodents. In the incisors and molars of the latter, AMTN is predominantly expressed in maturation-stage ameloblasts [AMTN expression is detected in ameloblasts as soon as enamel matrix deposition has started, and is maintained from secretion to late maturation stage. AMTN is no longer expressed in the reduced ameloblasts after tooth eruption. In salamander larvae, AMTN expression is detected in early stages of amelogenesis while in adults, the onset of expression is delayed and appears slightly later than in the lizard, during enamel matrix deposition. Then, in both larvae and adult salamanders, AMTN expression is maintained in ameloblast until maturation stage. The disappearance of AMTN signal in ameloblasts occurs earlier than in lizard but it persists longer in the inner dental epithelium along the dentin cone. This late AMTN expression in the cells facing the region lacking enamel along the tooth base could be related to the formation of the amphibian pedicel [AMTN gene nor the protein was ever detected beyond the cemento-enamel junction in the epithelial cells along the tooth root. Nevertheless, AMTN was found in Hertwig\u2019s epithelial root sheath cells entrapped in cementum [AMTN expression is detected earlier in secretory stage ameloblasts, and later in the cells of the inner dental epithelium along the tooth root. Immunolocalization of the protein should be performed to better understand the significance of the mRNA expression pattern.Differences in the spatial-temporal expression of loblasts . In liza pedicel ,57. Futu pedicel ,24, alth pedicel ,58. In rcementum . In lizaAMTN being expressed during the various steps of amelogenesis in lizard and salamander, we concluded that (i) AMTN must be considered an enamel matrix protein (EMP), as are AMBN, ENAM and AMEL, and (ii) this status was the ancestral condition in non-mammalian tetrapods. When comparing lizard and salamander to mammals, one could hypothesize that the differences observed both in the timing of AMTN expression and in the gene structure could be responsible for the differences in enamel microstructure  in these lineages. The absence of both Tomes\u2019s process and of an organized rod/interrod enamel structure in lizard and salamander, on the one hand, and in mice overexpressing AMTN under AMEL promoter, on the other hand, support this hypothesis [pothesis .AMTN expression and the gene structure changed in mammals could be visualized as the succession of several events that occurred early in mammalian evolution. One event probably occurred at the promoter level  resulting in late AMTN expression during enamel mineralization. Two other, probably independent events resulted in the loss of exon 2b encoding the SxE motif and in the intraexonic splicing of exon 8 eliminating the RGD motif. The occurrence of these events (in any order) may have led to important changes in ameloblast structure (differentiation of Tomes\u2019 processes) and in enamel organization (formation of prisms).The putative evolutionary scenario we propose to explain how the spatiotemporal A. platyrhynchos, we found that AMTN is also a pseudogene. As observed for EMP genes, AMTN underwent pseudogenization after the capability to develop teeth was lost in the common ancestor of modern birds, 80\u2013100 Mya [\u03c6 AMTN sequence that is now hardly recognizable from the non-coding gDNA. In turtles, the capability to build teeth was lost in the last common ancestor of chelonians, 250 Mya [C. mydas, AMTN also underwent pseudogenization, producing a \u03c6 AMTN, in which a few exons could be identified. These findings indicate that in the ancestors of these two sauropsid species AMTN had no other functions than being involved in amelogenesis and is, therefore, an enamel specific protein in toothed sauropsids. In addition, it appears that, after the loss of function, mutations in this gene accumulated more rapidly in birds than in turtles although teeth were lost three folds earlier in turtle than in the bird ancestor [In birds, pseudogenization of EMP genes is related to tooth loss ,31,60. I\u2013100 Mya ,61. Nume 250 Mya . In C. mancestor . This coancestor .AMTN pseudogenization in two xenarthran mammals, armadillo and sloth, in which teeth are devoid of enamel cover [In a previous study, we showed similar el cover . These rIn situ hybridization of AMTN during tooth formation in lizard and salamander revealed that AMTN is expressed in ameloblasts during all stages of amelogenesis (with some differences in both species). These findings suggest that AMTN is an essential EMP for amelogenesis in these two species. The differences in spatiotemporal expression of AMTN described in murine vs lizard and salamander teeth would then suggest that in sauropsids and amphibians AMTN could have different functions than in mammals.This is the first comparative study of AMTN sequence in representatives of various mammalian and non-mammalian vertebrates. AMTN was present in the last common sarcopterygian ancestor more than 420 Mya. Various loss or gain of coding exons were identified in coelacanth, amphibians, reptiles and non-placental mammals, indicating different evolutionary pathways in sarcopterygian lineages."}
{"text": "Bovine cutaneous fibropapillomas are benign hyperproliferative lesions induced by Bovine Papillomaviruses (BPVs). Bcl-2 is an important anti-apoptotic protein which is expressed in several cancer types. In contrary, p53 is a tumour suppressor protein that mediates cell cycle arrest, apoptosis and senescence in response to cellular stresses.Here, we investigated immunohistochemically and biochemically, the expression of bcl-2 and p53 in a subset of BPV positive fibropapillomas and bovine normal skin. Normal skin samples showed a weak signal for both proteins in the cytoplasm of the basal cells. Nine out of twelve (75%) tumour samples stained positive for bcl-2 throughout basal and parabasal layers, with most of cells showing strong cytoplasmic immunoreactivity. Nine out of twelve (75%) fibropapillomas were found to be positive for p53 expression, showing a strong cytoplasmic and perinuclear staining of p53 protein mainly in the basal and parabasal layers.Our data reveal an altered bcl-2 and p53 immunoreactivity in bovine cutaneous fibropapillomas, suggesting involvement of these two proteins in the cutaneous neoplastic transformation through an impaired apoptotic process.The online version of this article (doi:10.1186/1750-9378-10-2) contains supplementary material, which is available to authorized users. Bovine cutaneous fibropapillomas are benign hyperproliferative lesions induced by different BPVs types, infecting both the epithelial cells and the underlying dermis . These lBPV infection is supposed to be the most important causal factor in fibropapilloma development and our previous studies on these tumours have demonstrated alterations of GAP junction proteins and signaling transduction pathways . HoweverPapillomaviruses (PVs) are oncogenic viruses causing both benign and malignant tumours in humans as well as in several animal species. These viruses have developed numerous strategies in order to block host-mediated apoptosis, contributing to cancer development .in situ and mammary carcinoma [Programmed cell death, known as apoptosis, is a pivotal mechanism of cell death that plays an important role during diverse biological processes, including development, cell differentiation and proliferation . The regarcinoma .P53 is a tumour suppressor protein that mediates cell cycle arrest, DNA integrity and repairing, apoptosis and senescence in response to specific cellular stresses  . As a coP53 has a significant role in cell cycle control, therefore the loss of its suppressive function has been reported for different types of human neoplasia and animal tumours, such as BPV induced tumours \u201314. HoweIn this study, we investigated the expression of p53 and bcl-2 in bovine fibropapillomas according to the detailed \u201cMaterials and Methods\u201d in the Additional file Nine out of the 12 (75%) bovine fibropapillomas were found to be positive for p53 expression. In most samples, a strong expression of p53 protein was detected in the basal layer and parabasal layer, where cells with a cytoplasmic and perinuclear staining were recorded  did not expressed neither bcl-2 nor p53 proteins. The lack of reactivity might be due to the loss of antigenicity.Bovine cutaneous fibropapillomas are benign lesions known to affect cattle worldwide. BPV are considered as causative agents of these tumours, inducing the proliferation of both epithelial and dermal cells . ApoptosIn our study, bcl-2 positive samples showed a reduced number of TUNEL positive cells when compared to the bcl-2 negative fibropapillomas. This is in accordance with human counterpart, where in head and neck squamous cell carcinomas the number of apoptotic cells was noted to be reduced in bcl-2 positive tumours when compared to bcl-2 negative tumours . This fiNormally, a very low expression of p53 protein is detected in cells; in response to cellular stresses, however, the protein is stabilized and cell division is inhibited in order to respond to such damage .Our results indicate that p53 is weakly expressed in basal epithelial cells of normal skin samples. Accordingly, a similar p53 expression pattern in the basal epithelial cells has been recorded in human normal skin, suggesting that it may exert its physiological function in bovine healthy epithelium , 20, 21.Fibropapilloma samples showed a strong cytoplasmic and perinuclear p53 expression in the basal and parabasal cell layers. Cytoplasmic accumulation of p53 has been proposed as a considerable mechanism to disrupt its function as a tumour suppressor and its expression has been reported in different human and animal tumours , 23. RecHowever, the higher expression of p53 in epithelial component of fibropapilloma may be due to different stresses followed by a temporary increase of the normal p53 protein or accumulation of mutated p53, resulting in a permanent abnormal form of p53 that is not functional and/or resistant to degradation .Our results indicate that bcl-2 expression may promote the survival of the neoplastic cells possibly depending on p53 impaired expression.Despite a number of studies have higlighted a role for BPV\u2019s oncogenes in impairing some cellular pathways and leading to neoplastic transformation, nothing is known about a possible interplay among the viral oncoproteins and bcl-2 and p53 dysregulation. Thus, the unraveling of new pathways possibly involved in the pathogenesis of bovine cutaneous fibropapillomas is of a great importance to complete the molecular scenario triggering to the development of these tumours.in vivo carcinogenesis.In summary, the current study provides evidence for p53 and bcl-2 expression in a majority of BPV-induced bovine tumours, suggesting an involvement of these two proteins in bovine cutaneous fibropapilloma development. Further studies are needed to better understand bcl-2 and p53 function Materials and Methods.(DOCX 32 KB)Additional file 1:"}
{"text": "HER2 (p=.003)aberrations were more common in the p53 mutation group. No survival difference was observed between patients with p53 mutations and those with wild-type p53. In patients with wild-type p53 and other aberrations, patients treated with matched-therapy against the additional aberrations had longer survival compared to those treated with non-matched-therapy or those who received no therapy . Results were confirmed in a multivariate analysis (p= .0002). In the p53 mutation group with additional aberrations, those who received matched-therapy against the additional aberrations had survival similar to those treated with non-matched-therapy or those who received no therapy (p=.15). In conclusion, our results demonstrated resistance to matched-targeted therapy to the other aberrations in patients with p53 mutations and emphasize the need to overcome this resistance.P53 mutations are associated with invasive tumors in mouse models. We assessed the p53mutations and survival in patients with advanced cancer treated in the Phase I Program. Of 691 tested patients, 273 (39.5%) had p53 mutations. Patients with p53 mutations were older (p<.0001) and had higher numbers of liver metastases (p=.005). P53 mutations were associated with higher numbers of other aberrations; PTEN (p=.0005) and  The tumor suppressor protein p53 is activated in response to a variety of stress signals to ensure genome stability in cells. The role of p53 in guarding the cell reflects its ability to act as a potent transcriptional activator, regulating the expression of genes that inhibit cell cycle progression or induce apoptosis or senescence. By eliciting cell growth inhibition, p53 functions to prevent cancer development . In addiin vivo studies. Mice that carry mutant p53 develop different types of tumors with more invasive phenotypes than the p53-null mice in which the p53 protein is not expressed [Loss of p53 function occurs frequently in human cancers and results from mutations in the p53 gene or defects in the pathway that activates p53 . More thxpressed , 8. In axpressed , 9, 10. xpressed . In addixpressed , 12.Several studies have investigated the importance of p53 mutation status in predicting clinical outcome in various types of cancer. Some of the p53 mutations have been correlated with shorter survival or a poorer response to treatment in several cancers. In addiWe have previously reported the clinical characteristics and response to standard systemic therapy of 145 patients with documented p53 mutational status (n=66 mutated vs. n=79 wild-type tumors) who were referred to our department for participation in Phase I clinical trials. In the From May 2010 to April 2013, 691 patients were found to have tumor tissue tested for p53 mutation status. Of these, 273 (39.5%) had p53 mutations and 418 (60.5%) had wild-type p53. The patients' baseline characteristics by p53 mutation status are shown in Table No difference between the two groups was observed in sex, race, performance status, number of metastatic sites, number of prior therapies, serum albumin and lactate dehydrogenase levels, and Royal Marsden Hospital score. The most common tumor types seen were colorectal, head and neck, and breast cancers, which reflected the referral pattern of patients in the Phase I Clinic. Colorectal cancer was the most common malignancy in both groups .Of 273 patients with p53 mutations, 17 (6%) had more than one p53 mutation. The distribution of p53 mutations by exons, codons, and tumor types is summarized in Table When all screened patients were considered, p53 mutations in the hot-spot codons  were significantly more common in colorectal cancer  than in the other tumor types . When the analysis was limited to patients with p53 mutations, 24 (49%) patients with colorectal cancer had hot-spot mutations compared to 60 (27%) patients with the remaining tumor types (p=.0002). Mutations in the DNA-binding domain excluding the hot-spot codons were more commonly seen in ovarian and lung cancer .BRAF mutations in the patients with wild-type p53 compared with those with mutated p53 .We examined aberrations other than p53 in patients with p53 mutations and wild-type p53. Additional aberrations were found more often in the group with p53 mutations than in the wild-type p53 group (p=.002). The distribution of the number of other aberrations in the two groups is summarized in Table KRAS (16%), APC (12%), and PI3K (10%). The most common aberrations seen in the wild-type p53 group were KRAS (16%), PI3K (14%), and PTEN (9%). Overall, aberrations involved in the RAS/RAF/MEK pathway were more frequent in the p53 mutant group than in the wild-type p53 group . In contrast, no difference was observed between the two groups in aberrations involving the PI3K/AKT/mTOR pathway .The distribution of the common additional aberrations in the groups of patients with mutant and wild-type p53 is summarized in Table Of the 273 patients with p53 mutations, 195 (71%) were treated on various phase I clinical trials; among them, 20 (7%) patients received local therapy, including various hepatic arterial infusion and intra-peritoneal therapy regimens. The remaining 78 (29%) patients did not receive any treatment for various reasons. Of 418 patients with wild-type p53, 312 (75%) were treated on various phase I clinical trials; among them, 26 (6%) patients received local therapy. The remaining 106 (25%) patients did not receive any therapy. The reasons for which patients did not receive treatment were ineligibility or unavailability of a clinical trial , insurance issues , and personal reasons .Among patients with additional aberrations in the p53 mutation group, 37 (26%) patients received targeted therapy against the additional aberrations, 62 (43%) patients received non-matched therapy, and 44 (31%) patients received no therapy. In the wild-type p53 group, 78 (30%) patients received targeted therapy against the additional aberrations, 117 (45%) patients received non-matched therapy, and 63 (25%) patients received no therapy.There was no difference in overall survival between patients with p53 mutations and those with wild-type p53 . The overall survival of treated and untreated patients with p53 mutations is shown in Figure The overall survival of patients with p53 mutations by exon is shown in We performed survival analyses of patients with p53 mutations and additional aberrations. The median survival durations were 13.6 months in patients who received matched therapy (n= 37), 13.9 months in those who received non-matched therapy (n= 62), and 7.6 months in those who received no treatment n= 44)  had longer survival than those treated with non-matched therapy (n= 117) or those who received no treatment (n= 63)  confirmed this difference between the treatment groups (p=.0002). These findings translated into longer survival for the patients with a more number of additional mutations in the wild-type p53, but not the mutant p53 group .This analysis expands on our previous report on the characteristics and survival of patients with advanced cancer and p53 mutations. In the current study, we evaluated the effects of specific p53 mutations and additional aberrations on survival in patients referred to the Phase I Clinical Trials Program. P53 mutations were associated with higher numbers of other aberrations and resistance to targeted therapy. P53 status did not affect overall survival in this patient population, but matched therapy for other aberrations was associated with longer survival in patients with wild-type p53. P53 mutations varied by tumor type and were associated with higher proportions of PTEN and HER2 aberrations than wild-type p53.Overall, 39.5% of the patients analyzed had a p53 mutation. Patients with p53 mutations were older (p<.0001) and had higher rates of hepatic metastases (p=.005) than patients with wild-type p53. P53 mutations within exon 5 were more common in colorectal cancer, while mutations within exons 6, 7, and 8 were more common in lung, pancreatic, and ovarian cancer, respectively.Our findings of older age, more frequent liver metastases, and more frequent PTEN loss in the p53-mutated group in the current, more extensive analysis, were similar to those in our prior analysis .Our results are consistent with previously known data showing that the percentage of p53 mutation varies by tumor type and ranges from 10% to 80% . Our resThe observation that patients with p53 mutations had more liver metastases may be partially explained by the role of mutant p53 in promoting cell migration and invasion , as showThe distribution of various types of p53 mutations in our study varies by tumor type. This difference may contribute to the variable prognostic value of p53 mutations in various tumor types with p53 mutations .In our series, tumors with p53 mutations had more additional aberrations than tumors with wild-type p53 (p=.002) . In both groups, the overall survival duration was longer in patients who received treatment than in those who did not. However, among patients with wild-type p53 and other aberrations, survival was superior in those who received matched therapy against the additional aberrations compared to non-matched therapy. We have previously published evidence that identifying specific molecular abnormalities and choosing therapy based on these abnormalities is associated with a longer time to treatment failure in the phase I setting than that of previous systemic therapy. Furthermore, in the non-randomized setting, rates of response, time to treatment failure, and survival were higher with matched targeted therapy than with non-matched therapy .An intriguing finding in the current report is the observation that patients with wild-type p53 and other aberrations who received matched therapy against these aberrations had longer survival than those treated with non-matched therapy or those who received no treatment . This survival advantage was confirmed in multivariate analysis after adjustment for other covariates (p=.0002). In contrast, in the p53 mutation group with additional aberrations, the survival durations were similar for those who received matched therapy against the additional aberrations, those treated with non-matched therapy, and those who received no therapy .The exact role of p53 in the context of other molecular aberrations and their prognostic significance in various tumor types needs to be elucidated. Whether p53 mutations are driver mutations or contribute to the emergence of resistance to targeted agents needs to be evaluated. Resistance to therapy seen in tumors with p53 mutation requires the development of new agents or strategies. Several investigators have reported that p53-based cyclotherapy may represent a successful strategy. -30 This Our data suggest that the identification of driver mutations in patients with wild-type p53 and other aberrations and the selection of targeted therapy may contribute to improved survival. Recently published data in head and neck squamous cell carcinoma also demonstrated that PI3K/Akt/mTOR inhibition using PF-04691502 is enhanced with induction of wild-type p53 in human xenograft and murine knockout models . The autIn conclusion, our data add to the published data demonstrating that p53 mutations are associated with a poor prognosis and resistance to treatment, and they emphasize the need to develop agents or strategies to overcome this resistance. These findings further support the need to individualize cancer therapy and should be validated in carefully designed prospective trials.We reviewed all patients who underwent testing for p53 mutation status in the Department of Investigational Cancer Therapeutics at MD Anderson Cancer Center. Molecular profiling was performed on available tissue samples from consecutive patients with advanced tumors referred to the Clinical Center for Targeted Therapy. Patients were of various ages and had advanced or metastatic cancer that was refractory to standard therapy, that had relapsed after standard therapy, or for which there was no standard therapy available. All protocols required that participants have evidence of evaluable or measurable disease according to Response Evaluation Criteria in Solid Tumors (RECIST) guidelines , 33 and The p53 mutation status was determined by either polymerase chain reaction\u2013based or next-generation sequencing in a Clinical Laboratory Improvement Amendments\u2013certified laboratory, as previously described . The nexTreatment was selected as previously published . BrieflyPatients' characteristics were analyzed using descriptive statistics. Categorical data were described using contingency tables, including counts and percentages. Continuously scaled measures were summarized by median and range. The association between two categorical variables was examined using the chi-square test. Survival and hazard functions were estimated using the Kaplan-Meier method, and survival between groups was compared using the two-sided log-rank test. Survival was analyzed according to the type of p53 mutations and the treatment. Hazard ratios with and without adjustment for potential confounding variables were estimated using Cox proportional hazard regression analysis."}
{"text": "Progression through the cell cycle is one of the most important decisions during the life of a cell and several kinds of stress are able to influence this choice. p57 is a cyclin-dependent kinase inhibitor belonging to the CIP/KIP family and is a well-known regulator of the cell cycle during embryogenesis and tissue differentiation. p57 loss has been reported in a variety of cancers and great effort has been spent during the past years studying the mechanisms of p57 regulation and the effects of p57 reexpression on tumor growth. Recently, growing amount of evidence points out that p57 has a specific function in cell cycle regulation upon cellular stress that is only partially shared by the other CIP/KIP inhibitors p21 and p27. Furthermore, it is nowadays emerging that p57 plays a role in the induction of apoptosis and senescence after cellular stress independently of its cell cycle related functions. This review focuses on the contribution that p57 holds in regulating cell cycle arrest, apoptosis, and senescence after cellular stress with particular attention to the response of cancer cells. Cells can encounter different kinds of stress during their life and in turn have evolved a wide range of responses. Stress-activated signalling pathways such as ATM/ATR, JNK/SAPK, and p38 pathways are activated in mammalian cells by DNA damage, starvation, heat and osmotic shock, and oxidative stress. Depending on the kind, severity and duration of insult, and on the cell type, these responses can lead to different final outcomes, spanning from cell survival to cell death. Cell cycle delay or arrest is often the first safety step triggered in a stressed cell, followed by injury repair and thus restoration of cellular proliferation, or by the induction of cellular senescence or cell death. in vitro, called for this reason replicative senescence. Genotoxic stress and more generally prolonged activation of the DNA damage response pathways results in the so-called premature senescence. Interestingly, cells usually arrest cell cycle in G1 phase during replicative senescence and in G2 phase during premature senescence. Senescent cells often display a flat, enlarged morphology and exhibit an increase in the lysosomal \u03b2-galactosidase activity that can be used as senescence biomarker . Many senescent cells also display a characteristic senescence-associated secretory phenotype (SASP)  is considered a master regulator of the cell cycle during embryogenesis and tissue differentiation , 14, butp57 belongs to the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors (CKIs) along with p21 and p27. The CIP/KIP family counteracts cell cycle progression inhibiting all the cyclin CDK complexes throughout the cell cycle (for a review on CKIs see ). In parDue to its role in cell cycle control, p57 is involved in the regulation of many cellular processes such as embryogenesis and tissue differentiation. In muscle differentiation p57 participates in the regulation of cell cycle exit of differentiating myoblasts , 25. Durp57 is considered a tumor suppressor gene due to its ability to inhibit proliferation and the importance of p57 in the suppression of cancer is highlighted by its mutation/inactivation in the Beckwith-Wiedemann Syndrome, a cancer predisposing syndrome . RemarkaA cytoplasmic localization for p57 has been described in non-small-cell lung carcinoma and esophageal squamous cell carcinoma , 42, sug cdkn1c, is located in the imprinted domain kcnq1/kcnq1ot1. Maternal and paternal allele of an imprinted gene display, despite the fact that they have identical sequences, different epigenetic modifications, such as DNA methylation within CpG islands, histones acetylation, and methylation. The imprinting of a cluster of genes is regulated by specific sequences acting in cis, known as imprinted control regions (ICR). In particular, cdkn1c is expressed only by the maternal allele, while the paternal allele is silent  have been reported to downregulate the expression of cdkn1c . For exaFinally, p57 protein stability is regulated by both phosphorylation and ubiquitination . In partAll the three members of the CIP/KIP family play an important role in controlling cell cycle exit. In particular, p57 is a master regulator of the cell cycle during embryogenesis and tissue differentiation , 14, butA CDK inhibition-dependent mechanism has been described for the stress-activated protein kinase (SAPK) p38 signalling, which is activated in mammalian cells by several insults, such as osmostress, oxidative stress, ionomycin, and UV \u201358. It h2-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway with a CDK inhibition-independent mechanism. Indeed, p57 negatively regulates the JNK/SAPK signalling cascade through direct inhibition of JNK/SAPK, independently of its well-known inhibitory function on CDKs [On the other hand, p57 participates in the c-Jun NH on CDKs . Deletio on CDKs , while t on CDKs , 62. Pre on CDKs , 64. It L or Bcl-2 was found to counteract p57 proapoptotic effect. The molecular mechanism by which p57 promotes cell death was investigated by Vlachos and colleagues [c release into cytosol and consequent caspase-9 and caspase-3 activation. Mitochondrial pathway specificity was confirmed, as p57 expression was ineffective in promoting death receptor-mediated apoptosis stimulated with agonistic anti-FAS antibodies. The mechanism by which p57 triggers the mitochondrial intrinsic pathway has been linked to the ability of p57 to stabilize the actin cytoskeleton by Kavanagh and colleagues [p21 and p27, the other members of the CIP/KIP family, have been reported to play a role in apoptosis , 66 and lleagues . p57 ovelleagues . They delleagues . Further\u03b2-mediated apoptosis induced by cisplatin treatment [p57 proapoptotic effects were observed in different cell lines, including HeLa cervical cancer cells, SH-SY5Y neuroblastoma cells, SKOV3 ovarian carcinoma cells, and mouse embryonic fibroblast cells . In H129reatment . However\u03b2, lipopolysaccharide, tumor necrosis factor-\u03b1, insulin-like growth factor 1, and different forms of cellular stress such as high-density culture or serum withdrawal. They found p57 reactivation only in cell lines with unmethylated promoter but not in methylated cells, leading to different outcome ranging from no effect on cell growth to G2 arrest and apoptosis, depending on cell type and type of insult. Remarkably, exogenous overexpression of p57 in p57 promoter-methylated leukemic cell lines resulted in marked cell growth arrest and induction of apoptosis, while the overexpression of p57 in partially methylated cells only resulted in a moderate inhibition of cell growth and had no impact on apoptosis, suggesting that the epigenetic status of p57 promoter can influence the cell response to stress stimuli.Kuang and colleagues performed an interesting study on different leukemia cell lines that differ for p57 promoter methylation status . They anIt is worthy of notice that the proapoptotic effects of p57 described so far have been mainly reported in cancer cells after p57 overexpression or reinduction, while antiapoptotic activity of p57 has been observed in its physiological regulation of JNK pathway (see previous section) and during embryogenesis, suggesting that cellular context drives a major contribution to the final outcome. For example, antiapoptotic activity of p57 was reported in response to green tea polyphenols administration. Catechins are known to induce cell death in many types of tumor cells, but normal human epithelial cells were found to survive in the presence of polyphenols because of their ability to induce p57 expression. p57 was able to prevent green tea polyphenols-induced Apaf-1 expression thus avoiding apoptosis . Finally Hes1. p57 infected cells or Notch1- and Notch3-silenced cells, which upregulates p57, arrest growth with a senescent morphology, SA-\u03b2-gal staining, and p16 expression [In two human hepatocarcinoma cell lines (HepG2 and SNU398) p57 overexpression has been found to affect proliferation and morphology without affecting the apoptotic machinery. In these cells, p57 expression is regulated by neither miR-221 nor the methylation status of the promoter but instead by the Notch target genepression .\u03b2-gal activity was reported to be partially reversible withdrawing p57 forced expression. Although senescent cells are thought to be resistant to apoptotic cell death, in one of the astrocytoma cell lines induced to express p57 (U373), a small subset of cells (15% of the population) was described to undergo apoptosis. Notably, Bax levels were unchanged. Why this occurs and why this particular cell line responds in a different manner to p57 induction is an interesting yet unanswered question.In a similar manner, Tsugu and colleagues have shown that p57 induction in p57-negative human astrocytoma cell lines  can block the proliferation and alter the morphology, with cells becoming large and flat with an expanded cytoplasm . These fA pivotal role of p57 in the premature senescence of vascular smooth muscle cells has been shown by Valcheva and colleagues in vitamin D receptor knockout mice . InteresIs the final outcome dependent on p57 levels? in vitro studies and overexpression of any gene can lead to experimental artefacts, which is the physiological relevance of p57 induction in vivo?As many data come fromWhich is the grade of overlapping between the three members of the CIP/KIP family?Bearing in mind that stopping abnormal proliferation is a key goal of our scientific community, is the reinduction of p57 a promising approach for cancer therapy?Do cancer cells respond in a different way from normal cells to p57 overexpression?Initially identified as a cyclin-dependent kinase inhibitor, p57 has since been shown to have different cellular roles aside from cell cycle inhibition, such as cell migration and regulation of cell differentiation. Nowadays it is emerging that p57 plays a key role also in coordinating the cellular response to stress, being able to drive to both apoptosis and cellular senescence. Different mechanisms of action, both CDK inhibition-dependent and CDK inhibition-independent, have been disclosed and, as highlighted in this review, p57 is now implicated in the crosstalk between several different pathways, among which MAPK signalling, DNA damage response, mitochondrial apoptotic pathway, and cytoskeleton organization. The findings that p57 can induce cell cycle arrest, apoptosis, or cellular senescence depending on cell types and cellular context arise several questions:p57 is now emerging as a new master regulator of cell fate and the mechanisms through which p57 participates in the cellular response to stress have been just started to be dissected."}
{"text": "The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of alterations involve p53 missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may concomitantly gain novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in various model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 in various organ systems are reviewed and their limitations discussed.The functional importance of  Numeroueatments . In addi did not . Cells esiveness , 21. Mutisplatin . In Li\u2013Futations . These np53 knockin alleles in mice provided direct in vivo evidence for the GOF activities of mutant p53. Knockin mouse models that express mutant p53R172H and p53R270H proteins, which mimic hot spot mutations that correspond to amino acids 175 and 273 in human cancers, respectively, develop tumors that exhibit a GOF phenotype in vivo, with high metastatic capacity compared to tumors in mice inheriting a p53-null allele  incidence of metastasis in mice expressing a single, mutant p53 allele  and inhibited the function of p63, properties that were essential for the invasive nature of these cells . Mutant p53-dependent sequestration of p73 from an NF-Y complex permits this transcriptional complex to function at the platelet-derived growth factor \u03b2 promoter, resulting in expression of PDGFR\u03b2 and a prometastatic phenotype  breast cancer cells, Freed-Pastor et al.  p53, bind to and upregulate chromatin regulatory genes, including the methyltransferase MLL1, MLL2, and acetyltransferase MOZ, resulting in genome-wide increases of histone methylation and acetylation. Furthermore, upregulation of MLL1, MLL2, and MOZ was found in human tumors with p53 mutants, but not in WT p53 or p53-null tumors (p53. The emerging themes by which mutant p53 exhibits its GOF are (1) through formation of mutant p53 complexes with other proteins that modify their activities  and (2) by interaction of mutant p53 with other transcription factors  that bring a potent transcriptional activation domain to promoters not normally regulated WT p53 -on-chip experiments and expression arrays using SKBR3 breast cancer cells with the ic genes . Importar et al.  identifi changes . In this culture . Lastly,tant p53 , 44. Morl tumors . In summ3 Figure , 47.p53 proteins exhibit intrinsic differences. Some are classified as structural mutants  as the mutation alters the structure of the DNA-binding domain while others are classified as DNA-binding mutants  because they alter an arginine that directly interacts with DNA. Other mutants show partial defects. For example, the p53R172P mutation, albeit rare, is able to activate the cell cycle arrest but not apoptotic programs of p53  deposition, wound healing, and chronic inflammation, which were validated by staining with various markers. Lujambio and colleagues selectively deleted p53 in hepatic stellate cells, resulting in modifications to the tumor microenvironment (TME) and enhanced malignant transformation of epithelial cells   , 60\u201363. ID hosts . Hill etID hosts  further p53 proteins exhibit GOF activities, a growing body of evidence suggests that tumor cells may be addicted to mutant p53 expression. Experiments using siRNA knockdown of mutant p53 in cancer cell lines showed a higher apoptotic response to drug treatment in cells with knockdown of mutant p53  in 3D culture leads to phenotypic reversion to more normal, differentiated structures with hollow lumens  or whey acidic protein (WAP) promoters which are hormonally regulated and therefore do not simulate expression of mutant p53 from the endogenous locus   replete with p53-heterozygous stromal elements. CAFs, macrophages, T cells, neutrophils, endothelial cells, and so on remain heterozygous for p53 following conception (due to the presence of the STOP cassette) with undefined effects on tumor biology. Stroma is a requisite component of tumor initiation and growth, and as mentioned earlier, prostate tumor epithelium selects for p53-null stromal fibroblasts in p53\u00b1 mice, yielding a highly proliferative stroma that contributes to tumor progression. Given the powerful roles of p53 in cellular plasticity and embryonic stem cell differentiation, tumors that develop from and under conditions of p53 heterozygosity may differ in unappreciated ways from human tumors that initiate from WT p53 cells. A model that more closely mimics the events in sporadic tumorigenesis is sorely needed.Knock-in mice with germline mutations in tivities , 25. Yetus locus , 71. Curp53 carry the potential to inform us of essential mechanisms of cancer initiation and metastasis translatable to therapeutics in humans. However, many genetic mouse models used to study mutant p53 in vivo incorporate germline mutant p53 alleles that may alter normal and cancer biology in ways that compromise its relevance to human cancer. Innovation in genetic mouse models of mutant p53 is mandatory to more closely model human biology and to serve as translational biologic platforms to better evaluate and develop novel therapeutic agents in human cancers. Moreover, given the importance of the TME in cancer development and metastasis, mouse models that preserve the complex regulatory and tumor\u2013stromal interactions are mandatory to the development of effective, translational biologic platforms to target the TME toward therapeutic ends.Mouse models of mutant MK, YZ, and GL wrote and edited the manuscript. MK and YZ generated the figure.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."}
{"text": "About 15 years ago it was proposed that generation of the truncated protein p25 contributes to toxicity in Alzheimer\u2019s disease (AD). p25 is a calcium-dependent degradation product of p35, the principal activator of cyclin-dependent kinase 5 (Cdk5). The biochemical properties of p25 suggested that its generation would cause Cdk5 overactivation and tau hyperphosphorylation, a prerequisite for neurofibrillary tangle (NFT) formation. Whilst this model was appealing as it explained NFT formation, many laboratories could not confirm the finding of increased p25 generation in brain from AD patients. On the contrary, it emerged that p25 levels are reduced in AD. This reduction occurs primarily in the early stages of the disease. Further, p25 generation in the mouse hippocampus is associated with normal memory formation and p25 overexpression enhances synaptogenesis. Therefore, it transpires that p25 generation is a molecular memory mechanism that is impaired in early AD. I discuss the prospect that investigation of p25-regulated proteins will shed light into mechanisms underlying synaptic degeneration associated with memory decline in AD. Figure 1) was as follows: Abnormal APP processing generates amyloid peptides that oligomerize thereby enhancing calcium signaling (In the late Nineties Li-Huei Tsai\u2019s lab published a paper proposing that the truncated cyclin-dependent kinase 5 (Cdk5) activator p25 is specifically formed in sporadic Alzheimer\u2019s disease AD; . This fiignaling . This woignaling . p25 forignaling . Additiodownregulation of p25 expression in AD forebrain. This decreased p25 expression was not simply due to neuronal loss, as it was adjusted to the expression of a neuronal marker. However, the downregulation of p25 expression was not discussed by the authors, possibly because p25 expression was considered toxic in line with Tsai\u2019s lab. Instead, the key point of discussion was that p25 upregulation was not reproducible in postmortem AD tissue. It was realized that postmortem delay is a variable to consider, since p25 can be formed from degradation of p35 before the tissue is frozen (p = 0.06)   and in t = 0.06) . Recentl = 0.06) . In our  = 0.06) , but we  = 0.06) . Taken t = 0.06) .Motivated by the How can p25 expression be reduced in AD? A reduction in p25 expression indicates that p25 formation occurs under normal physiological conditions, and that such physiological p25 generation is impaired in AD. In agreement with this, spatial memory formation is associated with p25 generation in the mouse hippocampus . ImportaThe importance of p25 generation for spatial memory has been tested in various mutant mouse lines. First, mutant mice expressing low levels of p25 under control of the \u03b1CaMKII promoter which is active in postnatal forebrain, show improved spatial memory formation . Second,Overexpression of physiological levels of p25, or transient expression of high levels of p25 enhance spatial memory formation . In thesIn AD synapse loss, which precedes neuronal loss, correlates best with impaired memory formation . Thus, sCdk5 and glycogen synthase 3 (GSK-3) are thought to be the main tau kinases . In AD rFigure 1). Impaired p25 generation in AD is expected to contribute to synaptic degeneration in the disease. However, more studies are needed to establish the precise synaptic function of p25. For example, p25 generation could be a signal from synapse to nucleus to stimulate gene expression needed for long-lasting synaptic plasticity. Recent evidence also shows that the expression of p25-regulated synaptic proteins is downregulated in early AD hippocampus. Analysis of these synaptic downregulations promises to give insight into the mechanisms of early synaptic degeneration in AD. So far, it has emerged that p25-regulated proteins are downregulated at the pre- and post-synaptic side, suggesting that degeneration occurs equally at both sides of the synapse. This is consistent with the finding that oligomeric amyloid peptide binds to pre- and post-synapses and has the potential to induce dysfunction on both sides of the synaptic cleft (In recent years it has emerged that p25 is not an exclusively toxic molecule. Spatial memory formation leads to p25 generation in the hippocampus, where p25 can stimulate synthesis of synaptic proteins followed by synaptogenesis and synaptic strengthening. In the early stages of AD hippocampal p25 expression is reduced. Taken together, this suggests that p25 generation is a memory mechanism that is affected in early AD (ic cleft . It is hThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
{"text": "JASPAR (profile score threshold 80%) and TFSEARCH datasets were used to search transcriptional start sites for p53/p63 response elements. Remarkably, these data revealed 63 microRNAs that targeted p63. Furthermore, there were 39 microRNAs targeting p63 that were predicted to be regulated by p63. These analyses suggest a crosstalk between p63 and microRNAs. Here, we discuss the crosstalk between p63 and the microRNA network, and the role of their interactions in cancer.The transcription factor p63 is a member of the p53 gene family that plays a complex role in cancer due to its involvement in epithelial differentiation, cell cycle arrest and apoptosis. MicroRNAs are a class of small, non-coding RNAs with an important regulatory role in various cellular processes, as well as in the development and progression of cancer. A number of microRNAs have been shown to function as transcriptional targets of p63. Conversely, microRNAs also can modulate the expression and activity of p63. However, the p63\u2013microRNA regulatory circuit has not been addressed in depth so far. Here, computational genomic analysis was performed using miRtarBase, Targetscan,  The regulation of gene expression is implicated in multiple molecular mechanisms, which include transcriptional, translational, and post-translational regulatory mechanisms , 2. In aP63 is a member of the p53 gene family of transcription factors . Like otAlthough p63 has been shown to closely associate with cancer development, the underlying molecular mechanisms have not been fully elucidated. In fact, many studies indicate that transcriptional regulation of p63 is one of the most important mechanisms in various tumor processes by triggering the expression of oncogenes , 23. TApmiRNAs are 18- to 24-nucleotide non-coding RNA molecules . It regumiRNAs are canonically produced through a multistep process and transcription factors are recognized as key regulatory points for miRNA production . AbnormaThe human p63 gene belongs to the p53 family and plays a crucial role in maintaining genomic stability and suppressing tumor formation . AlthougThe p63 gene contains two promoters and produces two diametrically opposite groups of isoforms: those containing the transactivation (TA) domain (TAp63), and those lacking it (\u0394Np63) . \u0394Np63 iTAp63 is transcribed from the first transcriptional start site within the p63 gene and contains the transactivation domain and three different C-termini termed \u03b1, \u03b2 and \u03b3 . The higThe role of p63 in tumorigenesis is very complex. Thus, more work is needed to explain how the differential expression of the p63 isoforms could influence this process. Considering the fact that all p63 isoforms contain a DNA binding domain and are able to bind to thousands of gene promoters, studying their interaction with regulatory genes may not only increase our understanding of the role of p63 in tumorigenesis, but could also open the door to the development of innovative diagnostic procedures and targeted therapies.miRNAs are small, non coding RNAs with a length of 18 to 24 nucleotides . They plmiRNAs are encoded throughout the human genome. Most miRNAs are found within intragenic regions as well as protein-coding or non-coding transcriptional units . The proAs discussed previously, miRNA biogenesis is a complex multistep process, and each step of this process is tightly regulated. Mutation or aberrant expression of any component of the miRNA biogenesis machinery could contribute to a dysregulation in miRNA expression in tumors. The majority of miRNAs are transcribed by RNA polymerase II and III, and transcription factors significantly contribute to the activation of their expression by directly binding to the promoters of miRNAs. Recent evidence indicates that p63-mediated activation of miRNA transcription is an important event in the regulation of miRNA levels , 95.Dysregulation of miRNA expression is common in human cancers. However, the mechanisms underlying miRNA dysregulation are not clear. As described above, transcriptional regulation is one of the most important steps in the synthesis of miRNAs. Transcriptional activation and repression of specific microRNAs by \u0394Np63\u03b1 was shown to attenuate the expression of several proteins involved in cell death and survival and to regulate tumor cell resistance to cisplatin . In humaAnother important transcriptional program that has been identified involves post-transcriptional control of miRNAs by p63. The cropping step regulated by the Drosha/DGCR8 heterodimer is an important event in regulation of miRNA levels. There has been no direct study to prove that p63 can directly regulate the expression of Drosha. However, it has been reported that PY domain-containing proteins interact with WW domain-containing proteins. Interestingly, p63 contains a PY domain , while DFurthermore, p63 can control the expression of miRNAs by regulating the expression of miRNA transcription factors, as well as several key regulatory factors that allow for the correct maturation of primary miRNA. As an example of the former, the gene of the transcription factor early growth response 2 (EGR2) contains a p63 RE and is therefore a direct target of p63 . InteresAs discussed above, p63 has been defined as a regulator of miRNAs. Conversely, p63 expression can also be controlled by specific miRNAs in certain tissues. Recent studies identified p63 as one of the conserved targets of miR-203 \u2013114, andAdditionally, Papagiannakopoulos T et al. showed for the first time that miR-21 targets the tumor-suppressive protein TAp63, and confirmed that miR-21 is necessary for suppression of apoptosis in glioblastoma cells . Wang T Given these findings, p63 isoforms regulated by miRNAs can be divided into two classes: \u0394Np63-isoforms, which enhance cell proliferation and repress apoptosis and are repressed by miR-92 and miR-203, and Tap63-isoforms, which induce cell cycle arrest and apoptosis and are repressed by miR-21, miR-30 and miR-302  have been validated , 130. A It is now apparent that many tumors are dependent upon a dysregulation of p63 and miRNAs, providing a rationale for therapeutic intervention of p63 and miRNAs. Therefore, the p63/miRNA network represents a novel approach for treatment and is likely to lead to new therapeutic anticancer strategies."}
{"text": "Lymph node metastases indicate increased tumor burden and tumor cell heterogeneity and affect both the treatment strategies and the prognosis. In this study, expressions of NF-\u03baB, MMP-1, p53, and Ki-67 were between the primary tumors and lymph node metastases in 110 Dukes' stage C, CRC cases by immunohistochemical methods, related to patients' clinical outcomes. NF-\u03baB, p53, and Ki-67 expressions were significantly higher in the metastatic lymph nodes compared to the primary tumor tissues . In the metastatic lymph nodes NF-\u03baB expression was correlated with both p53  and Ki-67  expressions. The univariant and multivariant analyses showed that only \u201cpT stage\u201d preserved an independent prognostic significance for recurrence-free survival rates and 5-year overall survival rates (P < 0.001 for both). Metastatic cells can acquire different biological characteristics compared to their primaries. Elucidation of properties acquired by metastatic cells is important in order to better determine prognosis, reverse drug resistance, and discover new treatment alternatives.Colorectal cancer (CRC) is the third most frequent malignancy. Many factors such as NF- Colorectal cancer (CRC) is one of the most common malignancies that has high morbidity and mortality throughout the World, especially in developed countries , 2. ExisThe variations in the biological characteristics of tumor cells in the metastatic tissues have an influence on their ability to survive . A varie\u03baB signaling is of importance to cell proliferation, apoptosis, and creation of the inflammatory response and has been found to be implicated in the development of many cancers [\u03baB affects CRC development and especially carcinogenesis after chronic inflammations. Several studies have revealed that NF-\u03baB can be used as a marker of resistance to adjuvant chemotherapy in CRC [NF- cancers . There hy in CRC \u201314.\u03baB and MMP. NF-\u03baB activation has been reported to be involved in the regulation of some MMP types [There is also evidence that matrix metalloproteinases (MMPs) might be a modulator of tumor progression and spread, which affect the prognosis of the disease \u201317. In aMP types , 24.It has been shown that mutations of p53, as a well-defined tumor suppressor gene, contribute to the development of many cancer types and that p53 is a poor prognostic factor in many cancer types \u201330. It w\u03baB, MMP, p53, and Ki-67 production and the clinical behavior of metastatic tumor cells.Ki-67 is a proliferation marker, observed in all phases of cell cycle except G0. High Ki-67 expression is a poor prognostic factor in many tumors and there are correlations between Ki-67 expression and expression of other poor prognostic factors. In a previous study, Ki-67 expression was found to be higher in metastatic lymph node tissues compared to primary in breast cancer . In CRC,\u03baB, MMP-1, p53, and Ki-67 expressions in the primary tumor tissues and metastatic lymph nodes were compared in Dukes' stage C, CRC cases.A series of unique biological features may affect the ability of cancer cells to create metastasis. It is important to reveal these biological characteristics of metastatic cells in CRC in terms of development of new therapeutic strategies. However, to our knowledge, studies are needed on the variations of expression patterns of these markers in the metastatic lymph nodes in CRC cases. Therefore, in this study, NF-A total of 457 CRC patients were operated on in Adnan Menderes University Hospital, Aydin, Turkey, between August 1998 and December 2010. Of these, 152 with stage Dukes' C were evaluated in this study; however, 42 were excluded as either their paraffin blocks were not available for pathologic review and/or their clinical follow-up data were incomplete. None of the patients had received chemotherapy and/or radiotherapy in the preoperative period. There was no history of previous cancer, familial polyposis coli, or preexisting inflammatory bowel diseases. Preoperative evaluations, based on clinical examination, complete blood count, blood biochemistry, chest X-ray and/or computed tomography (CT), abdominal ultrasonography and/or CT, and bone scintigraphy did not show distant metastasis in any of the patients. Ethical approval was obtained from the ethical committee of the university (ADUBAPTPF-12.0141). Tissue samples from both the primary tumors and the metastatic lymph nodes were obtained in all 110 patients. The selected samples did not include the areas of necrosis, hemorrhage, inflammation, or technical deformation. The samples from the primary tumors were representative of the histological grade of each tumor and the samples from the lymph nodes (LN) were obtained from the largest area of metastases. Microscopic examinations of the haematoxylin-eosin- (HE-) stained slides obtained from the paraffin blocks were used to confirm the diagnoses.\u03bcm in thickness obtained from selected paraffin embedded blocks were put on positively charged slides. Second, all the sections were deparaffinized in xylene and dehydrated through a graded series of ethanol solution. Third, antigen retrieval was performed at 96\u00b0C  for 40 minutes in a thermostatic bath (PT Link). The sections were incubated with NF-\u03baB/p50 , MMP-1 , anti-Ki-67 monoclonal antibody MIB-1 , and anti-p53  for 60 minutes at room temperature. Positive and negative controls were added for each antibody and to each batch of staining. A streptavidin-biotin enhanced immunoperoxidase technique  in an automated system was used to show immunoreactions. The sections were incubated with DAB and counterstained lightly with haematoxylin to demonstrate binding. Finally, the sections were dehydrated and mounted onto the slides. The slights known to be positively immunostained were used as positive controls. Normal rabbit serum IgG was used to replace primary antibody as a negative control.All immunostaining was carried out at room temperature using DAKO Autostainer Universal Staining System . First, sections 4 Sections were reviewed by 3 pathologists , who were blinded to the patients' clinicopathological characteristics and follow-up information, and discrepancies (about 12% of cases) were resolved by joint discussion of the slides viewed with a multiheaded microscope. Immunostaining was scored (as described by Zhigang and Wenlv) based on the intensity of staining and the percentage of cells that stained positively . The intP < 0.05) to identify the independent factors of recurrence. Survival rates were calculated by the Kaplan-Meier method and compared by the log-rank test. Stepwise forward Cox regression model was conducted for parameters found to be significantly associated with survival by the log-rank test (P < 0.05) in order to identify the independent factors of survival. Values of P < 0.05 were considered significant in all analyses.All statistical analyses were performed with SPSS . The Spearman correlation test was used to determine correlations between the expression of biological markers in the primary tumor and the expression of these markers in the lymph nodes. The Chi-squared test or Fisher's exact probability test was used to compare the recurrence rates. Logistic regression analysis was conducted on the parameters found to be significantly associated with recurrence by the Chi-squared tests or Fisher's exact probability test . However, the rate of cells stained with MMP-1 was not statistically different between the primary tumor and the metastatic lymph nodes (P = 0.07).The rate of cells stained (moderate + strong) with NF-\u03baB expression was significantly correlated with p53  and Ki-67 expression  in the metastatic lymph node tissues. However, NF-\u03baB expression was not correlated with p53  and Ki-67  expression in the primary tumor tissues. MMP-1 expression was not correlated with any markers in the primary and the metastatic tumor tissues. Likewise, there was not a significant difference between p53 and Ki-67 expressions.NF-r = 0.518, P = 0.018) was significant. Similarly, the size of the primary tumor was correlated with Ki-67 staining in the primary tumors . Furthermore, expressions of the other markers in the primary tumors and metastatic lymph nodes were not associated with any clinicopathological parameters.The correlation between immunohistochemical Ki-67 staining in the primary tumors and the number of metastatic nodes (P = 0.01) and 5-year overall survival (OS) (P = 0.01) rates. No significant correlations were observed among other clinical parameters and the immunohistochemical staining scores of the studied markers. In the univariant and multivariant analyses only \u201cpT stage\u201d of the tumor was a significant independent prognostic factor for both RFS and 5-year OS rates   the 5-year overall survival rates were 46.8%. Recurrence was observed in 42 patients (38.2%) and 68 (61.8%) had died during the follow-up. The recurrence rates in pT2, pT3, and pT4 tumors were 22.2%, 33.3%, and 55.2%, respectively. The 5-year overall survival rates in pT2, pT3, and pT4 tumors were 69.9%, 55.3%, and 32.8%, respectively. There were statistically significant correlations between the pT stage and both recurrence-free survival (RFS)  .Although CRC is one of the most common malignancies, carcinogenesis and many factors influencing the clinical course of this disease have not been completely described. Understanding the pathogenesis and the factors affecting the development and the progression of CRC would result in better prediction of the disease course and selection of appropriate targeted treatments for patients refractory to classical chemotherapy and radiotherapy . It is o\u03baB has an important role in many physiological events such as apoptosis, inflammation, and cell proliferation. Its activation has a regulatory role in expression of more than 400 gene products associated with inflammation, cell survival, proliferation, invasion, and angiogenesis [\u03baB in hematological and solid tumors, previously [\u03baB have been shown to have a relation with the development and the progression of solid tumors. Aberrant NF-\u03baB has been shown to be effective through increased proliferation and antiapoptotic mechanisms in colon cancer development as in many other types of cancer. NF-\u03baB is known to be associated with CRC development by increasing inflammation [\u03baB expression was higher in the lymph node metastases than in the primary tumors, which is consistent with the results of other previous studies showing that NF-\u03baB plays a role in cancer development and progression [\u03baB expressions in CRC. Increased NF-\u03baB expression in lymph node metastases indicates a potential role of this molecule in the metastasis process of CRC. Although NF-\u03baB is thought to play a role in early stages of CRC development, the idea that it can also be involved in later stages and metastases is supported by the findings of the present study.NF-ogenesis \u201312 Many eviously . The subammation . In the gression . The res\u03baB. In one study, the rate of p53 overexpression was as high as 73% in primary CRC and 66% in distant metastases [\u03baB. Actually, the relation between NF-\u03baB and p53 is defined well. Increased NF-\u03baB and p53 expressions in metastatic cells lend support for the idea that these two molecules are multifunctional coregulators. The proliferation marker Ki-67 is utilized as a prognostic indicator, and it is suggested that a biomarker-based approach can be an effective strategy to improve results of adjuvant treatments [Another finding of the present study is that p53 and Ki-67 expressions were significantly higher in the metastatic cells than in the primary tumor cells. As in many other tumors, in CRC, mutations which may occur in the p53 gene may disrupt the activity of NF-tastases . The p53tastases , 27. Howtastases . The preeatments . The res\u03baB with MMPs suggesting that migratory genes may support NF-\u03baB in inflammation and carcinogenesis [\u03baB triggers many extracellular events like tumor angiogenesis, it is very likely to have a relation with MMP-1. In the current study, a positive correlation was not found between MMP-1 and NF-\u03baB expressions, and also MMP-1 expression was decreased nonsignificantly in the metastatic tumors. This may be partly related to the complex stromal structures of metastatic lymph nodes and their microenvironment [MMP-1 is frequently found in aggressive tumors. It may not be the basic factor in the initial stage of tumor formation and invasion; however, it plays an effective role in advanced stages of tumors. MMP-1 is the leading enzyme which plays a role in destruction of collagens 1, 2, and 3, the most common stromal structures between the tissues. It has an active role in various tumors such as gastric cancer, breast cancer, squamous cell carcinomas of the head and the neck, colon carcinomas, and adenocarcinomas of pancreas and lungs , 22. In ogenesis , 24, 34.ironment .\u03baB and both Ki-67 and p53 in the metastatic lymph nodes.The present study also revealed that Ki-67 expression was related to the size of the primary tumor and the number of metastatic lymph nodes. A significant positive correlation was found between NF-\u03baB signal pathway, in cancer cells so far. In the present study, NF-\u03baB, as a transcription factor involved in many physiological and pathological processes, was shown to play a role in CRC progression. It is of particular importance that NF-\u03baB expression was correlated with poor prognostic factors including p53 and Ki-67. At present, many targeted molecules, such as Bortezomib which increases apoptosis by reducing NF-\u03baB levels and decreases resistance to chemotherapy, are under research for the treatment of CRC [\u03baB, which inhibits nuclear translocation, can be developed. As in the present study, revealing changes in NF-\u03baB in primary and metastatic tumor cells in various types of cancers may accelerate the development of drugs against this molecule.Many potential target molecules have been studied to determine whether they could inhibit NF-t of CRC , 36. In CRC development is a multifactorial and multistep process. Activation of many oncogenes and inactivation of tumor suppressor genes play roles in this process. Many genetic and epigenetic changes are incorporated in the processes of normal colorectal mucosal epithelial transformation into carcinoma and tumor progression. CRC, with the exception of some well-defined familial syndromes, is mostly sporadic. Approximately 90% of CRC cases are thought to develop from colorectal adenomas with classic adenoma-carcinoma sequences . Changesp53 is a molecule in cell replication that arrests the cell cycle when there is DNA damage and when the damage cannot be repaired it leads to apoptosis. As a result of the mutation of p53, cells with damaged DNAs are likely to lead to cancer. Despite thousands of studies in literature, critical mechanisms in which p53 has been involved in carcinogenesis have not yet been fully explained. Mutated p53 has been observed in approximately 50% of all cancer cases and 60% of CRC cases, such that it is called \u201cthe guardian of the genome\u201d . MutatedIt is known that an increase in proliferation/apoptosis rate characterizes oncogenesis due to the proliferation of the damaged cells that lose their apoptosis functions as a result of the p53 mutation. In this study, a higher Ki-67 proliferation rate in metastatic lymph nodes than the primary tumor cells may represent the increased mitotic capacity of metastatic cells in CRC.\u03baB is a well-known transcription factor that has a role in development and progression of many cancers. NF-\u03baB is claimed to have a role in apoptosis, angiogenesis, and proliferation effects and development of cancer. Higher expressions of NF-\u03baB in adenomas than that of normal mucosa have been previously stated in colorectal cancer development [\u03baB, like p53 and Ki-67, has been found to have a higher expression in metastatic lymph nodes than primary tumors in CRC patients.In addition to playing an important role in inflammation and immune responses, NF-elopment , 41, 42.\u03baB can have a role in the development of CRC lymph node metastasis. These results showed that tumor cells in CRC lymph node metastasis may display a different biological behavior as compared to that of the primary tumor cells. In CRC patients, adjuvant chemotherapy applications affect the prognosis in lymph node metastases and in view of these results, more aggressive primary tumor cells with increased proliferation and decreased apoptosis rates seem to have caused the lymph node metastases.In CRC, many factors may have a role in tumor progression. According to the results of our study, p53 and NF-In recent years, with the integration of targeted treatments better clinical results are obtained in some selected cancer patients . In our \u03baB activity has a unique role in the course of CRC. Increased NF-\u03baB, p53, and Ki-67 expressions in metastatic tumors may have negative effects on either the natural clinical course or the response to treatments. Unfortunately, the data on microsatellite instability and KRAS mutations are (mostly) not available since this is a retrospective study. These markers would be useful since the presence of such lesions may interact with several pathways such as p53 mutations and also clinical outcomes. The finding that metastatic tumor cells in the lymph nodes have biological features differing from those of the primary tumor cells is particularly important in development of future treatment alternatives, such as NF-\u03baB inhibitors. The results of this study also indicate that in CRC progression many other markers' expressions need to be researched.In view of the results of this study, it seems that NF-"}
{"text": "The Editorial on the Research TopicHuman Tumor-Derived p53 Mutants: A Growing Family of Oncoproteinsp53 gene are collectively the most common event in human cancers. These do not merely reflect a loss of the tumor suppressive function of wild type (wt) p53 but are also selected during tumorigenesis for their acquired gain-of-function (GOF), together contributing to multiple hallmarks of cancer. Over 30\u2009years of extensive study into wt p53 provided a wealth of information about its regulation, functions, and contribution to cancer prevention. Albeit, with a significant delay, the interest in mutant p53 has been growing fast over the past decade with the realization that most cancer patients present tumors with mutant p53, and these particularly manifest in aggressive and metastatic diseases. The growing understanding of mutant p53 exposes attractive therapeutic opportunities with wide clinical applications, which coincidently raise many challenging questions concerning associated complexities. In this research topic, we assembled 12 reviews exposing some critical issues and discussing prospect development in this field.Mutations in the tumor suppressor Kim et al.). The Lozano group also emphasized the biological and biochemical differences between different mutants, even between different substitutions of the same amino acid, such as p53R172H versus p53R172P. Hence, not all p53 mutants are equal. While the abovementioned mouse models for inherited p53 mutations (the Li\u2013Fraumeni model), this represents a small fraction of p53 mutations in human cancers. This key point was discussed by the Lozano group highlighting the limitations of the current mouse models for sporadic p53 mutations in cancer. They discuss the problems with the current conditional mutant p53 models in which all cells are heterozygous for p53 from conception and hence do not faithfully mimic the role of mutant p53 in sporadic tumor development. These models lack the challenging context of the cells with wt p53 that normally comprise the tumor microenvironment and its inherent immune cells. There is a clear need for more sophisticated mouse models to better define the distinct roles of mutant p53 in these compartments. The Del Sal group  discussed the difference between the mouse and human mutant p53 exemplified by the p53R249S mutation, which exhibits GOF in human cells, but this had not been recapitulated in the relevant mouse model. A comprehensive list summarizing studies from the last decade is provided in the Del Sal review outlining which mutants GOF effects have been validated and the associated information.One of the most commonly used mouse models for cancer is the p53 knockout mouse. However, this model of p53 deficiency does not represent the majority of human cancers. A major leap in the understanding of mutant p53 regulation and GOF was derived from mouse genetics , 2. The Sabapathy discussed this important point in detail, emphasizing the timing and conditions under which dominant negative (DN) effects of mutant p53 occur, and when and how this would impact on tumorigenesis. His conclusion from the literature is that stress, whether acute  or chronic (activated oncogene), accumulates mutant p53; however, it is under the latter conditions that mutant p53 promotes tumorigenesis. Further complexity to this is the tissue specificity of the DN effect as learned from the KI heterozygote mouse models (Sabapathy). In the wake of the DN effect, there is often a loss of heterozygosity (LOH) of the wt allele. However, Walerych et al. discussed that LOH is also tissue specific, as exemplified by the work of Rotter and coworkers demonstrating that, in the embryonic stem cells of the KI mutant p53 mice, the LOH can be of either wt or mutant p53 alleles, potentially acting to control cell fate checkpoint (Sabapathy).Mutant p53 GOF requires the accumulation of the mutant protein and that, at least initially, it acts dominantly over the wt protein. Vijayakumaran et al. summarizes the differences and similarities in the regulation of wt and mutant p53. While both wt and mutant p53 are inherently labile proteins and accumulate in response to stress, only the mutant form remains stable. Intriguingly, wt and mutant p53 share many of their regulatory mechanisms. However, the loss of the key negative autoregulatory loops due to mutation in p53 result in the sustained accumulation of mutant p53 following stress conditions or exposure to oncogenic stress in cancer cells. This, together with a loss of specificity of additional E3 ligases toward mutant p53, provides an explanation for the accumulation of mutant p53. The additional complexity of p53 regulation, both wt and mutant p53, by microRNA (miRNA) is presented Vijayakumaran et al. This reveals the ways by which p53 can be deregulated in cancer but, at the same time, may define potential new therapeutic targets.A major requirement for GOF by mutant p53 is a constant stabilization of the mutant p53 protein, unlike the temporal accumulation of wt p53. The review by Giorgi et al. discussed the cytoplasmic apoptotic activities of wt p53, and the loss of these activities by mutations in p53. To date, there is no evidence for a GOF of mutant p53 directly regulating these activities . On the other hand, it was reported that mutant p53 proteins can aberrantly cooperate with known transcription factors by leading to disregulated gene expression. This results in increased proliferation, invasion, genomic instability, and chemoresistance. The interaction of mutant p53 with p53 family members p63 and p73 is key to some of its GOF. Ferraiuolo et al. review the intricate relationship between mutant p53 and p63 or p73. Mutant p53 proteins can also hamper tumor suppression transcriptional programs by binding to and displacing the p53 family members p73 and p63 from their consensus of target gene promoters . How are these plethora of activities achieved by mutant p53? At least two major mechanisms have been reviewed in this series. First, is by controlling gene expression through the alteration of specificity of certain transcription factors. Second, is by affecting chromatin remodeling through SWI/SNF and MLLs/MOZ . The effect of mutant p53 on MLLs/MOZ is achieved through ETS2, as reviewed in detail by Martinez. He discusses the mechanism by which mutant p53 protects ETS2 from degradation, and how this, in turn, affects the overall transcriptional effects of the ETS family and contributes to the oncogenic phenotype of mutant p53, such as increased nucleotide metabolic genes (Martinez). Bruno et al. reviewed the relationship of p53 with the cofactor Che-1/AATF. This provides an interesting example of a factor that acts as an activator and protector of both wt and mutant p53. In response to DNA damage, Che-1 induces the expression of wt and mutant p53, but activates wt p53 to induce growth arrest genes. In the case of mutant p53, it induces its expression and consequently the sequestration of p73 from apoptotic target genes, hence promotes survival .Beyond p53 family members, Haupt et al. reviewed the major tumor suppressive pathways, which are subverted by mutant p53, including PTEN, PLK2, and PML, which control the cell cycle and the latter also the circadian clock. Intriguingly, mutant p53 deregulates cellular metabolism including glucose, lipid, and nucleotide metabolism, ensuring the sufficient supply of building blocks to support tumor growth ; compounds that deplete mutant p53 expression, where HSP90, in particular Ganetespib, is the most advanced drug currently in phase III clinical trial; and explore other approaches, which are currently used for other oncogenes, including knockdown and read-through of premature termination . This review was complemented by two focused reviews on mutant p53 therapeutics. The first by Bykov et al., which focused on the mechanism of action by APR-246, including the refolding of mutant p53, the impact on mutant isoforms of p53 family members p63 and p73 and the effect on the cellular redox regulators, primarily glutathione and thioredoxin, to enhance oxidative stress. The potential of APR-246 as a single agent and in combination with DNA damaging agents is discussed, and the current clinical status of APR-246 and prospects are outlined . The second therapeutic review by Burgess et al. focused on MDM2/MDMX targeted therapies. This review provides a thorough overview of the current drugs and approaches to target p53 via MDM2 and MDMX pathways. They outline the clinical development of current MDM2 targeting compounds. Importantly, they discuss the major hurdle in this approach, which is severe cytopenias. Although, this approach has not been designed to target mutant p53, the relevance of mutant p53 to this therapeutic approach and the availability of appropriate biomarkers were discussed .A major consequence of mutant p53 GOFs is the acquired dependence of cancer cells on the expression of mutant p53. This dependence, which has been termed oncogenic addiction to mutant p53 has been discussed by multiple contributors to this series, highlighting its importance. Evidence for this addiction has been discussed by the Lozano, Sabapathy, and Del Sal groups. This addiction defines an Achilles Heal with important clinical implications. Overall, this series of reviews on mutant p53 expose the pivotal role of mutant p53 as an oncogenic driver and outline the fast advancement in our understanding of its regulation and oncogenic activities. Our deeper understanding of mutant p53 also highlights clear limitations, such as the differences between mutants p53 proteins and between mouse and human mutant p53. The lack of appropriate mouse models for somatic p53 mutations, which represents the most common event in human cancer is a major hurdle to our understanding of mutant p53 to the cancer cell versus the microenvironment. The series also reflects the excitement around the clinical opportunities and current clinical development but highlights the need of potent molecules to specifically target mutant p53, given its prevalence in human cancers. It has also been increasingly clear that mutant p53 proteins are not a single entity, but they behave as a family of oncoproteins whose deciphering and therapeutic tackling might impact enormously on the success of threatening the vast majority of human cancers.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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."}
{"text": "Patients harbouring this genetic anomaly are commonly resistant to standard therapy. Thus, various p53 reactivating agents have been developed in order to restore its tumour suppressive abilities. Small molecular compounds, especially, have gained popularity in its efficacy against myeloma cells. For instance, promising preclinical results have steered both nutlin-3 and PRIMA-1 into phase I/II clinical trials. This review summarizes different modes of p53 inactivation in myeloma and highlights the current p53-based therapies that are being utilized in the clinic. Finally, we discuss the potential and promise that the novel small molecules possess for clinical application in improving the treatment outcome of myeloma. TP53 encodes for p53 tumor suppressor protein. Deemed the guardian of the genome, p53 safeguards the integrity of the genome and ensures that the tissue homeostasis is kept in check. Under normal physiological conditions, cellular p53 is expressed at low levels, thereby turning off the activity of p53 network. With stress induction, p53 is stabilized by means of posttranslational modifications, such as phosphorylation and acetylation [Located at the chromosome 17p13.1,tylation . The subtylation .Thirty years of intensive research on p53 have yielded significant understanding of its structure and basic functions. The high percentage of patients with p53 germline mutations succumbing to a wide range of cancer in Li-Fraumeni syndrome , 2, 4, 5In MM, mutation of p53 gene is a rare occurrence at diagnosis; however, the incidence increases as the stage of disease advances, suggesting its essential role in disease progression \u20138. OveraDeletion of chromosome 17p13 region, which contains the p53 gene locus, is a recurrent cytogenetic abnormality in MM and has been associated with less favorable outcome \u201311. p53 Fifty percent of cancer harbours p53 mutations, while in the remaining 50%, the wild type p53 is deemed to lose its function via various mechanisms that affect the expression and activity of p53. The main inhibition mechanism of p53 has been described to be the amplification or overexpression of its negative regulator mouse double minute 2 homolog (MDM2). MDM2 is an E3 ubiquitin ligase which promotes proteasomal degradation of p53 as well as inhibiting the transactivation domain of the tumor suppressor protein \u201323. UndeMDM4, a homolog of MDM2, does not have E3 ubiquitin ligase activity but inactivates p53 by binding to and inhibiting the transactivation domain of p53. Due to its essential role in inactivating p53, MDM4 dysregulation in cancer has also been receiving important attention lately , 25. In  TP53 is also a subject of intense research of late. Deregulation of miRNAs in cancer is being rigorously explored and this has led to the hypothesis of the role of this group of noncoding genes in the pathogenesis of MM [On top of that, epigenetic regulation ofis of MM , 31. miRis of MM , 33. Impis of MM . Introduis of MM .In addition, epigenetic factors are also possible regulators of the expression and the activity of p53. For instance, the deregulation of p14ARF has been reported to be responsible in abolishing the integrity of the p53 pathway . ARF has TP53 gene itself has also been demonstrated in human myeloma cell lines [Hypermethylation of the promoter region ofll lines , 40. Revll lines , 40.Collectively, these findings describe the diverse mechanisms of p53 inactivation in multiple myeloma.Due to the fact that p53 is the nexus of various tumor suppressive pathways, it is imperative to study the means of reactivating or restoring p53 functions in human cancer in order to revert or rescue cells from resistance towards standard chemotherapeutic treatments. In fact, many anticancer drugs induce apoptosis through multiple pathways that are at least in part dependent upon functional p53 activation. In the late 1980s and early 1990s, the introduction of wild type p53 gene into a variety of human tumor cells has shown induction of efficient growth inhibition and apoptosis . In lineIn myeloma, a preclinical study demonstrated that adenovirus mediated delivery of wild type p53 could potently induce apoptosis in myeloma cells while sparing the normal hematopoietic cells and normal lymphocytes . FurtherThe use of conventional or high-dose chemotherapy has been a long-standing approach to treat MM patients. First line therapy for eligible patients remains autologous stem cell transplantation following high-dose chemotherapy. This is often preceded by induction therapy to decrease tumor load by utilizing a combination of treatments that often include chemotherapeutic agents such as alkylating compounds . In relaBortezomib was approved by the Food and Drug Administration (FDA) for MM treatment in 2008 . BortezoDespite the introduction of bortezomib shifting the paradigm of MM treatment, the disease remains incurable and resistance towards this remarkable drug still arises. Given this situation, novel therapeutics targeting the critical p53 pathway is of utmost importance in order to reactivate the tumor suppressor network to execute apoptosis, in the hope of improving the treatment outcome in MM. In view of this, novel p53-reactivating agents have been developed and these agents are relevant to the nature and pathology of MM. The following describes these p53-reactivating drugs and the potential they hold for clinical applications. Due to the fact that newly diagnosed cases of MM are often presented with wild type p53, therapeutic induction of p53 is an attractive potential treatment strategy for this disease. The conventional way of inactivating wild type p53 is through deregulation/overexpression of MDM2, which inhibits the transcriptional activity of p53 as well as increasing the rate of the p53 degradation. In view of this, the development of drugs to reactivate wild type p53 has focused on developing small molecule inhibitors to the MDM2-p53 complex.(i) Nutlin. The first reported and the most well studied MDM2 inhibitor is the nutlins [ nutlins , 41. Nut nutlins . Nutlin  nutlins . In othe TP53 are potential attractive targets for MDM2 inhibitor-based therapy. Nutlin-3 has been shown to be a potent inducer of apoptosis in cell lines deriving from hematological malignancies, including MM, ALL, AML, CLL, and Hodgkin's lymphoma [Because MDM2 inhibitors depend on p53 activation in cells expressing wild type p53, hematological malignancies that mostly retain wild type genotype oflymphoma , 58. In lymphoma . It was lymphoma . This eflymphoma . The mollymphoma . ImperatFurthermore, nutlin displayed wide synergy with various conventional chemotherapeutic drugs, namely, melphalan, etoposide, and velcade , 60, 61.As mentioned earlier, monoallelic deletion of p53 with the remaining allele being a wild type is a recurrent cytogenetic abnormality in MM patients and this group of patients often suffers from poor prognosis. It still remains inconclusive whether the single allele of wild type p53 in MM cells is actually still functional or is able to be reactivated by a nongenotoxic agent such as nutlin-3. Our lab has shown that, in WT/- cases with high p53 expression, nutlin-3 was able to induce a functional p53 pathway, but with a compromised activity compared to the WT/WT cells, whereas in WT/- cases with low or zero p53 expression, nutlin-3 showed no efficacy. The findings indicate a haploinsufficient activity of p53 in myeloma .The current evidence holds a lot of promise for nutlin-3 to be translated into the clinic as a treatment for MM with wild type p53. However, it must be kept in mind that sporadic mutations of p53 could arise from selective pressure in the cells upon prolonged nutlin treatment, rendering a state of resistance towards the drug . Further(ii) RITA (Reactivation of p53 and Induction of Tumor Cell Apoptosis). RITA is a small molecule compound identified in a cell-based screen. It has a reversed mechanism from nutlin, whereby RITA binds to the amino terminal on p53 domain instead of on MDM2 protein. This binding causes conformational changes of p53 that reduces the p53-MDM2 interaction and hence decreases p53 ubiquitination which then leads to p53 accumulation, MDM2 downregulation, and p53-dependent apoptotic pathway induction [ in vitro findings, mouse xenograft models of MM which were subjected to RITA treatment displayed tumor regression and lengthened survival [nduction . Antimyesurvival .The efficiency of RITA as an antimyeloma agent was further strengthened when RITA was found to be able to overcome resistance of MM cells towards MDM2 inhibitors such as nutlin-3 and MI-63 . In thisThe clinical translation of RITA as an antimyeloma agent was further highlighted by its synergistic relationship with nutlin in inhibiting the growth and killing of MM cells .(iii) Other Small Molecules Inhibitors of p53-MDM2. The importance of MDM2 inhibitors in hematological malignancies was emphasized when these compounds, MI-63, MI-219, and MI-319, showed preclinical efficacies [ in vitro and in vivo evidence of reactivation of the p53 pathway [ficacies . Like nuficacies , 68. Theficacies . On the  pathway . These r(i) PRIMA-1 (p53 Reactivation and Induction of Massive Apoptosis). p53 mutations are often associated with resistance to chemotherapy treatment in cancer [n cancer , 15. Then cancer . This con cancer . Since m in vivo level, whereby it potently inhibits the growth of tumor in human tumor xenograft model [PRIMA-1 has been shown to have good efficacy against various types of solid cancer cells, namely, breast cancer , small cft model , 76. PRIft model \u201379. Due ft model .The potency of PRIMA-1 in hematological malignancies came to light when it was found to have an antileukemic effect in CLL and AML cells , 81. Int(ii) MIRA-1. MIRA-1, structurally distinct from PRIMA-1, is a maleimide compound that targets mutant p53 with higher potency than PRIMA-1 [ PRIMA-1 . MIRA-1  PRIMA-1 . First i PRIMA-1 .Since drug resistance is ubiquitous in multiple myeloma, drug combination has been employed as a treatment regime to improve the treatment outcome. For instance, nutlin-3 has been shown to act in concert with various conventional chemotherapeutics to activate p53, in efforts to improve the treatment efficacy and reduce the collateral damage caused by chemotherapy. Nutlin-3 was reported to induce growth arrest in normal cells, and upon removal of the drug, cell cycle resumes . As chem in vitro and in vivo antimyeloma effects, promising results arising from drug combinations suggest that combined use is a more attractive therapeutic option. The fact that p53-independent activities are involved in the drug mechanisms denotes that further investigations in this aspect are strongly called for so that we could fully utilize the versatility of the drug while maximizing its killing capacity. Of course, understanding the precise mechanism of action of these drugs would also aid in the future design of a novel and improved compound for the treatment of multiple myeloma.As p53 is the bridging point of apoptotic mechanisms, reactivation of the p53 pathway itself confers an excellent therapeutic approach in treating cancer. The current perspective points towards the importance of utilizing small molecular agents to reactivate the wild type p53 or to restore the transcriptional activities of the mutant p53. Even though each agent on its own was reported to show potent"}
{"text": "Drug reaction with eosinophilia and systemic symptoms (DRESS) is a rare and severe adverse drug reaction. No pathognomonic test is available. The skin is the most frequent involved organ and easily sampled for a histopathological analysis. Recent series suggest that keratinocyte damage could be associated with either severe hepatic or renal involvement.To study the histopathologic findings in a large homogenous series of DRESS and their link, if there is any, with visceral involvement and severity.We describe in a retrospective large cohort, between 2005 and 2013, 45 patients diagnosed with a DRESS  and their cutaneous histological features.A facial edema in DRESS was associated with a definite case of DRESS (p=0.005) and a liver involvement (p=0.045). A younger age (50 years vs 66 years) was associated with a liver involvement (p=0.013). A severe neutropenia was associated with severe DRESS (p=0.012). Spongiosis and keratinocyte damage are the 2 most frequent epidermal changes noticed 55% and 53%. Pustules are not rare (26%). In the dermis a vascular alterations were frequent (88%) with a true leukocytoclastic vasculitis in 28%, a mild lymphocytic vasculitis in 13% and minor alterations in 44% of cases. In the dermis, there was a constant lymphocytic infiltrate mainly moderate in a perivascular distribution. In 57% the infiltrate is polymorphous with association of eosinophils neutrophil or atypical lymphocyte. In two third of cases, the changes are polymorphous and numerous, with no specific clear pattern. In the other one third of cases with few alterations, the presence of eosinophils, keratinocyte damage and spongiosis are usually present. A spongiosis was associated with non severe form of DRESS (p=0.041) and the absence of kidney involvement (p=0.023). A a higher of alterations per slides was associated a liver involvement (p=0.018). Our study do not support a prognostic value of keratinocyte damage.Histopathology of DRESS associated frequent spongiosis, keratinocyte damage, vascular alteration and polymorphism of the infiltrate. In two third of cases, the changes are polymorphous and numerous, in the other one third of cases with few alterations, the presence of eosinophils, keratinocyte damage and spongiosis are usually present. So cutaneous histopathology seems of little value to predict the visceral involvement."}
{"text": "Conformational and functional flexibility promote protein evolvability. High evolvability allows related proteins to functionally diverge and perhaps to neostructuralize. p53 is a multifunctional protein frequently referred to as the Guardian of the Genome\u2013a hub for e.g. incoming and outgoing signals in apoptosis and DNA repair. p53 has been found to be structurally disordered, an extreme form of conformational flexibility. Here, p53, and its paralogs p63 and p73, were studied for further insights into the evolutionary dynamics of structural disorder, secondary structure, and phosphorylation. This study is focused on the post gene duplication phase for the p53 family in vertebrates, but also visits the origin of the protein family and the early domain loss and gain events. Functional divergence, measured by rapid evolutionary dynamics of protein domains, structural properties, and phosphorylation propensity, is inferred across vertebrate p53 proteins, in p63 and p73 from fish, and between the three paralogs. In particular, structurally disordered regions are redistributed among paralogs, but within clades redistribution of structural disorder also appears to be an ongoing process. Despite its deemed importance as the Guardian of the Genome, p53 is indeed a protein with high evolvability as seen not only in rearranged structural disorder, but also in fluctuating domain sequence signatures among lineages. Proteins are dynamic, with a natural tendency to rearrange their conformational ensembles in response to the local environment . ConformRecognized as the Guardian of the Genome, yet infamous for its frequent implication in cancer; p53 is a versatile protein, known to perform numerous functions from DNA binding as a transcription factor to a regulator of apoptosis and beyond . With poFor every amino acid substitution, the conformational and functional ensemble may be altered, with plausible scenarios ranging from no change to gain-or-loss of function. While globular protein domains must fold to function, structurally disordered regions may be less constrained, challenging the common concept of structure being more conserved than sequence. Many possibilities to balance the fitness equation exist if some functions are benefitted and others slightly impaired. This could result in an expanded nearly-neutral network that would allow rapid sequence divergence . HoweverL. gigantea that comprises the four domain cassette, fall inside the hemichordate and early chordate group. B. floridae has two copies; one (XP_002598770) has the p53 DBD and OD and falls far from all vertebrate p53 domains in this phylogeny, the other (XP_002613954) has the entire four domain cassette. This four domain cassette protein forms the closest outgroup to the entire vertebrate p53 family in this phylogeny and is considered the last common ancestor of all p53, p63 and p73 proteins in vertebrates, in agreement with taxonomy and previous studies [Reported sightings of a p53 protein and perhaps even a p63/p73 protein in choanoflagellates and invertebrates, suggest that the evolutionary record of p53 predates the beginning of the animal lineage, Metazoa . Thus, ar XP_0026954 has t studies . In vertB. floridae but before sharks diverged [B. floridae, all vertebrate proteins in the p53 family have lost domains, but no domains have been added. Proteins in the p63 and p73 clades overall share the three domain composition of p53 DBD, OD, and SAM. TAD is not identified by Pfam (P. sinensis and B. mutus) or p73 (U. maritimus), and OD is not found in two sequences in the p53 clade. One is after a lineage-specific duplication in E. edwardii and the second is from the only bird representative found in data derived from bird genome data, P. humilis. Lastly, the N-terminus and linkers between domains are variable in length, and in some cases linkers are even absent.The gene duplication pattern resulting in three vertebrate proteins from one ancestral protein is consistent with two whole genome duplications that supposedly occurred at the time of early vertebrates, after the divergence of diverged . To furtdiverged , but the by Pfam . In the  by Pfam . For theG. gallus [P. humilis [G. gallus p53 has the p53 DBD and the OD but like many other reptiles, it lacks TAD. Further, these two bird sequences fall outside the reptilian clade as the outgroup to mammals and thus, we cannot conclude that these are the main p53 proteins in P. humilis or G. gallus. However, given that G. gallus and P. humilis are distantly related birds and that they fall close to their expected location in the p53 family phylogeny  between human and shark sequences in the p53, p63, and p73 clades respectively reveal 51.55%, 76.13% and 76.65% sequence identity. Consequently, p63 and p73 are more similar, with 61.81% sequence identity when comparing shark sequences and 59.24% sequence identity when comparing the human sequences. Further, pairwise sequence identity for shark p53 vs. shark p63 and shark p73, reveal 49.02% and 49.86% respectively. Interestingly, the same comparisons made with the human proteins, p53 vs. p63 and p73, reveal 40.99% and 42.82% pairwise sequence identity, respectively. In summary, the shark p53 family proteins have diverged less than the human counterparts, in accordance with the significantly slower divergence rate found in sharks compared to other vertebrates .L. chalumnae have the most ordered DBD among the entire vertebrate p53 family  substitution rates per site (SEQ) were inferred . For allWith a high degree and varying amount of disorder across the p53 family, an analysis of the secondary structure elements propensities was suitable. Mapped in a heat map context, similar to that for the disorder propensity, reveal multiple regions with secondary structure transitions between sequences in the same clade and in a clade-specific manner . To quanSince phosphorylation frequently modulates the conformations of disordered regions in a regulatory fashion, an analysis of predicted phosphorylation sites was conducted. Here the heat map shows the locations of predicted phosphorylation sites in a binary fashion. Since only Ser, Thr, and Tyr can be phosphorylated, the amount of Ser, Thr, and Tyr may also be important for how many phosphorylation sites are predicted. However, while there are significant differences in the fraction of Ser, Thr, and Tyr among the different clades  there is no significant difference in the fraction of sites predicted to be phosphorylated when comparing p53, p63 and p73 mean values . In all clades, about 5% of all sites are predicted to be phosphorylated . To quanRegions that are rapidly changing in disorder, secondary structure, and phosphorylation are likely less important for a conserved function. These rates are calculated for the entire vertebrate p53 family and clade-specific patterns are therefore indistinct. To gain resolution on the clade level, clade-specific rates were estimated  Figs. PlAll prediction methods applied are intentionally based on linear sequences and not on 3D structures since the repertoire of 3D structures, although quite impressive for the p53 family, may only provide a limited set of snapshots of the conformational ensemble in which these proteins exist. However, the structural context is valuable and site specific DOT as well as the site specific fractions of predicted disorder were mapped onto structures for TAD, p53 DBD, and OD  .For TAD, the MDM2 binding site is shown . Here, mFor p53 DBD, the tetrameric state with DNA bound is displayed for the p53 family,  but for For OD, the two different tetrameric states are displayed for the p53 family,  but for To investigate if phosphorylation may be one of the mechanisms utilized to differentiate the regulatory pathways of p53, p63, and p73 from each other, shared and clade-specific phosphorylation sites were identified using a 50% majority rule either within a clade or across the entire p53 family. In total, 66 phosphorylation sites were identified . Of thesUsing linear sequence predictors, properties of structural disorder (IUPred), secondary structure (PSIPRED), and phosphorylation sites (NetPhos) have been inferred. It is important to remember that these are predictions and cannot be perfect given that they are (i) independently aiming to predict traits that may depend on each other, (ii) using only the linear sequence context without considering long-range sequence contacts, and (iii) based on experimental data that may not reflect the dynamic nature of a protein sequence, e.g. one PDB structure is merely a snapshot of a conformational ensemble . The accTrichoplax adhaerens, have an MDM2 binding site [Caenorhabditis elegans [B. floridae . Both oInevitably, ongoing functional divergence is present in the p53 family, and especially in the p53 clade. The Guardian of the Genome gives the impression of still exploring its function and does not fit the picture of a resilient Guardian. Perhaps, a more appropriate way to refer to p53 is as a Gambler of the Genome?Three datasets were constructed: (i) the p53 protein family at the whole protein level in vertebrates, (ii) the p53 protein family at the nucleotide level in vertebrates, and (iii) the p53 protein family at the DNA-binding domain level in a representative set of vertebrate sequences and non-vertebrates. For (i), NCBI BLAST  was perfHomo sapiens, Bos taurus, Gallus gallus, Anolis carolinensis, Xenopus tropicalis, Latimeria chalumnae, Takifugu rubripes, Danio rerio, and Callorhinchus milii. Sequence identifiers for all vertebrate sequences are given in For major vertebrate taxonomic groups, a representative organism with sequence information for all three paralogs in the p53 protein family was selected from (i). Vertebrate organisms included in (iii) were: Monosiga brevicollis and Salpingoeca rosetta. The resulting topology was used to guide rooting the trees from the first two datasets by rooting on the branch containing both p63 and p73 clades and selecting the p53 clade as the outgroup.Sequences for datasets (i) and (iii) were aligned with MAFFT v7.123\u20131  using thTo assess the characterization of the structural properties of the proteins included in our phylogenies, the amino acid sequence of each protein  was used as input for different sequence-based predictors in order to predict structural disorder, secondary structure, phosphorylation sites and domain regions. Thereafter, for each prediction method, the predicted value for each residue in each protein sequence was mapped onto its corresponding site in the multiple sequence alignment. This resulted in three matrices for (i) structural disorder prediction, (ii) secondary structure predictions, and (iii) predicted phosphorylation sites. For (i) and (iii), the data predicted was continuous. For (ii), the data had three non-numerical categories. In order to analyze the transitions between order and disorder, between the presence of secondary structure elements and loops, and for presence or absence of phosphorylation sites, all matrices were represented as binary phyletic patterns (as described below). The phyletic patterns were individually analyzed in their phylogenetic context and transition rates were calculated.Structural disorder was predicted using IUPred ,62 versiSecondary structure was predicted using PSIPRED ,64 versiPhosphorylation sites for Serine, Threonine and Tyrosine residues were predicted using NetPhos  version Protein domains were predicted based on Pfam  version Rate4Site  was usedTo study the gain/loss transitions of structural properties in related proteins along their evolutionary history, a protocol that includes the estimation of evolutionary rates per site based on the phylogenetic trees and the binary matrices generated was adopted. GLOOME software  was usedInference methods implemented in R statistical software  were useConservation, here defined as the fraction of disorder per site from the binary matrices (gaps included), was calculated for the p53 family and the individual clades. Site specific rates and conservation of disorder were mapped onto representative PDB structures for the different domains. S1 FigOverview of the p53 family phylogeny including 74 representative species across Metazoa and Choanoflagellates, built based on their p53 DBD domains. Support values at the nodes indicate posterior probabilities. Nodes with posterior probability < 0.5 are unresolved.(PDF)Click here for additional data file.S2 Fighttp://tree.bio.ed.ac.uk/software/figtree/).(A) Circular representations of p53 DNA-based phylogeny and (B) its corresponding full-protein-based phylogeny. These consensus trees were obtained with MrBayes 3.2.2 after sampling trees for 15 million generations with the default burn-in phase (discarding the first 25% of trees) and using the 50% majority rule. Node circles show posterior probabilities ranging from 0.5 in red to 1 in white. Here proteins were colored by clade  with tip labels following the color guide from (PDF)Click here for additional data file.S3 Fig(A) Heat map showing Pfam domain predictions per protein into their corresponding multiple sequence alignment sites , all in the context of the p53 DNA-based phylogeny with tip labels colored according to the color guide. (B) In addition, individual domain architectures (labeled and colored as shown in Pfam domains box) were also included to highlight their actual lengths enforcing missing or broken domains. Figure generated with iTOL .(PDF)Click here for additional data file.S4 FigDistribution of structural disorder (grey) and order (blue) in full-length proteins and in p53 DBD domains sorted by p53 DNA-based phylogenetic tree with tip labels following the color guide. Furthermore, individual domain architectures (labeled and colored as shown in Pfam domains box) were also included. Figure generated with iTOL .(PDF)Click here for additional data file.S5 Fig2 = 0.41, and p-value < 0.05, concluding that linear correlation different to 0 is statistically significant).Scatter plot of the p53 DBD structural disorder percentage vs. the number of Pfam domains per protein from 74 hits, including invertebrates and vertebrates proteins. There is a positive correlation between these two variables Click here for additional data file.S6 FigBoxplots showing the fractions of serine, threonine and tyrosine residues per protein per clade compared to the fractions of sites predicted to be phosphorylated by NetPhos  using a (PDF)Click here for additional data file.S7 FigCombined profiles of normalized evolutionary rates per aligned site for family and clades (vertebrates set) comparing disorder-order transitions (DOT) with (A) amino acid substitutions (SEQ), (B) secondary structure elements-loop transitions (SLT), and (C) phosphorylation transitions (PT). Grey shaded areas delimitate Pfam domain regions.(PDF)Click here for additional data file.S8 FigCombined profiles of normalized evolutionary rates per aligned site for family and clades (vertebrates set) comparing amino acid substitutions (SEQ) with (A) secondary structure elements-loop transitions (SLT) and (B) phosphorylation transitions (PT). Grey shaded areas delimitate Pfam  domain r(PDF)Click here for additional data file.S9 FigHeat map for structural traits plotted in the order of the p53 DNA-based phylogenetic tree context, showing p53 protein names as boxes colored according to the color guide in (PDF)Click here for additional data file.S1 TableAccession numbers for the vertebrate datasets (i) and (ii).(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableAlignment sites following a 50% majority rule of sequences with phosphorylation predictions based on NetPhos phosphorylation prediction score cut-off = 0.75 (gaps included). Information displayed per clade (specific) and per family (shared). Shaded areas correspond to the majority rule (phosphorylation predicted for more than 50% of taxa per clade or for the family). Corresponding positions in the canonical human proteins  are shown.(PDF)Click here for additional data file."}
{"text": "Helicobacter pylori, a bacterial pathogen that commonly infects the human stomach and is strongly linked to gastric cancer. Besides H. pylori, a number of other bacterial species were recently discovered to inhibit p53. These findings provide novel insights into host\u2013bacteria interactions and tumorigenesis associated with bacterial infections.p53 tumor suppressor has been identified as a protein interacting with the large T antigen produced by simian vacuolating virus 40 (SV40). Subsequent research on p53 inhibition by SV40 and other tumor viruses has not only helped to gain a better understanding of viral biology, but also shaped our knowledge of human tumorigenesis. Recent studies have found, however, that inhibition of p53 is not strictly in the realm of viruses. Some bacterial pathogens also actively inhibit p53 protein and induce its degradation, resulting in alteration of cellular stress responses. This phenomenon was initially characterized in gastric epithelial cells infected with  Helicobacter pylori infection, which is currently the strongest known risk factor for adenocarcinoma of the stomach. This phenomenon, however, is not limited to H. pylori, and many other bacterial pathogens inhibit p53 using various mechanisms. Inhibition of p53 by bacteria is linked to bacterial modulation of the host cellular responses to DNA damage, metabolic stress, and, potentially, other stressors. This is a dynamic area of research that will continue to evolve and make important contributions to a better understanding of host\u2013microbe interactions and tumorigenesis. These studies may offer new molecular targets and opportunities for drug development.This review focuses on a novel aspect of host\u2013bacteria interactions: the direct interplay between bacterial pathogens and tumor suppression mechanisms that protect the host from cancer development. Recent studies revealed that various pathogenic bacteria actively inhibit the major tumor suppression pathway mediated by p53 protein that plays a key role in the regulation of multiple cellular stress responses and prevention of cancerogenesis. Bacterial degradation of p53 was first discovered in the context of  This interest originates from the protein\u2019s prominent role in tumor suppression that was eloquently paraphrased in the scientific literature as \u201cthe guardian of the genome\u201d . p53 is tp53) the most mutated gene in human tumors. p53 can also be inhibited by mutation-independent mechanisms. Inhibition of wild-type p53 by the SV40 virus was one of the first reported examples. SV40 is a small DNA tumor polyomavirus that induces cellular transformation in cell culture and an array of different tumors in animals. In infected cells, viral protein (SV40 large T antigen [T-Ag]) binds p53 and inhibits p53-dependent transcription, resulting in accumulation of inactivated p53 protein [Inactivation of p53 is a hallmark of tumorigenic changes. More than half of all tumors carry p53 mutations, rendering the p53 gene  has classified this bacterium as a Group 1 carcinogen. H. pylori infection is considered to be the strongest known risk factor for gastric cancer, and epidemiological studies have estimated that, in the absence of H. pylori, 75% of gastric cancers would not occur [Recent studies have found that it is not only viruses, but also some pathogenic bacteria, that actively inhibit p53 and induce its degradation. This phenomenon was initially described in gastric cells co-cultured with r pylori . H. pyloot occur .H. pylori infection is determined by interactions between bacterial factors and host cells. The most well characterized bacterial virulence determinants are the vacuolating cytotoxin A (vacA) and the cag pathogenicity island (cag PAI). The cag PAI is a 40 kb region of DNA that encodes a type IV secretion system (T4SS) that forms a syringe-like pilus structure used for the injection of a bacterial protein CagA (cytotoxin-associated gene A) into gastric cells. Following the delivery, intracellular CagA is localized to the plasma membrane and triggers complex alterations of the host signaling pathways [Pathogenesis associated with pathways , includipathways ,24. Oncopathways ,26.H. pylori infection results in conditions of cellular stress because the bacteria induce DNA damage and disturb normal cellular homeostasis (including aberrant activation of multiple oncogenic pathways), all of which are conditions that typically activate p53 [H. pylori is able to dampen activity of p53 protein by inducing its rapid degradation [H. pylori to suppress the p53 response was also demonstrated when DNA damage was experimentally induced by DNA-damaging agents [H. pylori but rather induced [H. pylori strains, with CagA-positive bacteria being more potent [vate p53 ,28. Howeradation . The abig agents ,29,30. T induced . The abie potent ,29. Althe potent ,30. Recee potent . Among oe potent \u201335. DownH. pylori infection, expression of p53 was changed in a bimodal fashion, with an accumulation after initial infection that was followed by a rapid down-regulation of p53 protein in gastric epithelial cells. A second peak of p53 was observed later, when gastritis (inflammation of the lining of the stomach) developed. These findings led to a hypothesis that, at a certain time, levels of p53 reflect a balance between p53 degradation induced by the bacteria and p53 induction caused by cellular stress [H. pylori-infected mice [The kinetics of p53 in infected cells in vivo appears to be complex. In infected Mongolian gerbils, which are commonly used for studies of r stress . A down-ted mice .H. pylori takes advantage of host mechanisms normally regulating p53 [H. pylori in vitro and H. pylori-infected animals and humans in vivo [H. pylori [H. pylori-infected cells [In contrast to small DNA tumor viruses, ting p53 ,35. The  in vivo ,35,37. I in vivo ,35,38. Aed cells . It remaH. pylori efficiently degrades p53 [H. pylori, may be particularly vulnerable to degradation of p53 [p14ARF tumor suppressor (termed p19ARF in rodents and p14ARF in humans), which functions upstream of p53, was found to be a critical modulator of p53 protein stability in infected cells , as ARF ades p53 . Loss ofades p53 . Given tn of p53 .H. pylori-infected cells [Among other cellular factors, ASPP2 protein (apoptosis-stimulating protein of p53), which normally activates p53, was identified to regulate p53 in ed cells . Buti etH. pylori varied greatly in their ability to degrade p53, but that, generally, isolates associated with a higher gastric cancer risk more strongly affect p53 when compared to low-risk counterparts [Recent studies suggest that bacterial degradation of p53 may contribute to gastric tumorigenesis. It was reported that clinical isolates of terparts .H. pylori inhibits p53 through multiple mechanisms, implying that inhibition of p53 activity is an important factor for successful infection. The bacteria not only induce degradation of p53, but also alter the expression profile of p53 isoforms [H. pylori with gastric epithelial cells, mediated via the cag PAI, induces N-terminally truncated \u0394133p53 and \u0394160p53 isoforms, which inhibit transcriptional and proapoptotic activities of p53, resulting in activation of NFkB. Induction of proinflammatory cytokine Macrophage Migration Inhibitory Factor (MIF) by H. pylori was suggested to inhibit p53 by decreasing its phosphorylation [H. pylori can facilitate mutagenesis of the p53 gene. Infection with H. pylori leads to aberrant induction of activation-induced cytidine deaminase (AID), which deaminates cytosine residues, leading to accumulation of p53 mutations in gastric tissues [isoforms . Interacrylation . It was  tissues . Interes tissues \u201348. SV40 tissues ,50.H. pylori  and blinding trachoma. Similar to H. pylori, C. trachomatis activates the PI3K/Akt pathway and increases phosphorylation of HDM2 (Ser166), leading to activation of HDM2 and proteasomal degradation of p53. Down-regulation of p53 allows Chlamydia to enhance activity of the PPP that provides bacteria with necessary metabolites, such as nucleotides precursors, and protects against oxidative stress by increasing the cellular NADPH pool [Chlamydia interferes with the host\u2019s response to genotoxic stress and may contribute to cancerogenesis in the female genital tract [A new and exciting development in this area is that other bacteria induce degradation of p53 using a similar mechanism to that of . pylori . Two res protein ,52. C. tal tract ,52.Shigella flexneri, which causes bacillary dysentery in humans. Infection with Shigella is accompanied by strong genotoxic stress and cellular damage [Shigella causes rapid degradation of p53 using two distinct mechanisms. During the early phase of infection, the bacterial virulence effector IpgD promotes activation of the host PI3K/Akt pathway and phosphorylation of HDM2 at serines 166 and 186, causing activation of HDM2 and degradation of p53. The second mechanism for p53 inhibition comes into play during the late phase of infection. p53 is proteolytically cleaved by the calpain protease system, in which activation is facilitated by the Shigella virulence effector VirA. The VirA activates calpain by promoting proteolysis of the calpain inhibitor calpastatin. Bergounioux et al. suggested that Shigella inhibits p53 to prevent apoptotic cells death that saves energy and preserves its own epithelial niche [Salmonella typhimurium infection [Enterobacteriaceae family, down-regulation of p53 protein was reported in studies of Neisseria gonorrhoeae, which is responsible for the sexually transmitted gonorrhea that may increase the risk of genital neoplasms [N. gonorrhoeae causes strong genotoxic stress and induces both single and double strand DNA breaks. The mechanism of p53 down-regulation is not fully understood, but Vielfort et al. reported that the bacteria can inhibit transcription of the p53 gene [Inhibition of p53 through the HDM2-dependent mechanism is also employed by enteropathogen nfection . Outsideeoplasms . Similarp53 gene .H. pylori, expression of the CagA virulence factor is sufficient to inhibit p53 and extend short and long term survival of gastric epithelial cells that underwent DNA damage [Chlamydia. As described above, degradation of p53 allows C. trachomatis to release inhibition of the PPP elicited by p53. When bacterial degradation of p53 was experimentally inhibited, the development and formation of infectious progeny was blocked, suggesting that metabolic control of p53 provides antibacterial protection. It is possible to draw a parallel between Chlamydiae and viruses since both are obligatory intracellular pathogens, which strictly rely on the host resources. Similar to viruses, inhibition of p53 allows Chlamydia to reprogram the host cell signaling to create a metabolic environment necessary for chlamydial survival and growth. To some extent, this may also be applied to obligate parasitic Mycoplasma bacteria, which inhibit activity of p53 [H. pylori. One proposed possibility is that inhibition of p53 helps H. pylori to compromise the gastric epithelial barrier, allowing the bacteria to acquire nutrients from the host or get access to the lamina propria. This concept is supported by recent findings showing that H. pylori inhibits activation of p53 induced by disruption of the adherens junctions, which stabilize cell\u2013cell adhesion [H. pylori adapt during the early phase of infection and prevent the host immune response [Inhibition of p53 may provide certain benefits to bacteria. One particular mechanism that may be targeted by bacteria is the p53 DNA damage response. Inhibition of p53 may allow bacteria to subvert the host cell cycle control and apoptosis mechanisms, resulting in inhibition of cell death and survival of host cells damaged by infection. This is in agreement with the findings of antiapoptotic and prosurvival effects produced by bacterial pathogens, which inhibit p53 ,29,52,53A damage . Besidesy of p53 . A more adhesion . It was response . The p53response . Among ds of the bacterial regulation of p53 hold the promise of a better understanding of pathogenesis and tumorigenesis associated with bacterial infections.Interaction of bacterial pathogens with the host cells induces DNAdamage, alters intracellular signaling, and profoundly affects normal cellular homeostasis. To prevent the cellular stress response, which may be detrimental to a successful infection, some bacteria have evolved to inhibit p53, a key component of the stress response machinery. Bacteria inhibit p53 through multiple mechanisms, including protein degradation, transcriptional inhibition, and post-translational modifications. Current research revealed that p53 has a role in controlling the bacterial infections and that inhibition of p53 may confer certain selective advantages to bacteria. Unfortunately, this may have grave consequences for the hosts, increasing the risk of tumor development. It is particularly relevant to prolonged chronic infections. Initial experiments with inhibition of protein degradation of p53 demonstrate that p53 activities can be restored in infected cells using specific chemical inhibitors. These findings may offer new and exciting opportunities for therapeutic targeting of p53 in infected cells. Future studie"}
{"text": "Ictidomys tridecemlineatus during multiple stages of the torpor-arousal cycle. Transcript and protein levels of p53 were both elevated in the skeletal muscle during early and late torpor stages of the hibernation cycle. Nuclear localization of p53 was also increased during late torpor, and this is associated with an increase in its DNA binding activity and expression of p53 transcriptional targets p21CIP, gadd45\u03b1, and 14-3-3\u03c3. The increase in p53 transcriptional activity appears to be independent of its phosphorylation at Ser-15, Ser-46, and Ser-392, consistent with an absence of checkpoint kinase activation during torpor. Sequence analysis revealed unique amino acid substitutions in the ground squirrel p53 protein, which may contribute to an increase in protein stability compared to nonhibernators. Overall, the study results provided evidences for a potential role of p53 in the protection of the skeletal muscle during torpor.The transcription factor p53 is located at the centre of multiple pathways relating the cellular response to stress. Commonly known as a tumor suppressor, it is responsible for initiating diverse actions to protect the integrity of the genome, ranging from cell cycle arrest to apoptosis. This study investigated the regulation of p53 protein in hibernating 13-lined ground squirrel Ictidomys tridecemlineatus). I. tridecemlineatus, an inhabitant of the central North American grasslands, typically hibernates from October to April, when seasonal changes that increase metabolic demand are met with a restriction on food supply. Hibernation in I. tridecemlineatus is characterized by cyclic arousal and reentry into a low metabolic state , 2019, 2 of MDM2 . Myotis brandtii bats contains unique amino acid substitutions at the highly conserved single-transmembrane domain, and this is thought to be associated with an altered expression of insulin-associated genes in the bats that is similar to those observed in the long-lived GHR\u2212/\u2212 mice [The cellular levels of p53 are typically determined by the rate at which it is degraded through the ubiquitin-mediated pathway, rather by the rate at which it is synthesized . While M\u2212/\u2212 mice . Sequenc\u2212/\u2212 mice .In this study, we reported the activation of the p53 transcriptional response during the torpor stages in the 13-lined ground squirrels. Increase in p53 activity appeared to be independent of the select posttranslational modifications examined in this study but rather a result of an increased p53 protein expression. We also identified two amino acid substitutions in the ground squirrel p53 protein sequence that were unique compared to other nonhibernating mammals; the K290R and S364P substitutions are of particular interest as these two residues contribute to the ubiquitin mediated degradation of p53 in human. In conclusion, our results show that the activation of p53 transcriptional response is mediated by its upregulation at the transcriptional and translational level, and further experiments will be needed to elucidate the significances of the ground squirrel specific amino acid substitutions, and whether these alterations contribute to changes in p53 stability compared to nonhibernating mammals."}
{"text": "The tumor suppressor p53 normally acts as a brake to halt damaged cells from perpetrating their genetic errors into future generations. If p53 is disrupted by mutation, it may not only lose these corrective powers, but counterproductively acquire new capacities that drive cancer. A newly emerging manner in which mutant p53 executes its cancer promoting functions is by harnessing key proteins, which normally partner with its wild type, tumor-inhibiting counterpart. In association with the subverted activities of these protein partners, mutant p53 is empowered to act across multiple fundamental cellular pathways (regulating cell division and metabolism) and corrupt them to become cancer promoting. Reliance on the tumor suppressive capacity of p53 is profoundly emphasized by its near universal malfunction in all cancers. P53 is the most altered gene in cancer. More than 50% of human cancers are afflicted with a p53 mutation. Severe consequences of p53 mutation include the failure to protect against cancer stimuli, compounded by the acquisition of new cancer promoting, \u201cneomorphic\u201d properties, referred to as \u201cGain of function\u201d (GOF), covered by other reviews in this series [reviewed in Ref. ].A particularly sinister GOF constitutes the subversion by mutant p53, of molecular partners of wild type (wt) p53, and this strategy forms the focus of this review. Specifically, mutant p53 conscripts proteins that normally partner with wt p53. This new association divests them of their anticancer activities and in place, they are corrupted to act as promoters of tumorigenesis . A numbProper cell cycle regulation is vital for normal cell function. Equally critical is the capacity to sense DNA damage and to interrupt the cycle to instigate repair or eliminate cells with irreparable damage, as appropriate. Wt p53 is a key dictator of cellular fate in response to DNA damage resulting from cellular stresses. Partnership with the tumor suppressor promyelocytic leukemia (PML) protein facilitates p53 stress responses. Specifically, wt p53 stabilization and activation in response to stress is promoted by PML, through temporal co-recruitment of post-translational modifiers of p53 , to fSignificantly, mutant p53 enslavement of PML defines a paradigm for mutant p53 disruption of tumor suppressive partners of wt p53. We identified that when p53 is mutated in cancer cells, its association with PML is constitutive, unlike the transient association with its wt p53 counterpart in response to stress. Importantly, PML facilitates mutant p53 to aberrantly transcribe targets in the context of hijacked transcription factor NF-Y . One trAt a higher level, cell cycle control is coordinated by the Circadian clock , and wt At a molecular level, the clock is comprised of at least nine interplaying proteins, and we will discuss only those pertinent to this review. The clock is positively activated in a cyclic fashion through the combined activities of the two transcription factors: CLOCK and BMAL1 Figure . As heteWt p53 controls the clock through negative regulation of Per2 expression Figure . In normPhosphatase and Tensin homolog (PTEN) is also a vital cell cycle regulator that has achieved its reputation as a tumor suppressor in the context of wild type (wt) p53. Pten curbs cell cycle progression and cell survival by suppressing PI3K\u2013AKT/PKB cell survival pathway . PTEN fuIn the context of mutant p53, in a diametrically opposing function, Pten promotes tumor growth . PTEN, iPolo-like kinase-2 (PLK2) is also a wt p53 target that contributes to cell cycle control. PLK2 is transcriptionally induced by wt p53 in response to the stress of DNA damage . PLK2 inRapid cell proliferation inherent in cancer growth is utterly dependent on the ready supply of \u201cmolecular building blocks.\u201d Recent studies have identified that fundamental metabolic processes normally regulated by wt p53 are extensively disrupted by mutant p53 to facilitate the supply of these necessities.Proper repair of DNA damage is orchestrated by wt p53, which not only temporally halts cell cycle progression to allow repair, but also actively facilitates the supply of constituents for the repair. Specifically, in response to DNA damage, wt p53 transcriptionally activates the small subunit of the ribonucleotide reductase (RRM2b) in a temporary manner, to facilitate the catalytic conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, which is an essential step for DNA synthesis. In contrast, when p53 is mutated, it constitutively upregulates RRM2b expression. Importantly, the mechanism of transcriptional activation of RRM2b is dependent on the status of p53: where wt p53 engages its REs in the intronic region and in contrast mutant p53 localizes to the promoter. Further, it has emerged that mutant p53 transcriptionally drives additional nucleotide metabolic genes, both in the salvage and new synthesis pathways, through co-recruitment with the transcription factor ETS2, to its target gene promoters. ETS2 engagement by mutant p53 is a recurring theme, as we discuss below for epigenetic regulation. Overall, mutant p53 upregulates nucleotide biosynthesis, which contributes to meeting the voracious demands of rapidly proliferating and invading cancers .Regulated glucose metabolism is vital for maintaining healthy, normal cell homeostasis, in contrast to the voracious consumption of glucose that feeds cancer cell proliferation and is inherent in the \u201cWarburg effect.\u201d Proper glucose regulation is then an important tumor suppressive capacity of wt p53. Wt p53 regulates glucose metabolism by restricting cellular glucose at three levels through : (1) supProfoundly, when p53 is mutated, not only are these points of regulating glucose metabolism lost but further glucose uptake is accentuated through a novel GOF. This disastrous mutant p53 GOF is the shunting of the glucose transporter, Glut1, to the cell membrane surface where it stokes glucose uptake by cancer cells . ElevateReliance on a mutant p53-dependent enhanced supply of glucose to foster cell proliferation defines a unique point of vulnerability in cancer cells. This appetite for glucose identifies a potential therapy target which is currently being extensively investigated . SpecifReactive oxygen intermediates perform important cellular functions including signaling; however, they are seriously damaging to normal cells if not properly contained and are linked to cancer [review in Ref ]. A mastA novel GOF of mutant p53 is its capacity to reduce Nrf2 protein levels (without impacting its mRNA), in response to oxidative stress. The consequence is low levels of Nrf2 target detoxifying genes and elevated levels of reactive oxygen species (ROS). Remarkably, in contrast to growth inhibition imposed on wt p53 cells subject to oxidative stress, those with mutant p53 tolerate elevated ROS, survive, and proliferate .When p53 is mutated, a radical shift in transcriptional activity occurs, which is conducive to cancer promotion. An altered repertoire of transcription factor engagement is emerging for mutant p53. While mutant p53 is not able to directly engage wt p53 response elements, it may instead directly bind its wt counterpart and impose a dominant negative effect over wt p53 functions, including depriving it of capacity to regulate transcription. Mutant p53 may also engage transcription factors that wt p53 does not, including the family members p63 and p73 and disrupt their functions. More specifically, the presence of arginine at codon 72 dictates the capacity of mutant p53 to sequester p73, where mutants with proline are incapable of this inactivation , 50.Beyond this negative regulation of wt p53 and its family members, mutant p53 may hijack transcription factor partners and disrupt their normal transcriptional activity (as mentioned above). Mutant p53 has been reported to engage NF-Y, NF-kappa B, SP1, E2F1, ETS1, ETS2, and SREBP. The outcome may be altered target engagement, or a change in the rate of transcription relative to a wt p53 context. These features of mutant p53 have been comprehensively reviewed recently , so we wAt a higher level, mutant p53 disruption of chromatin regulation is also now emerging. In order for wt p53 to access specific DNA responsive elements in the regulatory regions (upstream promoters or introns) of its target genes, it must coordinate with numerous chromatin regulators to expose appropriate regulatory elements and associated DNA to be transcribed . Wt p53 Mutant p53 can also alter transcriptional machinery, through distinct interactions from its wt counterpart. Wt p53 is able to physically interact through its core domain with the RNA polymerase II  and limit target gene expression . In contCorruption of the normal interactions between wt p53 and its molecular partners appears to lie at the heart of significant tumor promoting mutant p53 GOFs. Intriguingly, p53 mutations, which eliminate its function , are rare, in contrast to the frequent activating missense mutations. To an extent, which appears unequaled by any other gene, mutation of p53 confers an exceptionally wide range of fundamental new properties that promote deregulated cell growth. These findings provide new insights directing innovative and rational approaches to therapeutically targeting cancers with mutant p53, which have proven particularly resistant to treatment. The polarized functions of these key p53 partners, would also caution that p53 status be an important criteria to consider prior to adoption of therapies directed toward these targets.SH wrote the paper and prepared the figures. DR contributed to writing the paper and to preparing a figure. YH contributed to discussion and editing the paper.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."}
{"text": "The management of increased intracranial pressure (ICP) and monitoring of ICP is an important part of neurosurgery, but reference values for ICP have not been established differentiating between children and adults. The same estimated reference of 7-15 is used indiscriminately for children and adults, and does not take in to account that adults and children differ physiologically. The effect on ICP of a postural change from a supine to a upright standing position has been established, though the association between height and ICP in upright position has not been studied. The purpose of this study is to address if ICP values could depend on height.Patients having a shunt were excluded from the study in order to avoid the influence of mechanical siphoning. Forty-one patients undergoing diagnostic ICP monitoring for a suspicion of hydrocephalus or idiopathic intracranial hypertension (IIH) were included in this study. Data were consecutively and prospectively collected. Nine were children (age 7-17) and thirty-two were adults (age 23-85). ICP was measured in parenchyma by either a cable-based or a telemetric probe. Measurements included both a supine and a upright standing position, and changes in ICP were calculated.Mean height was 152.3 cm (range 122-180) for children and 169.4 cm (range 155-188) for adults. Height did not appear to have an impact on measured ICP in either supine (p = 0.15) or upright standing position (p = 0.28). Changing body position from supine to upright caused a significant decrease in ICP in both children  and adults median decrease 12.5 mmHg; p<0.001).In the entire group there was borderline significance between patient height and the calculated postural decrease in ICP occurring when the patient changed from a supine to a upright standing position (p = 0.053). When dividing into children and adults, the change in ICP correlated to height was significant in children (p = 0.022), but not in adults (p = 0.34).We did not establish a direct correlation between ICP and height. However, in children we found a relationship between height and ICP decrease going from supine to upright. A similar correlation was not found in adults. Our results may be important for approaches to physiological risks of overdrainage caused by shunting."}
{"text": "Scientific Reports6: Article number: 19222; 10.1038/srep19222 published online: 01122016; updated: 05132016.The authors of this Article have requested that it is retracted. In the western blotting analysis, the rabbit monoclonal antibody to human mutant p53 rather than wild type p53  was used. While in the immunohistochemistry detection, the mouse monoclonal antibody to both wild-type and mutant forms of human p53  was used. The authors believe that the use of antibodies for both wild type p53 and mutant p53 is not appropriate because it cannot reflect the overexpression status of mutant TP53. The results as described in the manuscript stated that the number of patients with overexpression of mutant p53 was 84/241 (34.9%), using the mouse monoclonal antibody to both wild-type and mutant forms of human p53 . Ninety (37.3%) pancreatic ductal adenocarcinomas (PDACs) showed a virtual absence of p53 immunolabeling compared with the adjacent normal tissues. These patients were also considered to be \u201cabnormal\u201d in p53 immunolabeling. In contrast, the normal immunolabeling of p53 was detected in 67 (27.8%) of the 241 PDACs. However, the authors later found that the number of patients with overexpression of mutant p53 was actually 113/241 (46.9%) in these same specimens when using the specific anti-mutant p53 antibody . This inconsistency could impact the subsequent analyses and the previous conclusion of this study could not be drawn. Therefore, the authors feel they cannot guarantee the accuracy of the results and conclusions described in this paper, and believe it is necessary to retract it."}
{"text": "Gamma-glutamylcyclotransferase (GGCT) is one of the major enzymes involved in glutathione metabolism. However, its gene locus was unknown for many years. Recently, the gene for GGCT was found to be identical to C7orf24, which is registered as a hypothetical protein. Orthologs have been found in bacteria, plants, and nematodes as well as higher organisms, and the GGCT gene is highly preserved among a wide range of species. GGCT (C7orf24) was also reported as an upregulated protein in various cancers. Although the function of GGCT in cancer cells has not been determined, the following important activities have been reported: (1) high expression in various cancer tissues and cancer cell lines, (2) low expression in normal tissues, (3) inhibition of cancer cell proliferation via anti-GGCT RNAi, (4) inhibition of cancer cell invasion and migration via anti-GGCT RNAi, (5) an epigenetic transcriptional regulation in cancer cells, and (6) an antitumor effect in cancer-bearing xenograft mice. Therefore, GGCT is promising as a diagnostic marker and a therapeutic target for various cancers. This review summarizes these interesting findings. The existence of human gamma-glutamylcyclotransferase (GGCT) has been known since around 1970. However, its amino acid sequence and gene locus were unknown for many years . RecentlMasuda et al. reported that C7orf24 is identical to cytochrome c-releasing factor (CRF21), which is a substance released into the cytoplasm when human leukemia cells U937 are treated with geranylgeraniol, an apoptosis inducer . They prAs mentioned above, a number of relationships between cancer and GGCT have been reported by some researchers and the role of this molecule in cancer has attracted attention. This minireview summarizes the role of GGCT in cancer cells and the possibility of using GGCT in cancer diagnosis and targeting treatment.GGCT is one of the major enzymes comprising the gamma-glutamyl cycle proposed by Orlowski et al.  2]. GGC. GGC2]. Glutathione is a tripeptide consisting of glutamate, cysteine, and glycine. Glutathione is found in cells at a relatively high concentration of 0.5\u201310\u2009mM and has a number of important functions such as an antioxidant and a detoxifying agent. Adequate amounts of glutamate, cysteine, and glycine are essential to maintain glutathione at proper levels . MeisterAlthough GGCT was isolated more than 40 years ago \u20133, its gSeveral studies concerning the distribution of GGCT in normal tissues were reported. Oda et al. examined the mRNA expression of GGCT in rats . They reThe intracellular distribution found by immunohistochemistry was in the cytoplasm and nucleus and these findings were almost consistent with a few other reports \u201314. CytoWe conducted proteome analysis with two-dimensional electrophoresis in bladder cancer to search for diagnostic markers for urothelial carcinomas , 16. FifAfter our report, other groups reported similar results using clinical samples , 17, 18.n = 30 in each cancer), and they reported significant decreased expressions were observed in renal and urothelial tumors [On the other hand, the results obtained by IHC were inconsistent in several types of cancers. Amano et al. examined 13 types of cancer specimens obtained by surgery  inhibitory concentration) with GGCT siRNA at a low concentration of 5\u2009nM in each cell line resulted in an additional 25\u201341% inhibition of proliferation over coadministration of docetaxel and control siRNA (manuscript in submission). The combined use of a functional inhibitor of GGCT with chemotherapy may become a promising treatment.We first reported that the administration of anti-GGCT siRNA inhibited cell proliferation in some cell lines, including bladder, prostate, lung, breast, and cervical cancers . SimilarA study of treatment targeting GGCT using an animal model was reported by Hama et al. . They prRecently, Yoshiya et al. developed probes \u201cLISA-4\u201d and \u201cLISA-101,\u201d which produce fluorescence as the result of an enzymatic reaction , 24. TheIt has only been a few years since the existence of GGCT was shown and accumulation of research results has not been sufficient. However, GGCT is a very attractive target molecule because its high expression has been observed in a wide range of cancer types. Inhibition of GGCT expression suppresses the proliferation of cancer cells, and mild adverse events are expected from a recent study. Further investigations are needed in the future."}
{"text": "To date, a total of 75 different disease-causing mutations have been published for the CTSC gene. A summary of recurrent mutations identified in Hungarian patients and a review of published mutations is presented in this update. Comparison of clinical features in affected families with the same mutation strongly confirm that identical mutations of the CTSC gene can give rise to multiple different phenotypes, making genotype\u2013phenotype correlations difficult. Variable expression of the phenotype associated with the same CTSC mutation may reflect the influence of other genetic and/or environmental factors. Most mutations are missense (53%), nonsense (23%), or frameshift (17%); however, in-frame deletions, one splicing variant, and one 5\u2032 untranslated region (UTR) mutation have also been reported. The majority of the mutations are located in exons 5\u20137, which encodes the heavy chain of the cathepsin C protein, suggesting that tetramerization is important for cathepsin C enzymatic activity. All the data reviewed here have been submitted to the CTSC base, a mutation registry for PLS at http://bioinf.uta.fi/CTSCbase/.Papillon\u2013Lef\u00e8vre syndrome  is an autosomal recessive condition characterized by palmoplantar hyperkeratosis and periodontitis. In 1997, the gene locus for PLS was mapped to 11q14-21, and in 1999, variants in the  Papillon\u2013Lef\u00e8vre syndrome  is a rare form of palmoplantar keratodermas. It was first described by Papillon and Lef\u00e8vre . The maiKeratoderma in PLS can present in the first 3 months of life, although palmoplantar hyperkeratosis generally first appears in years 1\u20134 Haneke . HoweverPeriodontitis and gingivitis result in the loss of primary and permanent teeth  was independently identified by two groups , this gene has nine splice variants. Of these, five occur in protein coding regions; the remaining four are noncoding transcripts.PLS is transmitted as an autosomal recessive condition affecting males and females equally. PLS was independently mapped to chromosome 11q14-21 by three groups , a lysosomal exo-cysteine proteinase belonging to the peptidase C1 family. Cathepsin C is an oligomeric enzyme composed of four identical subunits , also characterized by palmoplantar hyperkeratosis and periodontal inflammation, as well as arachnodactly, acroosteolysis, pesplanus, and onychogryposis , which is characterized by severe periodontal inflammation leading to tooth loss without the presence of skin symptoms  were reported in PLS cases, while only a few mutations (3%) were reported in HMS or AP1 cases. Note that some mutations were detected in two different disease entities: c.1040A>G p.Tyr347Cys was reported for AP1 and also for classic PLS families  literature search was performed to identify all known CTSC mutations. In addition, Hungarian pedigrees with PLS were screened for CTSC mutations and added to this article. All available information about mutation carriers have been uploaded to the CTSCbase, a mutation registry for PLS  list of locus-specific databases. The database can be visited at http://bioinf.uta.fi/CTSCbase/ and has been updated with data from the literature as well as unpublished variants identified in Hungarian PLS pedigrees.A PubMed , the sisters were found to carry the c.901G>Ap.Gly301Ser missense mutation in a homozygous form (data not published). The family was not aware of consanguinity. This mutation has also been previously published for a German patient with typical PLS skin symptoms , we have identified the c.748C>Tp.Arg250X homozygous nonsense mutation (data not published). Unfortunately, both of these patients were reared in state care and have no known relatives; therefore, investigation of the family was not possible. The fact that both individuals carry the same mutation raises the possibility that these patients are relatives. This mutation has also been previously published in the literature in a Turkish PLS family  and numbered with respect to the CTSC gene reference sequence (ENSG00000109861 corresponding to the CTSC gene transcript ENST00000227266). The 75 unique mutations \u2013 point mutations, small deletions, and insertions \u2013 are summarized in Figure To date, a total of 75 mutations have been identified for the n = 40), 23% are nonsense (n = 17) and 17% are frameshift (n = 13) variants. There are two in-frame deletions, one intronic splice-site variant and one point mutation in the 5\u2032 untranslated region (UTR) of the CTSC gene. The majority  of the mutations has only been reported once. Among these, 65% (n = 36) were present in homozygous form in the investigated patients, while 35% (n = 20) occurred in a compound heterozygous form. Recurrent mutations  occurred both in homozygous and in compound heterozygous forms and were detected in geographically distant, unrelated families, suggesting mutational clustering on the CTSC gene. However, there are reports suggesting that an initial founder effect and subsequent migration of carriers can lead to the presence of the same mutation in geographically distant and unrelated families  are located within exons 5\u20137, encoding amino acids 231\u2013394 in the heavy-chain region. Of the remaining half, 16% (n = 12) are located within exons 1\u20133 encoding amino acids 25\u2013134 in the exclusion domain, 12% (n = 9) are located within the second half of exon 7 encoding amino acids 395\u2013463 in the light-chain region, 13% (n = 10) are located within exon 4 and the first half of exon 5 encoding amino acids 135\u2013230 in the propeptide region, 3% (n = 2) are located in the 5\u2032 end of exon 1 encoding amino acids 1\u201324 in the signal peptide region and 3% (n = 2) are located within UTRs. Note, not all mutations have been identified by DNA sequencing.Known mutations that have been sequenced are unequally distributed on the CTSC mutations (n = 75) were reported in a homozygous form in PLS patients. Of these mutations, 85% (n = 64) were present only in homozygous form in PLS patients, while 15% (n = 11) were also detected in a compound heterozygous state. Among the homozygous mutations, 50% (n = 32) were missense, 25% (n = 16) nonsense, 23% (n = 15) frameshift mutations, and 2% (n = 1) were other types of mutations  of all CTSC gene mutations identified to date. Missense mutations occur in all coding regions of the gene; however, the majority occurs in exons 5\u20137, encoding the heavy-chain region of the cathepsin C protein  of the pathogenic mutations identified for the CTSC gene to date. Nonsense mutations occur in all coding regions of the gene; however, the majority is located in exons 5\u20137, encoding the heavy-chain region of the cathepsin C protein  of the mutations identified to date. Frameshift mutations occur in all coding regions of the gene; however, the majority is located in exons 4\u20135 encoding the propeptide region of the cathepsin C protein  of all identified CTSC mutations (n = 75) were detected in a compound heterozygous form. The most frequent  compound heterozygotes involved two heterozygous missense mutations. The combination of a heterozygous missense and a heterozygous nonsense mutation occurred in 22% (n = 5) of the cases, a heterozygous missense and a heterozygous frameshift mutation in 16% (n = 4), a heterozygous nonsense and a heterozygous frameshift mutation in 11% (n = 3), and two heterozygous nonsense mutations in 5% (n = 1)  of the mutations have been reported twice or more in different ethnic groups. One of the most frequently reported missense mutation, the c.815G>Cp.Arg272Pro variant, has been detected in Lebanese, Turkish, Saudi, Holland, Russian and French PLS patients , an exclusion domain (110 amino acids), a propeptide (96 amino acids), as well as heavy-(164 amino acids) and light-(69 amino acids) chain regions  search, the cathepsin C protein is highly conserved in vertebrates: the human cathepsin C shows 82% sequence similarity with the sequence from dog, 70% with turkey, and 63% with frog and zebrafish  of all CTSC gene mutations affect the heavy-chain domain and result in different positioning of its N-terminus. As the N-terminal region is involved in oligomer contacts with the N-terminal region of the light chain, the mutation may interfere with tetramer formation  affect this domain  of all CTSC mutations affect the exclusion domain, which blocks access to the active site and prevents substrates from binding any part except their N-termini. Thirteen mutations were detected in the exclusion domain; of these, six are nonsense variants, four are missense mutations, and three are deletions (two resulting in frameshift and one in an in-frame deletion).Sixteen percent (n = 10) of all CTSC gene mutations affect the propeptide fragment, which plays a pivotal role in the activation of the cathepsin C precursor. The majority of frameshift mutations are located in this domain  of all mutations affect the light-chain domain, which is important for tetramerization of the mature enzyme: four are missense mutations, two are nonsense variants and one is an in-frame deletion. Three common missense variants, rs151269219, rs28937571, and rs3888798 are also located in this domain  of all mutations are located in the signal peptide region, presumably affecting the translocation or secretion of the protein: one nonsense mutation and one frameshift variant  of the all 75 mutations are located within exons 5\u20137, encoding the heavy-chain region of the cathepsin C protein. Three types mutations accounted for 93% (n = 61) of CTSC gene mutations: missense 53% (n = 41), nonsense 23% (n = 17), and frameshift 17% (n = 13). In addition, the majority of missense, nonsense, and frameshift mutations occur in exons 5\u20137.Analysis of data reported for Hungarian PLS patients revealed 75 CTSC mutation location  suggested that mutations located outside coding regions are more likely to be associated with transgression of the lesions (Hart et al. CTSC gene mutations with little functional consequences are putative causes of more common types of early-onset periodontal disease (Hart et al. In general, no strict genotype\u2013phenotype correlations have been identified for PLS. Analysis of CTSC gene can lead to the development of HMS or AP1 as well as PLS. The common characteristic of these three entities is periodontal inflammation (Hart et al. Mutations in the CTSC gene can give rise to multiple different phenotypes: the c.1040A>G p.Tyr347Cys missense mutation can lead either PLS or AP1 (Toomes et al. CTSC mutation may reflect the influence of other genetic and/or environmental factors (Hart et al. Several reports indicate that identical mutations of the CTSC gene mutations has not yet resulted in the identification of genotype\u2013phenotype correlations. Future efforts might provide insight into these correlations and elucidate the mechanism of the different phenotypic variants \u2013 PLS, HMS, and AP1 \u2013 of the disease. We believe that, to improve molecular analysis of the CTSC gene, it is necessary to promote both better awareness of the PLS, HMS, and AP1 phenotypic variants of the same disease and better understanding of the underlying molecular mechanisms. The availability of the extended clinical findings from CTSC mutation carriers, as provided by the CTSCbase, is critical for furthering both our understanding of the disease and the development of causative therapies that will be more specific and effective than the symptomatic treatments currently available for patients with PLS, HMS, and AP1 variants.To date, the comparison of"}
{"text": "Adjuvant chemotherapy was administered for the uterine corpus cancer and the patient remains healthy 48 months after the surgery. These tumors may have become malignant due to the presence of p53 mutations.A malignant solitary fibrous tumor arising from the omentum is extremely rare. To our knowledge, this is the first case of a malignant solitary fibrous omentum tumor coexisting with uterine corpus cancer. A 62-year-old woman presented to our hospital with vaginal discharge. Endometrioid adenocarcinoma was diagnosed by endometrial curettage. In addition, a solid tumor in front of the uterus was detected following computed tomography and/or magnetic resonance imaging, which was suspected to be a primary (or secondary) malignant tumor arising from the omentum. Hysterectomy, bilateral salpingo-oophorectomy, omentectomy, and lymphadenectomy were performed. A malignant solitary fibrous tumor of the omentum and grade 3 endometrioid adenocarcinoma of the uterus were diagnosed by pathohistological analysis. Interestingly, the tumor cells were immunoreactive for  We present here the clinical course and immunohistological findings.A solitary fibrous tumor (SFT) is a rare neoplasm mainly originating in the pleura; however, extrathoracic SFTs have been increasingly described, such as those in the meninges, liver, upper respiratory tract, orbit, thyroid, salivary gland, and female genital tract . SFT of  p53. Tumor in the peritoneal cavity was hypercellular and exhibited a patternless arrangement of fibroblastic spindle cells. It was composed of markedly atypical cells with a high mitotic activity (>10 mitotic figures/10 high power fields (HPF);  p16  were administered for the uterine corpus cancer. The patient remains healthy 48 months after the surgery.A 62-year-old menopausal woman presented to our hospital with vaginal discharge. The uterus, which was slightly larger than a hen's egg, along with a movable goose-egg-sized hard tumor in front of the uterus, was palpable during the internal examination. Grade 3 endometrioid adenocarcinoma of the uterus was detected by endometrial curettage. Computed tomography (CT) and/or magnetic resonance imaging (MRI) detected 2 solid tumors in both the uterus and the peritoneal cavity, measuring approximately 4\u2009cm and 10\u2009cm, respectively. Bilateral ovaries could also be detected . The tumF);  p16 , and p5316  and are summarized in  p53 tumor suppressor gene plays an important role in the regulation of cell growth. Mutations in this gene have been reported in various malignant tumors and are thought to be involved in pathogenesis and progression [ p53 mutations are known to be associated with uterine corpus cancer development  [ p53 expression in SFT [ p16 and weakly positive for p53. Mosquera and Fletcher suggested that p53 and p16 overexpression in the anaplastic component of SFT are in keeping with their potential role in the dedifferentiation process previously identified in some sarcomas and carcinomas [ de novo occurrence of malignant SFT [ de novo due to p53 mutations.Thegression . In relarcinoma) . Howeverrcinoma) . There hn in SFT , 7. In orcinomas . Yokoi e"}
{"text": "The protective function of p53 (in its homotetrameric form) as a tumor suppressor is lost in more than 50% of human cancers.Despite considerable experimental evidence suggesting the presence of multiple p53 states, it has been difficult to correlate the status of p53 with cancer response to treatments and clinical outcomes, which suggest the importance of complex but essential p53 regulatory pathways.TP53 gene encodes at least 12 p53 isoforms, which are produced in normal tissue through alternative initiation of translation, usage of alternative promoters, and alternative splicing. Furthermore, some researchers have suggested that the formation of mutant p53 aggregates may be associated with cancer pathogenesis due to loss-of function (LoF), dominant-negative (DN), and gain-of function (GoF) effects.Recent studies have indicated that the expression pattern of p53 isoforms may play a crucial role in regulating normal and cancer cell fates in response to diverse stresses. The human As different isoforms or the aggregation state of p53 may influence tumorigenesis, this review aims to examine the correlation of p53 isoforms and aggregation with cancer. In particular, p53, as a transcription factor, is responsible for the induction or inhibition of gene expression by specific binding to p53-responsive elements (p53REs). These interactions usually occur as a tetramer to finely control proteins related to apoptosis, DNA repair, and cell cycle arrest, and serve to block the transformation of normal cells to cancerous cells by maintaining genetic stability. The p53 evidently is known active as a tetrameric form, and it binds with high affinity to DNA or interacts more efficiently with various other proteins in this conformation. The ability of p53 to distinguish various signals allows it to influence delicate and complex regulatory processes.p53 protein, named after its molecular weight, was discovered by David Lane in 1979 as a protein that is bound to the simian virus (SV40) large T antigen. A few years later, p53 was called \u201cthe guardian of the genome\u201d due to its ability to inhibit cancer by maintaining various cellular functions.In humans, p53 is composed of 393 amino acids, which can be classified into 6 domains: the transcription activation domain (TAD) (residues 1\u201367), which interacts with a variety of proteins and can be further subdivided into TAD I (residues 1\u201340) and TAD II (residues 41\u201367); the proline-rich region (residues 67\u201398), which is conserved in the majority of p53s; the central core domain (residues 98\u2013303), which includes the DNA-binding domain wherein more than 90% of cancer-causing p53 mutations are found in humans; a nuclear localization signal located at residues 303 to 323; and the tetramerization domain, located at residues 323 to 363; and the C-terminal basic domain (residues 363\u2013393), which contains a nonspecific DNA-binding domain that recognizes and binds to damaged DNA.TP53 gene. Furthermore, more than 90% of TP53 point mutations are located in the central core domain. Inactivation of p53-regulated pathways through point mutations dramatically increases susceptibility to cancer. Cancer can occur even when no p53 mutations are present, but the p53 pathway is disrupted. Many studies were conducted to elucidate the mechanisms underlying the above mentioned phenomena. Recently, numerous researchers have focused on the potential importance of different isoforms and the aggregation states of p53, which we will discuss as follows.The importance of p53 as a tumor suppressor is clearly evident, considering that more than 50% of all malignant tumors are caused by mutations in the 1.21.2.1TP53 gene is composed of 11 exons  through alternative initiation of translation, usage of alternative promoters, and alternative splicing.\u201313Figure p53 gene. The most abundant p53 isoform, the canonical p53 protein (p53\u03b1 or p53), has the full TAD sequence and the longest C-terminal domain. In addition, depending on the translation initiation site, 3 \u0394N variants, \u039440p53, \u0394133p53, \u0394160p53, can be expressed. These 4 N-terminal variants can be combined with 3 different C-terminal domains . p53 isoforms are not only expressed differently for different cancer types but they also have different transcriptional activities and tumor-suppressor functions that can affect various other biological functions. Currently, researchers reported the existence of various isoforms of p53, but their biological functions have not been fully investigated. In short, p53\u03b2 was reported to enhance the transcriptional activity of p21 through p53(\u03b1) pathway and through BCL2-associated X protein (BAX) promoters. p53\u03b2 can also lead to apoptosis through p53-independent manner. On the contrary, p53\u03b3 could enhance the transcriptional activity of only through BAX promoter. Interestingly, \u039440p53\u03b1 could influence the dominant-negative effect to p53(\u03b1), interfering transcriptional activities. In addition, interactions between \u0394133p53\u03b1 and p53(\u03b1) could regulate the gene expressions by arresting apoptosis, G1 cell-cycle arrest, and replicative senescence, and enhancing blood vessel formation, metastasis formation, and endothelial cell migration. Hence, the functions of various p53 isoform could strengthen or interfere the tumor suppressor activity. Recently, the expression patterns of p53 isoforms were investigated for their importance in regulating gene expression in cancer cells compared with that in normal cells. Hence, the expression of abnormal p53 isoforms, regardless of the mutation, may contribute to the development of cancer.The 1.2.2,17\u201325 Collectively, results from these clinical studies suggest that specific expression patterns of p53 isoforms may be associated with tumor progression, clinical response, and prognoses. The expression patterns of p53 isoforms were studied in various cell lines, such as colorectal adenoma, renal cell carcinoma, mucinous ovarian cancer, serous ovarian cancer, breast cancer, cholangiocarcinoma, acute myeloid leukemia (AML), squamous cell carcinoma of the head and neck (SCCHN), and melanoma cell lines. Fujita et al showed that the progression of colorectal adenoma to carcinoma could be predicted by the ratio of p53\u03b2 to \u0394133p53\u03b1. In renal cell carcinoma, p53\u03b2 was evaluated as a potential progression marker at both the mRNA and protein levels, and p53\u03b2 overexpression appeared to be associated with tumor progression. In addition, the expression of \u039440p53\u03b1 in mucinous ovarian cancer was found to improve recurrence-free survival rates. \u0394133p53\u03b1 and \u039440p53\u03b1 isoforms were assessed in serous ovarian cancer cases as potential biomarkers. p53\u03b1, p53\u03b2, and p53\u03b3 isoforms are expressed in normal breast cells, but following the expression pattern of the p53\u03b3 isoform alone may be sufficient to predict good prognosis. In a subsequent study, the expression patterns of p53 isoforms were analyzed in 30 cases of primary breast tumors. In 18 cases, loss of p53\u03b2 and p53\u03b3 expression was seen, whereas in 12 cases, overexpression of the isoform \u0394133p53 was seen. The expression patterns of isoforms in tumors differed from those in normal tissue, and could differ even between each tumor case. The expression of \u0394133p53 in cholangiocarcinoma correlated with poor clinical outcomes. In contrast, overexpression of p53\u03b2 and p53\u03b3 was correlated with improved responses to chemotherapy in AML. In SCCHNs, elevated levels of the p53\u03b2 isoform were found in a large number of samples. In a study on melanoma, elevated levels of mRNA for p53\u03b2 and \u039440p53 were detected in tumor cells but not in melanocytes or fibroblasts. The expression patterns of p53 isoforms in the various tumors mentioned above are summarized in Table p53 plays an important role in the growth and progression of cancer and its response to chemotherapy. Despite many studies, it was difficult to predict these factors with only p53 status. Many institutions have independently performed clinical studies trying to correlate the expression patterns of p53 isoforms, at the mRNA and protein levels, with various types of cancers. and Marcel et al suggested to measure the changes in p53 mRNA levels by using nested RT-PCR and quantitative real-time RT-PCR and p53 protein quantification by using western blotting, immunoprecipitation, and luciferase reporter gene assays.p53 isoforms could be found in various cancers and their compositions may vary. These dynamic changes of p53 isoforms may affect the main known function of p53 as tumor suppressor. Hence, understanding and monitoring p53 isoforms at the level of mRNA and changes in transcription landscape will pave the directions of future studies of p53 isoforms. In addition, Khoury et alAlthough diverse p53 isoforms were studied clinically by using various types of cancer models with mRNA and protein expression levels, the challenges in correlating p53 expression patterns with cancer prognosis and treatment outcomes may result from investigating only individual isoforms instead of profiling for all isoforms. Hence, further investigation of various aberrant p53 isoforms may help in understanding cancer development and progression, and may ultimately provide novel targets for more effective cancer therapies and tumor markers.1.31.3.1,29 Over the past 2 decades, many neurodegenerative diseases such as Alzheimer disease , Parkinson disease (\u03b1-synuclein), Huntington disease (polyglutamine-huntingtin), familial amyloid polyneuropathy (transthyretin), prion diseases , amyotrophic lateral sclerosis (superoxide dismutase 1), and type II diabetes (islet amyloid polypeptide) have been investigated to understand how misfolding or aggregation of their respective proteins induces pathological features. The fundamental paradigm that explains transmission of these aggregates in their respective diseases was pioneered by Stanley Prusiner in the 1980s and is now referred to as the prion hypothesis. Recent research trends in p53 aggregation are based on the prion hypothesis, which will be discussed as follows.Protein misfolding and aggregation occur in several human disorders, which are commonly referred to as protein misfolding diseases (PMDs). PMDs are characterized by the formation of protein oligomers, protofibrils, and mature fibrils, which can accumulate intra- or extracellularly in several tissues.1.3.2 At that time, aggregated p53 was incorrectly considered to be a quaternary structure, which might be produced in order to prevent rapid degradation. However, recently, research on the p53 aggregation has been actively conducted in the motif obtained prion concept. As noted above, many in vitro aggregation studies focused on the central core domain (hot spot mutation with more than 90% genetic modification), and transactivation and tetramerization domains. Ishimaru et al confirmed the formation of various aggregates, which were due to physical interactions of the central domain of p53 (p53C), by using AFM and thioflavin T binding. Later, similar amyloid aggregates formed by the transactivation and tetramerization domains were reported.\u201335 In particular, the p53 hotspot mutant R248Q appeared to self-aggregate more than wild-type p53. p53 mutant (R248Q) seemed to be located in the hotspot of the protein, influencing self-aggregation. In addition, p53 mutant (R248Q) could be influenced by various environment factors, pH, temperature, and pressure. It was reported that p53 mutant (R248Q) could become a seed for the p53 aggregations, including with wild-type p53. The immunofluorescence colocalization assay of breast tissue from biopsy revealed the deposition of p53 aggregates that such aggregates could alter and decrease the normal function of p53 of tumor suppressor function. Aggregation might be due in part to a decrease in the thermodynamic stability of protein conformations in the mutants, which might compensate for this instability by aggregating. A variety of techniques such as electron microscopy, X-ray diffraction, Fourier-transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), cell viability assays, and anti-amyloid immunoassays could be used to accurately determine the underlying physical mechanisms.Recently, interest in p53 aggregation has grown among researchers. In fact, the high-molecular weight form of p53 was already detected in 1991. It was also shown that full-length and N-terminally truncated protein (p53C) aggregates could be internalized by cells into the cytosol and induce coaggregation with endogenous p53 protein, further supporting the possibility of the prionoid nature of p53 aggregates. Other in vivo evidence has revealed the accumulation of p53 aggregates, which were detected using an amyloid oligomer specific antibody (A11) or a fibrillar-specific antibody (OC), in paraffin-embedded breast tumor biopsy sections and basal cell carcinoma (BCC) cancer samples.,39,40 A summary of the above-mentioned results can be found in Table Furthermore, it was shown that p53 aggregates may act as seeds for coaggregation with other p53 family proteins such as p63 and p73.Together, these results implicate p53 misfolding and aggregation in cancers and support the hypothesis of prion-like activity of p53 aggregates. As p53 aggregates could coaggregate with wild-type p53, they may have a cytotoxic effect on mammalian cells by hindering the normal functions of p53.1.4The tumor-suppressor protein p53 is inarguably important for cancer development and progression and the response to chemotherapy. Despite many studies, it is difficult to predict the therapeutic response and clinical outcome based on p53 alone. Indeed, the p53 pathway can be regulated  in a number of ways. As a result, considerable efforts were made to uncover the mechanisms of inactivation, for example, by p53 protein isoforms and aggregation. Two independent mechanisms have been proposed regarding the isoforms and aggregation of p53. Most of the in vitro methods used to induce p53 aggregation use a partial peptide of the p53C domain, which is similar to the N-terminal truncated p53 isoform . In addition, robust aggregation of p53 could be induced at low pH, which is thought to simulate the tumor environment.,41 The formation of this complex could be due to native functional interactions or non-native interactions, which might arise from the formation of aggregates or be due to other causes that inactivate p53. Moreover, in recent studies, the presence of p53 aggregation affected the response of ovarian cancer cells to chemotherapy, which agrees with earlier studies that showed an association between chemoresistance and the presence of shorter p53 isoforms.,42 These results strongly suggest a more robust relationship between p53 isoforms and aggregation. In Fig. TP53 gene or other proteins in the downstream pathways in the transcription or tranalation mechanisms could alter the expressions of mp53 or Ip53, resulting aggregations of wp53 and suppressing the normal functions of p53.Furthermore, p53 isoforms were found to form a complex with FLp53 in several studies.\u201348Breaking p53 aggregations similar with prion-like behavior could be the novel therapeutic target in the anti-cancer therapy, as well as other PMDs from protein aggregations, such as Alzheimer disease and Parkinson disease.We also propose that further investigations are required to determine the relationship between p53 isoforms and aggregation, especially to clarify their contributions to the regulation of the normal functions of p53. These studies could provide a comprehensive understanding of p53 regulation and functions, and could contribute to the future development of cancer biomarkers and pharmaceutical therapies."}
{"text": "ExIII knockout mice which still express the short isoform of p75NTR. To scrutinize the role of p75NTR in the hippocampus, we analyzed adult and aged p75NTRExIV knockout mice, in which both, the short and the full-length isoform are deleted. Deletion of these isoforms induced morphological alterations in the adult dentate gyrus (DG), leading to an increase in the thickness of the molecular and granular layer. Based on these observations, we next determined the morphological substrates that might contribute to this phenotype. The cholinergic innervation of the molecular and granular layer of the DG was found to be significantly increased in the knockout mice. Furthermore, adult neurogenesis in the DG was found to be significantly altered with increased numbers of doublecortin (DCX) positive cells and reduced numbers of apoptotic cells in p75NTRExIV knockout mice. However, cell proliferation as measured by phosphohiston H3 (PH3) positive cell numbers was not affected. These morphological alterations (number of DCX-positive cells and increased cholinergic fiber densities) as well as reduced cell death in the DG are likely to contribute to the observed thickening of the granular layer in p75NTRExIV knockout mice. In addition, Sholl-analysis of DCX-positive neurons revealed a higher dendritic complexity and could thus be a possible morphological correlate for the increased thickness of the molecular layer in p75NTR deficient animals. Our data clearly demonstrate that deletion of both, the short and the full-length isoform of p75NTR affects DG morphology, due to alterations of the cholinergic system and an imbalance between neurogenesis and programmed cell death within the subgranular zone.Analyses of mice carrying a deletion of the pan-neurotrophin receptor p75NTR have allowed identifying p75NTR as an important structural regulator of the hippocampus. Most of the previous analyses were done using p75NTR Neurotrophins exert many of their specific actions through tyrosine kinase (trk) receptors that bind the neurotrophins with high affinity and, in addition, all neurotrophins can signal through a low-affinity receptor, known as p75NTR  are viable and fertile; however they display several defects in the nervous system, including, among others, deficits in the peripheral sensory nervous system . Deletion of Exon IV results in a loss of both, the full-length and the short isoform of p75NTR  in both mutants is unaffected  the neurotransmitter supply in the hippocampus is affected, whether (2) spine densities of DG granule cells are altered, and (3) whether changes in adult hippocampal neurogenesis can be observed in p75NTRExIV mice.However, it can be speculated that deletion of p75NTR has also an impact upon the DG, since proNGF can signal via p75NTR and since proNGF seems to be capable of inhibiting adult neurogenesis in the DG  were analyzed in comparison to littermate controls (p75NTRExIV+/+) that were both obtained by crossing heterozygous p75NTRExIV mice.p75NTRFor the subsequent analysis, mice with an age of 4\u20136 months (\u201cadult\u201d) as well as with an age between 20 and 23 months (\u201caged\u201d) were used. All animal experiments were performed in accordance with German animal rights regulations and with permission of the Landesamt f\u00fcr Landwirtschaft, Lebensmittelsicherheit und Fischerei (LALLF) Mecklenburg-Vorpommern, Germany.2PO4, 10.73 g Na2HPO4 and 9.0 g NaCl in 1,000 ml distilled water, pH 7.2) followed by perfusion with 4% paraformaldehyde . Brains were removed and immersed in the same fixative for at least 5 days.Animals were killed with an overdose of ether and transcardially perfused with phosphate-buffered saline  stained 30 \u03bcm thick coronal serial sections, sampled at a random start position  and cut at 120 \u03bcm. Analysis of dendritic spines was conducted in a blinded procedure. Only one segment per individual dendritic branch and neuron was chosen for the analysis. Spines were analyzed in the dorsal, but not ventral, leaf of the DG because spine densities are different in these locations and no subdivision into different types of spines [filopodia, stubby, thin, and mushroom, which reflect temporal snapshots of a dynamic phenomenon . For statistical evaluation of two groups unpaired two-sided p \u2264 0.05. Data in figures were expressed as mean \u00b1 SEM; significant changes in the figures were indicated by an asterisk.For the analysis of more than two groups one-way ANOVA followed by a Tukey\u2019s multiple comparisons test was used. In case of the HPLC analysis, a two-way ANOVA followed by a Bonferroni post-test was used. Significance levels for all tests were set at ExIII knockout) causes a significant increase of the DG resulting from an increased width of the granular, but not the molecular layer in adults  as well as the granular layer  in adult p75NTRExIV knockout mice  nor in the molecular layer (p = 0.735).We have previously shown that functional deletion of the long isoform of the p75 receptor  and subgranular (p = 0.041) layer of the DG in p75NTRExIV knockout mice were significantly increased by more than 32%, as compared with age-matched controls  as well as subgranular layer (p = 0.0003) of the DG.We next examined whether and to what extent deletion of p75NTRs Figure . Thus, ds Figure . The ince Figure , in contExIII knockout mice are reduced as compared to controls . Aged p75NTRExIV\u2212/\u2212 mice also showed reduced rates of apoptosis, but due to the great variance observed in older animals, the difference was not significant from age-matched controls .We have recently shown that the rates of cell death in the hippocampus of p75NTRExIV\u2212/\u2212 mice was not different from the number determined in control mice . Thus, neither deletion of Exon III  as a marker Figure . In adulExIV\u2212/\u2212 mice harbor significantly more DCX positive cells within the DG than age-matched p75NTRExIV+/+ mice . This is in contrast to mice with a deletion of Exon III  as a marker for both mitotically active and postmitotic young neurons Figure . As showExIII\u2212/\u2212 mice  was not statistically different (t-test: p = 0.83) from the numbers determined in the respective controls  during aging from 17.33 to 14.81 spines per 10 \u03bcm and in p75NTRExIV\u2212/\u2212 mice an age-related decline in spine densities from 17.93 to 13.71 spines per 10 \u03bcm (p = 0.011) was found. Concerning the mean length of dendritic spines, subtle alteration in mean spine length was noted by comparing p75NTRExIV\u2212/\u2212 mice with age-matched controls; however, these effects were not significant  and neurogenin 3, which might contribute to the phenotypes observed. Activation of p75NTR was shown to up-regulate the expression of Hes1/5 and increased expression of these genes is sufficient to decrease the number of dendrites . However, due to hind limb ataxia p75NTRExIV\u2212/\u2212 mice are not well-suited for those behavioral tests  expressing cells, a lasting increase in the number of cholinergic neurons was observed (Boskovic et al., In summary, our data show that deletion of both, the short and the full-length isoform of p75NTR causes altered DG morphology with an overall thickening of the involved cell layers.Consequently, alterations in morphogenic substrates such as the quantity of cholinergic innervation as well as the amount of active neurogenesis and programmed cell death were evident. Thus, p75NTR is likely to play a role in regulating the cholinergic system and modulating adult neurogenesis by balancing neuronal differentiation and apoptotic cell death within the DG.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."}
{"text": "Humans have an impressive ability to augment their creative state . Though this \u201cthinking cap\u201d phenomenon is commonly experienced, the range of its potential has not been fully explored by creativity research, which has often focused instead on creativity as a trait. A key question concerns the extent to which conscious augmentation of state creativity can improve creative reasoning. Although artistic creativity is also of great interest, it is creative reasoning that frequently leads to innovative advances in science and industry. Here, we studied state creativity in analogical reasoning, a form of relational reasoning that spans the conceptual divide between intelligence and creativity and is a core mechanism for creative innovation. Participants performed a novel Analogy Finding Task paradigm in which they sought valid analogical connections in a matrix of word-pairs. An explicit creativity cue elicited formation of substantially more creative analogical connections . Critically, the increase in creative analogy formation was not due to a generally more liberal criterion for analogy formation . The use of an online sample provided evidence that state creativity augmentation can be successfully elicited by remote cuing in an online environment. Analysis of an intelligence measure provided preliminary indication that the influential \u201cthreshold hypothesis,\u201d which has been proposed to characterize the relationship between intelligence and trait creativity, may be extensible to the new domain of state creativity. Creativity research has frequently followed the form of intelligence research, treating creativity as a trait, i.e., a stable attribute of which some people have more and other people have less \u20133. A somOf particular importance to efforts aimed at augmenting creativity and innovation is state creativity in the context of reasoning. Creative reasoning, especially in the form of analogical reasoning, is frequently the basis of innovation in science and industry \u201326. AnalThis initial evidence suggests that the dynamism of state creativity can be channeled to improve reasoning, but important questions remain. Valuable creative insights in the sciences and industry cannot often be gained by simply assessing the validity of an already-formed analogy as in the study by Green et al.  or by chA further question concerns the relationship between state creativity and intelligence. The creativity-intelligence relationship has been a target of inquiry in the trait creativity literature. Much of this work has surrounded the \u201cthreshold hypothesis,\u201d originally proposed by Guilford . The thrAn additional unexplored question concerns the effectiveness of state creativity augmentation via remote online communication. This question is timely as online communication becomes an increasingly common medium for education, work, and research \u201347. StudTo contribute to the developing understanding of state creativity, and state creative reasoning in particular, we sought to address these questions. We employed a novel Analogy Finding Task devised to be sufficiently open-ended to detect individual differences in creative performance yet sufficiently constrained to enable sensitivity to inappropriate responding. The Analogy Finding Task tests participants\u2019 ability to find analogical connections among a large matrix of word pairs while avoiding combinations of word pairs that form invalid analogies. State creativity was measured, as in prior state creativity research  in April of 2015 and paid $2.00 for participation. Data were quality controlled to identify responses not indicative of appropriate attention to task instructions. Exclusion of a substantial number of participants is common for online data collection via Mturk and was anticipated for the present study. Thirty-seven Participants who did not complete the entirety of the survey were removed. Completion times were reviewed and 17 participants with times greater than two standard deviations above the mean  or less than one standard deviation below the mean  were considered to be potentially affected by inappropriate attention to the tasks and were removed. Note that two standard deviations below the mean would have been 0 minutes and thus not a meaningful exclusion criterion. IP Addresses were reviewed. In one case, multiple participants had the same IP address, and only the first completed survey from this IP address was included for analysis. Two participants who participated in the pilot phase of the study, described below, were also removed. A question to \u201cprove you are human\u201d appeared at the end of the survey. Three ostensible participants who did not accurately complete this question were removed. Lastly, quality control analogies were embedded within the Analogy Finding task itself. The task was broken into two matrices of word pairs (described below), and each matrix included 5 quality control analogies that were determined via a pilot study (described below) to be the most readily identifiable analogies in the matrices. In order to eliminate participants who were either not properly following instructions or not giving appropriate attention to task performance, seven participants who did not identify at least three quality control analogies in each matrix were removed from further analysis. The fully quality-controlled dataset included 90 participants . Written informed consent from all participants (main study and pilot study) was obtained according to Institutional Review Board guidelines. Study procedures were approved by the Georgetown University Institutional Review Board.One hundred and fifty seven participants were recruited online via Amazon Mechanical Turk . Second, we sought to confirm that a set of quality control analogies embedded in each matrix would be the most readily identifiable analogies in each matrix .A total of 296 participants were recruited for the pilot study. Based on similar exclusion criteria to the ones described above, the sample was cut down to a final N of 178 . Since we were interested in confirming that the quality control analogies would be the most readily identified items in the matrices, participants were not initially excluded for failing to identify quality control analogies.The Analogy Finding task consisted of two matrices of word-pairs . Each mahttp://lsa.colorado.edu). We used the LSA topic space of \u201cgeneral reading up to first year college (300 factors)\u201d and term-to-term comparison type. This measure of semantic distance corresponds to the cosine of the angle between vectors corresponding (in our usage) to the terms of each analogy within a given semantic space, which is derived through analyses of all of the contexts in which each word tends to be present or absent in that topic space ) Participants in the pilot study completed the two Analogy Finding Task matrices used in the main study, with the order of the matrices fully counterbalanced between subjects. Participants were randomly assigned to one of two conditions: they were either cued to be creative for both matrices (Cued condition), or were not cued to be creative in either matrix (Uncued condition).Creativity was assessed via the following outcomes measures: (1) the total semantic distance of valid analogies identified, (2) the number of valid analogies identified, which is related but not informationally identical to the total semantic distance, and (3) the number of invalid analogies identified. The change in these measures from the first/uncued matrix to the second/cued matrix was taken to measure the effect of the creativity cue in the main study. The total, rather than the average, semantic distance was used because averages were deemed likely to provide misleading representations of the ability to formulate creative analogies. For example, it was possible to identify very few analogies (as few as one) and yet to have a high average semantic distance.X2 = 99.10, p < 0.001, r = 0.75), and the least frequently identified quality control item was identified more often than the most frequently identified non-quality control analogy .The pilot study revealed that a set of analogies intended to be used as quality control analogies in the main study were, as hoped, the most frequently identified analogies. On average, participants (N = 178) identified 8.17 out of a possible 10 quality control analogies compared with 5.73 out of a possible 24 cross-domain analogies. This difference in frequency of identification was significant , 29 participants who failed to identify at least three quality control analogies in each matrix were removed. The reduced sample , showed no practice effect of performing a second Analogy Finding Task matrix after performing a first matrix. That is, there was no significant change on any outcome measure from the first matrix to the second in either condition  were more semantically distant than those identified by participants in the Uncued condition .M = 565.33 \u00b1 SD = 305.96) than in the first/pre-cue matrix (M = 457.69 \u00b1 SD = 247.09), t(89) = 5.48, p < .001, d = 0.39 (M = 8.38 \u00b1 SD = 3.52) than on the first/uncued matrix (M = 7.12 \u00b1 SD = 2.91), t(89) = 5.90, p < .001, d = 0.39. Critically, this increase in finding valid analogies was not accompanied by a greater number of \u201cfalse alarm\u201d invalid analogies identified in the second/cued matrix (M = 2.23 \u00b1 SD = 3.22) than in the first/uncued matrix (M = 1.98 \u00b1 SD = 3.42), t(89) = .97, p = .334. A repeated measures ANCOVA model taking number of false alarm analogies as a covariate demonstrated that the effect of the creativity cue on semantic distance was independent of false alarms, F = 31.08, p < .001, \u03b72 = .25. There was no main effect of false alarms in this model (p = .752), and no interaction of cue by false alarms (p = .266). Additionally, a linear regression model confirmed that pre-cue vs. post-cue change in the number of false alarm analogies was not predictive of the change in total semantic distance, \u03b2 = .10, t(88) = .965, p = .337.Consistent with our primary hypothesis, participants showed notable improvement on creativity measures following the creativity cue. Participants identified analogies that were more creative (greater semantic distance) in the second/post-cue matrix  of 39.54% \u00b1 SD = 22.89%. Verbal IQ was strongly correlated with the change in semantic distance from the pre-cue to the post-cue matrix . MRT score also showed a correlation with change in semantic distance that approached significance . A model in which change in semantic distance (effect of cue) was regressed on both Verbal IQ and MRT indicated that Verbal IQ remained significantly predictive, \u03b2 = .35, t(87) = 3.27, p = .002, whereas MRT was not significantly predictive in this model \u03b2 = .08, t(87) = .77, p = .441. First-order correlations of the two intelligence measures with all creativity outcome measures are reported in Participants showed an average estimated Verbal IQ of 114.19 \u00b1 SD = 4.24. The lower IQ group included 55 participants with average Verbal IQ = 106.22 \u00b1 SD = 10.72. A 2 X 2 ANOVA indicated an expected main effect of the creativity cue on semantic distance  = 39.43, p < .001, \u03b72 = .31), a main effect of IQ group  = 13.20, p < .001, \u03b72 = .13), and, most relevant to the threshold effect, an interaction of Cue by IQ group  = 9.51, p = .003, \u03b72 = .10) indicating that the creativity cue yielded greater increases in semantic distance in the higher IQ group.To test whether an IQ threshold effect could be observed for augmentation of state creativity in our data , the dataset was split into groups of participants with higher and lower Verbal IQ. We used Verbal IQ \u2265 120 as the threshold for inclusion in the higher IQ group based on prior research , 42, 44.r = .36, p = .007). By contrast, the higher IQ group showed no correlation between change in semantic distance and Verbal IQ . Fisher\u2019s r-z transformations suggested a difference between IQ groups for these correlation coefficients that approached significance (p = .053 one-tailed).In the lower IQ group, change in semantic distance (effect of cue) was significantly correlated with Verbal IQ , and formed a greater number of valid analogies. Critically, the increase in formation of creative analogies was not due to an increase in false alarm formation of invalid analogies, and invalid analogies were not more numerous after the creativity cue. This indicates that the augmentation of state creativity we elicited does not reflect an augmentation of divergence at the expense of reasonable constraint . In addition, the Analogy Finding Task was not easily susceptible to inappropriate use of outside materials; the correct answers could not be found in any external resource. Other confounds are more difficult to address, including potential interactions of varying testing environments with our creativity cue manipulation.Analysis of the relationship between a measure of Verbal IQ and augmentation of state creative performance indicated preliminary support for a threshold effect. We found an interaction of cue by IQ group on the semantic distance of analogies, and the correlation between IQ and cued augmentation of creative performance was substantially higher in the lower IQ group. This research is a first empirical exploration of the threshold hypothesis in the context of state creativity. Taken together, the findings provide a preliminary indication that the threshold hypothesis may extend into this novel domain.Some prior research has questioned the validity of the threshold hypothesis. Runco and Albert  concludeAugmentation of state creativity to improve creative reasoning is a promising research direction with potential to inform strategies for innovation in multiple contexts. Here, we extended prior evidence of successful conscious augmentation of creative state in response to an explicit creativity cue. The data provide new evidence that conscious state creativity augmentation can be extended to formation of analogies in an open-ended workspace, and that remote cueing of state creativity is effective in an online environment. A key element of the present findings was that increases in creative analogy finding appear to reflect \u201creal\u201d creativity rather than simply augmented divergence at the expense of sensible constraint. The present data also provide preliminary evidence that the threshold hypothesis, which has been influential in understanding their relationship between intelligence and trait creativity, may be extensible to the domain of state creativity. This work sets the stage for further work to test the boundary conditions of the efficacy of state creativity augmentation in reasoning, including exploration of this phenomenon in increasingly ecologically valid paradigms.S1 Appendix(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file."}
{"text": "For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation. Wild type (wt) p53 is a tumor suppressor, which plays a key role in the cellular stress response. Abrogating p53 function is a key event in human cancer, leading to deregulated cell cycle, genomic instability, resistance to stress signals, and ultimately leading to cancer development , 2. DysfMost p53 mutations are missense  and occur at its DNA-binding domain, which accounts for the improper DNA engagement and disruption of transcriptional activity. P53 mutants also gain new oncogenic functions \u00adincluding resistance to chemotherapies, enhanced cell growth, metabolism, and invasion . Dein vivo.p53 stability is tightly controlled by ubiquitin E3 ligases, which together with the enzymatic activities of E1 and E2, and in certain cases also E4 ligases coordinate the efficient degradation of proteins through the 26S proteasome machinery . The temin vivo , which cleave ubiquitin from proteins. Unlike E3 ligases, the role of DUBs in controlling mutant p53 stability is poorly explored. USP10 deubiquitinates and stabilizes both wt and mutant p53 . InhibitAlthough much of the focus in the field has been devoted to the 26S-mediated proteasomal degradation of p53 during post-stress recovery, the 20S proteasome has been identified as the destination of unmodified p53 that is inherently unstable, unless protected by the NADH quinone oxidoreductase 1 (NQO1). Specifically, inhibition of NQO1 (for example by dicoumarol) promotes p53 degradation through the 20S proteasome in an Mdm2-independent manner. Interestingly, mutant p53 interacts strongly with NQO1, rendering it resistant to NQO1 inhibitors [reviewed in Ref. ]. PertinFree ribosomal proteins are known to regulate the Mdm2/MdmX-p53 axis and activate wt p53, thereby inhibiting tumor proliferation [reviewed in Ref. ]. For exThe regulation of wt p53 degradation is modulated by PTMs. Wt p53 is extensively modified post-translationally in response to stress conditions, which lead to the stabilization and/or activation of p53 [reviewed in Ref. ]. CriticIn addition to mutant p53 itself, modifications of Mdm2/MdmX contribute to the protection of mutant p53 from these key inhibitors. In response to DNA damage, Mdm2 is phosphorylated by ATM and c-Abl, which compromises the ability of Mdm2 to degrade p53 , 84. ATMWhile wt and mutant p53 have distinct and opposing effects on cancer cells, many aspects of their regulation are shared. The majority of the positive and negative regulators of wt p53 that have been tested have a similar regulatory effect on mutant p53. Critically, however, the tightly controlled myriad of positive and negative auto-regulatory loops, which govern wt p53 levels, is uncoupled in the context of mutant p53. In addition, mutant p53 is recognized as a misfolded protein by the heat shock protein chaperons. Together, these contribute to the protection of mutant p53 from the well-coordinated recovery from stress conditions. This results in the chronic accumulation of active mutant p53, which exerts its gain of functions. It is therefore of prime importance to screen patients for p53 mutations prior to treatments, which are known to activate and stabilize p53. The identification of mechanisms that protect mutant p53, as shown by the chaperon HSP proteins, identifies novel approaches to expose mutant p53 to its negative regulators and drive its destruction. Future studies identifying the unique protectors of mutant p53 are a rational approach to define novel approaches to target mutant p53 in cancer cells.RV contributed to writing and editing the paper and prepared a figure. KT contributed to writing and prepared a figure. PJM contributed to writing. SH and YH contributed to writing and editing the paper.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."}
{"text": "However, there is some conflicting evidence.Juvenile-onset SLE (JSLE) is a severe auto-immune disease that can occur in children at any age. Variations regarding the extent of organ involvement, disease activity and damage are found in different age categories. Factors that may contribute to an earlier age of onset are gender, ethnicity and positive family history of SLE. It has been shown that JSLE has a more aggressive disease course compared to adult onset SLE. Correspondingly, it seems that disease severity in younger JSLE patients increases with decreasing age To determine the differences in gender, ethnicity and family history of SLE in JSLE patients with age of onset <10 years and >10 years respectively, and to characterize differences in disease characteristics  between these groups.Data on patient demographics, family history and disease activity (BILAG) and damage (SLICC) at diagnosis and last follow-up visit were collected from the UK JSLE Cohort Study database. Patients were divided into two age categories based on age of onset (\u226410 or >10 years) and were analyzed. BILAG scores of a subset of patients (n = 24) who were followed for five years were analyzed, to evaluate influence of disease duration.A total of 313 patients with JSLE, diagnosed using the ACR-criteria, were included in the analysis. Their data is summarized in the following table: The percentage of female patients was 80% in the <10 group and 84% in the >10 group (p = 0.423). However, the proportion of black children in the <10 group was higher (p = 0.073) and the proportion of Caucasian children in the <10 group was significantly lower (p = 0.047). Family history for SLE was not significantly different between the age categories (p = 0.121).Disease activity at onset was higher in the older group but not significantly (p = 0.105) and no significant differences were found in organ systems involvement. This is in contrast with most literature. Referral time might be a factor causing this and would be an interesting factor to study in this population. Disease activity and damage at follow up  were low in both groups, irrespective of disease duration. Five years after disease onset, disease activity in the <10 group (n = 15) was 1 \u00b1 3, in the >10 group (n = 9) it was 4 \u00b1 5 (p = 0.290). This indicates that for most patients in this cohort, their disease is probably managed well.In contrast to other published cohorts, there were no striking differences in demographics, family history or disease characteristics in this cohort between children diagnosed before or after the age of 10 years old.None declared."}
{"text": "This CH2 domain contains a very crucial S36 residue which is phosphorylated in response to oxidative stress and plays a role in apoptosis. Whereas p52Shc and p46Shc are ubiquitously expressed, p66Shc shows constrained expression. This adaptor protein has been shown to be involved in mediating and executing the post effects of oxidative stress and increasing body of evidence is pinpointing to its role in carcinogenesis as well. It shows proto-oncogenic as well as pro-apoptotic properties. This multitasking protein is involved in regulating different networks of cell signaling. On one hand it shows an increased expression profile in different cancers, has a positive role in cell proliferation and migration, whereas on the other hand it promotes apoptosis under oxidative stress conditions by acting as a sensor of ROS (Reactive Oxygen Species). This paradoxical role of p66Shc could be attributed to its involvement in ROS production, as ROS is known to both induce cell proliferation as well as apoptosis. p66Shc by regulating intracellular ROS levels plays a crucial role in regulating longevity and cell senescence. These multi-faceted properties of p66Shc make it a perfect candidate protein for further studies in various cancers and aging related diseases. p66Shc can be targeted in terms of it being used as a possible therapeutic target in various diseases. This review focuses on p66Shc and highlights its role in promoting apoptosis via different cell signaling networks, its role in cell proliferation, along with its presence and role in different forms of cancers. The Shc family of proteins consists four members ShcA, ShcB, ShcC and ShcD, of which the best characterized to date is ShcA ,2. ShcA,p46Shc and p52Shc are ubiquitously expressed, while p66Shc is expressed at different level in various tissues. The CH1 domain of p46/p52Shc has three tyrosine residues which are phosphorylated in response to a number of cell surface ligand activated receptors. p52Shc binds to phosphorylated receptors via the PTB domain or the SH2 domains in response to growth factors, and is phosphorylated on these tyrosine residues  within the CH1 regions. Grb2 (Growth Factor Receptor-bound Protein 2) forms a complex with SOS (Son of Sevenless) protein. The Grb2/Sos complex is recruited by the phosphorylated p52/p46Shc proteins. This Grb2/Sos complex is then docked onto these phosphotyrosine residues, via direct interaction with the Grb2-SH2 domain, which ultimately leads to the activation of Ras proteins . This coAnother adaptor protein E3b1 together with Eps8 also plays a role in regulating the phosphorylation, reducing ubiquitilation and increasing the stability of p66Shc protein through Rac1 . Sos1 cain vivo has been observed to increase metabolic rate as well as decrease fat mass and resistance to diet-induced obesity. p66Shc generated ROS regulates the effect of insulin and may lead to acceleration of aging by favoring fat deposition and as a result fat related disorders [p66Shc is also a part of a signal transduction pathway that is activated by increased intracellular ROS, leading to apoptosis. The oxidative stress resistance and lifespan of a cell and an organism is increased by mutation of this pathway. The accumulation of oxidatively damaged macromolecules occurs in virtually all aging mammals . In C. eisorders .Furthermore, depletion of p66Shchas been shown to lead to Warburg effect, causing enhanced glycolysis and increased allocation of glucose-derived carbon into anabolic metabolism. This role of p66Shc has been observed in mice that are deficient in p66Shc. These p66Shc deficient mice show resistance to diabetes and obesity. This altered metabolism was seen to be mediated by mTOR . This indicates towards an inhibitory role of p66Shc in anabolic metabolism, unlike other isoforms of Shc . Stimulap66Shc has also been shown to contribute to EAE  induced neuronal damage. It does so, most likely, through the opening of PT pore that triggers mitochondrial swelling and leads to neurodegenerative stress . In \u03b2-amp66Shc has a unique CH2 region at the NH2 terminal. This CH2 region is of around 110 amino acids and, like the CH1 domain, is rich in glycine and proline residues. The CH2 region contains the unique and all important serine phosphorylation S36 and S54) sites . The CH24 sites .Under oxidative stress the S36 residue of p66Shc is phosphorylated. Treatment with an iron-containing porphyrin, hemin increased the phosphorylation of p66Shc at the S36 residue. In hemin treated K562 erythroleukemic cells p66Shc was transcriptionally activated through the ARE (Antioxidant response element)-Nrf2 (NF-E2-Related Factor 2) pathway . In humaTill now most of the studies have demonstrated that p66Shc shows a pro-apoptotic and pro-oxidative role. Up to 30% extension in lifespan was seen in p66Shc knockdown mice. These knockdown mice also showed better handling of ROS and oxidative stress ,36-38. TThe S54 site in the CH2 domain of p66Shc is important for its stability . The pro2S inhibited the mitochondrial ROS production and as a result, phosphorylation of p66Shc and lead to sulfhydration of p66Shc on C59 residue [2O2 was also seen to be mediated by p66Shc [Recently it was seen that in SH-SY5Y cells or in the mice cortex, exogenous H residue . ROS arey p66Shc . Many sty p66Shc . The assy p66Shc .2O2. All these studies point to the fact that p66Shc is needed for bringing about apoptosis. Phosphorylation of p66Shc on the S36 residue has been shown to have a pro-apoptotic effect in different cell lines. Upon expression of p66S36A mutant into these p66Shc-/- cells, they are still not able to restore the pro-apoptotic responses [The increased levels of ROS act as the trigger to interfere with many cellular processes. These triggers result in inhibition of cell proliferation and induction of apoptosis. Apoptosis is the process of programmed cell death that takes place in multicellular organisms and comprises of many cellular events like cellular blebbing, nuclear fragmentation, chromosomal DNA fragmentation and ultimately cell death. p66Shc is known to have a role in apoptosis triggered by oxidative stress. The affirmation to this has been seen in a number of studies. Oxidative stress gives a stimulus to mouse thymocytes, peripheral blood lymphocytes and splenic T-cells and these cells then attain the ability to express p66Shc -48,50,51esponses -30.The gene encoding p66Shc has also attracted major interest in aging research in the recent years since it plays a vital role in apoptosis induced by oxidative stress. p66Shc is known to have a crucial role in the regulation of intracellular ROS levels . The accROS can either originate from exogenous sources such as ultraviolet and ionizing radiations, or it can be endogenous . ROS has been implicated in the damage of cellular proteins, lipids and DNA. This oxidative cellular damage is apparently the main origin of aging. It causes mutations and deletions in both nuclear as well as mitochondrial DNA ,44,53. VThe p66Shc-/- fibroblasts cells have altered bio-energetic properties vis-\u00e0-vis decreased resting and maximal oxidative capacity. This suggests that deletion of p66Shc may extend lifespan by shifting metabolic energy away from oxidative and toward glycolytic pathway . One can2O2 and UV. p53-/- MEFs also show resistance to UV-induced and H2O2-induced apoptosis [The tumor suppressor p53 is critically involved in oxidative stress-dependent apoptosis. p53 has a role in mediating the oxidative stress response of p66Shc, since p53 is up-regulated upon treatment with Hpoptosis . Increaspoptosis . p66Shc poptosis   help the cells to sense their location as well as that of the neighboring cells. When these cell-ECM interactions are incorrect, apoptosis takes place. This kind of location specific apoptosis is termed anoikis. Anoikis is a special case of programmed cell death that is initiated by cell detachment instead of the normal pro-apoptotic signals . AnoikisCells that lack p66Shc have been shown to escape anoikis. Anoikis in these cells was restored with the reexpression of p66Shc. p66Shc restores anoikis via RhoA activation. Transient as well as stable transfections with p66Shc lead to cell death in detached cells, but not in adherent cells. In contrast to apoptosis caused by oxidative stress, this ability of p66Shc to confer anoikis properties is not dependent on S36 phosphorylation of p66Shc . In addiCell proliferation, migration and adhesion are the main properties of a tumor cell. Tumor cells proliferate, migrate and adhere to target tissue. These steps allow the tumor cell to obtain metastatic phenotype. Cell proliferation and migration depend on the signals transmitted by growth factors and adhesion proteins . These pp66Shc also plays a crucial role in proliferation of epithelial cells. The processes of cell proliferation and apoptosis have to be well balanced. The balance of these two processes is governed by signal transduction machinery . Any imbARF1 has been found in complex with Grb2 and p66Shc upon EGF stimulation of the basal like breast cancer MDA-MB-231 cell line. The small GTPases ARF1 and ARF6 have been shown to be activated downstream of the EGFR and act as key regulator of growth, migration and invasion of breast cancer cells . Estrogep66Shc protein levels were seen to be closely correlated to the growth rate of prostate cancer cells. p66Shc protein levels were also seen to be significantly higher in prostate cancer adenocarcinomas as compared to the adjacent benign glandular cells ,97. p66SA consistent increase in the p66Shc protein levels were observed in ESCC  and EAC . The controls of ESCC showed a lower basal level of p66Shc expression . In the 2O2 [The expression of p66Shc was seen to be increased in all kinds of proliferating thyroid tissues, but not in the normal thyroid tissue of the same patient . This da2O2 .Aiolos, which is often expressed in lung cancers and correlated with reduced patient survival, has been seen to be inversely correlated with the expression of p66Shc in lung cancer tissue as well as in single cells . On the In ovarian cancer cells the level of ErbB-2 expression positively correlates with the level of p66Shc. ErbB-2 is a prognostic marker for ovarian carcinoma . In ovarIn colon cancer tissues, cancerous cells show higher levels of p66Shc proteins as compared to the adjacent non-cancerous cells . p66Shc A recent study has shown the regulatory role of p66Shc-derived mitochondrial ROS. The p66Shc-derived ROS has a regulatory role on growth factor signaling by induction of protein tyrosine phosphatase (PTP) oxidation. It was seen that p66Shcdeletion and down regulation lead to decrease in PDGF-induced signaling and migration, by decreasing PDGF-induced PTP oxidation . In summp66Shc being a candidate protein that controls both cell death as well as cell proliferation and plays a crucial role in regulating longevity and cell senescence, by regulating intracellular ROS levels. These counteracting properties of p66Shc make it a perfect candidate for further studies in various cancers and aging related diseases. p66Shc can be targeted in terms of it being used as a possible therapeutic target in various diseases, by figuring out how to use p66Shc protein for balancing the ROS levels in the cells and tipping the scales in our favor."}
{"text": "N- and C-termini and in the core domain. The phosphorylation sites are relatively less mutated than other residues in p53. To understand why and how p53 phosphorylation sites are rarely mutated in human cancer, using a bioinformatics approaches, we examined the phosphorylation site and its nearby flanking residues, focusing on the consensus phosphorylation motif pattern, amino-acid correlations within the phosphorylation motifs, the propensity of structural disorder of the phosphorylation motifs, and cancer mutations observed within the phosphorylation motifs. Many p53 phosphorylation sites are targets for several kinases. The phosphorylation sites match 17 consensus sequence motifs out of the 29 classified. In addition to proline, which is common in kinase specificity-determining sites, we found high propensity of acidic residues to be adjacent to phosphorylation sites. Analysis of human cancer mutations in the phosphorylation motifs revealed that motifs with adjacent acidic residues generally have fewer mutations, in contrast to phosphorylation sites near proline residues. p53 phosphorylation motifs are mostly disordered. However, human cancer mutations within phosphorylation motifs tend to decrease the disorder propensity. Our results suggest that combination of acidic residues Asp and Glu with phosphorylation sites provide charge redundancy which may safe guard against loss-of-function mutations, and that the natively disordered nature of p53 phosphorylation motifs may help reduce mutational damage. Our results further suggest that engineering acidic amino acids adjacent to potential phosphorylation sites could be a p53 gene therapy strategy.p53 protein has about thirty phosphorylation sites located at the  In adp53 phosphorylation ,7 at almGiven the functions controlled by p53 phosphorylation, it is expected that the specificity and dynamics of p53 phosphorylation are carefully regulated, and mutations would affect p53 phosphorylation pattern and function. Phosphorylated p53 mutants were found to accumulate in tumor tissues ,28. p53 C-terminal homo-oligomerization domain and becomes phosphorylated predominantly at Y99, and also at Y121 and Y240 . It. It46PDDF1B1/p62 .N-terminal transactivation domain (TA), a DNA-binding core domain, a C-terminal tetramerization and a regulatory domain. In addition, p63 and p73 also have a sterile alpha motif domain (SAM) at the end of the C-termini. Most of the N-terminal TA domain and C-terminal regulatory domain are highly flexible and have no well-defined structure. Therefore, p53 can also be classified as a typical intrinsically unstructured protein [The structures of p53/63/p73 contain an  protein ,100, whi protein ,101. The protein ,103.It was suggested that natively disordered proteins are more likely to be associated with cancer . p53 is p53 gene transfer in human glioma correlates with site-specific phosphorylation [Gene therapy to restore p53 function is among the clinical approaches currently under investigation . For exarylation ,104, sugrylation . It is krylation . While siYj=i+1, XiYj=i+2, and XiYj=i+3, which are equivalent to the dipeptide XY, tripeptide X \u00d7 Y, and tetrepetide X \u00d7\u00d7 Y motifs, respectively. Then we calculate the propensity of amino acid pair association by normalizing the frequencies of the overall counts of individual amino acid in the p53, p63, or p73 families:XiYj is the propensity of amino acid pair association in XiYj=i+1, XiYj=i+2, and XiYj=i+3 positions, respectively. NXiYj is the total number amino acid pair counts in XiYj=i+1, XiYj=i+2, and XiYj=i+3 positions in all sequences in each family, respectively. The NX and NY are total numbers of amino acid type (X and Y) in all sequences in each p53, p63, or p73 families [XiYj is similar to that used by Vonderviszt and Simon [XiYj/(NXY \u00d7 PX \u00d7 PY) to measure the dipeptide propensity . PXiYj in equation 1 changes more smoothly with the variation of NX and NY in the calculation of the dipeptide propensities across evolution [For each sequence in a protein family, we count the two amino acids (X and Y) in the positions of Xfamilies . Thus, and Simon , who usevolution .XiYj by adding PXiYj and PYiXj for non-diagonal elements in the correlation matrix.We calculate the degenerate dipeptide pair correlation propensity DPThus, we do not distinguish between XY and YX and label such amino acid combinations X/Y, and the propensity X/Y is obtained from the combined XY and YX counts.Disorder propensities were calculated using the webserver FoldUnfold . The aveWe use ClustalX 2.0  to alignp53 gene therapy.The existence of almost thirty phosphorylation sites in p53 poses a considerable evolutionary pressure on p53 to selectively bind to specific kinase with the right phosphorylation motif to respond to correct phosphorylation signals. What are the sequence and structural features in p53 phosphorylation motifs? Why and how p53 phosphorylation sites are rarely mutated in human cancer? In our study, we focused on the phosphorylation sites and nearby flanking residues and computationally examined the consensus phosphorylation motif pattern, amino acid correlations within the phosphorylation motifs, the propensity of structural disorder of the phosphorylation motifs, and cancer-related mutations observed within the phosphorylation motifs. Among the many factors potentially contributing to the safeguarding mechanism against mutations in p53, we found two patterns that appear unique and correlated with p53 mutations. p53 proteins have high propensities for acidic amino acids adjacent to potential phosphorylation sites. The negative charge near a phosphorylation site might make a mutation of the phosphorylation site less deleterious, since the motif already has a negative charge and may still have certain electrostatic interactions with other binding partners. The above conclusion is consistent with experimental observations. p53 phosphorylation motifs are mostly disordered. Even though cancer-related mutations largely decrease the disorder propensity of the phosphorylation motifs, the disordered nature of phosphorylation motifs might still help to compensate for mutational effects on p53 phosphorylation. Our computational results suggest that engineered acidic amino acids adjacent to potential phosphorylation sites could be a potential strategy in"}
{"text": "Drosophila have unraveled intriguing roles of Dmp53 in promoting cell division in apoptosis-induced proliferation, enhancing fitness and proliferation of the winner cell in cell competition and coordinating growth at the organ and organismal level\u00a0in the presence of stress. In this review, we describe these new functions of Dmp53 and discuss their relevance in the context of carcinogenesis.The canonical role of p53 in preserving genome integrity and limiting carcinogenesis has been well established. In the presence of acute DNA-damage, oncogene deregulation and other forms of cellular stress, p53 orchestrates a myriad of pleiotropic processes to repair cellular damages and maintain homeostasis. Beside these well-studied functions of p53, recent studies in  The p53 protein is the product of the tumor suppressor gene, TP53. It is a member of the p53 superfamily proteins that comprise TP53, TP63 and TP73. p53 is mutated or inactivated in more than half of human cancers. Early works on p53 have elucidated its canonical function in response to DNA damage. Specifically, in the presence of mild stress or damage signal, p53 blocks cell cycle progression and activates DNA repair machinery to promote cell survival and maintain genome integrity. However, when the damage is extensive, p53 drives cellular senescence or irreversible apoptosis to eliminate the potentially oncogenic cells with genomic instability and therefore reduces the risk of tumorigenesis , 2. In aDrosophila is instrumental to study the pleiotropic functions of p53 in both stress response and development [Drosophila melanogaster p53 (Dmp53) and found it to be the only member of the p53 family proteins in flies [Drosophila studies showed that after irradiation treatment, Dmp53 mediates apoptosis but not cell cycle arrest [C. elegans, the p53 homologue, cep-1 still harbors a SAM domain. However, studies from Banerjee and col. have challenged this view as Drosophila p53 is able regulate cell cycle upon energy deprivation: the \u201ctenured\u201d mutation disrupts the mitochondrial electron transport chain and causes reduced cellular ATP level, which leads to a G1/S checkpoint arrest that requires Dmp53 [Drosophila p53 carries the ancestral functions of all p53 family members . This ies Dmp53 . Specifies Dmp53 . These rDrosophila p53-mediated apoptosis, DNA repair and homologous recombination, but also were among the first to assign physiological and developmental functions to p53 [Since the cloning of Dmp53, studies in the fly primordial germ cells, imaginal discs and adult photoreceptors have not only elucidated the detailed mechanisms underlying s to p53 \u201319. Reces to p53 .Drosophila have unraveled intriguing roles of Dmp53 in promoting cell division in apoptosis-induced proliferation, enhancing fitness and proliferation of the winner cell in cell competition and coordinating growth at the organ and organismal level in the context of stress , which in turn induce immediate and local proliferation in the healthy surrounding cells [Drosophila [In response to damage or injuries in the developing tissue, apoptotic cells promote cell proliferation in the surrounding tissue to repair and regenerate , 23. Apong cells \u201326. Thisosophila \u201329 and iosophila . AiP is osophila .rpr or hid, but apoptosis is concomitantly inhibited by the expression of the caspase inhibitor p35 [AiP was first studied using \u201cundead cells\u201d, which are generated by subjecting the cells to a pro-apoptotic signal, such as itor p35 , 29. In itor p35 , 32. Theitor p35 \u201335. Impoitor p35 , 36. In itor p35 . This paCnidaria hydra [There is evidence that such Dmp53-controlled proliferation and AiP program are conserved throughout evolution. In the planarians, p53 regulates proliferation and self-renewal in adult stem cell lineages . Similaria hydra , in thatia hydra . Interesia hydra . In thisDrosophila p53 regulates multiple targets essential for apoptosis, DNA repair and proliferation [Drosophila, there are four annotated transcripts for Dmp53, which leads to the expression of two detectable protein isoforms: Dp53 and D\u0394Np53  that contains a putative p53 binding site, the authors showed that varying Dmp53 protein levels regulate N gene transcription. This study corroborates previous mouse studies showing that p53 binds Notch1 promoter and that p53 silencing resulted in reduced Notch expression [In this issue of Apoptosis, Busturia and col. have examined the contribution of Notch (N) signaling to AiP in relation to D\u0394Np53 . The autpression , 46. Thepression .dronc- and dmp53-dependent manner; while in differentiating tissues such as the posterior part of the eye disc, Hh is preferentially activated in the dying cells via the non-apoptotic activation of the effector caspases Drice and Dcp1 [While overexpressing D\u0394Np53 activates N and Wg expression in the developing wing, it is unclear how N, Wg, Dpp and Hh contribute to the different AiP paradigms. It was proposed that distinct programs of AiP are activated depending on the cellular state , 26. Forand Dcp1 . Thus, ddronc mutants [Drosophila p53, the induction of proliferation by Dmp53 is prompted by apoptosis upon stress. In the classic DNA damage response, Dmp53 rapidly activates rpr expression and hence the apoptosis cascade. A recent study by Shlevkov and Morata further cements the tight association between Dmp53 and the apoptotic cascade by showing that Dmp53 and JNK mutually activate each other and act upstream of the pro-apoptotic genes  and establish a positive feedback loop that amplifies the initial apoptotic stimuli [An interesting observation is that D\u0394Np53 induces mitogen expression and proliferation even when apoptosis is inhibited in  mutants , 36, 41. mutants , 36. How stimuli . Dmp53 a stimuli \u201350. Thus stimuli . Based orpr, hid and grim expression, inhibits DIAP1 and ultimately activates the initiator (dronc) and effector caspases  [Another interesting conundrum is why the AiP program is only induced in response to cell injury, but not in developmental apoptosis. In several fly developmental processes, apoptosis is required to reduce the primordial germ cell numbers in the embryo and eliminate the excess neuroblasts in the larva or the supernumerary interommatidial retinal cell during pupal development, and some of these processes are known to depend on Dmp53 \u201354. Likee, dcp1) , yet sucDrosophila imaginal wing disc upon growth challenge. Specifically, the authors hampered growth in the posterior compartment\u00a0of the wing disc, either by the expression of Ricin, a ribosome-inactivating toxin, or by the inhibition of insulin pathway. The cell division and growth rate in the unaffected compartment accordingly slow down to generate an adult wing that is normally proportioned, even though the final size is smaller than the wild-type wing , and reducing or removing Dmp53 drastically diminishes such Hippo phosphorylation, and consequently dampens Hippo pathway activation in the presence a cell-death signal. This work nicely demonstrates that Hippo acts epistatically to Dmp53 to control the activation of apoptosis. Now in the light of the finding that Dmp53 can coordinate organ size and systemic growth in a stress context, an intriguing question begs the answer: can Dmp53 act in concert with the components of the Hippo pathway to control and coordinate tissue growth? And vice versa, does the Hippo pathway play a role in AiP in the classic IR-induced damage model? We do not yet have answers. However, the analysis of yorkie function may hold some clues. It has been well-established that Yorkie activates Diap1 to limit apoptosis during tissue proliferation and growth [Drosophila ASPP1 homologue and Dmp53, which can activate the downstream apoptotic genes [The Hippo signaling cascade is one of the most conserved organ size control pathways in evolution \u201366. By pd growth . Recentld growth . Reducind growth , 68. Howic genes , 71. TheDrosophila is a genetically tractable model to explore the \u201cprimordial\u201d functions of p53 in addition to its well-known functions in mediating apoptosis in the presence of genotoxic stress. In fact, even such canonical Dmp53 function is more complex than we first realized, as studies begin to unravel the roles of Dmp53 isoforms in controlling AiP and programmed cell death at the same time in the same tissue. The molecular details of these processes require future investigation. Furthermore, studies in Drosophila pioneered many founding works in identifying the physiological functions of p53, including the recent discoveries of the role of Dmp53 in controlling organ size, system growth and cell competition in the presence of stress. From an evolutionary point of view, is this kind of growth-control function part of the \u201cprimordial\u201d p53 functions? Four years ago, a study from John Abram\u2019s laboratory discovered that Dmp53 mediates meiotic recombination in the germ-line, and proposed that such primordial function of p53 is the origin of the genotoxic stress response [Studies of p53 in the past 35\u00a0years have depicted the many facets of p53 functions that are forever evolving. We now recognize that besides being the classic \u201cguardian of the genome\u201d , p53 parresponse . Now an"}
{"text": "P53 is most well-known for its tumor suppressive function in differentiated cells. Its activities in embryonic stem cells (ESCs) are, however, less well understood. For many years it was thought that p53 is not active at all in ESCs and unable to elicit a DNA damage response in this cell type. In the last few years, it emerged that p53 may have some functions in ESCs. Nevertheless, it remained a mystery how its activity is controlled in ESCs. A recent report demonstrates that p53 activity is regulated by a novel RNA-containing negative feedback loop that promotes apoptosis specifically in ESCs. This study not only demonstrates unequivocally that p53 is active in ESCs, it further illustrates a novel mechanism of gene regulation\u2013by protein coding RNAs. Cell Stem Cell has shed light on the intricate pathway that regulates the pro-apoptotic function of p53 in ESCs [Embryonic stem cells (ESCs) are pluripotent and can differentiate into many cell types. To avoid passing mutations to daughter cells, ESCs have great need to maintain their genome integrity. The tumor suppressor p53 plays proverbial roles in regulating the genomic integrity of ESCs. Several studies have established that p53 induces ESC differentiation in response to DNA damage , 2. Howe in ESCs .Apela for further study that is repressed by p53 and highly expressed in ESCs. They found that Apela positively regulates p53-mediated DIA in mouse ESCs. Surprisingly, although Apela RNA has a putative coding region, this region is dispensable for the function of Apela in the DIA of mouse ESCs. Instead, Apela acts as a regulatory RNA. But as no direct interaction could be found for Apela and p53, how can this RNA regulate p53 function? To answer this question, the authors performed RNA-precipitation. One of the proteins that precipitated with Apela was the heterogenous nuclear ribonucleoprotein L (hnRNPL). This protein associates with p53 and inactivates the tumor suppressor protein by sequestering it from the mitochondrial department and by stimulating its degradation, the former but not the latter function is antagonized by Apela. In response to DNA damage, the relative interaction between hnRNPL and p53 is decreased, freeing p53 from its constraint and allowing its accumulation and initiation of apoptosis in ESCs. Huang and his colleagues speculate that Apela may facilitate a quick response of ESCs to DNA damage by skipping the translation step. As Apela is repressed by p53, p53, Apela and hnRNPL form a tri-element negative feedback loop in ESCs. Given the enrichment of Apela in ESCs, this negative feedback loop is most likely specific for the regulation of p53 activity in ESCs.To investigate the mechanism, Huang and his colleagues set out to test whether p53 induces apoptosis in mouse ESCs upon DNA damage and found that p53 is critical for DNA damage-induced apoptosis (DIA). Thus, the pro-apoptotic roles of p53 are conserved between stem cells and differentiated cells. They then used genomic approaches and identified a number of potential downstream targets that mediate p53\u2019s pro-apoptotic function. Since little is known about the mechanism of gene repression by p53, the authors decided to focus on one transcript called Because of its highly anti-proliferative activities, p53 needs to be kept in check and released from this constraint when its activity is needed. Therefore, several regulatory mechanisms have been implemented in cells that control p53 abundance and activity in stem cells as well as in differentiated cells including regulation of abundance and activity by posttranslational modifications , associa"}
{"text": "It has been shown that p53 has a critical role in the differentiation and functionality of various multipotent progenitor cells. P53 mutations can lead to genome instability and subsequent functional alterations and aberrant transformation of mesenchymal stem cells (MSCs). The significance of p53 in safeguarding our body from developing osteosarcoma (OS) is well recognized. During bone remodeling, p53 has a key role in negatively regulating key factors orchestrating the early stages of osteogenic differentiation of MSCs. Interestingly, changes in the p53 status can compromise bone homeostasis and affect the tumor microenvironment. This review aims to provide a unique opportunity to study the p53 function in MSCs and OS. In the context of loss of function of p53, we provide a model for two sources of OS: MSCs as progenitor cells of osteoblasts and bone tumor microenvironment components. Standing at the bone remodeling point of view, in this review we will first explain the determinant function of p53 in OS development. We will then summarize the role of p53 in monitoring MSC fidelity and in regulating MSC differentiation programs during osteogenesis. Finally, we will discuss the importance of loss of p53 function in tissue microenvironment. We expect that the information provided herein could lead to better understanding and treatment of OS. P53 is a guardian of cell differentiation.P53 regulates genomic stability, growth, proliferation, and immunoproperties of mesenchymal stem cells (MSCs).P53 is a negative regulator of osteogenic differentiation of MSCs.Loss of function of p53 in MSCs compromises their osteogenic differentiation and affects the properties of bone tumor microenvironment (BME) components, therefore it dictates the conditions for osteosarcoma (OS) development.in vivo and in vitro key molecules involved in the process of bone remodeling, in the context of loss of function of p53.To identify Are there any molecules produced by p53-null MSCs that could affect osteoclast properties and compromise bone homeostasis?How do they relate to the diagnosis and prognosis of OS?3 Having been discovered since 1979, p53 is the most studied member of the family with over 60\u2009000 papers so far published. This large mass of scientific data evidentiate a huge complexity for p53 functional program, ranging from the regulation of metabolism6 and mitochondria/oxygen radicals8 to the deeply analyzed DNA damage repair system,14 autophagy,16 and, last but not the least, its role in cell stem maintenance and lineage determination.18 Despite all these investigations, efforts, and advances in knowledge, many crucial intriguing points still remain unanswered to fully understand the physiological and pathological role of p53. These wide range of effects raise from several angles, including, for example, its regulation at the transcriptional level, at the level of micro-RNA,22 and splicing isoforms24 to its translational regulation and its stability/degradation at the protein level.29 In parallel to so much effort in understanding the function of p53, significant efforts are also underway on its potential clinical exploitation.37 Although being identified after ~20 years, already now, p63 and p73 show a similar complexity, and also the ability to interact with p53 at the structural and functional level,49 where the p63 function is highly relevant in skin formation and homeostasis,51 as well as in cancer53 and stem cell regulation.57TP53 belongs to the so-called \u2018p53 gene family' of transcription factors, which includes also the proteins p63, p73, and p53 itself.58 Thus, a single gene can exist in multiple isoforms with distinct biological functions.60 P53 protein, encoded by the TP53 gene in humans and the Trp53 gene in mice, is well known for its role as the \u2018guardian of the genome' and exerts a pivotal role in maintaining the genetic stability.63 It can prevent tumor formation by regulating cell cycle,64 apoptosis,65 senescence,66 and metabolism67 by binding to responsive elements on DNA (p53RE).68 Abnormal regulation of the p53 family has a critical role in tumorigenesis; indeed, TP53 mutations have been detected in over 50% of all human cancers.70The p53 family of transcription factors have several members including p53, p63, and p73. Each member of this family expresses unique mRNA variants resulting from alternative splicing, promoters, and transcription initiation sites.TP53 and/or the retinoblastoma gene RB1 have been reported to be the main causes of the development of sporadic OS.71In vitro experiments comparing MSCs with malignant OS cells, as well as in vivo studies using transgenic mice with targeting p53 and Rb  silencing in MSCs, have elegantly demonstrated that when p53 alone was deleted, the incidence of OS could reach 60%.72 Another function of p53 in suppressing tumor is to act as \u2018a guardian of differentiation'.59Silent mutations in the tumor suppressor gene 74Notably, p53 guards osteogenic, myogenic, adipogenic, hematopoietic, and neural differentiation of adult stem cells.75 Seven subtypes of OS have been characterized according to histological analysis of the osteoid matrix produced by aberrant osteoblasts: osteoblastic, fibroblastic, chondroblastic, telangiectatic, epithelial, small cell, and giant-rich cell.76 The abundant deposition of osteoid matrix and osteoblast-like features of the malignant cells are the dominant characteristics of the osteoblastic phenotype. This subtype manifests the highest incidence representing 75% of screened OS.78 Within OS of the osteoblastic subtype, aberrant preosteoblasts and osteoblasts produce their own osteoid mineralized matrix close to the area of growth plate (GP). Although chromosomal abnormalities have a decisive role in the development of OS,79 the karyotype is not essential for the subtype classification. OS frequently occurs in human patients with Li-Fraumeni syndrome and with hereditary retinoblastoma. Li-Fraumeni patients carry a germline p53 mutation in one allele compromising the function of p53.82 Different studies have reported that preosteoblasts and osteoblasts represent the cells of origin of OS.83 Importantly, cellular microenvironment is also decisive in determining the fate of stem cells and in promoting tumor formation.84 The osteogenic differentiation of p53-deficient or mutant MSCs can be affected by signals from BME and promote eventually OS. Intrabone inoculation of undifferentiated p53\u2212/\u2212 and p53\u2212/\u2212Rb\u2212/\u2212 MSCs generated osteoblastic OS and developed metastasis characterized by osteoid areas in the lung, spleen, and heart.33 These data suggest that, along with specific bone microenvironment conditions, undifferentiated MSCs with compromised p53 function can represent the cells of origin of OS  released during bone resorption. Insulin-like growth factors and transforming growth factor-beta (TGF-\u03b2) are examples of two coupling growth factors secreted during bone degradation, which have been proven to stimulate the osteoblast activity.88 However, MSCs and osteoblasts can also secrete molecules that can influence the osteoclast activity, and affect bone remodeling in the same cases.89 Indeed, throughout the lifetime bone remodeling is kept at a constant rate to balance bone formation and bone degradation, and to guarantee bone homeostasis. However, along with aging, this exquisite equilibrium is subjected to alterations mainly as a consequence of hormonal dysfunctions. Indeed, steroid hormone deficit enhances the resorbing activity of osteoclasts, which could terminate in an osteoporotic condition.90 Conversely, an increase in bone mass and bone density is representative of osteosclerosis and osteopetrosis conditions. Higher intake of bone in osteosclerosis is exclusively promoted by osteoblasts, whereas osteopetrosis is due to aberrant activity of osteoclast-mediated bone resorption.91 Notably, during the process of endochondral bone formation, which occurs until adolescence, the mesenchymal progenitor cells first differentiate into chondrocytes, which generate new cartilage on the GP. The chondrocytes will be slowly replaced by osteogenic progenitor cells and osteoblasts to produce the bone.84 Interestingly, under normal conditions p53 acts as a negative regulator of osteoblastogenesis by repressing the promoter activity of transcription factors required in the early phase of osteogenic commitment, such as Osterix,92 Cbf\u03b1-1, and Runx2 in osteoprogenitor cells93 .101 P53 can regulate key transcription factor genes involved in exclusive programs of differentiation and dedifferentiation of somatic cells, with an impact on stemness and development. Given that p53 is a tumor suppressor gene and gate keeper of cell differentiation, in this review we want bring to light the connection of p53 status in MSCs, BME, and OS development. Notably, p53-null MSCs exhibit accelerated growth rate and aberrant osteogenic differentiation compared with wild-type MSCs, which contributes to tumor bone formation. Indeed, distinct studies highlight that p53-deficient bone marrow-derived MSCs can proliferate faster, and appear to differentiate earlier during in vitro osteogenic differentiation compared with the wild-type MSCs.102 However, this \u2018tricky' appearance to differentiate earlier into osteoblasts reflects a more complex scenario; indeed, p53-null MSCs are impaired in achieving terminal differentiation towards mature osteocytes.92 MSCs represent a source of precursor for osteogenic progenitor cells and osteoblasts. P53 mutations that lead to defects in the control of cell growth of osteogenic progenitor cells represent the main source of sporadic OS.MSCs are a subset of adult progenitor cells that exist in almost all adult tissues . Adult MSCs have proven to be cells of mesodermal origin, which can give rise to skeleton, muscle, heart, spleen, and other internal organs.in vitro knockdown of p53 in mouse embryonic fibroblasts (MEFs), which are cells representative of an embryonic stage of development, induced higher expression level of Osterix and Runx273 but not of terminal differentiation markers such as Osteocalcin and Sost-Sclerostin.103 Conversely, p53 knockdown in multipotent bone marrow stromal cells (MBA-15), which resembles adult progenitor cells,104 promoted terminal osteogenic differentiation.73 Consistently, also after reintroduction of wild-type p53 in OS cell line, apoptosis and terminal differentiation were promoted.104 We can emphasize that p53 can regulate bone formation and the differentiation of early osteogenic precursors as demonstrated by the knockdown of p53 in MEF, and, furthermore, it can also promote terminal differentiation in MBA-15.The in vitro for several passages without being severely compromised in their properties.105 The induction of p21 or cyclin-dependent kinase (CDK) inhibitor p21Cip1/Waf1 mainly promoted by p53 is associated with cell-cycle arrest  and C/EBP-\u03b1 (CCAAT/enhancer-binding protein-\u03b1).114 Adipocytes and osteoblasts are both cells derived from multipotent progenitor cells. The undifferentiated status of the cells is kept by the repression of transcription factors that repress each other to preserve multipotency. However, upon appropriate stimulations, MSCs make sequential cell fate choices.115 The commitment of MSCs towards a specific mesenchymal differentiation program is coordinated also by p53, which regulates their multipotential state. In in vivo studies, p53 is upregulated in adipocytes from genetically obese mice in a fed state. Transgenic mice overexpressing functional p53 gain less body mass and adipose tissue when compared with wild-type mice; this has suggested an inhibitory role for p53, which may be exerted by changes in metabolism.116 Indeed, in the absence of functioning p53, a shift from oxidative phosphorylation towards glycolysis was observed.117 Furthermore, even myogenic differentiation can be monitored by p53. Distinct studies have shown an increase in p53 mRNA levels during myogenic differentiation in vitro.120 P53 might be involved in this process by regulating the retinoblastoma protein, Rb, which has a pivotal role in the differentiation of muscle through cell-cycle arrest and also by specific genes involved in the myogenic differentiation program.123 The association of p53 expression and cell-cycle regulators, which are target genes of p53, such as p21, was observed during the development of the mouse nervous system.125 Indeed, p53 monitors the differentiation of neural stem cells via its regulating pathways including cooperation with phosphatase and tensin homolog (PTEN).125 Notably, accumulation of mutant p53 in neural stem cells in the subventricular zone of the brain could generate aberrant neural progenitor cells and promote glioma formation.126 However, it is still unclear how p53 functions in the specific signaling context to regulate neural stem cells differentiation.127 In hematopoiesis, p53 induces hematopoietic stem cells to differentiate into proper mature blood cells and function in maintaining their quiescence.73 So far, the development of skeletal muscles and blood system has not yet been found abnormal in p53-null mice. Several in vitro studies have confirmed the role of p53 as a negative regulator in cell differentiation pathways, which reflects the complexity of the underlying mechanisms , necessary for metabolism adaptation, avoiding its proteasomic degradation through murine double minute 2 protein.134 Ablation of p53 increases the expression of HIF-1-\u03b1 in a hypoxia condition, which in turn induces the expression of vascular endothelial growth factor (VEGF) in tumor cells and promotes angiogenesis, neovascularization, tumor growth, and invasion. VEGF and HIF-1-\u03b1 are, furthermore, overexpressed in several types of human cancers, especially with HIF-1-\u03b1 in metastatic OS.135Interestingly, our previous work has proved that MSCs that lack p53 exhibit tumor-promoting characteristic through high secretion of nitric oxide (NO) and higher vigorous immunomodulation when compared with wild-type MSCs.84 Given the plasticity of MSCs to generate and differentiate into several cell types, including osteoblasts, it is not surprising that MSCs with p53 aberrations have been suggested to be the cells of origin for bone tumor, including OS, chondrosarcoma, Ewing's sarcoma, and sarcoma.75 We previously have discussed about the impaired osteogenic differentiation of p53-deficient MSCs; however, we here want to emphasize that p53-null MSCs also represent an important cellular component of tumor microenvironment. Interestingly, it has been demonstrated that in tumor BME, MSCs can support OS growth through the expression of CCL5 (chemokine ligand 5).136 Interestingly, CCL5/CCR5 (C\u2013C chemokine receptor type 5) axis can promote OS migration through the extracellular signal-regulated kinase pathway, which induces the nuclear factor \u03ba-light-chain enhancer of activated B cells (NF-\u03bab).136 Conversely, growth factors secreted from cancer cells and during bone resorption, such as tumor necrosis factor-\u03b1, TGF-\u03b2, bone morphogenetic protein 2 (BMP2), and interleukin-6 (IL-6), can promote osteoclast maturation by directing the expression of RANK (receptor activator of the receptor activator of nuclear factor-\u03bab) on the surface of osteoclast precursor cells increasing bone erosion. The increased bone degradation culminates in the release of BMP2 and TGF-\u03b2, which severely contribute to tumor growth and stimulate the osteoclast activity.137 TGF-\u03b2 can evoke MSC-secreted IL-6, which acts by promoting OS metastasis via STAT-3 .138 These data emphasize how the loss of function of p53 is a determinant in dictating the conditions that contribute to initiate OS: on the one hand, it can affect proliferation, immunoproperties, and compromise osteogenesis of undifferentiated MSCs, but on the other hand, it can affect the properties of BME components compromising the talk between BME and cancer cells, a further condition that supports OS initiation and development.During bone growth, several factors and extracellular matrix components secreted by mesenchymal progenitor cells and chondrocytes in the GP will recruit BME components to guarantee a balanced bone remodeling. The BME hosts different types of cells, including osteoblasts, osteoclasts, mesenchymal cell precursors, hematopoietic stem cells, chondrocytes, and endothelial cells, as well as fibroblasts stromal cells and immune cells. In this special scenario, p53 status has a determinant role . In the Wang et al.92 and other groups have proposed a negative role of p53 in regulating osteogenesis and other mesenchymal differentiation programs. We set the evidence that p53 mutational events occurring in undifferentiated MSCs or in osteoblasts at different stages of commitment can promote OS initiation72 as a consequence of alterations of osteogenic differentiation, bone remodeling, and bone homeostasis.139 Indeed, OS is a heterogeneous tumor that includes cells at different stages of commitment during osteogenesis.140 Interestingly, the osteosclerotic condition observed in p53-null mice imposes the phenotype of OS development.106 We summarize that p53 can affect osteogenic differentiation of MSCs and largely contribute to OS initiation: (1) it can promote or abrogate differentiation of multipotent progenitor cells acting as a negative mediator of transcription factors of early osteogenic differentiation; (2) it can regulate the genomic stability, growth, and proliferation of MSCs; (3) it can affect the immunoproperties of MSCs through growth factors and chemokine secretion; (4) it can affect the BME-regulating immune properties, growth, proliferation, and differentiation of microenvironment components. Further investigations on the molecular mechanisms through which loss of function of p53 can affect properties of MSCs and osteoprogenitor cells should be considered. This will ameliorate the knowledge of p53 function in the context of bone biology, and also will be helpful in identifying new strategies for targeting key molecules necessary for OS formation and survival.In this review, we aimed to bring to light that p53 has a pivotal role in keeping the balance between bone formation and bone degradation. Indeed, p53 not only regulates the genomic stability of MSCs but also their osteogenic differentiation functioning as \u2018bone remodeling surveillant' to prevent bone tumor initiation."}
{"text": "Upon activation, p53 leads to cell-cycle arrest and promotes DNA repair or induces apoptosis via several pathways. p63 and p73 are structural homologs of p53 that can act similarly to the protein and also hold functions distinct from p53. Today more than 40 different isoforms of the p53 family members are known. They result from transcription via different promoters and alternative splicing. Some isoforms have carcinogenic properties and mediate resistance to chemotherapy. Therefore, expression patterns of the p53 family genes can offer prognostic information in several malignant tumors. Furthermore, the p53 family constitutes a potential target for cancer therapy. Small molecules  have been objects of intense research interest in recent years. They restore pro-apoptotic wild-type p53 function and were shown to break chemotherapeutic resistance. Due to p53 family interactions small molecules also influence p63 and p73 activity. Thus, the members of the p53 family are key players in the cellular stress response in cancer and are expected to grow in importance as therapeutic targets. Human cells are constantly exposed to external and internal stressors, which cause damage to the integrity of the cell and to its genome. In order to guarantee the survival of the organism, cells have developed numerous strategies to adapt to stressors. In this review, we would like to discuss the influence of cellular stress on tumor development as well as strategies in cancer therapy targeting pathways involved in cell-cycle control and apoptosis. Special emphasis is put on the members of the p53 family.The development of cancer is a multistep process that involves a series of mutations in the progenitor cell . It enabWhile p53 has been known for more than three decades, two further members of the p53 family, p63 and p73, have been discovered more recently. The three genes exhibit a high degree of homology and there is increasing evidence that they have risen from the triplication of a common ancestral gene , 25. AllWhile p63 is crucially involved in craniofacial, limb, and skin development , p73 plaMalignant tumors often exhibit defects in apoptosis signaling pathways, resulting in tumor cell survival. Therefore, understanding the exact mechanisms of apoptosis can provide new strategies for the development of anti-cancer treatments. The extrinsic apoptosis signaling pathway is initiated by ligands such as TNF\u03b1, CD95L, and TRAIL binding to death receptors \u201338. The c from the mitochondrial intermembrane space  (core domain) is shared by most p53 isoforms and binds to response elements of target genes. A large number of p53 mutations occur within this region of the gene . The N-tp53 controls a large number of genes mediating G2/M and G1 cell-cycle arrest, DNA damage recognition, DNA repair, apoptosis, and senescence  Figure , 56. EveAnother important upstream regulator of p53 activity is p14ARF, a protein transcribed from an alternate reading frame of the CDKN2A gene locus that also encodes for the tumor suppressor p16INK4a , 68. p14c release , which plays an important role during neurogenesis. TAp73 knock-out mice show reduced levels of p75NTR and suffer from peripheral nerve defect, including myelin thickness and thermal sensitivity , which is essential for the oxidative pentose phosphate pathway . Cox4il p73 is rarely mutated in human cancer (<1%), but overexpression of p73 can be found in several malignancies, for example, in hepatocellular carcinoma , 162, neLarge-scale genome sequencing has shown that over half of human malignancies exhibit point mutations in the p53 gene impairing p53 function. Most p53 mutations are missense point mutations located within the DBD. Many of them lead to destabilization of folding of the domain at physiological temperatures and interfere with its DNA-binding ability . Certainin vitro and in vivo settings . RITA binds p53 and thereby induces conformational changes within the molecule that prevent MDM2 association , 196. In in vivo . The ant in vivo .Rational design led to construction of the spiro-oxindole MI-219, which is a highly specific small molecule inhibitor of p53/HDM2-interaction . Later, As an alternative to interfering with p53/MDM2-interaction, degradation of p53 can be prevented by inhibiting the E3 ligase activity of MDM2, and therefore, preventing ubiquitination of p53 . A serieThe tryptamine JNJ-26854165 (Serdemetan) effectively prevents p53/HDM2 from binding to the proteasome, thereby inhibiting degradation of p53 . In acutSIRT1, a nicotinamide adenine dinucleotide-dependent class III histone deacetylase, deacetylates p53 at Lys382, thereby reducing its activity . Hence, MET (APR-246), a compound that bears great structural similarities to PRIMA-1, but has higher activity than its predecessor, was discovered  in advanced prostate cancer and hematological malignancies, as well as a phase Ib/II clinical trial using this compound in addition to carboplatin in recurrent high-grade serous ovarian cancer are under way and will offer more insight into the effectiveness and practicability of mutant p53 reactivation .PRIMA-1 in vivo , 225. Alin vitro and in a mouse model (MIRA-1 (mutant p53 reactivation and induction of rapid apoptosis) is a maleimide-derived molecule and has no structural similarity with PRIMA-1, but it is equally able to restore p53 function leading to cell death via apoptosis with even higher potency than PRIMA-1 . By reesse model . To dateAlthough PRIMA-1 and MIRA-1 seem to have stabilizing effect on a great variety of p53 mutants, they are not able to restore normal protein configuration to the Phe176 mutant . This shCP-31398 was discovered by screening a library of more than 100,000 synthetic compounds for substances that effectively stabilize p53 conformation . InitialIn vivo, in a xenograft mouse model, tumor growth could be decelerated by intraperitoneal injection of RETRA (However, increased p53 activity bares risks for non-cancerous cells that might also be subject to apoptosis and further research is needed to find the adequate dose-response relationship, specific to the compound used . In an aof RETRA . AlthougMoreover, restoring p53 apoptotic function and modulation of p63 and p73 expression is often essential for sensitivity toward chemotherapeutic drugs or radiation, as lack of p53 and unfavorable expression patterns of p63 and p73 can lead to resistance toward treatment in different malignant tumors \u2013235. RecHowever, restoration of wild-type p53 might not be beneficial in all types of tumors. Jackson et al. showed that doxorubicin lead to cell-cycle arrest and senescence instead of cell death in breast cancer expressing wild-type p53, thereby promoting tumor cell survival and resistance to chemotherapy . This shThe p53 family plays a central role in cancer development and treatment response. Whereas p53 is often mutated in tumors, p63 and p73 function is preserved, yet altered by different expression patterns of their TA and \u0394N isoforms. Increasingly, these expression patterns are evaluated to estimate prognosis and adapt anti-cancer therapy. Nevertheless, the molecular mechanisms regulating the interplay between the different isoforms of the p53 family are only partly understood and are focus of current research. Identifying compounds that interfere with oncogenic signaling induced by certain p63 and p73 isoforms could be a novel approach in anti-cancer therapy.An increasing number of compounds that re-establish pro-apoptotic p53 function in cancer cells have emerged over the past decade. A variety of small molecules, which aim at increasing p53 function in cancers expressing wild-type p53, have been discovered. Among them are Nutlins, which are already undergoing clinical evaluation, RITA, tenovins, and many others.In tumors with underlying p53 mutation restoring wild-type activity of p53 has proven more difficult, but nevertheless feasible. PRIMA-1 and MIRA-1 are effective at inducing apoptosis via p53 in tumors that exhibit a great variety of p53 mutations. Yet, there are other small molecules, like PhiKan083, which are more specific and restore wild-type configuration of specific mutants only.in vivo studies and clinical trials have shown synergistic effects of small molecule treatment and chemotherapeutic drugs in a variety of malignancies. Especially cancer cells, which are resistant to chemotherapy due to impaired p53 function, become more susceptible to treatment.A number of Taking the approaches of p53 reactivation further, there might be new possibilities of targeting CSCs, which are often insusceptible to chemotherapy. Induction of p53 in these cells could lead to activation of pro-apoptotic pathways via differentiation.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."}
{"text": "The standard classification used to define the various cancer genes confines tumor protein p53 (TP53) to the role of a tumor suppressor gene. However, it is now an indisputable fact that many p53 mutants act as oncogenic proteins. This statement is based on multiple arguments including the mutation signature of the TP53 gene in human cancer, the various gains-of-function (GOFs) of the different p53 mutants and the heterogeneous phenotypes developed by knock-in mouse strains modeling several human TP53 mutations. In this review, we will shatter the classical and traditional image of tumor protein p53 (TP53) as a tumor suppressor gene by emphasizing its multiple oncogenic properties that make it a potential therapeutic target that should not be underestimated. Analysis of the data generated by the various cancer genome projects highlights the high frequency of TP53 mutations and reveals that several p53 hotspot mutants are the most common oncoprotein variants expressed in several types of tumors. The use of Muller's classical definition of mutations based on quantitative and qualitative consequences on the protein product, such as \u2018amorph', \u2018hypomorph', \u2018hypermorph' \u2018neomorph' or \u2018antimorph', allows a more meaningful assessment of the consequences of cancer gene modifications, their potential clinical significance, and clearly demonstrates that the TP53 gene is an atypical cancer gene. However, their relatively infrequent mutation and/or association with specific tumor groups could limit their value as biomarkers or therapeutic targets.6The development of next-generation DNA sequencing methods has led to a burst of information, including the release of the sequences of >5000 cancer genomes, an achievement inconceivable only 15 years ago.7 Therefore, drug discovery and development programs in academia and industry have mostly focused on kinase inhibitors and other approaches designed to inhibit activated oncoproteins, whereas relatively few therapeutic strategies to reactivate tumor suppressors have been developed to date.In general, cancer genome studies highlight oncogenes as the most promising targets for drug development mainly because these hyperactive protein variants appear to be more easily druggable than the products of tumor suppressor genes, inactivated by heterogeneous nonsense or frameshift mutations.et al.8 in the 1960s, showing that fusion of normal cells with tumor cells resulted in a normal phenotype. Loss of specific chromosomes from the hybrid cells led to reappearance of the tumor phenotype, suggesting the existence of critical genes able to suppress the tumor phenotype. The tumor suppressor gene concept was also supported by Knudson's epidemiological studies on familial and sporadic retinoblastoma and the subsequent loss of heterozygosity (LOH) analysis that identified chromosomal deletions in tumors.10 Haber and Harlow defined tumor suppressor genes as \u2018genes that sustain loss-of-function (LOF) mutations in the development of cancer'.11 This classification has evolved over time and novel classes such as caretakers, gatekeepers or landscapers have been added to take into account the function of novel genes and their association with tumor development.12 Although oncogene and tumor suppressor gene alterations may have direct consequences on cell growth, caretakers and landscapers act indirectly by promoting either genetic instability or an abnormal cellular environment that will foster neoplastic transformation. This classification, based on the diversity of the functional activity of the wild-type product, should not be confused with the consequences of their alteration.The first evidence for the existence of tumor suppressor genes came from cell fusion experiments performed by Harris 14 Although the two-box oncogene/tumor suppressor categorization remains a central concept in tumor biology, it is becoming increasingly obvious that it is difficult to put some genes into one box. As cancer research develops rapidly, it is essential to ensure highly flexible data classification so that labels do not paralyze the emergence of new concepts.For example, LOF is still the main characteristic of tumor suppressor genes, but the Haber and Harlow definition does not take into account the heterogeneity of the different protein variants or other activities such as dominant negative (DN) effects on wild-type protein or gain-of-function (GOF). Furthermore, for some genes such as Notch, this definition can be cell-type specific, as the spectrum of mutations is different in blood and solid tumors, with different consequences for the protein.versus inactivated, despite accumulating evidence that the majority of mutant p53 proteins are heterogeneous oncogenic proteins with multiple GOF activities and with potential as therapeutic targets.The status of TP53 as a tumor suppressor gene can be used as a paradigm. Wild-type TP53 clearly acts as a negative regulator of cell growth, but considering TP53 mutations solely as LOF mutations would prevent a full understanding of how TP53 mutations drive tumor growth, as TP53 status in human cancer is often defined in binary terms, wild-type In this review, we will show that, despite the important diversity of TP53 mutations, some specific p53 mutants are among the most frequent variants expressed in human cancer. In addition, we have gathered various lines of evidence accumulated over 35 years of research that illustrate how TP53 is an out-of-the-box entity. Finally, using Muller's classical definition of mutations based on quantitative and qualitative consequences on the protein product, we will discuss how this classification would be more appropriate to define the heterogeneous effects of cancer gene mutations.18 More than 80% of somatic and germline TP53 alterations are missense mutations that lead to the synthesis of a stable mutant protein that accumulates in the nucleus of tumor cells . Although their method can separate classical oncogenes from tumor suppressor genes in general, the TP53 gene would still straddle the boundary between the two classes of genes.Oncogenes are typically activated by missense mutations that target specific key residues of the protein . Tumor s19The strong selection to maintain expression of full-length p53 protein in tumors is highly suggestive of a vital role in transformation, including DN activity and/or GOF. As >90% of TP53 mutations are localized in the core domain of the protein (residue 100 to 300), all p53 protein isoforms are affected by these alterations.20 This vast scattering of TP53 mutations is due to the marked fragility of the core domain, which can be destabilized by amino acid substitutions at many different positions.21The distribution of mutations in the p53 protein is also unique among all cancer genes, including oncogenes and tumor suppressor genes. All but 7 residues of the 393 amino acid residue p53 protein have been the target of at least 1 mutation in human cancer, and in the core domain that contains the DNA-binding region, each residue has been found to be mutated at least 5 times in independent tumors, and up to >2,000 times for hotspot mutants.22 The data include genomic, epigenomic, transcriptional and proteomic information. Not surprisingly, TP53 was found to be the most frequently mutated gene in this new set of data  and homozygous  animals. This is an important finding because it rules out the possibility that the inability of mutant p53 to act as a transcription factor and induce sufficient levels of MDM2 protein, a p53-specific E3 ubiquitin ligase that targets p53 for modification and subsequent degradation by the 26\u2009S proteasome, is a cause for mutant p53 hyperstability.Terzian 30 The CHIP E3 ligase participates in p53 degradation by forming a large complex that includes HSP90 and HSP70. Mutant p53 forms stable complexes with both HSP, CHIP and MDM2, inhibiting the ligase activity of both. A large survey of tumor cell lines expressing wild-type and mutant p53 showed that stabilization of endogenous mutant protein is due to a complete lack of ubiquitination.31 Inhibition of HSP90, either via knockdown or by specific drugs, alleviates the formation of such complexes, leading to reactivation of both MDM2 and CHIP, and mutant p53 degradation.However, additional ubiquitin ligases have recently been identified that participate in the degradation of the tumor suppressor, including Pirh2 (p53-induced protein with a RING-H2 domain), Trim24 , COP1 (constitutive photomorphogenesis protein 1) and CHIP (C-terminus of Hsc70 interacting protein).35 Nuclear magnetic resonance, circular dichroism and X-ray diffraction analyses have confirmed and expanded this description and suggested that multiple thermodynamic stages are associated with the various mutations depending on their position.38 This heterogeneity extends to the biological activity of mutant p53 proteins.39 Analysis of the transcriptional activity of 2500 p53 mutants on 8 different target genes representative of different TP53 functions demonstrated heterogeneous penetrance of the various p53 mutants with >50% presenting only partial loss of activity.43The heterogeneity of p53 mutants was discovered 25 years ago with the description of the so-called \u2018structural' mutants, in which the DNA-binding domain is unfolded (e.g. p.R175H), and \u2018DNA contact' mutants, in which residues interacting directly with DNA are substituted .in vitro and in vivo systems . Further studies have largely supported this observation. Mutant p53 GOF includes enhanced tumorigenesis, metastasis, resistance to therapy and genomic instability. The genomic instability effect was further supported in a recent analysis of 3000 tumors showing that TP53 mutations were strongly associated with a high frequency of copy number changes.49 GOF in p53 mutants is highly heterogeneous, an observation that may be related to the various conformations of p53 mutants. GOF mechanisms may be the result of changes in the specificity of the DNA-binding activity of the p53 mutant, leading to the induction of novel transcriptional programs, or changes of its interaction with other cellular proteins directly or indirectly related to the regulation of gene expression. Several transcriptional programs are specifically activated by p53 mutants, most of them resulting in increase of tumorigenicity but whether this is mediated by a direct binding of the mutated protein to a specific DNA sequence or via an interaction with other transcription factors remains to be elucidated52 , an observation adding one more grain to the important relation between TP53 and metabolism. This observation is also in line with the study by Zhang et al.55 showing that p53 mutants stimulates the Warburg effect in cancer cells.Mutant p53 GOF has been extensively investigated in multiple 57 TP63 and TP73 are both expressed as many isoforms due to alternative usage of promoters for transcription and alternative splicing. Long isoforms (p73 or p63 containing the transactivation domain (TA-p73 or TA-p63)) are able to transactivate the same target genes as p53 and induce apoptosis, while short forms (amino-deleted p63 or p73 isoforms (DN-p63 or DN-73)) have an opposite activity via DN mechanisms. TP63 and TP73 are able to cooperate with TP53 to induce apoptosis, suggesting the existence of a complex network of interactions between the products of these three genes. p53 mutants with unfolded structure, but not DNA contact mutants, bind specifically to p63 and p73 and impair their apoptotic activity  increased the complexity of this network, as the two p53 homologs might also contribute to its oncogenic potential.An important issue that has never been fully analyzed is the consequence of accumulation of mutant p53 protein in tumor cell nuclei. Studies have shown that this accumulation may be as much as 10-100-fold higher than that of wild-type protein, which raises questions concerning the specificity of some mutant p53 activities and how some nonspecific squelching effects may be associated or confounded with these novel properties. Furthermore, this aspect also raises the issue of tumor heterogeneity, as p53 accumulation in human tumors is highly heterogeneous, even for single p53 variants, and could be associated with the genetic background of the tumor and the individual.63 Cells from mice deficient for the three major TP53 target genes, CDKN1A (the gene encoding p21), BBC3 (the gene encoding PUMA) and PMAIP1 (the gene encoding Noxa) (p21\u2212/\u2212puma\u2212/\u2212noxa\u2212/\u2212 mice) are deficient in their ability to undergo p53-mediated apoptosis, G1/S cell-cycle arrest, and senescence, but these animals also remain tumor-free.64 Taken together, these observations suggest that TP53 driver mutations are selected to impair specific TP53 pathways that remain to be identified, and only mutants defective in such transcriptional programs will be selected in human tumors.65The general idea that loss of transcription in mutant p53 is the driving force selected during tumorigenesis also needs to be reevaluated, as it is far from straightforward. Surprisingly, mice expressing p53 mutants transcriptionally defective for the three canonical pathways, growth arrest, senescence and apoptosis, are not prone to cancer.66 Homozygous TP53\u2212/\u2212 mice are highly prone to cancer, in particular T-cell lymphoma and sarcoma. Although these knockout mice supported the model of TP53 as a tumor suppressor gene, they were not fully satisfactory, since most cancers in humans are carcinomas, which were observed at very low frequency in TP53 knockout mice.68 Improvements in the production of transgenic mice led to the creation of knock-in mice expressing various hotspot mutants that include structural or DNA contact mutants and result in more \u2018human-like' tumors.70 Compared with TP53\u2212/\u2212 mice, these novel knock-in models displayed a higher degree of heterogeneity in the spectrum of tumor types with more frequent carcinomas. Furthermore, these tumors were highly invasive and metastatic, a feature absent in TP53\u2212/\u2212 mice. Two characteristics of these mice models support the notion of heterogeneous GOF for p53 mutants. First, the spectrum of tumors differed according to the various mutant alleles used in these mice. For example, the \u2018DNA contact' mutant p.R270H (p.R273H in humans) results in a high frequency of carcinomas, whereas the \u2018structural' mutant p.R172H (p.R175H in humans) predominantly results in osteosarcomas. Second, the tumor spectra of heterozygous TP53 p.R172H/\u2212 or TP53 p.R270H/\u2212 mice were different from those of TP53+/\u2212 or TP53\u2212/\u2212 mice, which argues against any differences due to a DN activity toward wild-type p53. Some p53 mutants such as p.R175P are defective in activating genes associated with apoptosis without impairing growth arrest.71 Onset of tumors in knock-in mice expressing this mutant (p.R172P in mice) is delayed compared with TP53\u2212/\u2212 mice or mice expressing hotspot mutations.72 In contrast to tumors expressing other p53 mutants, these tumors were mostly diploid, indicating that the growth arrest function of TP53 could be essential for maintaining genetic stability, but is not the primary tumor suppressor function of wild-type TP53.The first TP53 knockout mice were generated by removing exons encoding the DNA-binding domain, thus impairing the expression of all p53 isoforms.74 This is a major challenge, for several reasons. Mutant p53 is clearly a different kind of target compared with those of successful novel anticancer agents such as Herceptin (trastuzumab) and Gleevec (imatinib) that block critical oncogenic kinases overexpressed in various tumors. In the case of mutant p53, the main aim is to refold and reactivate a dysfunctional tumor suppressor  in cells.NSC319726 (ZMC1) is another mutant-specific p53 reactivator. Carpizo and co-workers81 PRIMA-1 and its structural analog PRIMA-1Met (APR-246) refold mutant p53, enhance expression of several TP53 targets, including Bax, Puma and Noxa, and inhibit human tumor xenografts in SCID mice and mouse tumors in syngeneic hosts. These effects are observed with a range of mutant p53 proteins. APR-246 synergizes with several chemotherapeutic drugs, for example, adriamycin and cisplatin, to induce mutant p53-dependent tumor cell death.82 Both PRIMA-1 and APR-246 are converted to the Michael acceptor methylene quinuclidinone (MQ) that binds covalently to cysteines in the p53 core domain83 MQ binding to p53 is sufficient for mutant p53 reactivation, as shown by protein transfer experiments with APR-246/MQ-treated recombinant p53. It is currently not clear to which p53 cysteines MQ binds, although docking simulations and functional studies in cells have indicated that C124 is one possible target.84 APR-246 has been tested in a phase I/II clinical study in patients with hematological malignancies or hormone-refractory prostate cancer.85 This study showed that APR-246 is safe and has a favorable pharmacokinetic profile. Biological effects consistent with p53 reactivation were observed in patient leukemic cells after treatment, and clinical responses were observed in two patients, including one AML patient with a p53 p.V173M core domain mutation.The small molecules PRIMA-1 (p53 reactivation and induction of massive apoptosis-1) and MIRA-1 (mutant p53-dependent induction of rapid apoptosis-1) were identified in a cell-based screen of the NCI Diversity set.86 thereby increasing cellular ROS levels. Moreover, Tessoulin et al.87 found that APR-246 can decrease GSH levels and thus impair the redox balance in multiple myeloma cells independently of p53.In addition to targeting p53, APR-246 has been shown to inhibit thioredoxin reductase (TrxR1) and convert the enzyme to an NADPH oxidase,74 Their mechanisms of action are not fully understood and they have not yet been tested in the clinic.Various other small molecule reactivators of mutant p53 have also been identified, including CP31398, Ellipticine, P53R2, SCH529074 and stictic acid reviewed in the study by Bykov and Wiman.88 Disruption of complexes between mutant TP53 and TP63/p73 by RETRA leads to restoration of expression of TP53 target genes and tumor suppression.In light of the growing evidence in favor of GOF activities of mutant p53, a more modest but still attractive aim for therapeutic targeting of mutant p53 is inhibition of the oncogenic properties of mutant p53 . This ma19 . Restoration of nonsense TP53 mutations will obviously require different approaches than those described above for missense mutations. Interestingly, aminoglycosides, for example, gentamicin and G418, have been shown to induce read-through of the c.637C>T (p.R213*) mutant p53 and expression of full-length p53.89 Although clinical use of these drugs is limited by their toxicity, the results nonetheless demonstrate that induction of read-through of premature stop codons in nonsense mutant p53 is feasible and suggest that high throughput screening for more efficient and less toxic read-through-inducing compounds should be carried out. Questions remain as to the activity of the full-length p53 protein induced on translational read-through and to what extent pharmacological induction of read-through of premature stop codons will induce read-through of natural stop codons. If these problems can be solved, induction of read-through could be a useful strategy not only for reactivation of nonsense mutant p53, but also for reactivation of other tumor suppressors that are frequently inactivated by nonsense mutations, for example APC and PTEN (phosphatase and tensin homolog gene).A significant fraction (8%) of TP53 mutations are nonsense mutations that give rise to expression of a truncated and inactive p53 protein. The c.637C>T (p.R213*) mutant is the most common nonsense mutation in TP53, and is actually more common than many missense mutations in human tumors90 suggested a classification of mutations based on quantitative analysis of wild-type \u2018characters'. He proposed the terms \u2018amorph, \u2018hypomorph', \u2018hypermorph', \u2018neomorph' or \u2018antimorph'. The significance of these terms was subsequently modified, as they were proposed at a time when the relationship between a gene and its product was not clearly established.90 These terms can now be used to classify cancer genes to more clearly understand the consequences of cancer gene alterations. This system has enormous benefits, as it is based on the outcome of the mutation regardless of the initial biological function of the gene. It could greatly facilitate the design of optimal drug development strategies.In Muller91 Other mechanisms, such as promoter methylation, loss of expression via microRNA dysregulation or frameshift and nonsense mutations, can also lead to total LOF, but in many cases it is difficult to exclude residual activities that will lead to heterogeneous hypomorphic variants, and for many genes it is likely that true amorphic variants are not as frequent as hypomorphic variants  mutation can be applied mainly to tumor suppressor genes, whose function(s) must be totally impaired to drive tumorigenesis, whereas a hypomorph mutation only leads to partial reduction of activity. From a genetic point of view, true amorphic alterations can be easily associated with genes that sustain biallelic deletions such as PTEN or retinoblastoma gene (RB1).variants . For the93 have been defined as disruption of the wild-type activity by the mutant polypeptide  or the potassium channel tetramerization domain containing 11 proteins (KCTD11) are co-deleted with TP53.96 Analysis of tumors at this transition from TP53Mut/wt to TP53Mut/\u2212 would be highly informative in this regard.Antimorphic or DN mutations, as subsequently described by Herskowitz,ypeptide . DN acti98 The boundaries between hypermorph and neomorph mutations are not clear cut and alterations in a single gene can lead to variants that can have either one or both characteristics.The \u2018hypermorph' label is the most explicit term to describe many protein variants expressed from mutated oncogenes, as most of them are hyperactive proteins. For amplified genes such as N-myc, MDM2 or CCND1 (the gene encoding cyclin D1), or genes upregulated via chromosomal translocations such as c-myc, this definition appears to be obvious at first glance, as it refers to the abnormal accumulation of wild-type proteins . Neverth99 As discussed in the previous section, several hotspot p53 mutants are obviously neomorphic with a marked diversity in the activity gained by each variant.Although chromosomal translocations leading to fusion genes and the synthesis of chimeric proteins with novel functional specificity are the most obvious and easy-to-demonstrate neomorph mutations, it is more difficult to demonstrate missense mutations acting as neomorph mutations. Nevertheless, neomorphic mutations have recently been demonstrated in isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2), which are mutated in several cancer types such as glioma and AML . In bothIn vitro, mouse in vivo and clinical studies all point toward the importance of selection for oncogenic p53 mutants in human tumors. Although classification of cancer genes into oncogenes or tumor suppressor genes can be very helpful, it is becoming increasingly obvious that the frontier between the two classes is not as clear as previously thought, and that some genes may straddle these two categories. Furthermore, tissue type, genetic background and many other factors also influence the phenotype induced by a specific mutation. Nonetheless, we can predict that, except for rare cases, most cancer gene alterations will give rise to products with pleiotropic activities. Using the classification based on Muller's proposition more than 80 years ago, it is possible to establish a comprehensive and meaningful categorization of the various cancer genes that illustrates how TP53 does not fit the classical definition of a tumor suppressor gene. Beyond the rhetorical aspect of this statement, there is a genuine need to avoid an overly simplistic, binary, \u2018wild-type versus inactivated' classification; defining p53 mutants as oncogenes with heterogeneous GOFs that affect multiple pathways must be considered. Designing drugs to target specific p53 mutants seems an attractive strategy, as several of these mutants are among the most frequent protein variants found in several cancer types and associated with the death of >120,000 patients worldwide. Indeed, small molecules capable of targeting hotspot mutants p.R175H or p.Y220C have been identified. Further drug discovery efforts should focus on other frequent p53 mutants, such as p.R273H, p.R248W and p.R249S. With drugs targeting a wide range of common p53 mutants in our future arsenal, our chances of efficiently fighting cancer may be greatly improved.100"}
{"text": "Although there are some studies about the mechanism and role of p53 aggregation and amyloidosis in tumors, there still exist some controversies. In this paper, we review the mechanism of p53 amyloid fibril structure and discuss the characteristics and effects of p53 amyloid aggregation, as well as the pathogenic mechanism leading to the occurrence of aggregation in tumors. Finally, we summarize the various inhibitors targeting p53 aggregation and prion-like behavior. In conclusion, a comprehensive understanding of p53 aggregation can expand our understanding of the causes leading its loss of physiological function and that targeting p53 aggregation might be a promising therapeutic strategy for tumor therapy.Similar to neurodegenerative diseases, the concept that tumors are prion like diseases has been proposed in recent years. p53, the most well-known tumor suppressor, has been extensively studied for its expression, mutation, and function in various tumors. Currently, an interesting phenomenon of p53 prion-like aggregation has been found in several tumors, and studies have found that its pathological aggregation may lead to functional alterations and ultimately affect tumor progression. It has been demonstrated that the mechanism of p53 aggregation involves its mutation, domains, isoform, etc. In addition to p53 itself, some other factors, including Zn Prion and prion like diseases, which result from the misfolding and aggregation of prion proteins, have been demonstrated in transmissible spongiform encephalopathies and various neurodegenerative diseases. The deposited key proteins can affect the function of the central nervous system or peripheral organs. Pathological aggregation of these key proteins can spread directly within or between cells, even between the same or different species of animals, leading to a gain of toxic function and ultimately to cell death \u20135. In reTo date, p53 aggregation has been found in various tumors, and wild-type p53 was found to be aggregated in neuroblastoma , 17, brep53 oligomerization occurs mainly through its oligomerization domain (OD) under physiological conditions. p53 dimerization is a normal event, and tetramer is considered the major active transcriptional unit. In nontetrameric forms of p53 (monomers and possibly dimers), p53 exists in the cytoplasm. The tetramerized p53 localize in nuclear for transcriptional activation . When DNVirtually, most proteins can aggregate under extreme chemical conditions, but only a small fraction of proteins aggregate in vivo under physiological conditions . The amyp53 amyloid aggregation affects the normal function of p53 in several ways, and various independent studies have shown that the formation of mutant p53 aggregates is related to loss of function (LOF), gain of function (GOF), and dominant negative effect (DN) , 39, 40 LOF: p53 amyloid aggregates are present in a variety of human and animal tumor tissues . The losCDK6, CDK4, CDC45, PCNA, E2F2, RAD51, TIMELESS and upregulate of genes including ERBB2, MAP2K1, EGF. As well as the EMT-associated genes, which contribute to tumor invasion and metastasis can be dysregulated by p53 amyloid formation [GOF: alterations in p53 mutant function may be associated with the GOF of \"emerging\" target genes. For example, the first reported mutant, R175H, activated the promoter of multidrug resistance gene 1 (MDR1), which is not targeted by wild-type p53 . Amyloidormation . Thus, pormation .DN effect: p53 acts as a tetramer in cells, and it is acknowledged that DN is caused by the combination of inactive mutant and wild-type p53 in a mixed tetramer, resulting in a reduced concentration of functional p53 in the cell. p53 aggregation provides an alternative hypothesis that the DN effect of conformationally unstable mutants is induced by mutation-induced coaggregation. When Milner et al. studied R248Q mutants, they found that the mixture of amyloid oligomers and protofibrils of R248Q, like seeds, accelerates the aggregation of wild-type p53, and the aggregation isolates not only functional p53 but also other tumor suppressors and p53 homologs . The seeActually, although LOF, GOF, and DN were tend to be studied separately, they are related to each other. In addition to losing the tumor-suppressing function of wild-type p53 (LOF), mutant p53 is also found to function in a tumor-promoting manner (GOF) through DN regulation of remaining wild-type p53 or independently of wild-type p53 , 40, 47.Many mechanisms have been reported for p53 amyloid aggregation. In the following, we focus on the mechanism of p53 amyloid aggregation in terms of p53 high frequency mutations, structural domains, and isoforms  as the core region of p53 are taken for hotspot mutations . p53 agg2+ of p53 leads to exposure of this segment, which can result in p53 self-aggregation. The process is an ordered process by which the aggregation-nucleating segment self-assembles into a nonnative state of \u03b2-structured molecular assemblies [Many studies have focused on the aggregation tendency of p53 domains. The three functional domains of p53, transactivation domain (TAD), DBD, and OD, have been demonstrated to form amyloid aggregates in vitro, and the highest aggregation propensity remains in the DBD region, which has the most hotspot mutations , 46, 58.However, by analyzing aggregates by restriction protein hydrolases and the effect of mutations on kinetics and agglomeration products, Wang et al. suggested that there is no unique aggregation sequence for p53 in vitro but rather multiple sites. The occurrence of p53 aggregation is not the contribution of an individual site but a collaborative network . Stindt p53 dysfunction may also be caused by abnormal expression of isoforms. The p53 gene consists of 11 exons . ThroughAs mentioned before, a partial peptide of the p53C structural domain can induce p53 aggregation in vitro, which is similar to the N-terminal truncated p53 isoforms , and isoforms can form a complex with full-length p53, leading to its inactivation , 63. AltThe kinetics of amyloid aggregation occur slowly with an extended lag period in vitro but accelerate dramatically once seeds are formed, and the thermodynamics are altered. In vivo, studies are much more complicated due to proteases, chaperone proteins, and some fibrillar stabilizing factors, such as extracellular matrix components and other proteins. They will incorporate into amyloid aggregates. Some factors that may contribute or accelerate the production of amyloid aggregates are as follows: the overproduction of wild-type amyloid , the breMutations in key proteins: Conformational changes in key proteins lead to their pathological aggregation, ultimately resulting in deposits and disease, which is the unified pathogenesis of conformational diseases . MutatioChaperone abnormality: Protein folding is a prerequisite for normal function, while misfolding affects biological functions or even generates harmful oligomers. It is closely related to the binding of molecular chaperones and proteases in the intracellular translation process . On the Hsp, well-known chaperones, are frequently overexpressed in various tumors . Protein2+ concentration, pH, temperature, and pressure, may destabilize the side chain interactions, the original folding structure may be opened, and a new structure or polymer may be formed under other conditions [Environment and other factors: polypeptide chains seem to have a universal ability to form amyloid fibrils, but different amino acid sequences have different propensities to form amyloid fibrils . The standitions \u201395. It inditions . p53 aggnditions , and thenditions .) [The effect of RNA:\u00a0RNA molecules could modulate of p53 aggregation and seeding was reported recently . It is p) .The concAs an important suppressor gene, p53 inactivation plays an important role in tumors, and scientists have investigated various approaches targeting p53 to recover the function of p53. For example, interfering with p53-MDM2 binding through small molecules or peptides , 89, 101Peptide inhibitor: The most notable therapy for targeting mutant p53 aggregation is ReACp53, which was developed by Wang et al. in 2016. ReACp53 is a sequence-specific peptide inhibitor that inhibits p53 mutant aggregation and tumor growth in a peptide-based approach. ReACp53 acts in the aggregation phase of p53 dynamic equilibrium to rescue the function of p53, and it targets the 252\u2013258 region of p53. The hydrophobic isoleucine in this aggregation nucleating segment is replaced by an arginine in ReACp53, which inhibits the tendency of aggregation in this region by shielding the segment and converting the folding to a functional, wild-type-like state . The inh2+ ion that is crucial for proper protein folding and function. Zinc-free p53 exhibits an accelerated aggregation of proteins [I is a novel designed bifunctional ligand. On the one hand, iodine in LI contributes to the interaction with the hydrophobic segment; on the other hand, LI, which possesses metal chaperone activity, can restore the binding of zinc in mutant p53. It was found that LI not only restores the function of p53 by regulating the aggregation of mutant p53 and interacting with the zinc-binding fragment but also restores zinc binding using the metal chaperone approach, which greatly increases the content of zinc in cells. It increases cytotoxicity in tumor cells and ultimately restores p53 function. Combined with oxaliplatin, LI increases the effectiveness of platinum-based therapy [Bifunctional small molecule: The DBD region of p53 contains a Znproteins , 113. Mu therapy .Protein mimics: Oligopeptide-based \u03b1-helical mimics have been reported to be effective in modulating the self-assembly of the key proteins, amyloid \u03b2-peptide in Alzheimer's disease , 115 andSince p53 aggregation proceeds through multiple sites, blocking one site may not be an effective strategy to stop its aggregation. Combining physical, chemical, and computational approaches to obtain information about the initial steps of protein aggregation revealed the existence of a unique precursor state molten globular phase of p53 . The proIn addition to pathogenicity, aggregation has been reported to be functional in bacteria, fungi, and higher eukaryotes. The prevailing view of protein aggregation in the field of neurodegenerative diseases is abnormal and harmful to cells. In 2018, a study reported that aggregates of the TDP-43 protein, which occurred in most cells of neurodegenerative diseases and are considered harmful, are beneficial to healthy muscle. We may change our mind that amyloid aggregation may have beneficial effects rather than simply being associated with disease .To date, there is still much controversy regarding whether tumors can be classified as prion diseases, and many questions need to be addressed with experimental evidences. In addition to p53, scientists have also searched for the presence of other related proteins that misfold or aggregate in tumors. PTEN, which has properties similar to p53, such as a high propensity of mutation, intrinsically disordered regions (IDRs), and oligomerization ability, has been reported to have aggregation behavior recently . ComputaOver the years, scientists have comprehensively investigated p53 in many aspects, and new p53 aggregation and prion-like characteristics in tumors have been proposed in recent years. The aggregated mutant p53 in cells, the interaction with its targets, the signaling with other cells, and the effects on tumor progression represent an emerging field and new therapeutic possibilities. Elucidation of the cellular biology and structural features of p53 amyloid aggregates will be an important topic in tumor biology and will provide new perspectives and directions for targeting p53 for tumor treatments."}
{"text": "The p62 protein, also called sequestosome 1 (SQSTM1), is a ubiquitin-binding scaffold protein. The expression of p62 was statistically higher in carcinoma compared to non-neoplastic mammary glands. Our results, although preliminary, are similar to the results of breast cancer, therefore, also in the cat, p62 could be considered a possible oncotarget.The p62 protein, also called sequestosome 1 (SQSTM1), is a ubiquitin-binding scaffold protein. In human oncology, although the interest in the function of this protein is recent, the knowledge is now numerous, but its role in tumorigenesis is not yet clear. This preliminary study aims to evaluate the immunohistochemical expression of p62 in 38 cases of feline mammary carcinoma with different grades of differentiation and in 12 non-neoplastic mammary gland tissues, to assess the expression level and a possible correlation with malignancy. The expression of p62 was statistically higher in carcinoma compared to non-neoplastic mammary glands: 28 feline mammary carcinomas (73.7%) had a high p62 expression score, three (7.9%) had a moderate expression, while seven cases (18.4%) had a low expression. The grade of the differentiation of the carcinoma was not correlated with the p62 expression. This study represents the first approach in feline oncology that correlates p62 expression in feline mammary carcinoma. Our results, although preliminary, are similar to the results of human breast cancer, therefore, also in the cat, p62 could be considered a possible oncotarget. The p62 protein, also called sequestosome 1 (SQSTM1), is a \u201cscaffold\u201d protein (capable of bringing other proteins together), fundamental in the process of autophagy, linked to ubiquitin, which polymerizes through an N-terminal PB1 domain and which can interact with ubiquitinated proteins through the C-terminal UBA domain ,4.In human oncology, the cellular mechanism of autophagy has only recently been studied to understand its possible involvement in the carcinogenetic process. Autophagy is a non-selective mechanism, responsible for the degradation of the majority of long-lived cellular proteins and cell organelles . VariousThe metabolism of cancer cells is very different from that of normal cells: the high rate of cell proliferation, the increase in cell size, and the simpler enzymatic mechanisms require much oxygen and nutrients with the consequent production of high quantities of toxic compounds. Cancer cells are therefore able to reprogram their metabolism and activate the autophagy process in the various stages of the tumor growth .Although the interest regarding the function of p62 is quite recent, in human oncology, several studies have been published. Nevertheless, the role of p62 in tumorigenesis is not very clear. p62 is considered a protein whose function arises at the crossroads between autophagy and apoptosis ,11,12, aIn veterinary medicine, knowledge of the role of p62 in tumors is still limited, therefore, the purpose of this study is to evaluate the immunohistochemical expression of p62 in feline mammary carcinoma with different degrees of differentiation and in non-neoplastic mammary glands to highlight how this protein is expressed and if there is a correlation with the degree of the malignancy of the tumor. Mammary neoplasia in cats is less common compared to dogs, but when it occurs, most of the cases are malignant and histologic grading has a prognostic value .This study included fifty cases: 12 post-mortem non-neoplastic mammary glands and 38 mammary carcinomas, of which 34 of the simple type and four of the ductal type were collected from 2012 to 2021 at the Pathology Unit of the School of Biosciences and Veterinary Medicine of the University of Camerino . From the same archive came reports and anamnesis of the cases. Ages ranged from 4 to 16 years, with a median age of 11.4 years, and all were European shorthair females. The specimens were fixed in 10% neutral buffered formalin, subsequently processed by an automatic histoprocessor, and embedded in paraffin. Serial sections (3 \u00b5m) were stained with hematoxylin and eosin (HE).2. To each tumor, a score from 0 to 3 was assigned, and thus they were subsequently classified as grade I or low grade (score of 0), grade II, or intermediate cancers (score of 1), and grade III or higher cancers (score of 2\u20133).All feline mammary carcinoma cases included in the study underwent a histological grading system, following the more recent system proposed by Mills et al.  based onImmunohistochemical analysis was carried out with a standard ABC-peroxidase method on 3 \u00b5m sections, as described by Di Cerbo et al. . The lab2 was analyzed for the semiquantitative evaluation. A score was then assigned to each sample according to positive cell percentage and their signal intensity. The scoring system used was that described by Mariotti et al., 2019. [As a secondary antibody, a goat anti-rabbit biotinylated antibody was used along with DAB (diaminobenzidine) as the chromogen. Normal mouse pancreas was used as a positive control , while r., 2019. Positive cell percentage was based on a 5-point scoring system: 0 = no positive cell, 1 = \u226420%, 2 = between 21% and 50%, 3 = between 51% and 70%, 4 = \u226571%. For each microscopic field, a count of immunopositive and negative cells was carried out and then transformed in percentage. The intensity of immunostaining was classified to a 4-point scoring system: 0 = no staining, 1 = low-intensity staining, 2 = moderate-intensity staining, 3 = high-intensity staining. In those samples where the intensity was heterogeneous, the score chosen was the predominant one in the sample itself. The overall score assigned to each case is derived by the multiplication of cell positivity and intensity signal score, with a minimum score of 0 and a maximum score of 12. The equivocal cases were evaluated by two authors (G.E.M. and F.M.) to establish the score. Scores between 1 and 2 were considered as a low expression of p62, those from 3 to 4 as intermediate, and those greater than 5 as high expression.p < 0.05.Data were analyzed using GraphPad Prism 8 software . All data are presented as the means \u00b1 standard deviation (SD). Differences between the score of non-neoplastic patients and those with mammary carcinoma and differences between feline mammary carcinoma cases with lymphovascular invasion were analyzed using a Mann\u2013Whitney test, while differences among different histological grades were analyzed using a Kruskal\u2013Wallis\u2019s test followed by Dunn\u2019s multiple comparison test. The level of significance was set at * Eight cases of non-neoplastic feline mammary gland tissue studied were negative for p62. Widespread negativity was observed in the epithelial cells of the mammary gland A; four cThe histological grading of the 38 cases of feline mammary carcinomas studied was as follows: five cases were low-grade GI (13.2%), 10 intermediate-grade GII (26.3%), and 23 high-grade GIII (60.5%).Twenty-eight cases (73.7%) had a high p62 expression score (\u22655), three (7.9%) had a score between 3 and 4, while seven cases (18.4%) had a low score between 1 and 2 . PositivThe correlation between the p62 expression score and the histological grade of the mammary carcinomas is reported in This preliminary study aimed to evaluate the immunohistochemical expression of the p62 protein in feline mammary carcinoma and feline mammary non-neoplastic tissue, taking into consideration tumors of different histological grades with different prognoses.In this study, we observed a high positivity of feline mammary carcinoma for p62. These preliminary observations could suggest a correlation between the expression of p62 and the carcinogenic process since in carcinomas there is a high expression of the protein as opposed to non-neoplastic mammary gland tissue. Although is not possible to draw a definitive conclusion about the expression of p62 in feline mammary carcinomas, the statistical difference observed between mammary carcinoma and non-neoplastic tissue is a result to be considered.In this study, no correlation was found between the expression of p62 and the different grades of mammary carcinoma, but the data obtained seem to show a certain tendency towards the loss of p62 from grade I carcinoma to grade III. We also wanted to evaluate the expression in tumors presenting lymphovascular invasion, a histological feature to be considered a statistically significant prognostic factor , and we In general, although the data are preliminary, they seem to agree, in terms of p62 expression levels, with those of female breast cancer. In fact, two studies demonstrated a higher expression of p62 in carcinomas compared to normal breast tissue ,26.Few studies on p62 and cancer have been performed in veterinary medicine. A study on canine cutaneous mast cell tumors demonstrated nuclear immunoreactivity in low-grade tumors and cytoplasmic immunoreactivity in high-grade tumors . In our A preliminary study on simple canine mammary carcinoma was more recently published and a reduction in p62 reactivity was observed as the degree of malignancy increased  .To date, both in human medicine and in veterinary medicine, the knowledge of the role of p62 in oncogenesis is still insufficient and not exactly delineated, partly due to the multiple activities of this protein, but from what we know so far, we can consider this protein as an independent tumor marker and not an oncogene .However, it has been shown that after oncogenic transformation, p62 is necessary for tumor initiation and tumor progression: the induced formation of Her2- breast cancer and the RAS-induced formation of lung cancer are hindered in p62 knockout animals ,30. FurtAnother very interesting study area, in which p62 could be involved, is the relationship established between neoplastic epithelial cells, the inflammatory response, and activated fibroblasts associated with epithelial cancer. Studies on human prostate cancer and breast cancer have suggested important metabolic differences between neoplastic cells and fibroblasts, as neoplastic cells are anabolic with a high expression of p62 while stromal cells are catabolic and lose p62 ,36. AccoThis study confirms the importance of the use of the cat as a translational spontaneous tumor model, especially for feline mammary carcinoma, whose analogies with human breast cancer are wildly acknowledged . This pr"}
{"text": "In the cellular response to stresses, the tumor suppressor p53 is activated to maintain genomic integrity and fidelity. As a transcription factor, p53 exhibits rich dynamics to allow for discrimination of the type and intensity of stresses and to direct the selective activation of target genes involved in different processes including cell cycle arrest and apoptosis. In this review, we focused on how stresses are encoded into p53 dynamics and how the dynamics are decoded into cellular outcomes. Theoretical modeling may provide a global view of signaling in the p53 network by coupling the encoding and decoding processes. We discussed the significance of modeling in revealing the mechanisms of the transition between p53 dynamic modes. Moreover, we shed light on the crosstalk between the p53 network and other signaling networks. This review may advance the understanding of operating principles of the p53 signaling network comprehensively and provide insights into p53 dynamics-based cancer therapy. Cells exhibit powerful information processing capabilities when they are exposed to various stresses such as DNA damage, hypoxia, and nutrition deficiencies ,2. In-deTranscription factors (TFs) are proteins that can bind to the promoters of target genes specifically to regulate their expression. Due to their versatility, TFs are usually considered as information processing centers. As one of the most important tumor suppressors, p53 mainly acts as a transcription factor to maintain genomic integrity by inducing a large number of target genes in cellular response to stresses . DiffereA fundamental strategy in understanding the underlying mechanisms of various dynamics is to seek out the corresponding regulatory networks in response to specific stresses. Based on the given functions of the network motifs, we can make some speculations about which kind of motif is responsible for the observed dynamics. The alternate activation of p53 and Mdm2 is responsible for p53 oscillations upon ionizing radiation (IR) . DiffereIn this review, we discuss recent advances in understanding the underlying mechanisms in the generation of p53 dynamics and the decision of cell fates in response to stresses. We summarize the latest findings in two aspects: encoding\u2014how DNA damage is encoded into p53 dynamics and decoding\u2014how cell fate is decoded from p53 dynamics. To give an overview of p53 response to DNA damage, we summarize the p53 signaling network including the damage types, sensors, upstream circuits, dynamics, transcription, and downstream circuits associated with different cell fates . MoreoveThe integrity of the genome is challenged by genotoxic stresses. Failure in cellular response to such stresses is closely related to carcinogenesis. During the cell cycle, cells are subject to transient and intrinsic DNA damage during mitosis. Extrinsic DNA damage agents mainly include antitumor drugs , etoposide, cisplatin, and NCS (Neocarzinostatin)) and irradiations. These damage agents can produce single- or double-strand breaks (SSBs or DSBs) in DNA. As the input to the p53 signaling network, different types of DNA damage activates the downstream pathways specifically, leading to different cellular outcomes .SSBs are breaks in one strand of the DNA double helix, mainly caused by UV radiation. They can be resolved by homologous recombination (HR) and alternative homologue-mediated SSB repair pathways . DSBs arCells sense DNA damage by activating several kinases including ATM, ATR, and DNA-PKcs, which are accountable for detecting damage lesions via the MRN (MRE11-RAD50-NBS1) complex and transmitting damage signals to the tumor suppressor p53. Upon DNA damage, ATM is specially activated by DSBs through phosphorylation, regulating DSBs repair and activating p53 . Based oATM, ATR, and DNA-PKcs are all members of phosphatidylinositol 3-kinase-related kinases (PIKKs) family. Although they can sense DNA damage independently, there is a crosstalk between them in some cases . DNA-PKcUpon DNA damage, p53 levels vary over time and exhibit rich dynamics . Lahav eIn response to UV-induced DNA damage, a single extended pulse of p53 is induced and its width and height increase with the UV dose . As a reNotably, the above results were observed in MCF7 cells, which were later found to be insensitive to a spectrum of antitumor drugs including etoposide, Nutlin-3a, and 5-Fluorouracil . Indeed,There exists another mode of two-phase dynamics of p53: cells exhibit a high-amplitude p53 pulse in the first phase and undergo low-amplitude p53 oscillations in the second phase . This kiDifferent p53 dynamics in various cell lines have inspired the investigation into whether such differences in p53 dynamics exist in different tissues or species. Lahav and coworkers observed that the radiosensitivity corresponding to p53 dynamics shows remarkable differences between tissues. The small and large intestines are relatively insensitive to p53 levels peaking at 2\u20133 h followed by decreasing in its levels, while lymphoid organs, including the spleen and pancreas, are more sensitive to radiation with sustained p53 expression . In anotThe mathematical modeling on p53 network provides a theoretical perspective to understand the generation mechanism of p53 dynamics. The negative feedback loop between p53 and Mdm2 governs p53 oscillations upon DNA damage . NeverthThe core status of the p53-Mdm2 loop in p53 regulation is challenged by the connection of p53 pulses to the pulses of the damage sensor ATM . p53-indLong-term measurements revealed that p53 oscillations exhibited marked heterogeneity, especially in the oscillation amplitudes . Based oThe generation of basal dynamics of p53 depends heavily on an excitable mechanism. A hallmark of an excitable system is that a transient input is sufficient for triggering a full response. Thus, similar to the case of cellular response to extrinsic damage, a complete p53 pulse will be initiated by transient damage during mitosis . BatchelThe coupling of two negative feedback loops is important for producing some specific properties of the p53 system, such as excitability . It has mdm2. This positive feedback loop may control the amplitude of p53 pulses [Interconnected positive and negative feedback loops (IPNFLs) are another prevalent coupling pattern in producing p53 pulses  Figure . The two3 pulses .In response to DNA damage, p53 is gradually activated to perform different functions and different feedback loops modulate p53 dynamics in this process. In modeling, it was assumed that there exist different forms of p53 based on its phosphorylation status ,16,19,47Moreover, other functional factors also play some roles in the encoding of p53 dynamics. Multiple miRNAs, including miR-605, miR-192, miR-29a, and miR-34a can directly or indirectly repress Mdm2, thus forming a p53-miRNA-Mdm2 positive feedback loop ,55. ThesIn response to stresses, p53 induces hundreds of target genes to modulate a wide range of cellular processes including cell cycle arrest, apoptosis, senescence, DNA repair, and metabolism ,60. ThisOver the past decade, much effort has been put into exploring the selective expression of the target genes by oscillatory p53. By experimental measurements, the amplitudes of p53 pulses were found to delineate promoter activation thresholds, while pulse frequencies were differentially filtered by the promoters of the target genes . These rUnder p53 oscillations, p21 mRNA tightly follows p53 pulses, while p21 protein has three different patterns: oscillation, slow, and rapid accumulation . The difAdditional mechanisms, including different activation thresholds and the presence of feedforward loops, should contribute to modulating the exclusive induction of targets under specific p53 dynamics . MeanwhiIn addition to the dynamic patterns of p53, there are multiple levels of regulation that can help p53 select targets to execute appropriate cellular responses . Upon DNThe acetylation status assists p53 in preferentially activating a specific cell fate, such as apoptosis, highlighting the importance of post-translational modifications in regulating the target selectivity of p53 . Gu et arylation . These mrylation ,76.The selective induction of p53 targets between cell cycle arrest, senescence, and apoptosis has been extensively explored. p53 leads to cell cycle arrest by inducing p21 ,78. The A great deal of modeling work has focused on the choice between survival and apoptosis in cellular response to stresses. In general, differential activation of BAX, PUMA, Apaf1, Casp3, and p21 can be exploited to indicate different cellular outcomes in the models ,82. BaseThe regulation of apoptosis becomes complex when multiple factors are involved. Activated E2F-1 induces ASPP, which promotes the expression of BAX to trigger apoptosis . p53-ind pathway . A Boole pathway . Similarp53 shows persistent pulses at low etoposide doses but exhibits monotonic increasing at rather high doses in U-2 OS cells . It is iIt is also important to investigate how p53 oscillations terminate after DNA repair. Recently, it has been proposed that p53 undergoes an intermediate state between early high-frequency oscillations and late low-frequency pulses and finally returns to a basal state  Figure c. It canIt was recently reported that the transition dynamics of p53 from oscillations to high rising in etoposide-treated U-2 OS and A549 cells may sequentially undergo damped oscillations and a rising with a moderate platform  Figure f. The trThe study of transition dynamics suggests that we can look for clues to kill cancer cells by amplifying p53 signaling. We can choose an agent that is sensitive to the specific cells. For example, MCF7 cells are sensitive to UV light but not to NCS and Etoposide . In factThe p53 network has a key role in response to cellular stresses, creating scenarios in which the p53 network couples with other signaling networks. The establishment of a coupling relationship is supported by several aspects: (I) coactivation by the same stimulus ; (II) sharing some key regulatory factors ; and (III) coregulation of the same cellular process. The coactivation of multiple signaling networks is the basis of network coupling, and the abundance of coregulators determines the degree of activation of individual networks, thus affecting the dynamic properties and functions of each network .Hypoxia can activate both p53 and HIF-1, thus causing an interplay between these two transcription factors. However, their activation is different and determined by the severity of hypoxia: HIF-1 is activated to promote glycolysis and angiogenesis upon mild and moderate hypoxia, while only upon severe hypoxia or anoxia, p53 is induced to trigger apoptosis . Our mod factors ,96,97. TIt was reported that the interference of NF-pathways . The inh and p53 . Furthernd NF-\u03baB . Indeed,p300/CBP . An expln of p53 . It remaE2F-1 is a vital transcription factor that primarily regulates the cell cycle progression from G1 to S phase. In early G1, its transcriptional activity is repressed by pRB, while in late G1, E2F-1 is released and activated as pRB is phosphorylated by the Cyclin/CDKs (cyclin-dependent kinase) complex, driving the cells from G1 phase to S phase . Due to It is a challenge to understand the coactivation of p53 and TGF-activity ; at the activity . These trylation ,108. TGF and Zeb . The mut and Zeb . Strikinl of EMT . It is all cycle . TherefoOver the last 20 years, the development in experimental techniques has advanced the measurement of p53 dynamics in individual cells. In general, p53 dynamics depend on the type of stimulus, cell, tissue, and species. In addition, the detection methods have also been found to impact the dynamics of p53 obtained by experiments . Both ex"}
{"text": "Gliomas are extremely debilitating malignant brain tumors with very limited response to therapies. The initiation and progression of gliomas can be attributed to several molecular abnormalities, such as mutations in important regulatory networks. In this regard, the mitogen-activated protein kinases (MAPKs) arise as key signaling pathways involved in cell proliferation, survival, and differentiation. MAPK pathway has been altered in most glial tumors. In glioma cells, the activation of p38 MAPK contributes to tumor invasion and metastasis and is positively correlated with tumor grade, being considered a potential oncogenic factor contributing to brain tumorigenesis and chemotherapy resistance. Hence, a better understanding of glioma pathogenesis is essential to the advancement of therapies that provide extended life expectancy for glioma patients. This review aims to explore the role of the p38 MAPK pathway in the genesis and progression of malignant brain tumors. Malignant brain tumors are rapidly growing cancer with high invasion potential to surrounding healthy brain structures, which include gliomas and neuronal or mixed neuronal-glial tumors . GliomasThe initiation and progression of gliomas can be attributed to several molecular abnormalities, such as mutations in important regulatory networks . The mitAmong the MAPK pathways, p38 mitogen-activated protein kinase (p38 MAPK) signaling was identified as a mediator of stress and inflammation responses . This paConsidering the importance of the signaling pathways involved in the genetic and molecular alterations in the onset of cancer, describing the role of the p38 MAPK pathway in tumor microenvironment and in the progression of gliomas becomes a key strategy to comprehend and propose new treatments for malignant brain tumors.Malignant brain tumors are aggressive and impact directly patients\u2019 life quality . GliomasGlioblastoma (GBM) is the highest grade of diffuse astrocytoma and is considered incurable . DespiteIt is well known that molecular characteristics inside the tumor microenvironment (TME) indicate the sensitivity of tumors to therapy. Initially, tumorigenesis is related to the gain of numerous genetic mutations, which provide malignant cells resistant to the growth-inhibitory and apoptotic signals . All GBMIn general, tumors are complex tissues composed of distinct cell types that interact with each other forming an intense signaling network . The gliHypoxia is considered a hallmark of GBM, and a critical factor associated with mutations in multiple signaling pathways that affect the tumor landscape . Solid tProtein kinases are enzymes that have the property to catalyze protein phosphorylation through the transfer of a phosphoryl group from ATP and GTP to threonine, serine , tyrosine , both serine/threonine and tyrosine  . The pho\u03b1, \u03b2, \u03b3, and \u03b4), and ERK5. Atypical MAPKs have nonconforming particularities and consist of ERK3/4, ERK7, and Nemo-like kinase (NLK)  .The activation of MAPKs and their downstream targets plays a pivotal role in different signaling cascades involved in transcription, development, differentiation, migration, cell death, and many other critical cellular functions . DetrimeMAPKs signaling cascades present recurrent contributions to oncogenesis, tumor progression, and drug resistance, which suggests that its pharmacological modulation can be a promising strategy in the development of cancer therapies . It is wDiscovered on a pharmacological screen in 1994, p38 MAPK is a signal transduction pathway that plays a key role in cellular adaptation to extracellular stimuli . Its actAfter extracellular signals, activation of the p38 pathway usually proceeds through a classical phosphorylation cascade, where a MAPKKK  phosphorylates and activates the MAPKKs specifics of p38 MAPK, MKK3, and 6, which in turn, mediate the activation of the different p38 MAPK isoforms . The p38The p38 MAPK family compromises four isoforms: p38\u03b1 , p38\u03b2 , p38\u03b3 , and p38\u03b4  . The fouIn recent years, the involvement of p38 MAPK in cancer has been widely described . This paSome studies have suggested a role for p38 MAPK in mediating pathways that lead to cell apoptosis and growth inhibitory signals, particularly in promoting cell cycle arrest and differentiation, which supports the idea that the stress-activated kinase is a tumor suppressor . HoweverMAPKs are expressed in various types of malignant brain tumors . Besidesin vitro and in vivo. Irradiation is often part of the GBM treatment scheme, although, in cells that present mutant PTEN it was described that irradiation can activate p38/Akt and PI3K/Akt signaling pathways, increasing MMP-2 expression and intensifying invasiveness  which activates the p38 MAPK pathway (Long noncoding RNAs (lncRNAs) are transcripts with more than 200 nucleotides that commonly do not encode proteins, many of them have been characterized as oncogenes or tumor suppressors in cancer . The MRC pathway .Nowadays it is known that, in tumor progression, gliomas orchestrate the immune system to a protumoral phenotype. The p38 MAPK is also correlated with immune and inflammation signaling, being responsible for the production of cytokines in the TME . Wang etin vitro  release . Other sin vitro . Figure Several small-molecule protein kinase inhibitors have been tested for cancer treatment, demonstrating interesting effects on the immune system. Among these, there is the EGFR tyrosine kinase inhibitor gefitinib, and other inhibitors focusing on multiple targets, like imatinib, sorafenib, and sunitinib . Regardlvia Nrf2 signaling inhibition, indicating that p38 MAPK/Nrf2 activation is a network involved in temozolomide glioma cells resistance  was used in combination with temozolomide, it reduced temozolomide antitumoral effects . BesidesSeveral clinical trials targeting p38 MAPK in cancer are currently underway. Ralimetinib (LY2228820), for example, is a potent and selective inhibitor of p38\u03b1 and p38\u03b2 and has been tested as a single agent and in combination with chemotherapeutic agents for the treatment of ovarian cancer, GBM, and metastatic breast cancer . An impoBecause of the wide-ranging controlling role of p38 in different cellular processes, the possibility of side effects resulting from an undesired pharmacological activity is a relevant concern for the p38 inhibitors. There are many discontinued p38 inhibitors that failed due to safety concerns, presenting adverse effects directed mostly to the hepatic system and skin . Other iMalignant brain tumors are debilitating diseases with a dismal prognosis and extremely limited response to therapies. The aggressiveness of GBM has been marked by several signaling pathway dysregulations that are implicated in cell proliferation, survival, migration, and angiogenesis. Tumor cells are heterogeneous, so it is also important to note that targeting a mediator involved in multiple pathways would constitute a more efficient therapy. MAPKs are shown to support numerous processes of cell survival. The p38 MAPK pathway is a key route capable to influence different frameworks of cellular survival mechanisms such as inflammation, proliferation, migration, invasion, and ROS signaling. Briefly, the p38 MAPK is likely to be an oncogenic factor contributing to GBM initiation, progression, and chemotherapy resistance. Hence, a better understanding of the GBM tumor microenvironment is essential to the advancement of therapies that provide extended life expectancy for patients. Thus, targeting key components of these signaling pathways using small-molecule inhibitors, such as p38 inhibitors and antibodies, could provide progress in the treatment of gliomas."}
{"text": "The p53 protein is a tumor suppressor encoded by the TP53 gene and consists of 393 amino acids with four main functional domains. This protein responds to various cellular stresses to regulate the expression of target genes, thereby causing DNA repair, cell cycle arrest, apoptosis, metabolic changes, and aging. Mutations in the TP53 gene and the functions of the wild-type p53 protein (wtp53) have been linked to various human cancers. Eight TP53 gene mutations are located in codons, constituting 28% of all p53 mutations. The p53 can be used as a biomarker for tumor progression and an excellent target for designing cancer treatment strategies. In wild-type p53-carrying cancers, abnormal signaling of the p53 pathway usually occurs due to other unusual settings, such as high MDM2 expression. These differences between cancer cell p53 and normal cells have made p53 one of the most important targets for cancer treatment. In this review, we have dealt with various issues, such as the relative contribution of wild-type p53 loss of function, including transactivation-dependent and transactivation-independent activities in oncogenic processes and their role in cancer development. We also discuss the role of p53 in the process of ferroptosis and its targeting in cancer treatment. Finally, we focus on p53-related drug delivery systems and investigate the challenges and solutions. The TP53 gene encodes a tumor suppressor protein called P53. The p53 protein was first identified in 1979 as a cellular protein that binds to the large T antigen of the simian virus (SV40) and accumulates in the nucleus of cancer cells. This protein contains domains of transcriptional activation, DNA binding, and oligomerization. The encoded P53 protein responds to various cellular stresses to regulate the expression of target genes, thereby causing DNA repair, cell cycle arrest, apoptosis, metabolic changes, and aging. Mutations in this gene and the functions of the wild-type p53 protein (wtp53) have been linked to various human cancers .According to studies, Eight TP53 gene mutations are located in codons that make up 28% of all p53 mutations. Mutations in these alleles appear in many human cancers and tissues. These alleles are called hotspots . MutatioThe p53 protein is vital in maintaining DNA stability and preventing cancer. Normally, wild-type p53 protein (wtp53) binds to a negative regulator and is inactivated or degraded. However, when DNA damage occurs in a cell, the p53 protein is induced, causing the cell cycle to stop. This gives the cells a chance to repair, but if the damage is severe, the cells will develop apoptosis. The p53 (mtp53) gene mutation has been linked to various cancers Many mutations occur in the DNA binding domain of the p53 gene. On the other hand, the successful interaction of wtp53 protein with transcription factors facilitates the repression or activation of appropriate target genes. This process is disrupted by the presence of the defective mtp53 protein. Thus, converting mtp53 protein to wild-type p53 protein could be a promising way to prevent or reverse tumor progression .Among these, we can mention some small molecular compounds, such as RITA , which target p53. This molecular compound directly binds to p53, disrupts the interaction between p53 and MDM2, and prevents p53 degradation. RITA increases tumor suppressor expression and suppresses oncogene expression through p53 reactivation; As a result, it causes cell death mediated by p53 .The p53 is a biomarker for tumor progression and a perfect target for designing cancer treatment strategies. In wild-type p53-carrying cancers, abnormal signaling of the p53 pathway usually occurs due to other unusual settings, such as high MDM2 expression. These differences between cancer cell p53 and normal cells have made p53 one of the most important targets for cancer treatment .In recent years, studies in this field have shown the structure, function, and role of p53 in tumor formation and development. These basilar studies strongly support the design and development of new approaches to targeting the mutant and wild-type p53 in cancer treatment. For example, delivery of wild-type p53 by adenovirus infection kills cancer cells and affects and suppresses tumor growth in preclinical and clinical trials . Also, min vitro and in vivo  are observed at the amino acid sites 175, 245, 248, 249, 273, and 282  Freed-P. MeanwhiAs mentioned, two hotspots and common cancer mutations in p53 protein are amino acid sites 175 and 273. R175H and R273H are more prone to aggregation than wild-type (WT) p53, and their pathological aggregation can lead to various cancers. A recent study investigated the dynamic and structural properties of the R175H and R273H mutants in the p53 core domain (p53C) using extensive all-atom molecular dynamics simulations. In this regard, the researchers found that in both R175H and R273H mutations, the \u03b2-sheet structure is well preserved; however, a larger hydrophobic surface and a higher loop flexibility than WT p53C were seen, thus predisposing proteins to accumulate in the cell. Also, an allosteric pathway has been identified through which the R273H mutation increases the flexibility of the N-terminal region around loop 2. These results indicate mechanical insights into the high accumulation tendencies of R175H and R273H mutants .In cancer biology, the vital point about p53 is that; the p53 mutant protein is found in 50% or more of 50% of human cancers. In addition to losing function, the mutant p53 can have a dominant-negative effect on the remaining wild-type p53 allele and subsequently inactivate it by losing heterozygosity (LOH). Also, some p53 mutations have additive functions, which will cause the tumor to grow. In cancers in which wild-type p53 is conserved, it is usually in regulatory genes that encode the up or down pathways of p53; changes are observed .Among p53 mutants, missense mutations not only cause the mutated p53 protein to lose its wild-type (LOF) function and gain dominant-negative activity but also increase the function of the mutated p53, leading to the tumor\u2019s more aggressive behavior and drug resistance .In the remainder of this section, we will discuss the relative contribution of wild-type p53 loss of function, including transactivation-dependent and transactivation-independent activities in oncogenic processes and their role in cancer development.The TP53 gene acts as a tumor suppressor and cellular stress sensor. In stress-free cells, p53 is targeted for degradation by E3 ubiquitin ligase MDM2 and kept at low levels. Stress signals, such as DNA damage, oncogene expression, and hypoxia relieve P53 from MDM2 inhibition. Mutations in human cancers that occur mainly in the domain of DNA binding and specific sequences; disrupt p53 activation. A mutation in the TP53 gene disrupts the binding capacity of DNA by disrupting the structure of the p53 protein; it also increases oncogenic function. The TP53 gene also encodes a transcription factor that is an essential factor in cancer prevention. Inactivation of this gene leads to the most common mutation in sporadic human cancers .Studies have shown that p53-R248Q expression reduces the motility and invasiveness of lung and breast cancer cells in a p53 transactivation-dependent manner. Indeed, in the cell lines expressing p53-R248Q, myosin light chain two expression decreases. This chain is a protein involved in actomyosin-based motility. Also, the expression of R248Q in Matrigel\u2019s absence significantly reduces the migration rate in a trans-dependent manner .The wild-type p53 regulates the expression of more than 2,500 genes as a transcription factor. The p53 helps respond to various stresses such as DNA damage, hypoxia, and oncogene activation. Also, depending on the severity and duration of stress, it leads to cell cycle arrest or apoptosis . Some ofThe wild-type p53 dysfunction can disrupt autophagy signaling. In this regard, the nuclear transactivation-dependent activation of autophagy by p53 is often impaired due to inactivation or deficiency of p53 in the cytoplasm. However, the tumor can stimulate pro-autophagic functions. The downregulation of the mTOR set translates this activity. The researchers found that p53 may inhibit autophagy using direct protein-protein interactions. Based on the findings of a study, gain-of-function (GOF) mutant p53 proteins inhibit the autophagic pathway and increase the proliferation of pancreatic and breast cancer cells. This reaction has been accompanied by stimulation of AMPK-mTOR genes and suppression of DRAM, Beclin-1, ATG12, and sestrin genes .Although most studies on p53-mediated tumor suppression mechanisms are based on the activation of p53 target genes, other aspects of this issue include the performance of p53 independent of the reaction, which has been reported recently. Induction of apoptosis in mitochondria can be considered the best function of p53 independent of transactivation. The p53 protein can bind members of the BCL-2 family of anti-apoptosis to replace them with members of the BCL-2 family of pro-apoptotic. It can also directly activate BAK and BAX, infiltrate the external mitochondrial matrix, and stimulate apoptosis  Ho et a.The p53 protein, in addition to mitochondria, has functions other than transactivation in other sites, such as the cytoplasm and nucleus , which cIn a study of mouse models of intestinal cancer based on the Wnt signaling pathway caused by an ApcMin/+ mutation or deletion of Csnk1a1 in the proximal intestine, cancer was suppressed in the presence of p53R172H. It can be said that this repression is based on a mutation in the DNA binding domain that is responsible for the trans-activation of the target gene and is related to the p53 \u2212/\u2212 model. This is related to the inhibition of TCF4 chromatin binding in the Wnt pathway and, in a sense, the suppression of this pathway and tumor-independent p53 activation. Further tests are needed to prove this mutant\u2019s neomorphic activity or presence in wild-type p53 .in vivo. p53R178E may provide a potential mechanism for apoptosis independent of the transactivation induced by p53R178E, and it can localize in the mitochondria  that can be regulated in various ways, including levels of transcription factors and changes in the activity of antioxidant enzymes. The wild-type p53 regulates ferroptosis through lipid peroxidation, amino acid metabolism, and the biosynthesis of glutathione and phospholipids . Also, tIt is important to note that p53 sometimes reduces cellular susceptibility to ferroptosis. Studies have shown that wild-type p53 protein activates p21 protein in a transcription-dependent manner and delays the onset of ferroptosis . ResearcThere are two main pathways in the induction of ferroptosis. The cysteine-glutamate transporter is the first pathway, which includes erastin, sulfasalazine, and glutamate. The second pathway in the induction of ferroptosis directly inhibits glutathione peroxidase (GPX) activity and includes RSL3 and DP17 . In the One of the roles of erastin is to influence the voltage-dependent anion channel (VDAC). This ion channel is located in the mitochondria\u2019s outer membrane . VDAC coC\u2212 system ; This was while the p533KR mutant cell mortality was very high (>90%). However, if the expression of SLC7A11 is excessive in p533KR mutant cells, treatment with erastin will significantly reduce the cell death rate (20%)  .SLC7A11 transcription were lost. This mutant model, called p534KR98, significantly reduces cell death and tumor inhibitory function during treatment with erastin. These results suggest that activating p53 by erastin may play an essential role in tumor inhibition by inhibiting transcription of SLC7A11 and stimulating the ferroptosis process  that inhibit SLC7A11 display significant radiation sensitizing effects on tumor organoids and xenografts derived from patients with p53 deficiency or mutation. Accordingly, activation of p53 and RT-induced ferroptosis leads to better clinical outcomes for RT in cancer patients, and it is suggested that these FINs be used in combination with radiotherapy to treat p53 mutant cancers (Radiotherapy (RT) breaks down two strands of DNA and produces reactive oxygen species (ROS); this causes the cessation of the cell cycle, aging, and cell death . Since t cancers .The frequency of p53 mutations in most cancers is about 50%, but this frequency varies depending on the type of cancer. For example, p53 mutation has been reported in cancers of laryngeal (40.4%), esophageal (43.1%), head and neck (40.6%), ovarian (47.8%), and colorectal (43.2%). However, the frequency of p53 mutation in a small number of cancers is less than 20%. For example, endocrine (14.6%), hematopoietic (12.7%), and cervical (5.8%) tumors have the lowest frequency of p53 mutations . TherefoProtein accumulation, instability, improper folding, and defective transport generally lead to cellular functions and processes dysfunction. Many of these protein accumulation diseases are caused by the wrong folding of the protein, possibly due to genetic mutations and environmental stress conditions . Accordiin vitro and in vivo. These peptides are for the region prone to p53 accumulation; amino acids 252\u2013258 are designed. For example, research has been conducted on treating different models of ovarian cancer in vivo and in vitro and the potential of these peptides in accumulating p53 mutants , which has an inhibitory effect on histone deacetylase (HDAC), can directly affect mutant p53 and lead to p53 phosphorylation in serine 15 and cessation of cell division in the G2/M stage and finally tumor cell death. It should be noted that its role in inhibiting ovarian cancer cell lines is well known .According to recent studies, zinc deficiency induces oxidative DNA damage and is involved in developing the mutated p53 . ResearcSome of these methods have limitations and may not be used to treat all cancers associated with p53 accumulation. For example, in the designer peptide method, if wild-type p53 is somewhat compacted or unfolded, wild-type p53 structures may also be targeted. If this occurs in normal cells in the body, it can lead to systemic toxicity. Also, in therapies based on small molecules, peptides are rapidly eliminated from the bloodstream due to their short half-lives, small molecular weight, and size; as a result, more optimization is needed . Hence, Another treatment method is the production of gold nanoparticles (AuNPs). These nanostructures provide a robust and non-toxic delivery system for gapmers in cancer cells and significantly reduce mutant p53 proteins. They overcome chemoresistance to gemcitabine. The formulation of AuNPs consists of a combination of a mixed polymer layer of polyethylene glycol (PEG) and PEI and a layer-by-layer assembly of bPEI through a sensitive linker. These nanoparticles can bind oligonucleotides through electrostatic interactions and release them as glutathione .in vitro. These findings suggest that combining synthetic mRNA NP delivery strategy with other cancer treatments can be very effective (The redox response for efficient delivery of p53-encoding synthetic messenger RNA (mRNA) is another nanoparticle (NP)-based engineered platform. Based on the findings, synthetic p53-mRNA nanoparticles significantly delay the growth of p53-null HCC and NSCLC cells by inducing apoptosis and cell cycle arrest. Also, p53 restoration significantly improves the sensitivity of these tumor cells to everolimus, a mammalian target of rapamycin (mTOR) inhibitor that is not expressed in HCC and advanced NSCLC. Furthermore, co-targeting the mTOR and p53 signaling pathways lead to distinct antitumor effects in several animal models of HCC and NSCLC ffective .In addition to the above, monoclonal antibodies can be potent tools against p53-specific mutations and maximize treatment potential with personalized or precision medicine approaches .Since the wild-type P53 protein has important and extensive roles and functions within the cell; as a result of mutations in this protein, which can occur due to various factors, causes various types of cancer. According to the findings, the p53 mutant protein is found in 50% or more of 50% of human cancers. Therefore, specialized therapeutic approaches to target P53 mutants or pathways associated with this protein have received much attention recently Although research in this area needs further investigation, it is hoped that it will lead to proper and effective treatments for various types of cancer and pave the way for future studies."}
{"text": "Antimicrobial stewardship (AMS) is a critical global intervention aimed at optimising antimicrobial use and decreasing antimicrobial resistance (AMR) with pharmacists playing a pivotal role within AMS teams. However, AMS is not comprehensively taught in pharmacy curricula and little is known about the relevance of pharmacists\u2019 training to meet AMS needs in South Africa.This study aimed to explore the attitudes, knowledge and perceptions of clinical pharmacists towards AMS participation and training in South Africa.This study was conducted among clinically practicing pharmacists in public and private healthcare sectors in South Africa.A quantitative exploratory research design was selected for this study. The study was conducted using a self-administered structured survey. Categorical variables were analysed using simple descriptive statistics. Mann\u2013Whitney and Kruskal\u2013Wallis tests were applied to determine differences between variables.p = 0.005), sector of employment (p = 0.01), position of employment (p = 0.015) and presence of AMS programmes (p = 0.004). Pharmacists indicated that their Bachelor of Pharmacy undergraduate studies inadequately prepared them for their role in AMS (median 4.3).Pharmacists demonstrated good attitudes knowledge and perceptions towards AMS (median 4.3). There was statistical significant differences in AMS participation between pharmacists of different years of experience  and workshops and insufficiently incorporated in undergraduate programmes.This study confirms that undergraduate pharmacy programmes inadequately prepare pharmacists for their role in AMS. Antimicrobial resistance (AMR) poses a major threat to the health of populations worldwide, puts achievements in medical advancements at risk and increases morbidity, mortality and the global economic burden  was approached to distribute the survey to its members. The SASOCP is a society formed in response to the resurgent interest in clinical pharmacy with 85 paid members at the time the survey was distributed. A sample size of 55 was obtained, yielding a response rate of 64.7%. Out of a maximum of 55 responses, not all respondents answered and rated each variable from questions two to nine. Hence, the number of responses received for each variable is reflected by a change in A self-administered structured survey was designed specifically for this study, incorporating questions from previous instrument designs of similar studies  of the University of the Witwatersrand, ethics clearance number M210937. A study information sheet explained the study in detail and the benefits and risks of participating in the study. Completion of the questionnaire was anonymous and data were confidentially maintained.n = 40) of the study population and male participants represented 27.27% (n = 15). The majority of the participants were between the ages of 30 and 39 years 45.45% (n = 25), have an M. Pharm qualification 65.46% (n = 36) with more than 10 years\u2019 experience 50.91% (n = 28). Pharmacists in the private sector represented 81.82% (n = 45) of the responses. The majority of participants were employed as pharmacists 32.73% (n = 18), followed by 29.09% (n = 16) clinical pharmacists and 27.27% (n = 15) ward pharmacists. Forty-one participants (74.55 %) indicated there is an AMS programme at their institution of employment while 25.45% (n = 14) indicated they work without an AMS programme.n = 51) agreed and/or strongly agreed on the definition of AMS, median 5 (IQR 5\u20135). A total of 47 (88.68%) agreed/strongly agreed that an AMS team should consist of a multidisciplinary team including a physician, infectious disease specialist, pharmacist and a microbiologist. Forty-five (84.88%) participants disagreed and/or strongly disagreed that a goal of AMS is to increase the use of antimicrobials while 39 participants (73.58%) agreed and/or strongly agreed that a goal of AMS is to reduce hospital stay. Majority of participants  agreed and/or strongly agreed that optimising antimicrobial use and reducing antimicrobial resistance is a goal of AMS. Participants unanimously agreed and/or strongly agreed that pharmacists play a critical role in AMS to promote optimal use of antimicrobials , educate healthcare professionals , lead and support AMS activities  and to recommend appropriate therapy or de-escalate . p = 0.33; 0.212 and 0.422, respectively). The various current positions of employment showed statistical differences p = 0.015 with ward pharmacists, senior pharmacists, assistant pharmacy managers and clinical pharmacists showing higher levels of participation compared with pharmacists (median 5.4) and pharmacy managers (median 6.2). Participants employed as pharmacists show low levels of participation in monitoring AMS duration (p = 0.015); monitoring antimicrobial consumption (p = 0.13); AMS rounds (p = 0.006); participating in AMS campaigns (p = 0.03) and screening AMS prescriptions in accordance with local guidelines before dispensing, (p = 0.006), medians = 5 compared with clinical pharmacists, ward pharmacists and senior pharmacists. Pharmacists with less than a year of experience showed higher levels of participation  followed by pharmacists with more than 10 years of experience . Pharmacists employed in private sector showed higher participation  compared with those employed in the public sector. Pharmacists working in an institution with an AMS programme demonstrated much higher levels of participation in AMS (median 8) compared with those working without an AMS programme (median 5.2), p = 0.004. Supplementary n = 11; 22%) rated \u2018participation won\u2019t make a difference\u2019 as a factor that somewhat limits participation.n = 40; 80%) and least likely to acquire this knowledge in undergraduate programmes . n = 48; 100%).n = 48; 100%). Followed by infectious disease , patient individualisation and surgical prophylaxis . All participants viewed agent of choice as moderately important . Only 28 participants (58.33%) rated standard treatment guideline compliance and team dynamics as a highly important, and 25 participants (52.08%) rated standard treatment guideline compliance as highly important. Supplementary Supplementary Pharmacists have a responsibility to take a prominent role in AMS programmes at institutional level. Previous studies found that a lack of education and training are key barriers to pharmacists\u2019 participation in AMS programmes and activities  compared with only 7.32% in the public sector. Well-established AMS programmes offer a structured and guided approach to AMS activities  were highly rated factor limiting pharmacists\u2019 participation. This could be because of doctors lack of knowledge and understanding about pharmacists\u2019 roles, competencies and activities .Universities worldwide have heeded this call and incorporated AMS training in undergraduate and postgraduate healthcare programmes; however, there is variation in the extent to which AMS training is included, as well as differences in pedagogies (Abubakar & Tangiisuran The study by Burger et al.  further Pharmacy schools should note that pharmacists were all in agreement that a comprehensive course in AMS and infectious disease would be most beneficial if taught in undergraduate programmes. Highly rated topics for inclusion in AMS curricula were infectious disease, dose optimisation, patient individualisation and surgical prophylaxis. The agent of choice was rated as moderately important. In order to be effective members of the AMS team pharmacists require skills and knowledge in antimicrobial use, infectious disease as well as leadership skills to implement AMS programmes and work effectively within a multidisciplinary team (Weier et al. Although it is important to create a solid foundation of AMS knowledge at undergraduate level, AMS education and training should also continue throughout health professional careers to shape and reaffirm their attitudes and behaviours to AMS (Majumder et al. The findings of this study suggest that clinical pharmacists, pharmacists involved in clinical work, and AMS in South Africa have good attitudes, knowledge and perceptions towards AMS and have good levels of participation in AMS activities. Despite the presence of AMS programmes, pharmacist indicated that many factors could limit their level of participation in AMS activities suggesting the need to enhance and develop AMS programmes. Furthermore, the study\u2019s findings confirm that AMS principles are insufficiently incorporated in undergraduate pharmacy programmes. However, informal education and training in the form of short courses, CPD activities, conferences and workshops are available, which pharmacists utilise to improve their knowledge on AMS.There are a few limitations to this study. Firstly, the study was conducted among SASOCP members, clinical pharmacists and pharmacists involved in clinical work and AMS who are not SASOCP members did not get an opportunity to participate in this study. Secondly, there was only a small representation from the public sector. The results from the public sector may differ with greater representation.As this study targeted SASOCP members and had minimum representation from the public sector, further large-scale studies are warranted to validate these results by including a greater number of pharmacists. This study highlighted that pharmacy schools in South Africa incorporate AMS training into their undergraduate curriculum; it is therefore recommended that a study be carried out to determine what pharmacy schools are teaching with regard to AMS. The findings from this study support the recommendation to pharmacy schools to implement systematic education and training for pharmacists, starting from instilling basic principles of AMR, AMS, infectious disease and interpersonal skill development in undergraduate programmes, to continuous education and development in the form of short courses and CPD. The Department of Health should redirect resources to improve AMS programmes in public sectors. Hospital administrators in private and public sectors are encouraged to monitor and support AMS programmes within their institutions. There is strong evidence that doctors are unwilling to work with clinical pharmacists; hence, a study should be conducted to evaluate the factors that influence interprofessional collaboration between doctors and clinical pharmacists.This study found that clinical pharmacists, pharmacists involved in clinical work and AMS in South Africa have good attitudes, knowledge and perception towards AMS. Pharmacists of all positions of employment participate in AMS with pharmacists employed as clinical and ward pharmacist\u2019s showing greater levels of participation. Masters programmes, short courses, CPDs, conferences and workshops are avenues available for pharmacists to acquire AMS knowledge; however, pharmacists are of the view that an undergraduate AMS programme will be most beneficial to bridge the current gap between education and practice."}
{"text": "Recently, astrocytes are fast climbing the ladder of importance in cognitive-related diseases. Perioperative neurocognitive disorder (PND) is a common consequence of anesthesia and surgery, which is widely investigated in elderly and susceptible individuals. There is no doubt that astrocytes also play an irreplaceable role in the pathogenesis of PND. Reactive astrocytes can be found in the PND model, with an altered phenotype and morphology, suggesting a role in the development of the diseases. As a prominent participant cell in the central inflammatory response, the inflammatory response is unavoidably a crucial pathway in the development of the disease. Astrocytes also play a significant role in the homeostasis of the internal environment, neuronal metabolism, and synaptic homeostasis, all of which have an impact on cognitive function. In this article, we discuss the function of astrocytes in PND in order to establish a framework for investigating treatments for PND that target astrocytes. Disordered neurocognitive functions after surgery and anesthesia is a heterogeneous set of situations, which include any form of the acute event (of postoperative delirium) and cognitive decline diagnosed up to 30 days after the procedure (a delayed neurocognitive recovery) and up to 12 months   (p. 11).There are several hypotheses on the pathogenesis of PND. The first, and the most extensively explored, is neuroinflammation  p. 11)..11] .It is of growing interest that astrocytes subject to different injuries develop into distinct subtypes. Astrocytes are classified into A1 and A2 states in the majority of studies; A1 exhibits a detrimental and proinflammatory phenotype, while A2 promotes repair ,23 .oup mice  (p. 13).Presently, many clinical and preclinical studies have confirmed the correlation between neuroinflammation and PND  (p. 13).\u2013C motif ligand 2 (CCL2) plays a vital role in the nervous system and is predominantly derived from astrocytes, while CCR2 (the main receptor for CCL2) is predominantly expressed in microglia. The surgery increases chemokine CCL2 release from the activated astrocytes, which promotes microglia activation and M1 polarization. M1 microglia is one of the phenotypes formed by stimulated microglia, which is considered to be destructive and can produce inflammatory mediators. Cellular interactions promote inflammatory responses and cause hippocampus neuronal injuries, jointly resulting in the development and progression of cognitive impairment [Recent evidence demonstrates that the Cpairment  (p. 13).pairment ,41 .pocampus  (p. 14).pocampus  (p. 14).pocampus  (p. 14).Synapses are specific structures that contact neurons and glial cells in the CNS. Synapses are involved in the transportation of ions and neurotransmitters as well as the exchange of neuronal information  (p. 14).Finally, astrocytes control neurotransmitter transmissions, see 2+-dependent exocytosis, but other mechanisms have also been proposed to mediate the release of glutamate from astrocytes as well [2+ in astrocytes and neuronal excitotoxicity induced by an excessive glutamate release [In contrast, recent studies have revealed that in addition to uptake, astrocytes release trace amounts of glutamate to the adjacent neurons. This release of astrocytic glutamate is plausibly mediated by Ca as well  (p. 14). release  (p. 15).2+ concentrations [2+ influx in hippocampal astrocytes, which leads to cognitive impairment [Acetylcholine is also known to be one of the major neurotransmitters of the central nervous system. Astrocytes in the hippocampus express both nicotinic and muscarinic cholinergic receptors ,63 (p. 1pairment  (p. 15).In addition, D-serine, which interacts with the NMDA receptor\u2019s glycine site and co-activates the NMDA receptor with the agonists  (p. 15),Firstly, astrocytes are a major component of the blood\u2013brain barrier (BBB). Astrocytes can send processes to the vasculature, and these processes terminate with endfeet, which covers up to 98% of the entire vascular surface ,70 . Since chronic isoflurane anesthesia reduces the expression of GJs-Cx43 in the hippocampus, the reduction in GJs-Cx43 levels may further lead to defects in astrocyte networks initiating or exacerbating neuroinflammation and ultimately leading to cognitive impairment in mice  (p. 15).Thirdly, in pathological conditions associated with the BBB breakdown, the endfeet of reactive astrocytes can express tight junction proteins to mount a parenchymal line of defense  (p. 15).Lastly, astrocytes secrete numerous paracrine factors affecting the barrier properties of the BBB, such as morphogens Sonic hedgehog (Shh), vascular endothelial growth factor (VEGF), apolipoprotein E (ApoE) and others ,82 (p. 1Astrocytes represent a major source of apolipoprotein E (ApoE) in the brain. The expression of the human APOE4 isoform is a risk factor for PND  (p. 16).The human brain accounts for only 2% of the overall body weight, but it consumes approximately 20% of energy at rest. Energy expenditure in the brain is boosted even more by various tasks  (p. 16).Firstly, astrocytes regulate cerebral blood flow  (p. 16).In addition to regulating blood flow, astrocytes can transport lactates to neurons, see In addition, astrocytes secrete glial cell line-derived neurotrophic factors (GDNF), which are fundamental in neurogenesis and learning. Amantadine reduces cognitive impairment following anesthesia and surgery by increasing the GDNF level  (p. 17).In summation, astrocytes can influence cognitive performance following anesthesia and surgery via a variety of mechanisms , while s"}
{"text": "Precision medicine is a powerful treatment modality for controlling tumor growth. However, given the substantial variability between cancer patients, identifying suitable targets is no easy task. Tumor protein p53 represents one of the most promising anti-cancer drug targets due to its high mutation rate and critical role in tumorigenesis. Despite this, current treatment strategies targeting p53 signaling have only seen modest clinical success presumably due to the complex signaling network surrounding p53 enabling cancer cells to have the ability to adapt to each treatment strategy. This review will focus on the multiple approaches to target p53 and will touch on some of the limitations that may be hindering their clinical success.While chemotherapy is a key treatment strategy for many solid tumors, it is rarely curative, and most tumor cells eventually become resistant. Because of this, there is an unmet need to develop systemic treatments that capitalize on the unique mutational landscape of each patient\u2019s tumor. The most frequently mutated protein in cancer, p53, has a role in nearly all cancer subtypes and tumorigenesis stages and therefore is one of the most promising molecular targets for cancer treatment. Unfortunately, drugs targeting p53 have seen little clinical success despite promising preclinical data. Most of these drug compounds target specific aspects of p53 inactivation, such as through inhibiting negative regulation by the mouse double minute (MDM) family of proteins. These treatment strategies fail to address cancer cells\u2019 adaptation mechanisms and ignore the impact that p53 loss has on the entire p53 network. However, recent gene therapy successes show that targeting the p53 network and cellular dysfunction caused by p53 inactivation is now possible and may soon translate into successful clinical responses. In this review, we discuss p53 signaling complexities in cancer that have hindered the development and use of p53-targeted drugs. We also describe several current therapeutics reporting promising preclinical and clinical results. Despite decades of research and rapidly advancing treatment modalities, cancer remains the second leading cause of premature death worldwide ,2. CellsThe tumor suppressor protein p53 has been called the \u2018guardian of the genome\u2019  due to iGenotoxic stress, such as chemotherapy and ionizing radiation, can also activate p53, leading to its accumulation and subsequent transcription of p53-regulated genes due to the convergence of the DNA damage response (DDR) pathway and p53 signaling ,45,46,47ARF in humans and p19ARF in mice) [Cell cycle progression indirectly leads to activation of p53, as transition through the G1 phase to S phase results in the activation of the cyclin-dependent kinases, CDK4 and CDK6, which phosphorylate retinoblastoma protein (pRb) causing dissociation of the pRb\u2013E2F complex. E2F, now free from inhibition, activates the transcription of multiple genes involved in the cell cycle progression from G1 phase to S phase. Interestingly, E2F also facilitates the transcription of ARF (p14in mice) , which iin mice) . Additioin mice) . p53 funin mice) .Activated p53 oligomerizes to form tetramers, which stabilize the binding interaction with p53-response elements in DNA and enable the expression of p53-regulated genes to initiate various cellular programs . p53 regSince its critical role as a tumor suppressor was uncovered ,77,78,79R175H has been found to bind and sequester the tumor suppressors p73 and p63 [+/\u2212 and p53\u2212/\u2212 mice [p53 is the most mutated protein in cancer, with nearly 50% of all tumor cells displaying some form of p53 mutation , with mu and p63 , both of and p63 . GOF mup\u2212/\u2212 mice ,95,96. G\u2212/\u2212 mice  and incr\u2212/\u2212 mice .p53 is implicated in virtually all the Hanahan and Weinberg \u201challmarks of cancer.\u201d Activation of p53 in response to DNA damage, proliferation, and oxidative stress leads to cell cycle arrest and activation of apoptotic pathways . p53 is R172H mutation  did not lead to tumor regression as it does in p53-null mice. Mutant p53R172H was demonstrated to bind to the restored WT p53 and exert a dominant negative effect that prevented the expression of pro-apoptotic proteins but not proteins involved in cell cycle arrest [Tumor suppressors evolved to prevent malignant transformation, with oncogenic stimuli leading to their activation. As such, cancer cells are constantly activating the pathways that evolved to trigger p53 signaling. However, with p53 function lost, they exist in a state of perpetual p53 activation. This led to the hypothesis that if normal p53 function were to be restored, cancer cells would be primed to arrest their growth and undergo apoptosis. Support for this method of treatment was strengthened following reports that replacement of WT p53 into p53-null cell lines resulted in arrested growth, increased apoptosis induction, and caused tumor regression in vivo ,109. p53e arrest . Cancer e arrest ,125. Unfe arrest ,127, ande arrest ,129. A pe arrest ,131. CliDespite the limitations surrounding restorative p53 gene therapy, it does indicate that there is potential that p53 reactivation could result in more meaningful clinical responses if a targeted approach is taken. In the case of cancer cells with WT p53 that present with overexpression of a negative regulator, there are several treatment modalities being explored for each specific indication. Perhaps the most promising and extensively studied are inhibitors of the MDM2-p53 interaction. The discovery that the small molecule nutlin-3a could bind to MDM2 and release p53 from its inhibition resulting in apoptosis and hindered tumor growth in vivo  spurred The variability in therapeutic responses to MDM2 antagonists can be partially explained due to the engagement of multiple p53-dependent pathways apart from apoptosis. Efeyan, A., et al.  demonstrTargeting the MDMX-p53 interaction represents an alternative strategy for restoring p53 activity as it may result in fewer side effects than MDM2 antagonists. Hematopoietic stem cells recover from short-term p53 restoration in mice lacking MDMX, presumably due to functional MDM2 retaining the ability to regulate p53 activity . MDMX tap = 0.13) [Cancer cells possessing mutant forms of p53 can be targeted using compounds that reverse the conformational changes acquired by mutation, thus restoring p53 function. The most clinically advanced compound, PRIMA-1, can preferentially inhibit the growth of cancer cell lines with the R273H DNA contact mutant or the R175H conformation mutant . Upon en = 0.13) . NeitherR175H. Hsp40 can then bind to mup53R175H and stabilize the unfolded protein, restoring its function [Other mup53-selective compounds are in preclinical development. PK7088 targets cancer cells harboring a Y220C p53 mutation, the ninth most common p53 mutation. PK7088 binds mup53 in a pocket created by the Y220C mutation and slows its denaturation . This refunction . The profunction .As if understanding endogenous p53 signaling was not complicated enough, even understanding p53 mutation is not a straightforward topic. Contrary to most tumor suppressor genes that undergo a loss-of-function following mutation, p53 can lose its endogenous function while simultaneously gaining novel tumor-promoting capabilities. DNA contact mutants and conformational mutants alter the DNA binding domain, which can alter the DNA elements recognized by p53 and result in the expression of tumorigenic genes involved in apoptosis resistance, proliferation, angiogenesis, and metastasis . AdditioR248Q slowed tumor growth and prolonged survival. The authors also demonstrated that Hsp90 stabilized mup53, and adding the Hsp90 inhibitor, ganetespib, increased median survival in tumor-bearing mice possessing either the R248Q or the R172H (R175H in humans) p53 mutants by 59% and 48%, respectively, with no effect on p53-null mice. Ganetespib-mediated inhibition of Hsp90 results in selective degradation of mup53 that is accompanied by apoptosis induction, thus providing evidence that elimination of GOF mup53 represents a viable treatment option (In some cases, cancer cells possessing a GOF mutation in p53 are dependent on it for their survival, and knockdown of mup53 abrogates tumorigenic potential in vitro and in vivo ,163. In t option D. Histont option D [164,16t option . PatientPerhaps one of the more obvious strategies to inhibit GOF mup53 would be via knockdown with siRNA. However, there is potential that siRNA may still recognize WT p53 in healthy tissue leading to off-target effects. Martinez et al.  demonstrRather than trying to correct the problems brought on by p53 mutation, an alternative approach is to capitalize on it and utilize the dysfunction for treatment options. The earliest example of this is ONYX-015, a replication-competent adenovirus that selectively infects and lyses cancer cells lacking functional p53. By removing the E1B gene that normally functions to inactivate p53 in infected cells , the adeGOF mutations in p53 can result in unique gene expression signatures relative to WT p53 possessing counterparts . BecauseAn alternative approach for targeting p53 dysfunction that is of particular interest to our group is targeting p53 transcriptional activation ,191. TypVarious oncogenes have been demonstrated to directly bind to the p53 promoter and facilitate its expression, for example, c-Myc  and NF-\u03baRather than targeting specific aspects of p53 inactivation, we propose taking a multifaceted approach to target the p53 network. The Hsp90 inhibitors mentioned above give a great example of this. SAHA and ganetespib facilitate degradation of mup53 protein that abrogates tumorigenic potential in animal models of cancer but failed to perform in the clinic ,166,167.An alternative strategy for targeting the p53 network relies on the use of gene therapy. The WT p53 adenovirus expression vectors have arguably demonstrated the most clinical success of any p53 targeted therapy thus far, but they still face many limitations, primarily low treatment responses and cancer cell adaptation to the WT protein . An addiDespite the complexities of p53 signaling, precision treatment using p53 remains a rapidly growing field in cancer therapy because so many aspects of tumorigenesis are influenced by p53. Unfortunately, despite promising preclinical data, most therapeutic strategies directed toward the modulation of p53 signaling have had low to moderate clinical success. There are a few compounds that show promise though, such as SAHA and ganetespib , that we"}
{"text": "We enrolled 133 patients . The median cFGF23 was significantly higher in HD vs. PD patients (p = 0.0017) and not significantly higher in patients without UO (p = 0.12). We found a negative correlation between cFGF23 and the UO volume (p = 0.0250), but the correlation was not significant when considering the type of dialysis treatment. Phosphorus , type of dialysis , and creatinine  were significant and independent predictors of cFGF23 levels. In conclusion, cFGF23 was significantly higher in HD than in PD patients. We found a significant negative correlation between cFGF23 and the residual UO volume, but the correlation was not significant considering the type of dialysis. Our study reveals that dialysis modality is an independent predictor of FGF23 levels. In particular, PD is associated with lower FGF23 levels than HD.Several studies investigated the role of fibroblast growth factor 23 (FGF23) in the regulation of renal phosphate excretion in chronic kidney disease (CKD). However, patients with residual urine output (UO) seem to control their serum phosphorus levels better. Our aim was to determine whether FGF23 levels are influenced by dialysis modality and UO. We performed a cross-sectional study in hemodialysis (HD) and peritoneal dialysis (PD) patients. The C-terminal FGF23 (cFGF23) levels were determined in plasma with a two-site enzyme-linked immunosorbent assay. The UO collection referred to an mL/day measurement. All  Mineral and bone disorders (MBDs) are prevalent in chronic kidney disease (CKD), especially in the advanced stages, representing a severe complication and an important mortality risk factor in both hemodialysis (HD) and peritoneal dialysis (PD) patients .The discovery of the \u201cfibroblast growth factor 23 (FGF23)-bone-kidney axis\u201d has led to several investigations to better understand this complex endocrine network which plays a crucial role in MBD development [FGF23 is a 32 kDa glycoprotein secreted by osteocytes in response to increased levels of 1,25 dihydroxy vitamin D 2D) ,5 and thDespite the fact that no phosphate sensor has yet been identified, most authors agreed that, in CKD, the reduced nephron mass causes a decreased renal phosphate excretion that leads to increased FGF23 secretion from the bone to compensate for hyperphosphatemia . It was The growing attention on FGF23 is partly due to the finding of its correlation with cardiovascular mortality and CKD progression. Concerning cardiovascular risk, a significant correlation was found between the FGF23 level and arterial calcification and stiffness , valve cSome studies have shown that both HD and PD with residual urine output (UO) have better maintenance of phosphate balance than those without UO ,18,19. MOn the other hand, there is no consensus about the phosphate balance in PD and HD patients, as some studies have shown better phosphate control in PD patients ,21, whilThe purpose of the present study was to examine the influence of dialysis type and the presence of residual UO on FGF23 levels.We performed a cross sectional-study in two groups of patients: HD and PD patients. We enrolled patients that underwent HD or PD treatment for at least 3 months with age \u2265 18 years and who agreed to participate in the study between October 2014 and October 2015.Inclusion criteriaAge \u2265 18 years;Patients in chronic dialysis HD or PD (\u22653 months);Informed consent was taken before enrollment.Exclusion criteriaDementia;Patients with missing or lacking anamnesis;Hepatic encephalopathy;Heart failure New York Heart Association (NYHA) functional classification IV;Acute myocardial infarction (IMA);Acute coronary syndrome (ACS);Lower respiratory tract infection (LRTI);Systemic inflammatory response syndrome (SIRS);Received immunosuppressive agents;Pregnant.This study was performed with the ethical principles of the Declaration of Helsinki. The protocol was approved by the Ethics Committee for Clinical Trials (CESC) of the province of Vicenza and the ethics committee of San Bortolo Hospital (N.41/14). All participants were informed of the objectives of the study and signed for their informed consent.The data collected from patients were: age (years), gender, hypertension, diabetes mellitus, and causes of CKD classified by 4 categories .Blood samples in EDTA were collected during the outpatient visits in the PD patients or prior dialysis treatment after a long interdialytic interval in HD patients. The plasma was immediately centrifuged and stored at \u221280 \u00b0C.\u00ae, SIEMENS Healthcare Diagnostics Inc., and Newark, Tarrytown, NY, USA). Parathyroid hormone (intact PTH) and 25 hydroxy vitamin D (25-(OH)D3) in serum were measured with the chemiluminescent immunoassay, CLIA , Milano, Italy) as routine laboratory exams. We determined the urea, enzymatic creatinine, calcium, and phosphorus (P)  levels were determined in plasma with a two-site enzyme-linked immunosorbent assay, ELISA . Two hundred microliters of plasma were used to assay the samples in duplicates. The samples with values greater than the highest standard were diluted by 1:10 or greater with the 0 pg/mL standard or optional sample diluent and reassayed. The result obtained was multiplied by the dilution factor. The residual urine output (UO) presence was collected from patients, and the quantity of UO (UO volume) was referred to as one day (mL/day).The continuous variables were summarized with the median and range (minimum-maximum) or the mean and standard deviation (\u00b1SD) as appropriate, and the categorical ones were summarized with the numbers and percentages of patients in each category.t-test or the Mann-Whitney U test was applied to compare the continuous variables according to their normal or non-normal distribution. The categorical variables were compared with the chi-square or Fisher\u2019s exact test. The correlations between the continuous variables were evaluated with Spearman\u2019s rank correlation (R), and the 95% confidence interval (CI) was calculated using Fisher\u2019s z transformation.All continuous variables were evaluated for normality with a Q-Q plot and with the Shapiro-Wilk test. The student\u2019s p < 0.05 and were considered in a multivariable linear regression model with a backward selection method at the 5% level of significance.A univariable linear regression analysis was used to evaluate the relationship between the FGF23 natural log-transformed and potential predictors. All variables formed statistically significant results with a p-values were two-sided, and the statistical significance was set at p < 0.05. The statistical analyses were performed with SAS 9.4  for Windows.All reported We enrolled 133 patients : 58 HD patients  and 75 PD patients . The principal cause of CKD for all patients  was secondary kidney disease . The residual UO was preserved in 93 (69.9%) patients. The baseline characteristics are summarized in p = 0.0017) (as represented in p = 0.0041) and 25-(OH) D3 (p < 0.0001) were significantly different.The cFGF23 levels were significantly higher in HD compared to PD patients (p = 0.0250)   between cFGF23 and the UO volume ( = 0.56) .p = 0.12).The residual urine output (UO) was preserved in 93 (69.9%) patients. The median cFGF23 level was higher in patients without UO  than in those with a preserved UO  without reaching the statistical significance (p = 0.0983) , there was not a significant difference (p = 0.0005), phosphorus (p = 0.0016), and 25-(OH) D3 (p = 0.0096).The other laboratory variables that resulted in significant differences between patients without and with residual UO were creatinine (p < 0.0001), urea , and phosphorus , and were inversely correlated with UO volume . A significant association was also found with the type of dialysis . The multivariable regression analysis showed that phosphorus , type of dialysis , and creatinine  were significant and independent predictors of cFGF23 levels.To identify the factors associated with cFGF23 levels, we performed a linear regression analysis . In the Mineral and bone disorders are common in the CKD population, especially in the advanced stages; they represent a severe complication and an important risk factor, mortality-related, in patients requiring renal replacement therapy  .Hyperphosphatemia is frequently found in patients with CKD, as the progressive reduction in nephrogenic mass leads to a significant marked decrease in the ability of renal phosphorus excretion (which is taken with diet). FGF23 has a phosphaturic action, as it inhibits the reabsorption of phosphorus at the level of the proximal renal tubule. Hyperphosphatemia results in increased secretion of FGF23 from bone . In advaSome studies have shown that dialysis patients with residual UO have a better phosphorus balance than those without UO ,18,19. CIn our study, 133 patients were enrolled , of which 58 patients had HD  and 75 patients had PD . The residual UO was preserved in 93 (69.9%) of 133 patients. Of the 75 patients with PD, 66 had UO (88%), and of the 58 patients with HD, 27 had UO (46.6%). In our cohort, the percentage of HD patients with a residual UO was higher than the percentage shown in other studies . NonetheThe prevention of hyperphosphatemia in CKD patients using the dietary restriction of phosphate is mandatory. However, dialysis patients have a high risk of protein-energy wasting (PEW) due to multiple causes, such as inflammation, metabolic acidosis, endocrine disturbances, protein losses into the dialysate, and catabolism. Since proteins contain 15 mg of phosphate per gram, a compromise may be necessary, and at least 1.0\u20131.2 g protein/kg body weight/day is recommended for stable HD and PD patients . MoreoveIn a PD patient, despite an adequate intake of protein (>1 g/kg/day), when their residual renal function decreases, the need for protein decreases.How could we explain why we found more controlled phosphorus values in patients without residual diuresis? Below we have formulated some hypotheses:Some unconsidered aspects in the study, such as compliance with a diet low in phosphorus and adherence to chelation therapy, may have altered the obtained results; it could also be due to the fact that patients with preserved diuresis, precisely for this condition, felt freer (making a mistake) not to follow an adequate diet poor in phosphorus and therefore paradoxically this determined that they had higher median phosphorus values than the dialyzed patients without residual diuresisIn regards to FGF23, the analysis of Bi and colleagues [p = 0.0017)    and not  = 0,12) .The serum FGF23 values in HD patients are higher, probably because compared to the PD patient group, they had a lower percentage of residual UO, although they still had good control of phosphorus. This also explains why the FGF23 values did not change significantly despite the improved phosphorus control.p = 0.0007) proved to be an independent predictor of serum FGF23 levels, and this could be explained by what was already published, i.e., that high values of phosphorus directly stimulate the secretion of FGF23 due to its phosphatic action.In our study, phosphorus (p = 0.0250) (as represented in p = 0.0415). However, the correlation was not significant when considering the type of dialysis   between cFGF23 and the residual UO volume ( = 0.56)  and in tAs also described in the previous work on the re-classification of uremic toxins and their role in CKD , althougThe principle behind the removal of urea toxins in PD is the diffusion of these from the blood through the peritoneal membrane into the peritoneal fluid, which is then eliminated after a certain period of time in the peritoneal cavity. The use of a highly permeable membrane together with better preservation of residual renal function determines that urea toxins are eliminated more efficiently through the peritoneal membrane and that the plasma and tissue levels of these molecules are lower in PD patients than in HD patients . Moreove(1)The cohort was obtained from a current study. Therefore, our studied population was not specifically chosen ad hoc; despite this, we defend the quality of the data obtained as they allowed us to confirm the data from the literature and provided us with points of reflection even for new investigations. What could be especially interesting is obtaining more data about the UO collection;(2)The two groups (patients in HD and patients in PD) were different in number and characteristics;(3)We only used single urinary output data and a single biohumoral examination data point;(4)The compliance with the phosphorus binding therapy and a low phosphorus diet was unknown.In conclusion, cFGF23 was significantly higher in HD patients than in PD patients, and it was higher in patients without residual UO. We found a significant negative correlation between cFGF23 and the residual UO volume, but the correlation was not significant when considering the type of dialysis, as to say that in the relationship between the residual UO and FGF23, the type of dialysis represents a confounding factor. Our study found that the dialysis modality is an independent predictor of FGF23 levels. In particular, PD is associated with lower FGF23 levels than HD."}
{"text": "We do not fully understand the resolution at which temporal information is processed by different species. Here we employed a temporal order judgment (TOJ) task in rats and humans to test the temporal precision with which these species can detect the order of presentation of simple stimuli across two modalities of vision and audition. Both species reported the order of audiovisual stimuli when they were presented from a central location at a range of stimulus onset asynchronies (SOA)s. While both species could reliably distinguish the temporal order of stimuli based on their sensory content , rats outperformed humans at short SOAs (less than 100 ms) whereas humans outperformed rats at long SOAs (greater than 100 ms). Moreover, rats produced faster responses compared to humans. The reaction time data further revealed key differences in decision process across the two species: at longer SOAs, reaction times increased in rats but decreased in humans. Finally, drift-diffusion modeling allowed us to isolate the contribution of various parameters including evidence accumulation rates, lapse and bias to the sensory decision. Consistent with the psychophysical findings, the model revealed higher temporal sensitivity and a higher lapse rate in rats compared to humans. These findings suggest that these species applied different strategies for making perceptual decisions in the context of a multimodal TOJ task. Rapid perceptual decision-making is often key to survival, for example a rat might need to quickly respond to the rustling of leaves to escape a lurking cat. To achieve rapid decisions, the brain needs to process the incoming sensory information, often requiring integration across multiple modalities, to generate an accurate percept and plan and execute the appropriate motor action, often all happening within several hundred milliseconds. The temporal precision of perceptual decisions vary across species; for instance electric fish can resolve 1 \u03bcs temporal disparity  while ecWe know that intersensory interactions occur when information from different senses arrives temporally or spatially coincident or near coincident in the brain . The braDiscriminating the order of stimuli is a key aspect of temporal processing, which is commonly measured using the well-known temporal order judgment (TOJ) paradigm. In this paradigm, the interval between the onset of two stimuli, also referred to as the stimulus onset asynchrony (SOA), is systematically varied, while requiring the participant to report which side the first of the two stimuli appeared , or whicPrevious studies have shown that both rats and humans accumulate evidence for perceptual decisions . In multFundamental similarities and differences are also observed in temporal processing in these species. For example, both rats and humans can detect temporal regularities in sound . But, inHere, by using the TOJ paradigm in rats and humans, we compare the temporal precision with which these species can detect the order of presentation of simple stimuli across two modalities of vision and audition. To this end, we designed similar audiovisual TOJ paradigms in which rats and humans were required to report which stimulus modality was presented first. Our goal was firstly to better understand how multiple sensory inputs are labeled and reported in their relative temporal occurrence in humans and rats, and secondly to investigate and directly compare the limits of temporal integration across these species. Finally, we performed drift-diffusion modeling on the human and rat data to isolate the contribution of various parameters including evidence accumulation rates, lapse and bias to the sensory decision. This data allows us to better understand how the profile of multisensory processing and labeling generalizes across species. To date, non-invasive methods such as electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) have provided insights about neural underpinnings of multisensory temporal processing in humans . Howeverad libitum access to food and free access to water for 2 h each day after each training or testing session. Their weight was monitored throughout training to ensure that they continued to gain weight at a normal rate. In human study, ten participants  aged 26-36 years (mean age of 31.6 years) were recruited to take part in this experiment. All participants were right-handed with normal or corrected to normal visual acuity and reported normal hearing. One participant was one of the authors of this paper, two of them had experience in psychophysical experiments and the others were naive to such experiments and the purpose of this study. All gave written informed consent prior to taking part in this study. Human experiment was conducted in line with the Declaration of Helsinki  was presented from a 5 mm white transparent LED located in the center of the front wall, 6 cm (center to center) above the nose-poke hole. Both stimuli had the same duration (20 ms). All procedures including stimulus presentation, measurement of behavioral responses and reward delivery were controlled by a MATLAB program and through an Arduino interface board. During the training and testing sessions the chamber was lit using a 9 W red LED bulb for video recording.Rats were trained and tested in a custom-made wooden operant chamber, measuring 35 cm (length) \u00d7 25 cm (width) \u00d7 35 cm (height) which was placed in a dark and sound-attenuating box. The chamber had a central hole (1.5 cm depth) in the middle of its front wall, 4 cm above the floor to serve as a nose-poke area. The rats received liquid rewards (sucrose 5%) following correct responses in both training and testing sessions from two copper spouts, positioned on either side 5 cm away from the nose poke area. The spouts and nose poke used a capacitive sensor for detecting lick contacts. The capacitive sensors were connected to an arduino interface and were read by a MATLAB program at a high temporal resolution (>1 sample per ms). Animals\u2019 contact with the spout or the metal piece at the end of the nose-poke hole, resulted in a sharp increase in the capacitance of the circuit. Applying a simple threshold allowed precise detection of behavior (nose-poke or lick at the spout). The auditory stimulus  was presented from a speaker  5 cm (center to center) above the nose-poke hole while the visual stimulus  stimuli. To this end, both stimuli were presented from a central point in front of the subjects . We deteRats were initially habituated to the chamber, and learned to receive the reward from both spouts. Assignment of the modalities to the left/right spouts started at this stage such that responses (licks) at each spout were accompanied by presentation of a stimulus in predetermined modality. For example, a rat would receive an auditory tone each time it licked at the right side and would see a visual flash each time it licked at the left spout.In this stage rats were trained to learn the assignment of modalities to left and right sides. For each modality, the left/right assignment was counterbalanced across rats. Rats initiated each trial by a nose poke into the central aperture  right, wAt this stage, we initially presented bimodal stimuli with long SOAs  and then progressively reduced the SOA to 15 ms across training. Here, only correct first choices were rewarded. Incorrect first choices resulted in the termination of the trial without any reward. No other punishment was applied. In initial stages, reduction of SOA was performed through 50 ms steps, down to 100 ms SOA. In the final stages, SOA was reduced from 100 to 75, 50, 25, and then 15 ms. Across the initial stages, passing through steps was dependent on reaching the criterion performance (>75% correct first choice for two consecutive days) in individual rats. However, for SOAs lower than 100 ms , there was no criterion applied. In each session only the most recent SOA was applied. Following this stage, rats were required to choose the reward spout, corresponding to the side of the modality of the first stimulus. A 3-s inter-trial interval (ITI) was applied between trials. The ITI was measured after the rat responded to the previous trial. For testing humans in this study, participants sat in front of an experimental panel similar to that used for rats. The distance between the experimental panel and the participant\u2019s eyes was \u223c80 cm. For collecting responses (thumb presses in p < 0.05).The asynchrony between the onset of bimodal stimuli varied from 15 to 200 ms in rats and from 25 to 200 ms in humans. The minimum SOA that rats could distinguish better than chance was 15 ms with an average performance of 58 \u00b1 3% (mean \u00b1 standard deviation) across rats . In our d\u2019) at each SOA using the following equation in each subject:A common analytic approach was used for rat and human experiments. In both experiments, subjects reported the modality of the first stimulus. To quantify the behavioral performance at various SOAs, we applied the Receiver Operating Characteristics (ROC) analysis from Signal Detection Theory . Accuracd\u2019 values indicate better temporal discriminability. The hit and false alarm rates for individual SOAs are depicted in the d\u2019, accuracy and reaction time.where, H is the hit rate, FA is the false alarm rate, and Z is the inverse of the cumulative Gaussian distribution function. Higher For analysis of reaction time, we only included correct trials in which responses were generated sooner than 4 sec after stimulus presentation. The values beyond this limit were considered as potentially not a direct response to that trial and were therefore excluded from analysis for both species. The number of excluded trials was relatively small for most humans and rats . We excluded incorrect trials in calculation of reaction time because these trials are unlikely to represent a correct reaction to the stimulus. In each condition for each rat, , we pooled trials from all testing sessions and calculated the median of the distribution as reaction time for that condition. We have also represented reaction time without exclusion of long outliers, and for both correct and incorrect trials in x value at the midpoint of psychometric function which indicates point of subjective simultaneity (PSS). The PSS is the temporal difference between two stimuli at which the subject is equally likely to report either modality as being first. In addition to slope, as an index of sensitivity we also estimated just noticeable difference (JND), where possible. The estimated value (mean \u00b1 SEM) was 0.067 \u00b1 0.02 and 0.081 \u00b1 0.01 in rats (n = 5) and humans (n = 10), respectively. Note that JND can reveal sensitivity in a broad range of SOAs  whereas the slope can be more informative at very short SOAs.A sigmoidal function (Equation 3) was fitted to responses at various SOAs using nonlinear least squares regression for all subjects to characterize the psychometric function . Paramet1. All SOAs  were used in the same fitting, consistent with the most common approach in the literature . Drift diffusion represents a class of integrator models, in which evidence in favor of each choice is accumulated until one of two decision thresholds (boundaries) is reached and a decision is made. The DDM was implemented using PyDDM simulatorterature .d\u2019 and reaction time in order to examine the effect of SOA and to elucidate species differences. For reporting statistical details where the sphericity (Mauchly test) was violated, degrees of freedom were corrected according to Greenhouse-Geisser Correction in ANOVAs. For statistical comparison of psychometric parameters  and parameters derived from drift-diffusion modeling between two species, we used an independent-samples t-test in either case. In independent-samples t-test we checked homogeneity of variances using Levene\u2019s test and in cases that homogeneity was violated we accordingly adjusted statistical details. For all statistical analysis, we used an IBM SPSS statistical package. The p value < 0.05 was considered as significant. We wrote code in MATLAB (R2021b) for extracting data and analysis including the sigmoidal fits and estimation of the fit parameters.Our approach for statistical analysis was to find out which factors affected performance in discrimination of temporal order of audiovisual stimuli and to examine whether these factors differ across the two species. To this end, we applied a mixed repeated measures ANOVA to either We implemented a similar TOJ task in rats and humans to test the temporal precision with which these species detect the order of presentation of simple stimuli across two modalities of vision and audition . Each trd\u2019) in both species for all SOAs. We used hit (H) and false alarm (FA) rates  in each subject to quantify d\u2019 at each SOA using the equation d\u2019 = Z (H) \u2013 Z (FA), where Z is the inverse of the cumulative Gaussian distribution function. Higher d\u2019 values indicate better temporal discriminability. d\u2019 gradually increased from 25 to 100 ms SOA and reached a plateau in SOAs longer than 100 ms. In humans, on the other hand, the increase in d\u2019 by increasing SOA revealed a more linear trend. We used a mixed-measures ANOVA  to examine the effect of SOA on d\u2019 value and to examine any statistical differences between humans and rats. The effect of Species was not significant  = 0.17; p = 0.68, 2\u03b7p = 0.01). This indicates that, when collapsed across the SOAs, the average d\u2019 and therefore, the overall temporal discriminability was comparable between humans and rats. However, the main effect of SOA was highly significant  = 78.62; p = 0.001, 2\u03b7p = 0.83), showing that the interval between stimuli determined their temporal discriminability. Post-hoc analysis with Bonferroni correction revealed significant differences between all pairs of SOA. The highest p value was 0.009 indicating difference between 50 and 75 ms SOAs. Importantly, there was a significant interaction between the SOA and Species factors  = 19.13; p = 0.001, 2\u03b7p = 0.54) indicating that the relationship between discriminability and SOA does not follow the same pattern in two species. Rats exhibited higher d\u2019 in short SOAs  whereas humans outperform rats at higher SOAs (>100 ms)  = 3.69, p = 0.004). On the other hand, at 200 ms SOA, d\u2019 was significantly higher in humans (t (11.93) = \u22123.67, p = 0.003).Here we aimed to determine the similarity and differences of performance across the two species by measuring temporal discriminability (>100 ms) . To quanF  = 4.08; p = 0.06, 2\u03b7p = 0.20), its interaction with Species  = 0.07; p = 0.79, 2\u03b7p = 0.005) and SOA  = 0.39; p = 0.6, 2\u03b7p = 0.024) were not significant. These findings indicate that in both species, order of modalities did not affect discrimination accuracy across the range of SOAs.To further investigate the effect of modality on performance, we included accuracy of choices in visual-leading  rate) and auditory-leading ) trials in a mixed-measures ANOVA using SOA (6 SOAs) and Modality  as within-subject factors and Species as a between-subject factor. The main effect of Modality  = 9.28; p = 0.01, 2\u03b7p = 0.37). However, given the differences in the apparatus and the type of response required from the two species, the absolute differences in reaction time are hard to interpret. The main effect of Modality was significant  = 8.3; p = 0.01, 2\u03b7p = 0.34): we observed shorter reaction times in visual-first trials. Interaction between the effects of Modality and Species was not statistically significant  = 1.67; p = 0.21, 2\u03b7p = 0.09)  = 4.15; p = 0.04, 2\u03b7p = 0.21), which means that the temporal interval between stimuli had an effect on reaction time. Importantly, there was a significant interaction between the SOA and Species factors  = 5.73; p = 0.02, 2\u03b7p = 0.26) indicating that SOA affected reaction times differently in the two species. Interaction between SOA, Species and Modality  = 0.8; p = 0.45, 2\u03b7p = 0.05) was not significant. Also, no significant interaction was revealed between the effects of Modality and SOA factors  = 0.66; p = 0.51, 2\u03b7p = 0.04). Visual inspection of data  and auditory-first  choices were positively correlated with SOA. In humans, however, a significant but negative correlation was found in both the visual-first  and auditory-first  choices. Overall, these findings indicate that while for humans, reaction times increased with task difficulty, the opposite relation was observed in rats.We next asked whether reaction times were also systematically different between the two species. In a decision process, a trade-off often exists between speed and accuracy, whereby difficult decisions often correspond to longer reaction times . We calc = 0.09) . The mai of data  revealedt-test: t (16) = 4.75, p = 0.001) which indicates that rats delivered more unattended or random responses specially in visual first trials compared to humans even when detecting temporal order of stimuli was easy. However, the lower lapse rate was similar in two species (independent samples t-test: t (16) = 1.52, p = 0.15). There was a significant difference in slope of the psychometric function fitted to human and rat data (independent samples t-test: t (16) = 4.64, p = 0.001) indicating that slope of psychometric function was different in two species. This revealed temporal sensitivity of rats was higher than humans. Therefore, rats compared to humans are able to distinguish stimuli with smaller temporal intervals from each other. In other words, rats had higher sensitivity to changes in SOAs. No significant difference existed between PSS of humans and rats, determined by independent samples t-test (t (11.04) = \u22120.908, p = 0.38). In rats, the average PSS (\u22124 ms) was not different from zero (one-sided t-test: t (7) = 0, p = 0.5), showing that they were unbiased to auditory and visual stimuli. Finally, in humans, the average PSS was +19 ms which was not statistically different from zero (one-sided t-test: t (9) = 0, p = 0.5), revealing no evidence for a modality bias in humans.In this section, for obtaining performance measures and examining the similarities and differences between the two species, we applied psychometric function to responses of individual rats and humans. To this end, we fit a cumulative Gaussian sigmoid curve to accuracy rates for rats and humans. These methods allowed us to isolate key parameters of the performance including (i) the temporal sensitivity which is captured by the slope of the sigmoidal function (ii) the interval at which the two stimuli are perceived as simultaneous (PSS) and (iii) the lapse rate which represents the probability of delivering unattended responses, regardless of the difficulty of stimulus. drift rate reflects the rate of sensory information accumulation. The decision threshold determines the amount of sensory evidence that needs to be accumulated before the choice is made. The nondecision time is the portion of reaction time which is not related to the decision process . The leak reflects the amount of sensory information that is lost over time. Finally, we fit a collapsing bounds parameter (tau) to capture the rat\u2019s impulsivity where less evidence is needed at longer reaction times. t (16) = 5.58, p < 0.001) and the leak rate (t (16) = 2.91, p = 0.001). Rats also exhibited a trend towards a lower decision bounds (thresholds) compared to human participants (t (16) = 1.71, p = 0.11). Altogether, having a faster collapse of bounds and rate for information leak along with lower bounds could explain the earlier observation that rats had significantly faster reaction times across all SOAs, whereas humans showed longer reaction times with short SOAs. As rats\u2019 bounds collapse more quickly, they tended not to delay their response at short SOAs despite the reduced evidence. This potentially explains why despite having higher lapse rates, rats exhibited a superior performance at low SOAs. Unlike the classic global dot motion paradigms, where sensory evidence continuously flows in, here the information is provided in a single time at the onset of the trial. Therefore, any delays in the decision can potentially corrupt the evidence through leakage. It is, therefore, unsurprising short SOAs where reaction times were longer corresponded to inferior performance in humans.So far we have found that rats have higher performances at short SOAs compared with humans, but exhibited lower performances at longer SOAs. There were no systematic differences between the two species in the point of subjective simultaneity. One difference that stood out in the psychometric function was the high lapse rate of rats compared to humans. The more impulsive behavior in rats, that may be partially due to task demands, could explain their high lapse rate. This is also consistent with the reaction time data where rats showed systematically faster responses and did not decrease their speeds with increasing task difficulty. To provide a more quantitative understanding of the underlying mechanisms that determined these sensory decisions in rats and humans, and to incorporate decision accuracy and reaction time into a single framework, we used drift diffusion modeling . Drift dd\u2019) systematically decreased by decreasing the SOA in both species. However, rats showed greater sensitivity at short SOAs (shorter than 100 ms) whereas humans showed greater performance at long SOAs (longer than 100 ms)  . We founvia increasing accuracy. For the current experiment, correct responses in rats were accompanied by a sucrose reward, therefore, incentive anticipation of reward probably may have promoted unattended impulsive responses  in the visual-leading and auditory-leading trials responded to the left and right sides, respectively. However, we did not observe any systematic differences in reaction times, and lack of bias in the PSS implies that overall performance was not influenced by nonspecific factors such as handedness or any potential intrinsic left/right bias in subjects. Only a modality-dependent bias was observed for reaction time, such that the average reaction time was shorter in the visual choices. Because visual choices were mapped to the left hand, therefore shorter response times in the left/visual choices (compared to right-auditory choices) suggests the presence of a modality effect rather than a handedness bias which would have been expected to favor the right choices in right-handed individuals. Future studies could adjust the human and rat paradigms to remove some of the methodological differences here including the extent of training and the presence of feedback. These studies could also record signals in the form of EEG from humans and neural activity in behaving rats undertaking this task to investigate the neuronal mechanisms that underlie perceptual decisions in such multimodal contexts.By implementing a similar psychophysical paradigm in rats and humans, we found that both species could reliably distinguish the temporal order of audio-visual stimuli based on the modality label. Rats showed superior performance at difficult trials, with SOAs shorter than 100 ms, whereas humans showed superior performance at easier trials, with SOAs greater than 100 ms. Rats exhibited a high temporal precision by performing better than chance even at the 15 ms SOA. Overall, rats produced faster responses compared to humans and did not increase their reaction time as difficulty increased. Despite their higher temporal precision, rats\u2019 overall performance was hampered due to a high level of lapse rate and this was most evident at the easy trials with long SOAs. Despite a few similarities, our findings indicate that rats and humans apply different strategies in terms of speed and accuracy in resolving perceptual decisions when the difficulty of decisions varies across trials.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Kerman University of Medical Sciences. The patients/participants provided their written informed consent to participate in this study.FM, MA, and EA designed the initial project. FM performed the experiments. All authors contributed to the data analysis and interpretation. FM, SG, and EA designed and built the experimental apparatus and wrote code to control the stimuli and monitor the behavior. MT performed the initial modeling. FM and EA drafted the first version of the manuscript. All authors contributed to the subsequent revisions."}
{"text": "GNAQ and GNA11 are involved in the process of carcinogenesis and are mutated in 80\u201390% of these tumours. Historically, there has been speculation as to whether these two genes are involved in the progression of primary uveal melanoma to metastatic disease, in addition to the oncogenic process itself. For this reason, both genes have been the subject of multiple research studies. Additionally, due to their high mutation rate in uveal melanoma, these genes and the downstream signaling pathways in which they are involved have been postulated as interesting therapeutic targets. This review aims to provide a current comprehensive view of what we know about GNAQ and GNA11 genes on oncogenesis, prognosis and therapeutic opportunities in uveal melanoma.The development of uveal melanoma is a multifactorial and multi-step process, in which specific and recurrent mutations arise early. Among the recurrently mutated genes, GNAQ and GNA11 genes are mutated in almost 80\u201390% of uveal melanomas in a mutually exclusive pattern. These genes encode the alpha subunits of the heterotrimeric G proteins, Gq and G11; thus, mutations of these genes result in the activation of several important signaling pathways, including phospholipase C, and activation of the transcription factor YAP. It is well known that both of them act as driver genes in the oncogenic process and it has been assumed that they do not play a role in the prognosis of these tumours. However, it has been hypothesised that mutations in these genes could give rise to molecularly and clinically distinct types of uveal melanomas. It has also been questioned whether the type and location of mutation in the GNAQ and GNA11 genes may affect the progression of these tumours. All of these questions, except for their implications in carcinogenesis, remain controversial. Uveal melanoma has a distinctive genetic profile, and specific recurrent mutations, which make it a potential candidate for treatment with targeted therapy. Given that the most frequent mutations are those observed in the GNAQ and GNA11 genes, and that both genes are involved in oncogenesis, these molecules, as well as the downstream signalling pathways in which they are involved, have been proposed as promising potential therapeutic targets. Therefore, in this review, special attention is paid to the current data related to the possible prognostic implications of both genes from different perspectives, as well as the therapeutic options targeting them.The  BRAF or NRAS oncogenes, which are typically mutated in CMM [Uveal melanoma (UM) is the most common intraocular malignancy in adults and the second most prevalent type of melanoma after cutaneous malignant melanoma (CMM) ,2. This d in CMM ,6,7,8. Id in CMM . In factThe genetic analysis of tumoral samples in UM is crucial for metastatic risk prediction, as well as for patient management and follow-up. Historically, several histopathological, clinical and radiological parameters with prognostic value have been considered to evaluate the risk of metastasis. Nevertheless, the presence of specific somatic cytogenetic and genetic biomarkers can estimate more accurately the progression to metastatic disease. Among these specific alterations, cytogenetic studies highlighted that tumours with monosomy 3 (M3) or gain of 8q are associated with poor prognosis. Monosomy 3 is present in nearly 50% of UMs acting as an independent risk factor for metastasis that strongly correlates with significantly reduced disease-free survival (DFS). This chromosomal aberration has become the most significant prognostic parameter in UM ,10. The BAP1 gene (encoding BRCA1-associated protein 1 and located in chromosome 3). The biallelic inactivation of this gene occurs in approximately 50% of primary UMs, combining M3 and a deleterious somatic mutation in the second BAP1 allele. BAP1-inactivated UMs are at a high risk of metastasis [Another recurrent genetic alteration that is strongly associated with a bad prognosis is the inactivation tastasis . GNAQ) and its paralogue guanosine nucleotide-binding protein alpha-11 gene (GNA11). Those two genes are mutated in nearly 90% of these tumours in a generally mutually exclusive pattern [GNAQ and GNA11 genes encode protein members of the q class of G-protein alpha subunits involved in mediating signals between G-protein coupled receptors (GPCRs) and downstream effectors [GNAQ and GNA11 genes arise early in UM and are even present at the very early stages corresponding to benign melanocytic lesions [GNA11 were found more frequently than GNAQ mutations in patient cohorts with metastatic UM (MUM) [In addition to those genetic alterations with strongly prognostic values, UM frequently exhibit other chromosomal aberrations and somatic mutations. Indeed, the most common detected somatic mutations in UM are located in the guanosine nucleotide-binding protein Q gene  ,22. GNAQ and GNA11 genes in UM was discovered, there has been speculation as to whether or not they might be involved in the genetic prognosis of the disease. Additionally, due to their high mutational rate in UM, both genes have also been the subject of therapeutic studies as they act as the switch of the MAPK/ERK signalling pathway, which is constitutively active in these tumours. Currently, important efforts are being addressed to develop effective therapies to prevent metastatic disease, which is actually the principal determinant factor of a patient\u2019s survival. Therefore, the inactivation of GNAQ/GNA11 mutants has been proposed as a potential strategy to treat UM, leading to a rapid expansion of clinical trials [Since the role of l trials ,24,25,26GNAQ and GNA11 mutated genes in the disease oncogenesis, prognosis as well as target therapeutic options for UM. In this review, we present and update the recent discoveries and studies about the role of GNAQ and GNA11 genes are located on chromosomes 9q21.2 and 19p13.3, respectively. They are paralogous genes with a sequence homology of approximately 90% and have a coding region comprising seven exons. The proteins encoded by these genes have similar molecular weights , and consist of 359 amino acids that comprise the \u03b1q and \u03b111 nucleotide-binding subunits of heterotrimeric G proteins [The proteins . GNAQ/GNA11 mutations is approximately 80\u201390% of the UM cases [GNAQ mutations have been reported to range from 24.2 to 53.3% and those of GNA11 mutations from 24.2 to 60% [The frequency of UM cases ,20,21. I2 to 60% ,29,30,312 to 60% ,33. ThisGNAQ and GNA11 in UM showed an exclusive pattern of somatic mutations in both genes [GNA11 mutations in codon 209 leads to glutamine to leucine (p.Q209L) and proline (p.Q209P) substitutions [GNAQ gene, a one-base change at codon 209 (CAA) can predict the substitution of glutamine by leucine  and proline , in most cases [GNAQ and p.Q209Y, p.E234K, p.E221D in GNA11, have also been described [The pioneering sequencing studies of th genes . Subsequth genes . Mutatioitutions ,32,34. Titutions . Contrasst cases ,28. In eescribed  , and CC > TT transitions at codons 182-183, which predict the replacement of arginine to cysteine (p.R183C) or histidine (p.R183H). Likewise, the few mutations reported to affect the codon 183 of GNAQ (CGA) are caused exclusively by G > A transitions [GNAQ are p.P170S, p.I189T, p.Q176R, and p.P193L, which achieve an overall frequency of 8.9% in some series [Overall, the frequency of mutations in the exon 4 of nsitions . Other me series   comprise the largest family of cell surface receptors encoded by the human genome. While cancer-related mutations in GPCR signalling are less common than those in receptor tyrosine kinases, comprehensive sequencing of the human cancer genome has revealed that roughly 20% of human cancer mutations are associated with altered GPCR signalling . GPCRs hFollowing activation, proteins belonging to the family of regulators of G protein signalling accelerate the G subunit\u2019s intrinsic GTPase activity, reverting the G protein to an inactive GDP-bound state and so promoting the formation of the inactive heterotrimeric protein complex. The p.R183 and p.Q209 mutations are located in the Gq/11 proteins\u2019 switch I and II domains, respectively. These mutations transform G proteins to a constitutively active state by decreasing their GTPase activity .BRAF mutations [GNAQ mutations [GNAQ/11 mutation is present [Primary and metastatic UMs showed that high levels of phosphorylated MEK and ERK proteins occur in the absence of utations . It is wutations . Then, iutations . These putations . Subsequ present .In vitro analysis using protein kinase C (PKC) inhibitors of various specificities support the concept that the mutant GNAQ protein increases UM proliferation through PKC activation. Wu et al. discovered that enzastaurin, which is a potent PKC\u03b2 inhibitor [GNAQ cell lines at considerably higher rates than in wild-type GNAQ and GNA11 cell lines [GNAQ mutations. This fact indicates that PKC signalling is crucial in mediating the oncogenic effects of mutant Gq in UM [nhibitor , inducesll lines . This efGq in UM ,45. InteIn vitro investigations of the GNAQ mutant in UM cell lines demonstrated that the suppression of PI3K-alpha and P13K-beta had little effect on proliferation, indicating that PI3K is not the primary growth promoting factor . HoweverGNAQ and GNA11 lead to YAP/TAZ activation under physiological conditions. In vitro cell lines and human samples with GNAQ or GNA11 p.Q209 mutations showed higher amounts of YAP dephosphorylation and nuclear localisation in comparison to cell lines with wild-type GNAQ and GNA11, confirming these findings [The Hippo-YAP pathway is a regulator of cell contact inhibition, proliferation and death . Yu et afindings . Moreovefindings .GNAQ-p.Q209 mutant model [A parallel investigation by Feng et al. further defined the molecular mechanism of YAP activation using a nt model . Other rnt model . GNAQ sint model . Dynamicnt model . Actin pnt model .GNAQ and GNA11 genes in the prognosis of these tumours. Firstly, it was suggested that several lines of evidence indicate that GNA11 mutations may have a stronger effect on melanocytes than those mutations in GNAQ [GNA11 p.Q209 than GNAQ p.Q209 mutations in UM metastases. These mutations were also more common in locally advanced primary tumours, specifically in those originating from ciliochoroidal region, which is per se a prognostically adverse feature [Prognostication of UM patients can be achieved by analysing the mutational tumour status. Since the initial UM sequencing studies, there has been controversy about the possible involvement of the  in GNAQ . The fir feature . This ob feature .GNAQ mutations and those with GNA11 mutations in their study, even though they observed a trend toward increased survival among patients with tumours carrying a GNA11 mutations [Several years later, Van Raamsdonk et al. also speculated that the differences observed in mice may be a functional consequence of the different mutations rather than a real difference in function between GNA11 and GNAQ proteins. In contrast, these authors observed no significant differences in survival among those patients with utations , which cGNAQ were not suitable to predict DFS in patients with UM [GNAQ mutations have been shown to have similar frequencies at different clinical stages of UM progression, and to be independent of chromosomal aberrations, acting as an early oncogenic event [One year prior to this, Bauer et al. described that mutations in  with UM . This waic event .GNAQ and GNA11 genes are not associated with patient outcome to an equal extent. They studied samples from 92 ciliary body and choroidal melanomas and correlated the mutational status of both two genes with DFS and other parameters. The authors concluded that the univariate analysis of patients with tumours harbouring mutations in GNAQ or GNA11 genes was not significantly lower than in wild-type tumours. They also examined whether mutations in these genes affected the prognosis of patients with M3 tumours by using a survival analysis for changes in chromosome 3 stratified for GNAQ and GNA11 mutations. Again, no significant effect on the DFS in tumours with M3 and the presence of mutations in GNAQ or GNA11 was observed [In 2013, Koopmans et al. published that GNA11 mutations in MUM, and a poorer disease-specific survival of GNA11-mutant tumours in a cohort of 30 UM patients with metastasis [Subsequently, numerous sequencing studies on UM tumour samples have been published. The results were always diverse and controversial, since although both genes showed some correlation with other prognostic features or different trends in relation to OS, no specific and reproducible pattern was ever observed among the different cohorts of the patients studied . In 2014tastasis .GNAQ mutations were associated with the absence of ciliary body involvement and greater largest basal diameter; both of these are defined as bad-prognosis characteristics. On the other hand, GNA11 mutations were not associated with any of the analysed clinicopathologic features with prognostic value [GNAQ/GNA11 mutations in Chinese patients affected by UM, finding that metastasis-free survival was not significantly associated with the GNAQ/11 mutations in the Kaplan\u2013Meier analysis . Those results indicated that GNAQ and GNA11 mutations were not significantly associated with metastasis [Later, Decatur et al. found that ic value . In the tastasis .GNA11 mutations, 2 harboured GNAQ mutations and only 1 of them had no mutations in neither of those 2 genes. Once more, regarding these results, the authors proposed a bias towards GNA11 p.Q209L mutations in metastatic disease [GNAQ and GNA11 mutations was also published by Staby et al. in 2018, whom found no significant differences in the prevalence of GNAQ and GNA11 mutations between the patients with or without metastatic disease. Despite this, these authors found that GNAQ mutations showed a tendency to be inversely associated with progression to metastatic disease [On the other hand, in 2018, Kennedy et al. described that in a patient cohort composed of 36 patients with primary UM, where 9 patients developed metastasis, 6 of them harboured  disease . However disease .GNAQ and GNA11 and patient survival could be demonstrated in most of the studies published to date. This is because, although most of them showed a trend supporting that mutations in the GNA11 gene are likely to lead to more aggressive disease development, this tendency has rarely been accompanied by statistically significant results. Nonetheless, very recently, Piaggio et al. demonstrated, for the first time, that UM with mutated GNA11 has worse prognosis than those with mutations in GNAQ . They analysed the association between GNAQ and GNA11 mutations with disease-specific survival, gene expression profiles, and cytogenetic alterations in 219 primary UMs from three different cohorts . In their study, these authors concluded that GNA11 mutated UMs have worse prognosis, and it is associated with high risk cytogenetic, mutational and molecular tumour characteristics that might be determined, at least in part, by differential DNA-methylation. Therefore, to date, this is the only published study in which statistical results support the existence of an association between mutations in the GNA11 gene and worse disease prognosis in patients with UM [GNA11 gene in terms of prognosis had been observed, could be due to a reduced number of analysed samples, or due to the insufficient follow-up time of patients.As it can be observed, no correlation between the presence of mutations in  with UM . TherefoIn most types of tumours, immunological characteristics, such as inflammation, infiltrate, and HLA molecules expression, correlate with the prognosis of the disease. In UM, it has been described that inflammatory phenotype, which is characterised by the presence of immune cells, such as T lymphocytes, macrophages, and an increased HLA expression, is closely related to bad prognosis ,57,58.GNAQ/GNA11 mutational status, to the specific type of mutation (p.Q209P/p.Q209L), and whether or not mutations in those two genes are responsible for different degrees of inflammation. As result, they found no differences in the expression of inflammatory markers, such as HLA expression, or levels of infiltrating leukocyte, according to the presence of GNAQ/GNA11 mutations. They also found no significant differences between the GNAQ/GNA11 or p.Q209L/p.Q209P mutations for the survival of patients [GNAQ and GNA11 do not play a direct role in the regulation of inflammation, and that the type and location of mutations in these genes do not appear to affect the progression of UM.In 2019, van Weeghel et al. tried to investigate whether the expression of different types of HLA molecules in UM is similarly related to M3 or might be related to the patients . These oGNAQ and GNA11 genes exhibit similar mutations rates in primary UM; however, less is known about the prevalence and significance of mutations in these genes in MUM. Indeed, most of the published investigations regarding somatic mutations in UM focused on the evaluation of elapsed time from the initial diagnosis and treatment of the primary UM to the development of metastasis or death.Many studies had showed that GNAQ and GNA11 in the prognosis of UM had reported that the distribution of GNA11 and GNAQ mutations differs between primary and MUM, with a GNA11 to GNAQ ratio of 0.7 in primary UM, and 2.6 in MUM [GNA11 mutations were considerably more frequent than GNAQ mutations in these specimens. Additionally, they found that patients with GNA11-mutant tumours had poorer disease-specific survival  and OS , than those with tumours lacking GNA11 mutations. Thus, they proposed that the survival data, combined with the predominance of GNA11 mutations in metastasis, raises the possibility that GNA11-mutant tumours may be associated with a higher risk of metastasis and poorer prognosis than those tumours bearing GNAQ-mutants [The pioneering studies on the role of 6 in MUM . Griewan-mutants .GNAQ and GNA11 mutations, the frequency of mutations in MUM specimens, and the commonly mutated GNAQ/GNA11 genes in survival after development of systemic metastasis. They found similar rate frequencies for both genes in patients, where mutations in GNAQ and GNA11 genes were observed in 44.8% and 47.1% of patients, respectively [GNAQ and GNA11 genes themselves [Recently, Terai et al. investigated the possible existence of a correlation between metastasis-to-death in MUM patients with ectively . This reectively ,19. Furtemselves .GNA11 to GNAQ ratio was 1.2. Contrary to other studies, the GNA11-mutated tumours demonstrated a longer average time to first metastasis (GNA11 vs. GNAQ: 77.8 vs. 43.1 months) and a better OS (79.8 vs. 33.7 months). Nonetheless, they discuss that the differences between their results, and those from other investigations, could be due to the ratio of metastatic vs. primary tumour examined, and also to the small number of samples [Subsequently, Isaacson et al. reported that in their cohort of patients, the  samples .GNAQ and GNA11 genes [Targeted therapy is a therapeutic modality that refers to the drugs designed to interfere with a specific molecular pathway that is believed to play a critical role in tumour development or progression . As it w11 genes ,20,62, tThe growing relevance of this therapeutic strategy is now evidenced by the numerous relevant papers that regularly report the latest results and knowledge on UM-targeted therapies ,74,75,76The main action of GDIs is to prevent the release of GDP, i.e., they inhibit GDP/GTP exchange, which leads to the G protein remaining in an inactivated state, and therefore, blocking its signalling. Two molecules stand out within this group, FR900359 and YM-254890 .The first of these, FR900359, is a cyclic depsipeptide derived from the plant Ardisia crenata, which selectively inhibits G\u03b1q, G\u03b111 and G\u03b114, and is inactive in cells lacking these proteins . It was GNAQ mutant cells were less sensitive than GNA11 mutant cells to YM-254890 [On the other hand, the YM-254890 molecule is a cyclic depsipeptide isolated from Chromobacterium sp. QS3666  also witM-254890 .Despite the fact that heterotrimeric G\u03b1q/11 G proteins have been known for decades, their selective inhibition is still considered a challenge. The reasons include the high affinity of GDP/GTP for the G-protein, as well as the fact that its high intracellular levels hinder biochemical competition, and that this \u201cmutation-nonspecific\u201d type of inhibitor can block both wildtype and oncogenic protein forms .It is well known that G\u03b1q/11 activation leads to the stimulation of the downstream signalling pathway MAPK, which contributes greatly to UM carcinogenesis .GNAQ/11 mutant UM [One of the most important intermediate effectors in this pathway are the MEK enzymes. Within the subgroup of MEK inhibitors, one of the best known and most tested molecules is selumetinib (MEK1/2 inhibitor). Ambrosini et al. demonstrated that treatment with selumetinib (AZD6244) regulated the expression of the genes involved in proliferation, cell invasion and drug resistance in tumour tissues of patients with metastatic utant UM . Thus, butant UM .In 2014, the results of a clinical trial (NCT01143402) in patients with advanced UM were presented. In this study, selumetinib as monotherapy was compared to chemotherapy (temozolomide or dacarbazine), showing only a modest improvement in progression-free survival (PFS) and response rate, with no improvement in OS . SubsequFollowing the previous approach, Decaudin et al. evaluated the potential of drug combinations to increase the efficacy of selumetinib in UM cell lines and patient-derived xenograft models (PDXs) by first assessing the combination of selumetinib and dacarbazine. They observed that this chemotherapy agent did not improve the in vitro or in vivo antitumour efficacy of selumetinib, which is consistent with the results of the Carvajal et al. clinical trial. Thus, they tested other combinations of selumetinib with docetaxel (chemotherapy agent), AZ6197 (ERK inhibitor), and vistusertib-AZD2014 (mistusertib-AZD2014), with the latter two appearing to be the most effective in UM PDXs . A multiGNAQ mutation), and four had stable disease for \u226516 weeks (including two who received treatment for >40 weeks) [Another orally administered selective MEK1/2 inhibitor is trametinib (GSK1120212). Falchook et al. conducted a trial (NCT00687622) including 81 patients with CMM, and 16 with advanced UM and. Although efficacy was mainly observed in BRAF-mutated CMM, among the trametinib-treated UM patients, two achieved 24% tumour shrinkage (one of whom had a 0 weeks) .Phospholipase C beta (PLC\u03b2) is able to activate several protein kinase C (PKC) isoforms and RasGRPs . In UM, GNAQ-mutated UM cells. Down-regulation of these PKC isoforms resulted in enhanced antitumour action through the induction of apoptosis and G1 cell cycle arrest on GNAQ mutant UM cells, compared to those wild type [GNAQ/11 mutations that induces G1 cell cycle arrest [In an experimental study, the molecule enzastaurin was shown to decrease the expression and/or phosphorylation of several PKC isoforms including \u03b2II, \u03b5 and \u03b8 in ild type . In the e arrest ,95.GNAQ mutations through the PKC/NF-\u03baB and PKC/ERK1/2 pathways specifically, by the inhibition of the expression of PKC isoforms \u03b1, \u03b2, \u03b4, \u03b5, and \u03b8 [In a similar preclinical approach, it was shown that the PKC inhibitor, AEB071 (sotrastaurin), was able to significantly reduce the viability of UM cells harboring \u03b5, and \u03b8 . Based o\u03b5, and \u03b8 . However\u03b5, and \u03b8 ,97.In the same way, it was shown that AEB071, together with CGM097 (MDM2 inhibitor) showed an additive effect enhancing the anti-proliferative and apoptotic effect, as well as the inhibition of tumour growth . SubsequGNAQ/11 mutant UM cells observed that the combination of LSX196 with trametinib (MEK1/2 inhibitor), showed a strong synergistic effect in reducing cell viability, while the combined treatment of this first molecule with VS-4718 (FAK inhibitor) reflected a more limited synergy [A promising novel PKC inhibitor is the LSX196 molecule, later registered as IDE196 (darovasertib). An in vitro study with  synergy .GNAQ/11 mutations, including MUM, CMM, and colorectal cancer among others, with an approximate completion date of late 2022\u20132023, presented in July 2021 a preliminary robust 57% 1-year OS in monotherapy and early partial responses, in combination with binimetinib (MEK inhibitor) and crizotinib (c-MET inhibitor), in the group of patients with MUM. More specifically, a partial response in 22% of cases, and tumour shrinkage in 79% was observed with darovasertib and binimetinib combination therapy .Several clinical trials are currently underway for evaluation. A phase I/II study, (NCT03947385) designed to characterise the safety and anti-tumour action of darovasertib in patients with solid tumours harbouring Another clinical trial (NCT02601378), with an estimated completion date of June 2022, aims to characterise the pharmacokinetics/pharmacodynamics, tolerability, safety and antitumour activity of LXS196 as a single agent and in combination with HDM201 (siremadlin), an MDM2 inhibitor, in patients with MUM. In 2019, Kapiteijn et al. published preliminary analyses suggesting tolerable toxicity and encouraging clinical activity of LXS196 as monotherapy in these patients .GNAQ oncogene is able to control the Hippo pathway through a cytoplasmic protein tyrosine kinase called focal adhesion kinase (FAK). They detailed that G\u03b1q activates FAK through a non-canonical TRIO-RhoA signalling pathway, which in turn positively regulates the yes-associated Protein (YAP) by tyrosine phosphorylation of MOB1, inhibiting the Hippo kinase cascade and promoting tumour growth in the UM [Feng et al. demonstrated that the n the UM . Therefoin vivo, that the pharmacological combination of MEK-ERK and FAKi showed a negative synergistic action on cell growth, as well as cytotoxic effects leading to tumour regression [It is well known that FAK can be stimulated when tumour cells are exposed to other types of tyrosine kinase inhibitors, implying therapeutic resistance . On the gression . Two exaPI3K/AKT/mTOR is a downstream signalling pathway that can also be activated and deregulated in the UM due to oncogenic G\u03b1 action. Within this pathway, mTOR inhibitor RAD001 (everolimus) is one of the most studied drugs, both alone, and in combination. It showed reduced cell line viability and significantly delayed UM growth in preclinical studies , whereasEverolimus was also studied together with the PI3K inhibitor GDC0941, which synergistically increased apoptosis in several UM cell lines compared to monotherapies, and enhanced the antitumour effect of each agent alone in UM PDXs. Evaluation of the combination of everolimus with the PKC inhibitor AEB071 (sotrastaurin) demonstrated greater activity than single molecules, inducing cell death and observing tumour regression in several UM PDXs .GNAQ/GNA11 UM cell lines, and similarly, inhibits tumour growth in vivo in a GNAQ mutant xenograft model [Following the strategy of these synergistic effects, the PI3K\u03b1 inhibitor alpelisib (BYL719) was studied in combination with sotrastaurin. In the first study, it was observed that the combination of BYL719/AEB071 decreases cell viability and induces apoptosis in ft model . In the ft model .GNAQ mutant cell lines and xenograft models, but no impact on apoptosis [Finally, molecules of this pathway have been tested in combination with MEK inhibitors. AZD8055 (mTOR inhibitor) and MK2206 (AKT inhibitor) in combination with selumetinib (MEK1/2 inhibitor) showed, in preclinical studies, a synergistic inhibition of viability in poptosis ,109.On the other side, GSK2126458 (PI3K inhibitor) was combined with trametinib (MEK inhibitor), a trial that showed a higher rate of apoptosis with the combination therapy compared to each PI3K and MEK inhibitor alone . In thisADP rybosylation factor 6 (ARF6) is a GTPase that triggers, in the presence of the oncogenic G\u03b1q mutation, multiple downstream signalling pathways, such as Rho/Rac/YAP and PLC\u03b2/PKC. The pyrazolopyrimidinone compound NAV-2729 was identified as a promising direct inhibitor of ARF6, reducing UM cell proliferation and tumorigenesis in a mouse model. Therefore, ARF6 is a potential therapeutic target for patients with oncogenic GNAQ-driven UM .As previously mentioned, c-MET inhibitors are also targets of study. Within this group, cabozantinib obtained, in a first trial (NCT00940225), encouraging results from a PFS and OS point of view . HoweverGNAQ/GNA11 genes in UM and their putative therapeutic opportunities. It seems clear that both genes play an important role in the oncogenic process of UM, but their role in the prognosis of these tumours remains controversial.In this review, we have attempted an interrelated assessment of the possible prognostic implication of mutations in the GNAQ and GNA11 genes are only involved in the early development of UM, playing a lesser role in its progression. Nevertheless, even in the absence of statistically significant results, several investigations have suggested a more aggressive course of tumours with the mutated GNA11 gene, due to the general observation of a trend towards longer survival among patients with tumours carrying GNAQ mutations. Irrespective of these results, it should be noted that most of the published investigations regarding somatic mutations in UM focused on the evaluation of time from the initial diagnosis and treatment of the primary UM to the development of metastasis or death. Moreover, there is even less knowledge about the role of these mutations in the advanced state of UM, and even less, in MUM. Regarding this, the few published studies that focuse on assessing the role of these mutations in MUM observed similar rate frequencies for both genes in patients with metastasis, and suggested that the survival of MUM patients could be predicted according to the mutation (p.Q209 vs. p.Q209L) rather than the mutated gene [GNA11 have worse prognosis than those with mutations in GNAQ [Most published studies agree that ted gene . After a in GNAQ .GNAQ and GNA11 genes are involved in various signalling pathways that are essential for the proliferation of tumoral cells in UM. This, in combination with its high mutational rate of these tumours, makes them promising targets and regulators of the therapeutic response in UM. Despite the relatively good response of the primary UM to treatment, almost 50% of patients will develop metastatic disease [The  disease ,117. Fur disease . It is fGNAQ/GNA11 signalling pathways could support the idea that these genes are not really involved in UM prognosis.Today, there are still important limitations that hinder and slow down the progress in the development of new adjuvant therapies. Overall, it is worth noting that many studies are in the preclinical phase, that trials include a small sample size of patients, and that the absence of randomisation is common, as well as the fact that molecular knowledge on UM is mostly based on primary tumour samples, and not so much on metastatic specimens ,105. MorGNAQ/GNA11 genes, and more sequencing studies involving a larger number of tumour samples at different follow-up times to generate preliminary findings that will require further clinical validation.Ultimately, to overcome these obstacles and obtain consistent conclusions on the benefit of different targeted therapies, it will be necessary, in the near future, to develop protocolised in vivo studies in advanced UM that consider key aspects such as the individualised molecular-genetic profile of each patient. This includes more studies to investigate the functional and prognostic relevance of oncogenic mutations in GNAQ gene, GNA11 gene, MAPK signalling pathway, targeted therapy in uveal melanoma, genetic basis of UM, sequencing studies in UM. The results of that search form the basis for publishing this article.This article was developed in the context of an intensive literature review. The considered publications were mainly searched in PubMed using key words or sentences such as the following: UM prognosis, driver mutations in UM, oncogenic mutations in UM, G-protein coupled receptors in UM,"}
{"text": "Vaccine hesitancy has seen an uprising over the decades, even though there have been many advances regarding vaccine-preventable diseases. Of late, vaccine hesitancy has resurged towards the coronavirus disease 2019 (COVID-19) vaccine. The SARS-CoV-2 virus has major effects on the human body and has led to the development of different vaccines, which have been shown to provide immunity against the novel coronavirus. Dentists are at an increased risk to COVID-19 because of the nature of their work. It is imperative to have high vaccination coverage for this group.The aim of this study is to determine vaccine hesitancy and drivers associated with vaccine hesitancy among dental students at a university in South Africa.A dental school in South Africa was chosen as the setting for this study.An analytical cross-sectional study was conducted by means of an anonymous, online, validated questionnaire to determine vaccine hesitancy.n = 172) students were vaccine not hesitant. The main concerns regarding the vaccines were identified as safety and efficacy of the vaccine. Pressure by family or friends and the university to get vaccinated was evident.Of the 205 dental students participated, 83.9% (Vaccine hesitancy is high despite mandatory vaccination policies in South Africa. Specific drivers contributing to vaccine hesitancy were identified as doubt in the efficacy and safety of the vaccine.This study has highlighted the prevalence of vaccine hesitancy among dental students at University of the Western Cape, prior to compulsory vacccination implementations. According to MacDonald , vaccineNations worldwide are trying to reduce the spread of the COVID-19 virus by enforcing different preventative measures such as physical distancing and wearing of face coverings as well as lockdown and travel restrictions. Regarding the latest omicron variant, there has been a reduction in COVID-19-related deaths and morbidity in South Africa  Oral Health Centres. The secondary aim is to determine the drivers associated with vaccine hesitancy in this population.n = 416) were included in this study and recruited from September 2021 to October 2021. A convenience sampling technique was utilised to recruit participants, as this sampling technique is simple and cost effective  between vaccine hesitancy and whether the participants felt that vaccines were generally effective , participated in this study. Thirty-three participants (16.1%) were vaccine hesitant. Among the participants, there were no statistically significant associations between vaccine hesitancy and demographics, including the year of study, age or sex . Internaffective .p < 0.001) (A majority of the participants did not have vaccine hesitancy . There w< 0.001) .n = 103) with only 4.4% (n = 9) of students admitting \u2018excellent\u2019 knowledge (n = 125), \u2018health officials\u2019 (n = 125) and \u2018social media\u2019 (n = 122). Although 77.1% (n = 158) of participants believed that the vaccine is necessary to combat the pandemic, 27.8% (n = 57) of participants did not fully understand the concept of herd immunity   .n = 140) did not feel pressured to get vaccinated; however, the pressure felt from the university (n = 46) followed by family and friends were reported (n = 39). Almost 45% (n = 90) of participants said that they have doubts regarding the COVID-19 vaccine, with concerns regarding the safety (n = 97) of the vaccine being the major contributing factor. Concerns regarding the efficacy (n = 67) and mistrust in the development of the vaccines (n = 65) were also evident. Interestingly, dental students having doubts regarding the vaccine, 81.5% (n = 167) would recommend the vaccine to their family and friends.Most students , p = 0.031, of experiencing vaccine hesitancy compared to participants who did not experience vaccine hesitancy, respectively  adjusted odds of experiencing vaccine hesitancy,  = 0.008 . In addiesitancy . Furtherectively .There is a very high rate of vaccine hesitancy (16.1%) prevalence in this population. Among the UWC\u2019s dental students, 29.3% do not believe that the vaccines available can prevent COVID-19 and its associated complications, which is similar to a study conducted among 248 dental students from three schools, which reported that 54% of dental students were concerned that the vaccine may not be effective , which is applicable to the age of the sample population. Information on social media is, however, not controlled and may provide misinformation, driven by spectator value and excitement, contributing to doubt and uncertainties among students. A study that was done in France indicated that vaccine acceptance and practices were better when information is obtained from healthcare providers as opposed to receiving information from relatives or from the Internet (Charron, Gautier & Jestin Social media was also identified as a primary source of knowledge (A study done by Kelekar et al.  conducteAccording to the authors\u2019 knowledge this type of study has not been conducted before in Africa. A limitation of this study is the small sample size. This could be because of non-response bias, as vaccine-hesitant participants would be less likely to participate.Vaccine hesitancy among undergraduate dental students at this healthcare facility is astonishingly high. Students still expressed doubts regarding the efficacy, safety, long-term side effects and effectiveness of the vaccines. Further research should be conducted to determine the feelings of students and to record any side effects experienced by dental students, after mandatory vaccination implementation on dental students in South Africa."}
{"text": "Cellular senescence and apoptosis were historically thought of as two distinct cell fate pathways. However, many of the proteins involved are integral to both pathways. In particular, the ability of p53 to regulate both senescence and apoptosis meant it was seen as the decisive factor in these decisions, yet questions remain about its ability to select on its own the most appropriate cell fate according to each situation. Therefore, cell fates are no longer considered fixed endpoints but dynamic states that can be shifted given the right combination of activation and/or inhibitions of cofactors.When a cell is damaged, it must decide how to respond. As a consequence of a variety of stresses, cells can induce well-regulated programmes such as senescence, a persistent proliferative arrest that limits their replication. Alternatively, regulated programmed cell death can be induced to remove the irreversibly damaged cells in a controlled manner. These programmes are mainly triggered and controlled by the tumour suppressor protein p53 and its complex network of effectors, but how it decides between these wildly different responses is not fully understood. This review focuses on the key proteins involved both in the regulation and induction of apoptosis and senescence to examine the key events that determine cell fate following damage. Furthermore, we examine how the regulation and activity of these proteins are altered during the progression of many chronic diseases, including cancer. Over time, cells are subject to a variety of external and endogenous stresses. Faced with these stresses, cells mount an appropriate response with two main end points\u2014survival or death. Survival may mean permanent cell cycle arrest in the form of senescence or repair and continued proliferation after a temporary arrest . Howeverp53 is one of the most well-studied proteins and has a key role in DNA damage repair, cell cycle arrest, apoptosis and senescence. Its domain structure is critical to its ability to participate in the different molecular mechanisms . The N-tIn unstressed cells, p53 is maintained at low levels by the E3 ubiquitin-ligase MDM2. However, when stress is detected, p53 is phosphorylated, which prevents its binding to MDM2 and stabilises its levels . Once stThe DNA damage response (DDR) is a complex pathway that has evolved to detect DNA damage and facilitate its repair. In humans, two proteins central to DDR are ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and RAD3 related (ATR). ATM is primarily involved in responding to double-strand breaks whilst ATR senses stalled or stressed DNA replication forks and is particularly important in detecting DNA damage in the S phase ,11. OnceApoptosis is a well-characterised programme, first described in 1972 as being a morphologically distinct form of cell death . FurtherThe intrinsic or mitochondrial apoptotic pathway is triggered by stresses including oxidative stress, irradiation, DNA damage and treatment with cytotoxic drugs. Crucial in regulating the intrinsic pathway is the BCL-2 family of proteins, which can be divided into three groups based on their function: firstly, the multi-domain anti-apoptotic proteins, BCL-2, BCL-xL, BCL-w, MCL-1 and BFL-1/A1; secondly, the pro-apoptotic effector proteins BAK, BAX and BOK; and finally, the pro-apoptotic BH3-only proteins, BAD, BID, BIM, BMF, HRK, NOXA and PUMA . The relIn response to internal damage, effector proteins BAK or BAX are activated at the outer mitochondrial membrane. Detailed structural and biochemical analysis has determined multiple steps required for both BAK and BAX activation ,23, inclThe extrinsic route to apoptosis, or the death receptor (DR) pathway, is mediated by transmembrane receptors that are members of the tumour necrosis factor (TNF) receptor protein superfamily . These rp53 principally serves as an apoptotic regulator by modulating the expression of key proteins in both the intrinsic and extrinsic pathways, including BCL-2 family proteins, death receptors CD95 and Death Receptor 5 (DR5), APAF-1 and Caspases . AdditioAlthough initiation of apoptosis is often thought of as the point of no return, some cancers have found ways to reverse or halt the process and thus change cell fate, a process known as anastasis . SeveralIn addition to cell death via the canonical apoptotic pathways, p53 has been reported to also be central to several forms of non-apoptotic cell death. One example of this is an alternative p53 driven cell death pathway first identified in experiments where caspase activity was inhibited using the inhibitor ZVAD.fmk . In thisSenescence was first observed in normal human cultured cells . Human fA senescent cell may be identified as being in a state of irreversible cell cycle arrest, though metabolically active while displaying phenotypic changes. Other forms of growth arrest exist, such as quiescence and terminal differentiation, which are distinct from senescence in terms of the pathways triggered and the resultant phenotype. Senescence can be activated by numerous cell stressors, such as DNA damage, hypoxia, oncogene activation and nutrient deprivation, with most mitotic cells in the body, including cancer cells, able to undergo senescence ,49. If tSenescence can occur due to perturbation of the balance between transcriptionally inert heterochromatin or the more open transcriptionally active form euchromatin. The extent to which each conformation exists depends largely on histone modifications such as acetylation or methylation. The nuclei of senescent cells contain areas of facultative heterochromatin known as senescence-associated heterochromatin foci (SAHF), which appear as punctate foci upon DAPI staining . SAHF arSenescence can be caused by stresses such as ionising radiation due to an accumulation of double-strand breaks leading to the activation of ATM and a DDR  53]. Th. Th53]. Oncogene-induced senescence (OIS) occurs when normal cells undergo senescence in response to oncogene activation and acts as a tumour-suppressive mechanism that prevents benign lesions from progressing to malignant tumours . RAS proWhilst the traditional definition of senescence implied that the cell cycle arrest had to be irreversible, some studies are now proposing that cell fate decisions can be changed. Modulating the key proteins involved in cell cycle arrest, such as p53 and p21, may allow cells to escape senescence. Whilst p53 levels in a non-stressed state are low, the pulsatile dynamics of p53 in stress may allow the cell options of quickly switching to sustained levels of p53 and arrest or a drop in levels and return to proliferation. This subtle balance may be therapeutically exploited in the right context to push tumour cells into senescence or potentially remove senescent cells from aged tissues. Additionally, there is growing evidence that the p53\u2013p21 cell cycle arrest pathway is not simply unidirectional but that the dynamics of p21 may affect cell fate decisions following stress. For example, overexpressed p21 has been shown to render glioma cells resistant to chemotherapy-induced apoptosis by allowing the repair of damaged DNA . Moreove\u2212/\u2212 cells display increased p53 levels without drug-induced DNA damage, which also correlates with an increased expression of p14ARF that may promote p53 stability by binding to MDM2 [Several studies have revealed functions for p21 aside from cell cycle arrest, which may contribute to determining cell fate. For instance, HCT116/p21 to MDM2 . These c to MDM2 . All thiMutant p53 can also cause normal cells to evade senescence . Near seAlthough therapeutic strategies that cause senescence have been developed over the last few decades, a growing number of studies have shown that TIS may not be a therapeutic endpoint as first thought. A small population of cells can evade growth arrest following a clinically relevant dose of chemotherapy and may contribute to a drug-resistant phenotype, aided by the cytokines and other factors secreted by the cells that have undergone TIS . In partThis suggests that a tumour cell turned senescent may sometimes not be therapeutically relevant and may sometimes even contribute to resistance and relapse. One strategy to bypass this issue would be to first push tumour cells into senescence, followed by the selective killing of these cells using specific compounds . This \u201cone-two punch therapy\u201d approach was demonstrated in liver cancer cells containing p53 mutations induced to senesce by inhibiting CDC7 kinase, which was followed by sertraline treatment to induce apoptosis in these cells through mTOR inhibition . AdditioAt first glance, it would appear that senescence and apoptosis are two distinct cell fate pathways. However, many of the proteins induced are integral to both pathways. The ability of p53 to regulate both senescence and apoptosis makes it an important protein in tumour suppression, as confirmed by the fact that it is mutated in about 50% of all cancers . The majARF, which in turn is stabilised by oncogenic c-MYC transcriptionally upregulating USP10. p14ARF stabilises p53 by sequestering MDM2 in the nucleolus and prevents MDM2 from binding and ubiquitinating p53 [p53 activation in response to oncogenic signalling is prevalent in pre-malignant and malignant cells . OIS canting p53 . In additing p53 . Conversting p53 . p21 hasting p53 ,79. p21 ting p53 . p21-medting p53 . It has ting p53 .As mentioned above, p53 regulates apoptosis by both transcription-dependent and independent mechanisms, the latter requiring p53 to be transported out of the nucleus ,83. FromIt has been observed that p53 binds to BAK and aids in oligomerisation. The binding of p53 to BAK is through the H2 helix, Loop 1 and Loop 3 within the DNA binding domain (DBD) of p53 to the N-terminal side of BAK, specifically the electropositive residues R280 (H2 helix), R248 (L3 loop) and K120 (L1 loop). These residues then interact with the electronegative residues E24, E25 and D160 on BAK , and p532O2), superoxide anion radical (O2\u2022\u2212), nitric oxide (NO), hydroxyl ion (OH\u2212) and hydroxyl radical (OH\u2022), the ions and radicals being highly reactive due to the presence of an unpaired electron. ROS are normally a by-product of mitochondrial oxidative phosphorylation in the electron transport chain (ETC) [2\u2022\u2212 and may be converted to H2O2 by superoxide dismutases (SODs), which act as the first line of defence in controlling ROS levels [ROS have been known as chemical entities for over 100 years, and their importance in biological systems has been studied since the 1950s . The reain (ETC) . Not allS levels ,93. AnotS levels . It has S levels . As ROS S levels .The connection between senescence and intracellular oxidants can easily be seen by the application of low concentrations of hydrogen peroxide to human diploid fibroblasts, which causes them to enter a senescent-like growth arrest . AdditioThe anti-apoptotic BCL-2 family of proteins also plays important roles in regulating both apoptosis and senescence, influencing whether a cell undergoes cell death or survives. After oxidative stress, an increase in BCL-2 expression is observed during the growth arrest, but these levels decline during apoptosis . MoreoveCell fate decisions in response to stress were initially seen in a simplistic way as being mainly proportional to the damage exerted. Whether a cell that survived after the necessary repairs entered senescence or died seemed to depend on the magnitude of the insult. However, a more complex picture emerged over the years, particularly with the discovery of the many different functions of p53. For a while, p53 was seen as the decisive factor in these decisions until questions emerged about its ability to select on its own the most appropriate cell fate according to each situation. This gave rise to two antagonistic models. In the first one, p53 was proposed to induce either apoptotic or arrest genes in a selective way, mostly through differential post-translational modifications (p53 smart), while the alternative (p53 dumb) suggested that p53 would induce both sets of genes simultaneously and other factors would help determine which one would dominate .Currently, the most accepted view is likely an amalgam of both hypotheses: cell fate decisions are still not completely understood but are seen as the result of the interaction of many different factors, probably with p53 still at the centre of an intricate network, but not alone at the commanding seat. The conjunction of these modulators is what eventually determines which p53 function eventually prevails . Among tIndeed, identifying the factors that modulate cell fates is particularly relevant in the context of cancer treatments in order to improve therapeutic outcomes. Senescence, once seen as a safe tumour-suppressor mechanism, is now a fate to be avoided since it is considered no longer irreversible or innocuous. Importantly, the accumulation of senescent cells after therapy has been shown to create a microenvironment in the tumour that enhances cancer cell survival and eventually fosters relapse . ConvertMore research is needed to fully understand how cell fate decisions are made and can be modulated. This could lead to a better understanding of an intricate network of biological events but also to improved therapies for many cancers. For this, it will be important to keep p53 centre stage but open the focus enough to include the many helpers that end up contributing to these processes."}
{"text": "Any women with newly diagnosed Stage I\u2013III breast cancer scheduled to receive taxane-containing chemotherapy was eligible. The primary outcome was development of grade 2 or higher CIPN during chemotherapy graded by the clinician before each chemotherapy cycle (NCI-CTCAE v5 criteria). We measured p16 expression in peripheral blood T cells by qPCR before and at the end of chemotherapy. A multivariate model identified risk factors for CIPN and included taxane regimen type, p16Age Gap, a measure of discordance between chronological age and p16 expression, and p16 expression before chemotherapy. Participants with higher p16Age Gap\u2014higher chronological age but lower p16 expression prior to chemotherapy - were at the highest risk. In addition, higher levels of p16 before treatment, regardless of patient age, conferred an increased risk of CIPN. Incidence of CIPN positively correlated with chemotherapy-induced increase in p16 expression, with the largest increase seen in participants with the lowest p16 expression before treatment. We have shown that p16 expression levels before treatment can identify patients at high risk for taxane-induced CIPN. If confirmed, p16 might help guide chemotherapy selection in early breast cancer.Identifying patients at higher risk of chemotherapy-induced peripheral neuropathy (CIPN) is a major unmet need given its high incidence, persistence, and detrimental effect on quality of life. We determined if the expression of  CIPN can limit post-treatment quality of life, particularly important for patients receiving chemotherapy for stages I\u2013III breast cancer5 since most receive a neurotoxic taxane and will have long-term survival. Unfortunately, up to 30% of these patients experience moderate to severe CIPN8. Symptoms occur predominantly in the hands and feet and include burning or shooting pain, paresthesia (numbness/tingling), pain perception abnormalities like allodynia and hyper- or hypo-algesia, temperature sensitivity, weakness, and, rarely, ataxia. In addition, multiple studies have shown that moderate to severe CIPN can be dose-limiting, raising concerns about compromised treatment efficacy10.Chemotherapy-induced peripheral neuropathy (CIPN) is among the most debilitating, common, and persistent chemotherapy toxicities11, with symptoms persisting five or more years post-treatment5 that dramatically impact quality of life14. Numbness in the feet can increase the risk of falling, particularly consequential in older patients where fractures can lead to inpatient rehabilitation and loss of independence17. Unfortunately, drug therapies to treat or prevent CIPN are largely ineffective18, especially among patients receiving taxanes19. Patients with severe, persistent, and painful CIPN may also be prescribed opioids, with a risk for opioid addiction22.Even with dose-reductions, over 50% of patients receiving weekly paclitaxel experience limited recovery from CIPN26 and/or selection of a less neurotoxic agent. In Stages I\u2013III breast cancer, neurotoxic taxanes (docetaxel and paclitaxel) are commonly used in both the adjuvant and neoadjuvant settings. Paclitaxel confers a much greater risk of CIPN than docetaxel27, therefore docetaxel may be preferred in at-risk patients. In addition, several preventive pharmacotherapies are under development28.Given the potential severity of symptoms and lack of effective treatments, CIPN prevention becomes critical. Available strategies include cryotherapy31, though there is no consensus about their relative importance or application in CIPN prevention2. Recently, cellular senescence was found to positively associate with cisplatin-induced peripheral neuropathy in mice, and depletion of senescent cells abolished neuropathy32.Identifying patients at-risk of CIPN is essential for prevention. Epidemiological risk factors include diabetes, obesity, and age39. Senescent cells undergo permanent growth arrest, are resistant to apoptosis, and secrete both inflammatory and pro-fibrotic cytokines, disrupting tissue function and homeostasis41. Recent studies elegantly demonstrated that induction of senescence in just the immune compartment, and T cells specifically, can induce both senescence and organ damage in tissues throughout the body43. Expression of p16INK4a (p16) mRNA in peripheral blood T lymphocytes has emerged as a key biomarker of senescence and a measure of senescent cell load44.Cellular senescence is a fundamental mechanism of aging and plays a causative role in nearly all chronic age-related diseases and physical declinep16 expression would associate with the risk of CIPN.Cellular senescence has not been evaluated as a risk factor for peripheral neuropathy in humans. We hypothesized that 27. Overall, 29% of participants experienced grade 2 or higher CIPN, of which 82% received paclitaxel and 18% docetaxel-based therapy. In a univariate analysis, none of the patient or clinical characteristics (except for chemotherapy regimen) were different between the CIPN and no CIPN groups, including age , diabetes  and p16 expression .Characteristics of 152 study participants with early-stage breast cancer receiving taxane-containing chemotherapies are shown in Table p16 and comorbidities may not be independent (see Methods for details). Taxane type (paclitaxel vs docetaxel) was also included given the difference in CIPN incidence between these two agents. The optimal model from these variables (Model 1) is shown in Table p16 expression before chemotherapy, chronological age, arthritis, and osteoporosis, contributed to model performance.Since no variables in Table p16 and age in the univariate vs. multivariate analyses prompted us to develop a measure called p16Age Gap, the difference between an individual patient\u2019s pre-treatment p16 and population-average p16 levels by age. First, we developed a method to convert p16 expression from log2 arbitrary units into years, and then directly compared p16-based age and chronological age (see Methods). Figure p16, with p16 converted to years (p16Age), and the difference between p16 and chronological age (p16Age Gap). p16Age Gap can also be thought of as a residual in the p16/chronological age regression model. A negative p16Age Gap suggests that an individual has p16 expression levels below an age-appropriate population mean; a p16Age Gap around zero suggests that p16 expression is similar to the population mean; and positive p16Age Gap signifies p16 expression above the population mean.Differences in the behavior of p16 (log2), p16Age, p16Age Gap, and chronological age in this study cohort are shown in Fig. p\u2009=\u20090.94, Student\u2019s t-test) , or older participants who are molecularly younger (negative p16Age Gap). p16Age Gap is strongly associated with p16Age  Fig. , demonstp16, and taxane shown to achieve similar performance to Model 1 (see Arc and BIC). When variables were analyzed for their individual contributions to the CIPN outcome (see Methods), p16Age Gap contributed 41% to the model as an individual predictor and 52% when considered with pre-chemotherapy p16 expression. Patients with a negative p16Age Gap  were at a higher risk for CIPN . Interestingly however, higher pre-chemotherapy p16 expression was also associated with a higher risk of CIPN in a multivariate model .To assess the role of this new measure, p16Age Gap, as a predictor of CIPN risk, we built a second regression model Table , Model 2The probability of CIPN in patients receiving paclitaxel- versus docetaxel-based chemotherapy was derived from regression Model 2 and shown in Fig. In addition to the regression model, we used the same variables to build a neural network to predict CIPN (see Methods). The relationship between the probability of CIPN and p16Age Gap as defined by the neural network algorithm is shown in Fig. p16 expression may be at higher risk of CIPN. We previously showed that p16 expression prior to chemotherapy is inversely correlated with the magnitude of chemotherapy-induced p16 increase45  as patients whose p16 did not change  as well as patients with high p16 exression levels were at the highest risk for CIPN. Although our findings of higher CIPN risk for those with age-inappropriately low p16 (negative p16Age Gap) initially seemed counterintuitive, we observed that patients with lower baseline p16 expression are more likely to have a larger chemotherapy-induced increase in p1645  is associated with highest CIPN risk  to Model 2 did not change model performance . This reduction in velocity might be perceived by the brain as more severe CIPN. Nerve conduction velocity studies might verify this hypothesis but are beyond the scope of this study.Peripheral neurotoxicity mechanisms of anticancer drugs are not fully understood and are likely to be complex. One hypothesis that could explain our findings centers on age-related changes in nerve conduction velocity. Because nerve conduction velocity is slower in older patientsp16 expression is modulated by p16 levels prior to chemotherapy. Tsygankov et al showed that p16 expression plateaus in late middle age after increasing exponentially with age in early to mid-adulthood47. Thus, participants with lower baseline p16 levels may have the capacity for a larger increase in p16 following chemotherapy, while participants with higher baseline p16 levels may have already reached a maximum threshold for senescent cell accumulation, suggesting that p16 levels cannot increase further without causing morbidity. p16Age Gap, measured at baseline, can therefore be interpreted as a predictor of patients that will accumulate more senescent cells in response to chemotherapy, leading to higher p16 expression and CIPN. Regardless of mechanism, our results, once validated, would allow identification of high risk patients, who could then consider CIPN prevention options like cryotherapy, substituting docetaxel for paclitaxel, or chemotherapy regimens without taxanes. Such high-risk patients would also be ideal candiates to be included in trials evaluating CIPN preventive strategies.Presently, it is unclear why chemotherapy-induced 32. However, in this study p16 expression was measured in dorsal root ganglia and not in blood. Although these discoveries may lead to ways to prevent CIPN through depletion of senescent cells (i.e. senolytic therapies), much work remains to understand human senescence and identify safe and efficacious senolytic therapies.While our study provides evidence for a connection between p16, senescence, and CIPN in humans, a causal relationship between senescence and CIPN has been demonstarted in mice. Removal of the p16+ senescent cells in cisplatin-treated mice completely reversed symptoms of CIPN48.In this study, we did not find diabetes, obesity, or age to be important contributors to multivariate models of CIPN. A larger study is now underway  that will provide the opportunity to validate p16Age Gap findings, further interrogate comorbidities such as diabetes and age as risk factors, and analyze other variables not available in this study such as taxane dose intensity and metabolism. There is also great interest in genetic polymorhisms as predictors of CIPN with different chemotherapeutic agents, including taxanes, which may ulimately improve risk predictionp16 versus age but, as noted above, the best fit between p16 and chronological age is likely non-linear later in life. This may explain the extreme negative p16Age Gap values for some patients in our analysis. We are currenlty conducting a study to build a computational model of senescence in multiple patient cohorts to provide a better estimator of p16Age Gap. But regardless of the absolute value for the p16Age Gap, low p16 expression in patients with higher chronologic age is a significant risk factor.In addition to the sample size, another limitation of this study is that measurement of p16Age Gap was calculated using linear regression of 27. Patients with age-inappropriate low p16 may be offered docetaxel regimens, possibly in combination with other efforts to reduce CIPN such as cryotherapy, or closer monitoring for CIPN symptoms26. Additionally, anthracycline regimens that lack taxanes but have similar efficacy but different toxicity profiles, might be preferable for some pateints where even moderate risk of loss of function due to neuropathy may be unacceptable .Our ongoing studies are designed to validate this p16Age Gap-based model for CIPN prediction for early-stage breast cancer patients and advance it into a lab-developed test to be used clinically to obtain a CIPN risk score. The planned studies will use patient reported outcomes (EORTC-QLQ-CIPN20) in addition to clinician-assessed (NCI-CTCAE) toxicity. If validated, this score may help to guide chemotherapy selection, given that regimens for this indication usually employ one of two different taxanes (paclitaxel or docetaxel) with similar efficacy but different risks of CIPN incidenceIn summary, measures of senescent cell load could ultimately help guide clinicians to avoid dose-limiting toxicities and improve quality of life in breast cancer patients. These observations may also be clinically relevant in other cancers that are treated with neurotoxic chemotherapies.7 and had p16 mRNA expression measures were included in this analysis. All patients who were offered and consented to adjuvant or neoadjuvant chemotherapy were eligible to participate. Studies were led by the University of North Carolina with REX Healthcare, Ohio State University, MD Andersen, and Duke University participating, and were approved by the IRB of participating sites. The study was performed in agreement with the guidelines of the International Conference on Harmonization, the ethical principles in the Declaration of Helsinki, and all applicable regulations. All patients provided written informed consent before participation in any study-related activities.Women newly diagnosed with stage I\u2013III breast cancer, enrolled in NCT02167932 or NCT02328313, who received a chemotherapy regimen containing a taxane7. For weekly regimens toxicity data was collected every other week so that all toxicity reports were either biweekly or triweekly. Briefly, CIPN symptoms were graded by an oncologist using the NCI-CTCAE v5 system. Symptoms were graded as none (0), mild (1), moderate (2), severe (3), or life-threatening (4). Clinicians assessed patients prior to each cycle of chemotherapy. In this study, measures to prevent CIPN such as cryotherapy or prescription medications were not captured as they were not widely utilized when the study was conducted.Chemotherapy regimens were classified as paclitaxel- or docetaxel-containing. CIPN toxicity data was collected as describedp16 mRNA expression was analyzed by real-time qPCR as described51, using reagents provided by Sapere Bio (SapereX). Positive and negative controls were included in each run; overall precision of p16 measurement  was 0.8 Ct. Each measurement was performed once on each sample.Peripheral blood samples were collected prior to starting chemotherapy and again at the end of chemotherapy, T cells were isolated, and p16 expression in 633 subjects52. The p16 value corresponding to the participants\u2019 chronological age at the start of chemotherapy was then subtracted from p16Age to calculate p16Age Gap.P16 expression levels were converted into equivalent years of aging (p16Age) using a linear regression formula derived from analysis of Chi-square tests, Fisher exact tests, and Student t tests were used to compare patient and clinical characteristics. All tests were two-sided with statistical significance set at 0.05. Analyses were conducted using SAS/JMP 15.1 software .p16 prior to chemotherapy, comorbidities, and their interactions were tested, as variables may not be independent. Comorbidities considered were obesity (BMI\u2009\u2265\u200930), diabetes, peripheral circulatory issues, osteoporosis, arthritis, high blood pressure, emphysema, and liver or kidney disease. Coronary heart disease and stroke were not used due to low prevalence in the CIPN group. Variables and their interactions were considered in the regression model and retained by forward stepwise addition to minimize the Akaike information criterion (AICc). The resulting CIPN probabilities were calculated and plotted. To determine the importance of each variable we calculated indices measuring the importance of factors in a model, in a manner independent of model type and fitting method. The fitted model is used only in calculating predicted values. This method estimates the variability in the predicted response based on a range of variation for each factor. If variation in the factor causes high variability in the response, then that effect is important to the model. Calculations assumed each variable was independent. In this analysis, for each factor, Monte Carlo samples were drawn from a uniform distribution defined by minimum and maximum observed values. Main Effect of importance reflects the relative contribution of that factor alone. Total Effect of importance reflects the relative contribution of that factor both alone and in combination with other factors. To mitigate overfitting when building a neural network model, 1/3 of the data set was reserved as a validation set using a holdback function and learning rate of 0.1. The model was built using a TanH activation function to fit one hidden layer with three nodes. The resulting probabilities of CIPN were calculated and plotted.To generate a multivariate linear regression model of CIPN risk, chronological age, race, p16 and CIPN incidence, p16 expression measured prior to chemotherapy was subtracted from p16 expression measured at the end of treatment and stratified into 2 groups: p16 increase  and no increase (p16 change\u2009\u2264\u20090.4). Fisher\u2019s exact test (2-sided) was used for group comparison.For the association between chemotherapy-induced change in Further information on research design is available in the Supplementary MaterialReporting Summary"}
{"text": "Four decades of p53 research since the discovery of this tumor suppressor have demonstrated that p53 prevents tumorigenesis by maintaining genomic stability and eliminates cancer cells by inducing cell growth arrest, necrosis, apoptosis, and ferroptosis . p53 actKung et al. gracefully described a canonical mechanism by which oncogenic stress induces p53 activation with some new information and thoughts. Specifically, the oncogenic c-MYC or RAS signaling induces the expression of ARF, an alternate open reading frame encoded by CDKN2A, which in turn activates p53 by interacting with and inhibiting MDM2 activity. Importantly, they also summarized several potential therapeutic strategies targeting the ARF-MDM2-p53 cascade, including small molecules, peptides, and the proteolysis targeting chimera (PROTAC) tactic. In a research study, Han et al. reported that the PARP inhibitor olaparib, a targeted therapy for cancers with BRCA1/2 mutations or homologous recombination deficiencies, can induce p53 activation via RPL5/RPL11-mediated inhibition of MDM2 by triggering nucleolar stress, demonstrating an additional action mode of PARP inhibitors by targeting the nucleoli and activating the p53 pathway.Blocking the MDM2-p53 feedback loop has been considered as a promising strategy to treat cancers harboring wt p53 for decades, although it has been quite challenging as there is not an applicable drug targeting this loop as an anti-cancer therapy in clinic use. In the issue, a review article by TP53, is the most frequently mutated gene in human cancers. The cancer-derived mutations of p53 include missense, frameshift, truncation, and deletion. Most of the p53 mutants are missense mutations that often occur in the DNA-binding domain of the p53 protein. These mutants not only lose their tumor inhibitory activity, but also exert a \u201cdominant-negative\u201d effect on the functions of wt p53. Remarkably, several hotspot mutants, such as mt p53-R175H, G245S, R248W/Q, R249S, R273H/C, and R282W, acquire GOFs to further promote tumor growth via diverse mechanisms (Madrigal et al. in this issue, mt p53 regulation of microRNA expression involves both transcription-dependent and -independent mechanisms. Mt p53 was recently found to associate with replicating chromatin and PARP1 to facilitate aberrant DNA repair (Annor et al. in this issue demonstrated that oligomerization of mt p53-R273H is not required for its chromatin association, though oligomerization of wt p53 is indispensable for its tumor suppressive activity. In a prospective essay by von Grabowiecki et al., the authors proposed a provocative idea that mt p53 might promote endosomal trafficking of a plethora of proteins involved in tumorigenesis and cancer progression by regulating Rab11-FIP1, which is supported by some recent studies as cited in this article and will await further validation.The p53-encoding gene, chanisms . Althougchanisms . As descA repair . An inteCui et al. offered a comprehensive review on the progresses of recent studies on the coordinated regulation of p53 and mTOR pathways in response to the physiological and genotoxic conditions. This is further consolidated by another review by Nagpal and Yuan, who elegantly collected numerous previous and new findings on the role of basally expressed p53 in restraining anabolic metabolism to prevent fast cell proliferation under non-stress conditions. In accordance, the tumor suppressive function of p53 has been also attributed to its activity to regulate glucose metabolism, lipid metabolism, amino acid metabolism, and iron metabolism in cancer cells, which are nicely illustrated in a review by Yu et al. Moreover, p53 has been shown to be involved in the regulation of recycling and clearance of metabolites, nutrients, and cellular debris. This line of information on wt and mt p53\u2019s new functions is systematically reviewed by Rahman et al. They offered another comprehensive and updated view on the roles of wt and mtp53s in the regulation of autophagy signaling and provided new insights into the therapeutic potential by modulating p53-mediated autophagy.Over the past years, growing evidence has revealed the crucial role of p53 in the maintenance of metabolic homeostasis and the prevention of cancer-associated metabolic remodeling . MammaliNagpal and Yuan described that basally expressed p53 is required for the maintenance of immune homeostasis. In addition, Shi and Jiang offered a detailed review on various mechanisms underlying wt and mt p53 regulation of inflammation and immunity. For example, p53 prevents inflammation-associated cancer development by suppressing NF-\u03baB and STAT3 signaling pathways (Zhang et al. showed that p53 mutation is associated the increased production of chemokines, leading to infiltration of different immunocytes in breast cancer. This suggests a complex tumor microenvironment in mt p53-harboring cancers. The development of vaccines targeting p53 has been an old, yet unsolved, topic, as both wt and mtp53 epitopes can be presented on the cell surface for T cell recognition (Zhou et al. offered a thoughtful review on the recent progresses of vaccination of p53 or its peptides and discussed the possibility and application of p53-targeting vaccines to cancer treatment. Collectively, these review and research articles as published in this issue not only show recent progresses in various regulations and roles of wt and mt p53 in cancer development, progression, and immunology, but also provide more new insights into the p53 anti-cancer functions and mt p53\u2019s oncogenic activities. Importantly, these articles also offer new thoughts and suggestions for targeting mt p53 as anti-cancer therapies, such as new potential approaches for developing p53 vaccines.The roles of wt and mt p53s in the regulation of inflammatory and immune responses have been a hot topic recently. To update this area of research, this issue has also collected several review and research articles. For instance, the review article by pathways , while mpathways . Moreoveognition . To updaAlthough the canonical functions of p53 as a key regulator of cell cycle, DNA repair, and apoptosis have been well documented, increasing studies have been continuingly unveiling novel roles of p53 in metabolic remodeling, immune surveillance, and cancer therapy, making this magic molecule as the most attractive research target as well as a promising therapeutic target for developing anti-cancer therapies in the future. We are deeply grateful to these authors who have made great efforts to our better understanding of new functions of p53 and new insights into this still mysterious molecule by contributing their comprehensive review and elegantly-designed research articles to this special issue."}
{"text": "Hidradenitis suppurativa/acne inversa (HS) is a chronic inflammatory disease of the pilosebaceous unit leading to formation of painful, inflammatory nodules, abscesses and tunnels in apocrine gland-bearing areas of the skin. Pain and drainage are the most important symptoms associated with reduction of quality of life in HS. On the other hand, an overlooked symptom in quality of life studies is itch, despite the fact that several studies have reported its importance. Various theories have tried to explain the pathogenesis of itch in HS, such as the presence of mast cells in the cell infiltrates and elevated Ig E levels in the lesional skin. Smoking and advanced stage of disease have been found to be associated with increased intensity of itch. A PUBMED search was conducted to perform a systematic literature review using the term \u201chidradenitis suppurativa\u201d [all fields], the keywords \u201cpruritus\u201d, \u201citching\u201d, \u201citch\u201d [all fields] and with \u201cAND\u201d as operator. Mast cells and mTor signaling were found to be raised in both lesional and perilesional skin. Itch as a presenting symptom has been found in 35\u201382.6% of patients across multiple studies. It often co-presents with pain and may be misinterpreted as burning, stinging, tickling, tweaking, prickling, etc. The presence of itch is associated with reduced quality of life, depression and impairment of social life. Brodalumab, a monoclonal antibody against IL-17A receptor, produced significant improvements in itch, pain, QoL and depression in patients with moderate to severe HS. Statins have shown some reduction in itch intensity score. Further studies are required to gain a better understanding of the etiopathogenesis and optimal therapeutic modalities for itch in HS that will allow clinicians to better address issue and reduce its impact on quality of life. Hidradenitis suppurativa/acne inversa (HS) is a chronic debilitating inflammatory skin disease of the pilosebaceous unit . It is cThe clinical features reported to most reduce the QoL of HS patients are pain and malodorous purulent discharge from skin tunnels. In a study conducted on 1795 subjects, pain was found in 83.6% cases [A PUBMED search was done for systematic review of literature using the term \u201chidradenitis suppurativa\u201d [all fields]; the keywords \u201cpruritus\u201d, \u201citching\u201d and \u201citch\u201d [all fields]; and \u201cAND\u201d as operator.The search yielded 105 articles; whereas after the removal of duplicates, 48 articles were left. A total of 28 articles were excluded due to eligibility. Finally, 20 articles were selected for the study. The selection of articles was performed independently by SBL and NSS. All articles were reviewed by the senior author, VRS, who also had the final decision in case of disagreement between the initial selectors .The presence of itch in patients with HS ranged from 62.1% to 77.5% in the selected studies ,19,25. TItch in HS has also been found to adversely affect QoL. In a study by Riis et al., a significant association was found between loss of utility and itch . A studyThe exact pathogenesis of itch in HS is not understood. In the studies by the Rotterdam group  has recently been identified as an important mediator of inflammation in HS. It has also been found to be significantly elevated in atopic dermatitis and cutaneous T-cell lymphoma, wherein it is likely to mediate the infiltration of eosinophils, basophils and T cells. Both conditions are also associated with high itch scores ,34.Itch is an important symptom in HS . In seveIn a retrospective data analysis conducted on 145 patients, itch was reported by 82.1% of the patients and correlated with Hurley stage . Vossen In a study evaluating the prodromal symptoms in HS, 20% patients experienced itching at least 24 h before the onset of any visible signs . ThroughEsmann et al.  studied Studies to assess the efficacy of various treatment modalities in HS associated itch are yet to be conducted. Few authors have associated pain with itch, and the alleviation of pain may reduce itch . Many toBrodalumab, a monoclonal antibody against IL-17A receptor, produced significant improvements in itch, pain, QoL and depression in patients with moderate to severe HS . StatinsJAK inhibitors have recently been used in treating HS successfully. INCB54707, a experimental molecule which is 52 times more selective for JAK1 than JAK2, has been tried in two multicentric phase 2 studies, wherein a single dose of 90mg oral treatment resulted in a hidradenitis suppurativa clinical response (HiSCR) (defined as at least a 50% reduction in inflammatory lesion count with no increase in abscesses or draining fistulae compared to baseline) score of 88% at week 8. A dose-dependent significant improvement in hidradenitis suppurativa quality of life scores was documented in response to the JAK1 inhibitor .Recent studies have shown that JAK inhibitors can reduce itch symptoms in patients with atopic dermatitis. It is suggested that JAK inhibitors act by blocking neuronal JAK1 signaling in non-inflammatory settings. JAK inhibition with tofacitinib were tried in five patients with severe chronic inflammatory pruritus off-label; all patients showed reduced itch scores, despite having failed to respond to multiple other treatments. These findings suggest the potential use of JAK inhibitors in alleviating itch symptoms in HS .p = 0.0001) at 24 weeks, which was statistically significant. Improvement in DLQI was significant in 64% of patients. In addition, depression, which was earlier noted to be severe in 11 patients, became nonsevere in 7 of the 11 patients [Studies have demonstrated the efficacy of metformin in treating recalcitrant HS. Metformin at a dose of 500 mg 3 times daily for 24 weeks resulted in an average reduction in Sartorius score by 12.78 (patients . Arun anpatients . The mecpatients .Despite a lack of complete understanding of itch in HS, it is an important aspect of the disease, reported in 62% to 75% of patients. Further study to gain a better understanding of the etiopathogenesis and optimal therapeutic modalities for itch in HS will allow clinicians to better address this aspect of the disease and reduce its impact on quality of life."}
{"text": "SQSTM1/p62 plays a key role in the autophagic machinery and may serve as a marker for autophagic flux in vivo. We investigated the role of p62 in neurodegeneration, analyzing its concentrations in the CSF of AD and FTD patients. We recruited 76 participants: 22 patients with AD, 28 patients with FTD, and 26 controls. CSF p62 concentrations were significantly increased in AD and FTD patients when compared to controls, which persisted after adjusting for age . In female FTD patients, p62 positively correlated with the neurodegenerative biomarkers t-Tau and p-Tau. A significant correlation between CSF p62 concentrations and several clinical features of AD was found. Our data show that p62 is increased in CSF of AD and FTD patients, suggesting a key role of autophagy in these two disorders. The levels of p62 in CSF may reflect an altered autophagic flux, and p62 could represent a potential biomarker of neurodegeneration.Several studies have revealed defects in autophagy in neurodegenerative disorders including Alzheimer\u2019s disease (AD) and frontotemporal dementia (FTD).  Neurodegenerative disorders such as Alzheimer\u2019s disease (AD), frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and Parkinson\u2019s disease (PD), are characterized by the progressive deposition of misfolded and aggregated proteins that lose their physiological functions and acquire neurotoxic effects . In receSQSTM1, OPTN, and VCP are directly involved in protein degradation pathways as their products contribute to recruitment of ubiquitinated proteins to the autophagosome, a key component of autophagy [Autophagy is characterized by a complex cellular process that leads to the clearance of misfolded or damaged proteins and dysfunctional organelles . This coutophagy .SQSTM1 gene and involved in several functions, including autophagy, apoptosis, inflammation, and cell survival, suggesting a role as a signaling hub that regulates cell viability in response to cytotoxic stress [SQSTM1 gene were initially described in Paget\u2019s disease of bone [Human p62 is a scaffold protein of 440 amino acids, coded by the c stress ,11. Muta of bone , a chron of bone  and FTD  of bone ,15. In n of bone .Cerebrospinal fluid (CSF) is enriched in brain-derived substances, and CSF biomarkers might represent a valid instrument to assess neuropathology in vivo. At present, however, there are no established CSF biomarkers for monitoring brain autophagy in vivo. In 2017, Au et al. examined p62 concentrations in the CSF of 30 children with severe traumatic brain injury (TBI) and found that p62 concentrations were significantly increased, suggesting an impairment of the autophagic flux .To date, a few studies have investigated CSF p62 concentrations across different neurodegenerative disorders. Thus, the primary aim of this study was to evaluate whether p62 concentrations were detectable in the cerebrospinal fluid of patients with AD or FTD, comparing them with a control population. In addition, we investigated correlations between this autophagy biomarker and clinical or radiological characteristics of AD and FTD.Seventy-six consecutive patients referred to the \u201cRita Levi Montalcini\u201d Department of Neuroscience of the University Hospital Citt\u00e0 della Salute e della Scienza di Torino  were selected for the study. Twenty-two patients with AD  and 28 patients with FTD  were enrolled.GRN, MAPT, SQSTM1 (that encodes p62), and C9orf72 genes.Clinical diagnosis of probable AD was established according to joint Alzheimer\u2019s Association Workgroup and National Institute of Aging guidelines  and the Twenty-six cognitive-spared patients  with neurological conditions other than neurodegenerative disorders were included as a control group, including patients with suspected acute polyneuropathy (n. 7), muscular dystrophy (n. 3), multiple sclerosis (n. 3), and normal pressure hydrocephalus (n. 13). The demographic and clinical features are summarized in g for 10 min at room temperature, subsequently transferred into aliquot tubes, and stored in a \u221280 \u00b0C freezer.CSF sampling and collection were performed according to previously published protocols and guidelines . After oThe CSF p62 levels were evaluated using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Cloud-Clone Corp). The limit of detection was 0.1 ng/mL for p62, and samples below the level of detection were assigned a value of 0 ng/mL. All samples were assayed in duplicate. CSF concentrations of A\u03b242, t-Tau, and p-Tau181 were also evaluated using the ELISA method with available commercially Innotest Kits (Fujirebio), according to the manufacturer\u2019s instructions.p value of <0.05 was considered significant in biochemical and clinical comparisons.All statistical analyses were performed using SPSS version 20 . Continuous variables were presented as means \u00b1 standard deviations or medians and range, and categorical ones as count and percentage. We compared diagnostic groups  using ANOVA for normally distributed variables and Kruskal\u2013Wallis and Mann\u2013Whitney U tests for non-normally distributed variables, as appropriate. Correlations between p62 and CSF biomarkers were also evaluated using Pearson\u2019s test, also adjusted for age. The statistical models were adjusted for possible confounders. A two-tailed No difference in education, comorbidities, or smoking habits was found between cases and controls. The gender distribution was comparable in the control and FTD/AD cohorts. Patients with AD and FTD did not differ in age, whereas control subjects were younger than patients with dementia .p = 0.011) (p = 0.01). The p62 concentrations were not different in AD males with respect to male controls, whereas female AD patients showed higher p62 levels when compared to female controls  (p = 0.779).The p62 concentrations were found to be significantly different between AD patients and controls  , which p= 0.044) . No signp < 0.001) (p = 0.008). When analyzing the gender effect, p62 concentrations were found to be significantly different between male FTD patients and male controls , and between female FTD patients and female controls .Crude p62 levels were significantly increased in FTD patients when compared to controls  , which pp = 0.002) and p-Tau in the female FTD subgroup alone   .p = 0.041) (p = 0.033), and depression . Furthermore, p62 concentrations were negatively correlated with temporal atrophy in the AD subgroup , mainly in the male subgroup . No correlation between p62 levels and MMSE was found  a. In AD as found b. FinallNo correlations between p62 levels and the collected clinical characteristics of FTD patients were found. In particular, no correlation between p62 CSF concentration and neuropsychological test results was found . In addition, we did not find any correlations between p62 levels and severity of the disease.SQSTM1 gene; we only found the single-nucleotide polymorphism rs186996560 in the SQSTM1 in a FTD patient. In our FTD group, p62 levels were elevated in patients both carrying and not carrying mutations in known causative genes in respect to controls. No significant correlation was found between p62 levels and the presence or absence of gene mutations (p = 0.317).In this cohort of patients, we did not find any mutations in the To our knowledge, this is the first study to investigate CSF p62 concentrations as marker of autophagy in patients with dementia. We showed that CSF p62 concentrations were significantly increased both in AD and FTD patients in comparison to controls. Furthermore, in female patients with FTD, we found that CSF p62 concentrations correlated with markers of neurodegeneration like p-Tau and t-Tau. In patients with AD, several neuropsychological characteristics of the disease showed a significant correlation with the investigated biomarker of autophagy. Taken together, our data support the hypothesis that CSF p62 concentrations may be a useful in vivo biomarker of neuronal autophagy.Increased CSF p62 concentrations in patients with neurodegenerative disorders may have several neurobiological explanations. First, p62 is involved in several cellular processes relevant to aging, including protein degradation , mitophaThe different forms of frontotemporal dementia are characterized by the aggregation of insoluble proteins within cells. The accumulation of aggregates is often due to genetic mutations in their coding genes. Most inclusions in FTD are characterized by immuno-reactivity, and the neurons and glia of FTD patients are characterized by p62 accumulations.MAPT gene mutation. In addition, we found that that CSF p62 levels correlate with biomarkers of neurodegeneration in FTD, and p62 concentrations were positively correlated with t-Tau and p-Tau. Many studies have shown that autophagy deficiency can cause tau accumulation [It has been shown that p62 represents a substrate for macroautophagy; hence, p62 levels increase when macroautophagy is inhibited, and its concentration decreases when macroautophagy is induced . In thismulation ,33. Incrmulation . In mousmulation . In a remulation . P62 mayThis is the first study to demonstrate an increase in p62 concentrations in the CSF of patients with FTD and AD; in view of the limited samples, the results obtained and their conclusions should be interpreted with caution, and further studies are needed to confirm our data. Autophagy represents a dynamic process, and our results may change according to different stages of neurodegeneration, so studies involving subjects at different stages of disease are desirable. Finally, a further investigation of other different autophagic biomarkers may be crucial for a better understanding of the autophagic machinery in neurodegenerative disorders.Our study showed a significant increase of CSF p62 concentrations in patients with Alzheimer\u2019s disease and frontotemporal dementia, supporting an important role of autophagy alterations in these neurodegenerative conditions. In addition, a significant correlation between increased autophagy and clinical characteristics of dementia was found. However, due to the relatively small number of subjects examined, replication studies in larger series are warranted in order to confirm our data."}
{"text": "Sexuality is a growing field in the context of the management of chronic diseases and cancer in particular. Cancer treatments and the traumatic nature of the cancer experience frequently elicit considerable sexual difficulties.To assess the prevalence of sexual dysfunction (SD) in patients with cancer, and to determine the associated factors.This was a cross-sectional study, conducted over 1 month, involving 100 cancer patients followed in the oncology department at the Habib Bourguiba University Hospital in Sfax (Tunisia). General, clinical and therapeutic data were collected using a pre-established questionnaire. Sexual function was assessed with the \u201cFemale sexual Function Index\u201d and the \u201cInternational Index of Erectile Function\u201d.These results showed that half of the patients were female, and 70% of them were married. Their mean age was 51.96, and 68% of them were unemployed. Unemployment in men and treatment with chemotherapy were statistically associated with erectile dysfunction (p=0.049 and p= 0.001 respectively). treatment with radiotherapy was statistically associated with decreased desire in men (p=0.048). Depression correlated with a decreased orgasm (p=0.032) and erectile dysfunction (p=0.043) in men, mean score of IIEF (p= 0.019) and with a decreased sexual arousal (p=0.006) in women.Sexual dysfunction is common in cancer patients. They can be of iatrogenic or psychological origin and can depend on the dynamics of the couple relationship. Training to raise awareness of the importance of sexuality first among cancer patients should be considered given the lack of communication between doctors and patients regarding sexuality issues.No significant relationships."}
{"text": "Laryngeal Squamous Cell Carcinoma (LSCC) is a squamous cancer with 2.4% new diagnoses each year, accounting for 25% of head and neck cancers, and has a high mortality rate. A deeper understanding of key mechanisms and knowledge of the putative target molecules of theranostic relevance are required. The receptor for neurotrophins p75NTR has been shown to be highly expressed in cancer stem cells (CSCs) of squamous epithelia, in LSCC as well as in other cancers. However, whether its cleavage product p75ICD expression, known to finely regulate the survival/death balance in neurons, is also a master regulator in cancer and LSCC in particular has not been directly addressed so far. To resolve this question, we performed a preliminary study using a limited number of LSCC specimens and studied p75ICD presence and expression pattern in LSCC specimens, showing that p75ICD may be a promising target in LSCC, requiring further investigation.We investigated the p75 Neurotrophin Receptor (p75NTR) expression and cleavage product p75NTR Intracellular Domain (p75ICD) as potential oncogenic and metastatic markers in human Laryngeal Squamous Cell Carcinoma (LSCC). p75NTR is highly expressed in Cancer Stem Cells (CSCs) of the laryngeal epithelia and it has been proposed as a marker for stemness, cell migration, and chemo-resistance in different squamous carcinomas. To investigate the clinical significance of p75NTR cleavage products in solid tumors, full-length and cleaved p75NTR expression was analyzed in laryngeal primary tumors from different-stage LSCC patients, diagnosed at the Policlinico Umberto I Hospital. Molecular and histological techniques were used to detect the expressions of p75NTR and p75ICD, and ATP Binding Cassette Subfamily G Member 2 (ABCG2), a CSC marker. We found regulated p75NTR cleavage during squamous epithelial tumor progression and tissue invasion. Our preliminary investigation suggests p75ICD expression and localization as possible features of tumorigenesis and metastaticity. Its co-localization with ABCG2 in squamous cells in the parenchyma invaded by the tumor formation allows us to hypothesize p75NTR and p75ICD roles in tumor invasion and CSC spreading in LSCC patients. These data might represent a starting point for a comprehensive analysis of p75NTR cleavage and of its clinical relevance as a potential molecular LSCC signature, possibly helping diagnosis, and improving prognosis and personalized therapy. Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth-most-common cancer worldwide, and Laryngeal Squamous Cell Carcinoma (LSCC) accounts for approximately 10% of all HNSCCs [Despite recent progress in the treatment of HNSCC, including surgery, radiotherapy, and medication-based therapy , a major impact on the survival rate is still lacking and it has remained substantially unchanged over the last 30 years. The general survival rate for LSCC is 61% at 5 years, but, analyzing by tumor stage, the cure rates increase up to 80\u201390% in case of limited disease T1 and T2). In case of local advanced diseases (T3 and T4) or regional lymph node involvement, the survival rate is <50% at 5 years [. In caseIn line with this, there is an unmet medical need for innovative treatment strategies, which are only possible through the investigation of the molecular mechanisms underlying LSCC development and progression, and the discovery of targetable signaling. In particular, characterization at the cellular and molecular levels of chemotherapy-resistance and invasion key players will be critical for the design of new drugs, as well as for the improvement of the clinical management of LSCC patients.Accumulating evidence over the last decade pinpointed the p75 Neurotrophin Receptor (p75NTR) as a potential malignant marker in cancer. For example, p75NTR has been implicated in melanoma ,4,5,6, aHowever, the described biological significance of p75NTR modulation may vary in different types of tumors, as it exerts malignant effects, such as the invasion depth of the cancerous tissue and lymph node metastasis in esophageal cancer . On the In prostatic tissues, p75NTR is absent in hyperplastic benign epithelial cells, and it is specifically overexpressed in prostatic cancer cells, where it correlates with high-risk tumors with an unfavorable prognosis (Gleason score > 7) . MoreoveOf interest for the present study, p75NTR may undergo regulated cleavage, generating intracellular fragments able to translocate to the nucleus harboring specific signaling activity, as already demonstrated for the pro-apoptotic activity of the p75NTR Intracellular Domain (p75ICD), released upon the \u03b3- and \u03b1- secretases\u2019 sequential cleavages of the p75NTR in neurodegenerating neurons of Alzheimer\u2019s Disease-affected brains .p75NTR-regulated cleavage has been hypothesized to also occur in some cancer types and in tumor cellular models. For example, p75NTR enzymatic maturation has been shown to be critical for glioma invasion and spreading .However, direct proof of the regulated cleavage of p75NTR leading to the generation of p75ICD in cancer cells is still lacking in most malignant tumors, including LSCC.Here, we used the specific anti-p75ICD antibody, which does not cross react with the p75NTR Full Length (p75NTR FL) molecule, to investigate its expression and localization in primitive LSCC and its nodal metastasis in a small group of patients representative of the diverse stages of LSCC.Furthermore, since p75NTR is also present in Cancer Stem Cells (CSCs) and it is co-expressed with stemness markers, such as CD133 in LSCC , and witThe present study firstly detected the expression pattern of p75ICD and compared it to the p75NTR FL in LSCC, and revealed that p75ICD was particularly upregulated in neoplastic squamous cells expressing ABCG2 and was strongly associated with tumor progression. Moreover, p75ICD expression was cytosolic in stages II-III and became nuclear in neoplastic epithelia and in ABCG2-positive CSCs.Although these results are arising from pilot study, and therefore need to be further explored in a larger patient group, our findings indicate that p75ICD generation by p75NTR-regulated cleavage might represent a relevant marker of tumor invasion and metastasis in LSCC, and support a role for p75NTR/p75ICD in the regulation of CSC proliferation.We evaluated laryngeal specimens from LSCC surgically removed at the Otolaryngology Unit, DAI Testa-Collo, Policlinico Umberto I University Hospital, 00161 Rome, Italy, from April 2016 to April 2018. All patients included in the study were affected by LSCC and subjected to open partial horizontal laryngectomy or total laryngectomy. Specimens were processed and evaluated at the Section of Pathology of the same hospital, where a definitive histological diagnosis was provided.The inclusion criterion was confirmed laryngeal cancer treated with surgical therapy. The exclusion criteria were previous radiation therapy on the head and neck region and neoadjuvant therapy. All patients gave written informed consent for inclusion in the study. The use of these clinical materials was approved by the institutional ethics committees (RIF. 6129).The stage of LSCC was classified according to the 8th edition of the American Joint Committee on Cancer . Relapse-free survival was defined as the interval between the date of surgery and the date of recurrence or last follow-up available.The study was performed on a total of 8 LSCC patients . In 3). Different tumor characteristics were evaluated, including the location , involved side , size, pattern of growth (exophytic or endophytic), ulceration, depth of invasion, spread to extralaringeal tissues, color, and features of the mucosa near the neoplasia. Data regarding the presence/absence of neo-plastic residual on resection margins was evaluated through ink application. Different sections of each tumor were sampled and collected so as to be representative of the maximum tumor invasion and of the interface between the tumor and adjacent normal mucosa. The pathological evaluation also included the characteristics of the lining epithelium of the mucosa adjacent to the tumor . All the lymph nodes that could be found from every specimen were also sampled for histological evaluation. Four-micrometer-thick tissue sections were cut from each paraffin block and used for hematoxylin\u2013eosin staining  . Each spn = 1), stage III (n = 1), stage IVa (n = 1), and stage IVb (n = 1) LSCC samples. Antigen retrieval was achieved by using sodium citrate buffer (pH 6.0) for 30 min at 37 \u00b0C on deparaffined sections. The slides were then cooled to Room Temperature (RT) and washed three times with phosphate-buffered saline (PBS) solution. For autofluorescence quenching, the slides were incubated with ammonium chloride  for 30 min, and then blocked with 10% normal donkey serum . The overnight incubation (4 \u00b0C) with primary antibodies  was followed by incubation with the appropriate combination of secondary antibodies. In particular, highly cross-adsorbed donkey anti-mouse-546 , donkey anti-rabbit-546 , and donkey anti-rat-488  were used for double immunofluorescence (IF) . Nuclei were counterstained with 1 \u00b5g/mL 4\u2032,6-diamidino-2-phenylindole .Four surgical specimens of the larynx  were selSlides were coverslipped using ProLong Diamond mounting medium with anti-fading . Slide imaging was performed with the laser-scanning confocal microscope (Olympus FV1200) using a 20\u00d7 air and a 40\u00d7 (NA = 1.25) oil immersion lens, and visualized with FV10-ASW software . An UV diode laser operating at 405 nm, an Argon laser at 488 nm, and a HeNe laser at 543 nm were used as excitation sources. To entirely reconstruct the longitudinal tumor tissue, 14 confocal Z-stacks (1024 \u00d7 1024 pixels) were collected at 0.29 \u00b5m intervals in a 4 \u00b5m total optical depth, and then converted into max projection images. Omission of the primary antibody was routinely performed as a control for antibody specificity.Images for direct comparison were collected using the same parameters and were analyzed with the help of FIJI open-source software .In order to compare the expressions of p75ICD and p75NTR in the resected specimens at different stages, they were evaluated by means of a semi-quantitative immunohistochemical assessment based on the intensity of immunoreactivity and its distribution ,19. The For Western Blot (WB) samples, 50 \u00b5g of protein was separated on precast 4\u201312% gel. At the end of electrophoresis, the proteins were stained with Ponceau Solution. Non-specific protein-binding sites on the nitrocellulose blots were blocked with PBS containing 7% non-fat milk (PBS\u2013milk) for 1 h at RT. The primary antibodies were diluted in PBS at the following dilutions: 1:1000 for p75NTR C-terminal, 1:200 for ABCG2 (BXP-53: sc-58224), and 1:1000 for GAPDH antibodies. The incubation of the nitrocellulose blots with these antibodies was performed overnight (4 \u00b0C) at RT. The blots were then washed three times with PBS and incubated for 4 h at RT with the appropriate peroxidase-conjugated antibodies diluted 1:1000 in PBS\u2013milk. Following PBS washes, the peroxidase activity of the nitrocellulose-bound secondary antibodies was detected with the Clarity West-ern ECL chemiluminescent reagents (BioRad Laboratories), imaged by Chemidoc XRS+ Image System (BioRad Laboratories), and bands were quantified by gel densitometry using the Fiji (ImageJ 1.53c) software . All the whole Western blot figures can be found in the We analyzed the p75NTR FL level and cleavage by using a C-terminal-specific anti-p75NTR antibody able to recognize both the p75ICD and the p75NTR FL by a biochemical assay. The p75NTR FL level was found to be detectable and high in all LSCC cases, while it was almost undetectable in the paratumoral tissue . MoreoveThe ABCG2 expression was also investigated in the same samples to compare the p75NTR and CSC marker expression levels in the different LSCC stages, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control protein for normalization. ABCG2 is a family member of the ABC transporter, and its positive expression in laryngeal carcinoma has been related to tumor differentiation, age, sex, and presence of loco-regional lymph node metastasis. It has been previously suggested that the abnormal expression of ABCG2 may be related to the invasion and metastasis of laryngeal carcinoma, thus providing a good reference basis for the evaluation of LSCC invasivity .The analysis of the tumoral marker in stages III and IV, with  and without  lymph nodes metastases, confirmed that LSCC cancer showed the maximal ABCG2 expression detectable by WB at stage III and further increased at stage IVa just before metastatization, being downregulated in the more advanced stages (IVb) as compared with previous tumoral stages.Interestingly, initial p75ICD positivity could be observed at stage IVa, corresponding to the highest ABCG2 level. p75ICD was still high with an apparent slight increase at stage IVb of LSCC tumors.As mentioned in In line with the histological characteristics of LSCC tissue , p75ICD-p75ICD was weak in stage II, while a progressive increase in the number of p75ICD-positive cells was noticeable from stages III to IVb, where a further increase in the single cell expression level was found in cells with enlarged nuclei, most of them also positive for the ABCG2 cancer marker. p75ICD was co-expressed by ABCG2-positive cells from stage II onward, while p75ICD/ABCG2 co-localization could not be detected in stage II stromal cells.Indeed, the p75ICD staining could be cytosolic, as well as nuclear, while ABCG2 staining was mainly cytosolic and punctuate, in line with its expression in extracellular vesicles , aberrant epithelium (2), the carcinoma invasive front (3), and carcinoma in situ (4) .In As reported in Zones 3\u20134, p75ICD expression was prominent in cancer cells with a highly dysmorphic appearance and enlarged nuclei, and was more evident in Zone 4 as compared with Zone 3.Note that not all of the p75ICD positive cells with aberrant morphology also expressed ABCG2.Representative pictures of the co-localization of the p75NTR (red) and ABCG2 (green) in Zones 1\u20134 from a stage III LSCC patient are shown in p75NTR FL and its C-terminal fragment p75ICD showed not only differential expression levels, but also distinctive intracellular localization. In fact, while the p75NTR FL was found to be mainly cytosolic a, the p7Interestingly, the p75ICD staining was also found to be nuclear in stage II epithelial cells b.The present pilot study investigated the occurrence and, eventually, the pathological significance of the p75NTR-regulated cleavage by \u03b1- and \u03b3-secretases, and the consequent release of the intracellular p75ICD fragment in LSCC. In particular, the study addressed the question of whether the distribution of p75ICD fragments might differ from the p75NTR FL, the expression of which has been largely described in several cancer types ,14,16, aNGF and its receptors (TrkA and p75NTR) are implicated in cancer growth, CSC proliferation, and immune system evasion . p75NTR Here, we used the specific anti-p75ICD antibody that does not cross react with the p75NTR FL molecule to investigate its expression and localization in primitive LSCC and its nodal metastasis in a small group of patients representative of the diverse stages of LSCC. Furthermore, since p75NTR is also present in CSCs and is co-expressed with stemness markers, such as CD133 in LSCC , and witTo resolve this lack of information, we first asked whether p75NTR undergoes regulated cleavage also in squamous cancers, such as LSCC, as it has been extensively reported in brain neurodegeneration and gliomas . We founIn fact, most of the findings reported by the literature do not allow discriminating between the two forms of the p75NTR, lacking the critical knowledge to target the p75NTR signaling for LSCC therapy. A deeper understanding of the opposite p75NTR and p75ICD-mediated biological functions could also help in explaining some apparently contradictory effects of p75NTR in cancers. In fact, behind the cancer-specific developmental mechanisms, p75NTR has been reported to be pro-apoptotic in gastric cancer  and prolIn order to specifically investigate p75ICD\u2019s biological action by immunofluorescent labeling in LSCC specimens, we resorted to a validated and specific anti-p75ICD antibody , and a cThus, we performed double IF staining for p75NTR/ABCG2 and p75ICD/ABCG2, and scored immunopositivity grading by the semiquantitative assessment method based on weak (+), medium (++), and strong (+++) immunolabeling .First of all, we observed that the two molecules had distinct patterns of expression  in the fThen, we investigated p75ICD expression at different LSCC stages (II-IV) and found a progressive increase in the number and intensity of p75ICD-immunolabeled cells from stage II toward stage IV ; Table 2Additionally, we explored p75ICD expression and localization in the four different zones of the single LSCC tumor specimen ; Table 3Additionally, we studied the p75NTR level and distribution in adjacent sections and found that p75NTR expression increased in aberrant , Zone 2 These observations are partially in line with the study by Li and collaborators, in which the immunohistochemical localization of p75NTR was reported in normal, paratumoral mucosa, and LSCC without any significant difference in the p75NTR level between dysplastic epithelia and LSCC. Unlike the specific antibodies used in this study and directed against the extracellular domain (p75NTR FL) and, specifically, the ICD domain (p75ICD) of the p75NTR, Li\u2019s study reported data obtained using an anti p75NTR C-terminal antibody, which virtually recognized p75NTR and all of its fragments, including the p75ICD . This fact might account for the difference in the trend and distribution, and also for the lack of correlation between p75NTR expression, histopathologic grading, and lymph node metastases. The same authors observed that \u201cwith the deterioration of differentiation, the ratio of p75NTR-positive cell increased partly, but the coloration intensity of each tumor cell degraded, with very faint cytoplasm coloration of p75NTR\u201d . Since wIn accordance, we found that the p75ICD pattern of expression in the different LSCC zones correlated with ABCG2 expression better than the p75NTR profile, as reported in Furthermore, p75ICD vastly co-localized with ABCG2-positive cells, some expressing low-intensity ABCG2 immunolabeling, and others morphologically abnormal with both intracellular and extracellular ABCG2 staining . Note thBased on the critical role of p75ICD nuclear translocation for its functions in the central nervous system, the subcellular localization of p75ICD (cytosolic versus nuclear) was also assessed in the resected specimen from LSCC stages II-IV. In agreement with the augmented nuclear p75ICD staining reported in neurodegenerating neurons of Alzheimer\u2019s Disease patients, we found prevalent p75ICD nuclear localization in aberrant epithelia from LSCC tumor stage II b, as welThe p75NTR has been demonstrated to be a central regulator of cancer invasion in highly invasive glioma , and itsTherefore, our study showing that the p75NTR and expression patterns differ in a stage-specific and zone-related manner, and that p75ICD is co-localized with the metastatic marker ABCG2 and shows a prevalent nuclear expression in aberrant and highly dysmorphic cancer cells, led us propose p75ICD as a good candidate prognostic factor in LSCC. Moreover, the presence of p75ICD in ABCG2-positive cancer cells surrounded by putative ABCG2 positive vesicles (as visible in Larger studies or meta-analyses will eventually help to reach sufficient statistical power to assess the utility of p75ICD as a prognostic factor in LSCC.Of note, p75NTR expression has been suggested by others to be prognostic when in combination with the marker CD44 in esophageal, hypopharyngeal, and oral carcinomas, as well as in HNSCC . DespiteAn in-depth study and revelation of the molecular mechanisms controlling normal epithelia and those events underlying its transformation toward metastatic SCC is of foremost importance in the quest for novel biomarkers improving early diagnosis and personalized formulation in the clinical treatment of LSCC.Although these results arose from a pilot study, and therefore need to be further explored in a larger patient group, our findings indicate that p75ICD generation by p75NTR-regulated cleavage might represent a relevant marker of tumor invasion and metastasis in LSCC, also supporting a role for p75NTR/p75ICD in the regulation of CSC proliferation.p75NTR was implicated in the control of the apoptotic machinery in neuronal cells and was indicated as a good pathological marker for the progression and spreading of both central nervous system cancers and non-neuronal carcinoma, such as LSCC. Although the exact mechanism is not clear, it is highly likely that p75NTR may predispose tumor cells to perineural invasion, conferring responsivity to NGF-producing Schwann cells and neurons, as reported in oral cancer and pancreatic cancer ,33. FurtOverall, it is advisable to foster our knowledge on p75NTR cleavage control at different stages of tumor development and specifically investigate p75ICD as a novel prognostic factor possibly improving diagnosis and instructing personalized treatment in LSCC, as well as other squamous cancers."}
{"text": "Literature suggests that health professionals (HPs) can play an important role in promoting physical activity (PA) in cancer patients . This study had three purposes: (1) analyzing HPs' knowledge and opinion about PA for cancer patients; (2) examining practices, barriers and roles that they identify when considering PA promotion in their patients; (3) determining factors that may enhance practices in this field.This study was designed in two phases and took place in Wallonia, Belgium. The first phase was qualitative: 12 HPs were interviewed about their experiences with PA in cancer patients. Then, in the second (and quantitative) phase, an online survey was created based upon the results of the first phase and upon the relevant literature. We sent this second-phase survey to professional organizations, hospitals and medical health centers and received 68 responses.In both phases of the study, HPs seemed to be aware of the benefits of PA for cancer patients and considered that they have to play a role in promoting PA in that specific population. However, only 25% (n = 17) of HPs were familiar with the official PA guidelines. According to our results, HPs discuss the topic of PA with more than 7 out of 10 patients (7.11 \u00b1 2.61). Lack of time and lack of knowledge about PA were identified as barriers to PA promotion. Finally, other relationships that may help to explain the role of HPs in PA promotion were uncovered: clinicians who used more information (e.g. PA benefits) (p = >0.001), exercised referrals (p = 0.012), and prescribed PA (p = 0.007) had higher intervention rates.Our findings suggest that improving HPs' knowledge about PA guidelines and how it can be proposed to cancer patients could help them to promote PA and to encourage their patients to consult a PA specialist, and eventually adopt a physically active lifestyle."}
{"text": "Tp53 gene. These mutations cause a disturbance in cellular response to stress, and eventually, cancer development. Apart from the full-length p53, at least twelve isoforms of p53 have been characterized. They are able to modulate p53 activity under stress conditions. In 2020, almost a half of million people around the world were diagnosed with renal cancer. One genetic disturbance which is linked to the most common type of kidney cancer, renal cell carcinoma, RCC, occurs from mutations in the VHL gene. Recent data has revealed that the VHL protein is needed to fully activate p53. Disturbance of the interplay between p53 and VHL seems to explain the lack of efficient response to chemotherapy in RCC. Moreover, it has been observed that changes in the expression of p53 isoforms are associated with different stages of RCC and overall survival. Thus, herein, an attempt was made to answer the question whether p53 and its isoforms are important factors in the development of RCC on the one hand, and in positive response to anti-RCC therapy on the other hand.p53 is a transcription al factor responsible for the maintenance of cellular homeostasis. It has been shown that more than 50% of tumors are connected with mutations in the  Consequently, kidney cancer is the 16th most common cancer globally . Kidney cancer  is one of the most common tumors, aside from bladder cancer of the urinary system . In 2020globally A. The stglobally B. In 202There are several types of kidney cancer. The most common type, however, is renal cell carcinoma (RCC) which arises from renal tubular epithelial cells, and it accounts for approximately 90\u201395% of all kidney cancer cases ,2. The mVHL gene result in the deregulation of expression of the hypoxia-inducible factor (HIF), which plays a crucial role as a transcription factor in oxygen regulation . M. MTp53 gTp53 gene/p53 protein (Tp53/p53) observed in renal cancer? Surprisingly, the level of mutations in Tp53/p53 seems to be quite low in kidney cancer compared to other cancers. According to the Catalogue Of Somatic Mutations In Cancer, COSMIC, those mutations are observed in 8.75% of total mutated samples from kidney  treatment has been varied [Are p53 isoform expression patterns associated with renal cancer development? Earlier studies using tissue samples of 45 RCC patients at different tumor stages and 25 non-neoplastic tissues have shown that expression of almost all p53 isoforms changed during cancer development and progression . An incrn varied . Thus, tn varied .More recently, it has been shown that p53\u03b2 is associated with better recurrence-free survival, RFS (the length of time after primary treatment for a cancer ends that the patient survives without any signs or symptoms of that cancer), and overall survival . AnalysiIt emerged that changes in p53 isoforms are somehow associated with different stages of RCC; in particular, a high level of p53\u03b2 seems to be an important molecular indicator of better prognosis for patients. Analyses of p53 isoform patterns in RCC patients could help predict how cancer will progress and what treatment is potentially effective.Tp53 gene is infrequently mutated in RCC, the presence of a mutated version of p53 influences clinical prognosis and overall survival [It has been shown that the crosstalk between p53 and VHL is a pivotal element in the DNA-damage response mediated by p53 in RCC . VHL-delsurvival ,39,40,41survival . It seemsurvival ,65. The survival , and by survival , which csurvival ,63,65. A"}
{"text": "The larvae of green lacewings and brown lacewings are called \u2018aphidlions\u2019, as they consume aphids. They play also an economic role as biological pest control. Aphidlions have, mostly, elongated spindle-shaped bodies, and similarly to most lacewing larvae, they possess a pair of venom-injecting compound jaws, also called stylets. Fossils that have been interpreted as aphidlions are known from amber of different ages . In this study, new aphidlion-like larvae are reported from about 100 million-year-old amber from Myanmar and about 35 million-year-old Baltic amber. The shapes of head and stylets were compared between the different time slices. With the newly described fossils and specimens from the literature, a total of 361 specimens could be included in the analysis: 78 fossil larvae, 188 extant larvae of brown lacewings, and 95 extant larvae of green lacewings. The results indicate that the diversity of head shapes stays about the same over time besides a certain increase in diversity of the head shapes in brown lacewing larvae. In certain other lacewings, a distinct decrease in the diversity of head shapes was observed in the larvae.Aphidlions are larvae of certain lacewings (Neuroptera), and more precisely larvae of the groups Chrysopidae, green lacewings, and Hemerobiidae, brown lacewings. The name \u2018aphidlion\u2019 originates from their ecological function as specialised predators of aphids. Accordingly, they also play an economic role as biological pest control. Aphidlions have, mostly, elongated spindle-shaped bodies, and similarly to most lacewing larvae they are equipped with a pair of venom-injecting stylets. Fossils interpreted as aphidlions are known to be preserved in amber from the Cretaceous (130 and 100 million years ago), the Eocene (about 35 million years ago) and the Miocene (about 15 million years ago) ages. In this study, new aphidlion-like larvae are reported from Cretaceous amber from Myanmar (about 100 million years old) and Eocene Baltic amber. The shapes of head and stylets were compared between the different time slices. With the newly described fossils and specimens from the literature, a total of 361 specimens could be included in the analysis: 70 specimens from the Cretaceous, 5 from the Eocene, 3 from the Miocene, 188 extant larvae of Chrysopidae, and 95 extant larvae of Hemerobiidae. The results indicate that the diversity of head shapes remains largely unchanged over time, yet there is a certain increase in the diversity of head shapes in the larvae of Hemerobiidae. In certain other groups of Neuroptera, a distinct decrease in the diversity of head shapes in larval stages was observed. The group Holometabola, with well-known representatives, such as beetles, bees, butterflies, or flies, represents a large share of animal biodiversity, especially in terrestrial ecosystems. Moreover, some smaller lineages are part of this diversity, such as Neuroptera, the group of lacewings. Neuroptera is generally understood as having been part of the early radiation of Holometabola, and as more diverse and species-rich in the past ,2,3. ForMany holometabolans spend most of their lifetime in their larval stages; this is also true for many lacewings. Hence, a supposed loss of biodiversity within a holometabolan ingroup should also be expected to manifest in a loss of the morphological and ecological diversity of larvae. Indeed, the Cretaceous has provided numerous lacewing larvae with peculiar morphologies, clearly not represented in the extant fauna ,18,19,20Furthermore, a loss of diversity was demonstrated with quantitative measures of morphology for the larvae of silky lacewings  and of A type of lacewing larva that is quite common in Cretaceous amber is that of the aphidlion. The term is used for extant larvae of the two groups Chrysopidae (green lacewings) and Hemerobiidae (brown lacewings). The name \u2018aphidlion\u2019 refers to the habit of larvae to consume aphids ,27. HencOlder studies still indicated a sister-group relationship between Chrysopidae and Hemerobiidae, mostly based on morphological characters e.g., ,31), but, but31])The mouthparts of aphidlions differ from those of the more commonly known lacewing larvae, such as those of antlions, in being simpler. Similarly to other lacewings, each upper jaw (mandible) and lower jaw (maxilla) form a stylet . These sChrysopa slossonae can sneak across ants guarding their prey, as they use the wax of their pre as part of the camouflaging cloak [Many larvae of Chrysopidae camouflage themselves by attaching different objects to their back. This camouflaging cloak or package can be composed of rather different objects, including the remains of consumed prey, snail shells , or planng cloak . Fossils of aphidlions with a very modern appearance are known from ambers from the Miocene ,49,50 anWe here summarise all known occurrences of aphidlions and aphidlion-like lacewing larvae in the fossil record and report numerous new specimens from different ambers. Similar to earlier studies, we compare the morphological diversity of the heads of these larvae over time in order to recognise possible decreases of diversity.Data for the analysis originated from different sources. Numerous specimens were based on images from the literature or databases (image repositories). Other specimens were directly inspected and documented. Extant specimens of aphidlions came from two zoological collections: the Zoologische Staatsammlung M\u00fcnchen (ZSM) and the Centrum f\u00fcr Naturkunde Hamburg (CeNak), Leibniz-Institut zur Analyse des Biodiversit\u00e4tswandels (LIB). Fossil specimens, all preserved in amber, came from various collections: the Palaeo-Evo-Devo Research Group Collection of Arthropods, Ludwig-Maximilians-Universit\u00e4t M\u00fcnchen (PED), the Senckenberg Forschungsinstitut und Naturmuseum, Frankfurt/Main (SMF Be), and collections of some of the authors, namely C.G. (CCGG), Ch.H. + H.-W.H. (CCHH), P.M. (BUB), T.W. (Weiterschan BuB), and J.W. (F xxx BU CJW) . Polarisers were placed on flashes and in front of the lens to provide cross-polarised light ,58.All images are composite images ; a stackOutlines of all head capsules and stylets were redrawn in Adobe Illustrator CS2 or Inkscape. The more accessible half of the head capsule and the more accessible stylet were outlined. The stylet was artificially rotated forward so that the tip was in a straight line with the proximal insertion on the head capsule. Afterwards, the drawn half of the head was mirrored.In cases in which the head was partly concealed by the thorax (the head was retracted) we only considered the visible or free part. This area reflects the functional part of the head, but also considers that for cases of fossils only the outer visible part can be considered. For many  extant specimens, often the entire head is additionally available, either in the literature or on cleared mounted specimens. Yet, to have an even dataset we did not consider this type of information, as it was not available for some of the other specimens.The analysis was performed in SHAPE. The procedure was outlined in . The app(1)Specimen 4819 (BUB 3060) is preserved in Myanmar amber. It is accessible in a dorsal B,C and v(2)Specimen 4821 (BUB 3066) is preserved in Myanmar amber. It is accessible in a ventral view A,B. No c(3)Specimen 4822 (BUB 3347) is preserved in Myanmar amber. It is accessible in a dorsal B,C and v(4)Specimen 4823 (BUB 3358) is preserved in Myanmar amber. It is accessible in a dorsal A and ven(5)Specimen 4824 (BUB 3359) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(6)Specimen 4820 (BUB 3361) is preserved in Myanmar amber. It is accessible in a dorsal C and ven(7)Specimen 4825 (BUB 3379) is preserved in Myanmar amber. It is accessible in a dorsal C and ven(8)Specimen 4826 (BUB 3393) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(9)Specimen 4827 (F 3196 BU CJW) is preserved in Myanmar amber. It is accessible in a dorsal A,B and l(10)Specimen 4829 (PED 0038) is preserved in Myanmar amber. It is accessible in a dorsal E,F and v(11)Specimen 4828 (PED 0034) is preserved in Myanmar amber. It is accessible in a dorsal C and ven(12)Specimen 4830 (PED 0065) is preserved in Myanmar amber. It is only accessible in a lateral view A\u2013C, henc(13)Specimen 4832 (PED 0248) is preserved in Myanmar amber. It is accessible in a dorsal E,F and v(14)Specimen 4831 (PED 0149) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(15)Specimen 4833 (PED 0251) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(16)Specimen 4834 (PED 0252) is preserved in Myanmar amber. It is accessible in a dorsal to dorso-lateral A,B and v(17)Specimen 4835 (PED 0253) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(18)Specimen 4836 (PED 0315) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(19)Specimen 4837 (PED 0323) is preserved in Myanmar amber. It is accessible in a dorsal E,F and v(20)Specimen 4838 (PED 0330) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(21)Specimen 4839 (PED 0375) is preserved in Myanmar amber. It is accessible in a dorsal D and ven(22)Specimen 4840 (PED 0427) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(23)Specimen 4841 (PED 0433) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(24)Specimen 4842 (PED 0441) is preserved in Myanmar amber. It is accessible in a dorsal C and ven(25)Specimen 4843 (PED 0455) is preserved in Myanmar amber. It is accessible in a dorsal B,C and v(26)Specimen 4844 (PED 0518) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(27)Specimen 4845 (PED 0541) is preserved in Myanmar amber. It is accessible in dorsal C and ven(28)Specimen 4846 (PED 0580) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(29)Specimen 4848 (PED 0642) is preserved in Myanmar amber. It is accessible in a dorsal A,B, vent(30)Specimen 4849 (PED 0666) is preserved in Myanmar amber. It is accessible in a dorsal B,C and v(31)Specimen 4850 (PED 0667) is preserved in Myanmar amber. It is only accessible in dorsal view A,B. No c(32)Specimen 4851 (PED 0696) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(33)Specimen 4852 (PED 0715) is preserved in Myanmar amber. It is accessible in a dorsal F and ven(34)Specimen 4854 (PED 0782) is preserved in Myanmar amber. It is only accessible in dorsal view A,B. No c(35)Specimen 4855 (PED 0793) is preserved in Myanmar amber. It is accessible in a dorsal C,D and v(36)Specimen 4856 (PED 0807) is preserved in Myanmar amber. It is accessible in a dorsal H and ven(37)Specimen 4853 (PED 0754) is preserved in Myanmar amber. It is only accessible in dorsal view F,G. No c(38)Specimen 4857 (PED 0837) is preserved in Myanmar amber. It is accessible in a ventral A,B and a(39)Specimen 4858 (PED 0901) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(40)Specimen 4859 (PED 0952) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(41)Specimen 4860 (PED 0983) is preserved in Myanmar amber. It is accessible in a dorsal A,B and l(42)Specimen 4861 (PED 0989a) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(43)Specimen 4862 (PED 0989b) is preserved in Myanmar amber, in the same amber piece as PED 0989a. It is accessible only in a dorso-lateral view F,G. No c(44)Specimen 4863 (PED 1000) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(45)Specimen 4864 (PED 1223) is preserved in Myanmar amber. It is accessible in a dorsal K and ven(46)Specimen 4865 (PED 1229a) is preserved in Myanmar amber. It is accessible only in a ventral view A,B. No c(47)Specimen 4866 (PED 1229b) is preserved in Myanmar amber, in the same amber piece as PED 1229a. It is only accessible in ventral view E,F. The (48)Specimen 4867 (PED 1258a) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(49)Specimen 4868 (PED 1258b) is preserved in Myanmar amber, in the same amber piece as PED 1258a. It is accessible in a dorsal G and ven(50)Specimen 4869 (PED 1287) is preserved in Myanmar amber. It is only accessible in a dorsal view A,B. Many(51)Specimen 4870 (PED 1301) is preserved in Myanmar amber. It is accessible in a dorso-lateral C and ven(52)Specimen 4871 (PED 1311) is preserved in Myanmar amber. It is accessible in a dorsal E,F and v(53)Specimen 4872 (PED 1322) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(54)Specimen 4873 (PED 1323) is preserved in Myanmar amber. It is accessible in a dorsal C,D and v(55)Specimen 4874 (PED 1333) is preserved in Myanmar amber. It is accessible in a ventral A,B,D and(56)Specimen 4875 (PED 1335) is preserved in Myanmar amber. It is accessible in a lateral A\u2013C and p(57)Specimen 4876 (Weiterschan BuB 11) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(58)Specimen 4877 (Weiterschan BuB 31) is preserved in Myanmar amber. It is accessible in a dorsal A,B and v(59)Specimen 4703 (SMF Be 2021) is preserved in Baltic amber. It is accessible in a dorsal C, ventra(60)Specimen 4702 (CCGG 7615) is preserved in Baltic amber. It is accessible in a dorsal view A,B. A pr(61)Specimen 4704 (SMF Be 1861) is preserved in Baltic amber. It is accessible in a dorsal view C,D. No c(62)Specimen 4753 (CCHH 1786-3) is preserved in Baltic amber. It is accessible in dorsal A,B and vAs an expanded basis for the shape analysis, numerous new aphidlions and aphidlion-like larvae are presented here. The terminology follows earlier studies ,23,24,25The analysis of the head shape resulted in four effective principal components . They toPC1 explains 79.69% of the overall variation. It is strongly influenced by the relative length of the stylets. A low value indicates shorter stylets, a high value indicates longer stylets .PC2 explains 9.24% of the overall variation. It is strongly influenced by the posterior rim of the head capsule. A low value indicates a concave posterior rim, a high value indicates a convex posterior rim .PC3 explains 3.90% of the overall variation. It is strongly influenced by the anterior rim of the head capsule. A low value indicates a convex anterior rim, a high value indicates a concave posterior rim .PC4 explains 2.55% of the overall variation. It is strongly influenced by the proximal region of the stylets at the transition to the head capsule. A low value indicates a concave shape, a high value indicates a convex shape .2-value of 0.40. For type 2 larvae, only two specimens were available, providing no coefficient of determination. For type 3 larvae, the correlation is quite strong with an R2-value of 0.88. For type 4 larvae, the correlation is weaker with an R2-value of 0.63. Finally, for Hemerobiidae-type larvae there is a strong correlation with an R2-value of 0.95.As PC1 was so strongly dominating , we looked for a possible correlation of it to size. We did this for different types of larvae (explained in more detail in the discussion section). For type 1 larvae , such a correlation is weak with an RAs outlined, aphidlions are larvae of Chrysopidae and Hemerobiidae, and these two groups seem not to be closely related to each other. What does that mean for the term aphidlion? What does it in fact refer to?We already know that, from the ecological functional side, the term refers to a larva with specific feeding habits. Yet, apparently it also refers to a certain combination of morphological characters, otherwise we could not recognise a fossil as \u201caphidlion-like\u201d. These characters include a simple head with curved, rather slender, but toothless stylets, well-developed antennae and labial palps, and a simple spindle-shaped body.Most of these characters very likely characterise a rather basal node within Neuroptera, which seems so far unnamed, and is namely the group of lacewings excluding Coniopterygidae, Nevrorthidae, Sisyridae, and Osmylidae (following the phylogeny of ). When aSimple-curved toothless stylets would become derived (\u201clost\u201d) in the lineages of Dilaridae, Mantispidae + Berothidae, and Myrmeleontiformia. A spindle-shaped body is retained in most lineages; within Myrmeleontiformia, a broader shape has evolved . Thi\u201cAphidlion\u201d is therefore likely a kind of ecological category, but it also refers to a rather unspecialised terrestrial predator that likely represents the ancestral condition of larval morphology and ecology for a large group within Neuroptera. Within the group, the larvae evolved more specialised morphologies and, coupled to this, strategies, such as pit traps Larvae of type 1 have relatively long protrusions on the anterior and posterior trunk, which are all about the same length. Many of the not-that-well-preserved specimens may represent type 1 larvae, yet they cannot be reliably interpreted.(2)Larvae of type 2 have relatively long protrusions on the anterior trunk, but even longer ones on the posterior trunk.(3)Larvae of type 3 have relatively long protrusions on the anterior trunk, but rather short ones on the posterior trunk.Many of the Cretaceous fossils bear protrusions on the dorsal side, similar to many fossil larvae that have been generally interpreted as relatives of Chrysopidae ,9,10,11.Another type of larvae, type 4, lacks dorsal protrusions. These larvae have shorter lateral protrusions and relatively long stylets. Their trunk region is often much better preserved than those of the first three morphotypes. None of these larvae seem to bear a camouflaging cloak.The last distinct type has a slender body, no protrusions, and shorter stylets. This feature is reminiscent of the larvae of Hemerobiidae. Therefore, we will use \u201cHemerobiidae-type\u201d as a reference. Details further supporting or contradicting this interpretation will be given in the following.Ankyloleon, [The stylets of all new specimens are gently curved and rather slender. In many ingroups of Neuroptera, the stylets are more or less straight , or curved, but shorter and stouter . In some neuropteran ingroups, the stylets are proximally straight, but curved distally . Simpleyloleon, ,16 unlikyloleon, ; some layloleon, ). FurtheThe head capsule shape of most of the new fossils clearly differs from that of long-nosed antlions (larvae of Psychopsidae) and long-necked antlions (larvae of Crocinae). Long-nosed antlions have a prominent labrum region projecting forward from the head capsule , which iOther prominent head structures are the antennae and the labial palps. The antenna of many fossils, especially of types 1\u20133, bears a prominent distal seta e.g., A. Such aThe labial palps of larvae of Chrysopidae consist of three elements, with the second one being the longest. The labium of larvae of Hemerobiidae often has a proximally conjoined part projecting forward, which seems rather unique within Neuroptera. Moreover, in Hemerobiidae, the palps have three elements, with the third one being the longest; in some modern larvae this element appears swollen .Yet, the case is in fact a bit more complicated. Extant larvae of Chrysopidae have empodia only in larval stages 2 and 3 ,38, but Some extant larvae of Chrysopidae have dorsal processes and use these to attach a camouflaging cloak . Larvae The structure of the processes in the new fossils differs from those of other groups, but also from those in the modern-day larvae of Chrysopidae. Still, it seems most parsimonious to assume that all Cretaceous specimens discussed here with such processes are closely related to Chrysopidae, basically following earlier interpretations of comparable fossils e.g., ,10,11).,11.10,11As to be expected in such a larger sample, there is quite some variation within the material. It seems likely that the sample contains different larval stages (see further below for this aspect), but probably also different species. As the state of preservation in many of these larvae is at best challenging, we refrain from erecting formal species based on these specimens. We hope that expanding the data set further in the future will allow for a stricter phylogenetic approach, as performed in Badano et al. ,15.Most specimens reported here seem to be relatives of modern green lacewings (Chrysopidae). Types 1\u20133 are characterised by the prominent processes of the back and hence are quite similar to the modern aphidlions of Chrysopidae. Moreover, type 4 specimens strongly resemble modern green lacewing aphidlions, but are clearly of a non-camouflaging type. The differences within these specimens (as outlined above) may indicate that this type in fact represents at least two different species. Indeed, the fossils of the Hemerobiidae-type possess characters that further support an interpretation of these larvae as at least closer relatives of these lineages.In earlier quantitative studies of lacewing larvae, no real effects of shape and size could be recognised , but alComparing the head shapes of the different groups included here reveals that all have large overlapping areas when plotting them in two dimensions. No group separates strongly from the others, yet certain differences can be well recognised . Extant The fossil larvae plot largely within the range of green lacewing aphidlions . Only a The comparison of the different time slices is partly complicated by the fact that the sample sizes for the Miocene and Eocene are much smaller than those of the Cretaceous and especially the modern fauna . The difWe can therefore mostly recognise that the fossils from the Miocene and Eocene fall within the area of the morphospace occupied by modern larvae. It is, however, noteworthy that the range of the green lacewing aphidlions in the Miocence and Eocene spans more or less the entire space occupied by modern green lacewing aphidlions in the PC1. This large range indicates that although the overall number of specimens is rather low, the overall morphological diversity of green lacewing aphidlions in the Eocene/Miocene was already about as large as today. Moreover, for the Cretaceous, the area occupied by modern green lacewing aphidlions was already largely occupied, at least along the PC1 .The larvae in the Eocene and Miocene already have a very modern appearance and are indeed likely representatives of modern lineages. The Cretaceous larvae are not only similar in head shapes, but also partly in possessing structures on the back to carry a camouflaging cloak. Yet, in the details these Cretaceous larvae are quite different from the modern ones. For example, the head is much more distinctly set off in the fossils; moreover, the exact morphology of the camouflage-cloak-carrying processes is quite different .These morphologies indicate that the fossils performed a very similar ecological role, yet are not closely related to the modern forms. We therefore assume that the Cretaceous species, probably entire lineages, went extinct, possibly at the end of the Cretaceous, and became ecologically replaced by the modern green lacewings before the Eocene. This combination of a severe extinction of some lineages in combination with a diversification of another lineage, basically substituting the extinct forms, is generally addressed as \u201cfaunal turnover\u201d. Yet, this expression is usually used to address taxonomic changes of a community.For brown lacewing aphidlions (Hemerobiidae), the case is different. The area exclusively occupied by modern brown lacewing aphidlions (not overlapping with green lacewing aphidlions) is basically unoccupied by fossils, besides a single specimen from the Eocene . The basIt is unlikely that this observation is due to a preservation bias. Brown lacewing larvae nowadays have a quite similar lifestyle to green lacewing larvae in the aspects of living on plants and hunting their preferred prey there. With this, they should not have had a lower preservation potential. One could speculate that larvae with such a morphology back in the Cretaceous had a different lifestyle that is making a preservation in amber less likely (see discussion in ). This wIt is well established that lacewings were more diverse in the past ,2, whichAs apparent in the here presented data set, the most common types of aphidlion larvae in the Cretaceous are those with protrusions on the back (types 1\u20133) that allow them to carry a camouflaging cloak . It is sLarvae of type 4 have only short protrusions on the abdomen segments. These may still have acted as a support for a camouflaging cloak, as observed in other lacewing larvae (cf. ). Hence,(1)A certain loss of very extreme forms, most likely coupled to a loss of specific life habits A loss of many further forms, especially of camouflaging larvae; yet this loss is factually compensated by:(3)A diversification of the modern lineages of green lacewings and their larval forms.In summary, we observe in the case of Chrysopidae and their closer relatives almost no change in head shape over the last 100 million years. Nevertheless, in detail we observe:In Hemerobiidae, the case is less clear due to a significantly smaller sample size. Still, the results indicate an increase in larval brown lacewings after the Cretaceous."}
{"text": "Non-alcoholic fatty liver disease (NAFLD) presents the most common chronic liver disease globally; it is estimated that 25.24% of the world\u2019s population has NAFLD. NAFLD is a multi-factorial disease whose development involves various processes, such as insulin resistance, lipotoxicity, inflammation, cytokine imbalance, the activation of innate immunity, microbiota and environmental and genetic factors. Numerous clinical studies have shown that the Mediterranean diet produces beneficial effects in NAFLD patients. The aim of this review is to summarize the beneficial effects of lycopene, a soluble pigment found in fruit and vegetables, in NAFLD. Non-alcoholic fatty liver disease (NAFLD) presents the most common chronic liver disease globally. It is estimated that 25.24% of the world\u2019s population has NAFLD with the highest prevalence rates in the Middle East and South America . NAFLD iIn NAFLD pathogenesis, mitochondrial dysfunction is characterized by structural alterations, such as the depletion of mitochondrial DNA and morphological and ultra-structural changes and functional alterations, involving the respiratory chain and mitochondrial \u03b2-oxidations . In factp < 0.01) between the baseline and the end of treatment. In a study of non-diabetic patients with NAFLD, the subjects underwent 16 weeks of MD, 16 weeks of washout and 16 weeks of low-fat diet . LY. LY58]. Several studies have shown that the white adipose tissue is an endocrine organ capable of releasing numerous adipokines and pro-inflammatory factors. It has been shown that high levels of adipokines, including IL-6 and TNF-\u03b1, can contribute to the reduction in lipid oxidation leading to lipotoxicity and insulin resistance. TNF-\u03b1 is a key modulator of adipocyte metabolism owing to its direct role in glucose homeostasis and lipid metabolism. In addition, both IL-6 and TNF-\u03b1 promote the production of leptin by adipose tissue, with an increase in inflammatory cytokines and the induction of the absorption of cholesterol by macrophages. In a study conducted by Luvizzotto et al., supplementation with LYC was observed to reduce leptin levels and restore plasma IL-6 concentrations significantly in obese rats .Moreover, some LYC metabolites are bioactive, particularly apo-10\u2032-lycopenoic acid (ALA), the major metabolite of LYC known to suppress hepatic steatosis and inflammation by stimulating sirtuin 1 (SIRT1). A study conducted by Chung et al. showed that ALA supplementation increased hepatic SIRT1 protein with a concomitant increase in the deacetylation of NF-\u03baB p65, which in turn decreased the hepatic protein levels of IL-6 and TNF\u03b1 by a mechanism not yet known . The prop < 0.05). In a study conducted by Jiang et al., 65 rats with NAFLD were divided into a control group and three groups treated with 5, 10 and 20 mg with LYC, respectively [p < 0.01). Furthermore, the serum levels of AST and ALT were significantly decreased in a dose-dependent manner after the LYC treatment. Regarding the serum lipid levels, a remarkable reduction in the concentrations of LDL-C and TG in dose-dependent manners was observed, showing the lipid-lowering effects of LYC. Additionally, this study reported a significant increase in SOD and glutathione (GSH) levels in rats treated with LYC. Finally, the results of this study showed the decreased expression of TNF-\u03b1 and CYP2E1, involving a reduction in inflammatory response [p = 0.002), and serum LDL levels (p = 0.008) and TC concentrations in the liver were normalized (p = 0.001). Regarding oxidative stress enzymes, it was observed that SOD and CAT activities were increased in the group on a normal diet supplemented with LYC. The macroscopic analysis found a complete recovery of injured livers. In a study conducted by Negri et al., 61 obese children with fatty liver were enrolled; all participants underwent a washout low carotenoid diet for two weeks, and then they were randomly assigned to a restricted calorie regimen (RCR) (Group 1) or RCR supplemented with tomato juice and basil and oregano extracts in extra virgin olive oil (Group 2) for 60 days [p < 0.01) accompanied with the loss of subcutaneous and visceral fat and decreased liver size. Additionally, the serum levels of insulin decreased by 34% in group 2. Total cholesterol showed a decrease in group 2 of 6.7%. Regarding the LYC antioxidant activity, it was detected that GSH levels increased in both groups after being exposed to LYC. Instead, the anti-inflammatory activity was ascertained by the decrease in IL-4 and increase in adiponectin.Several studies have been carried out to verify the effects of LYC on NAFLD . In a stectively . After tresponse . In a stresponse . After i 60 days . Subsequp < 0.01 and p < 0.05, respectively). Another study by Wang et al. showed that LYC can inhibit hepatocarcinogenesis induced by NASH [p < 0.01), suggesting that both supplementations have anticarcinogenic actions. In addition, a reduction in hepatocytes, which exposed the proliferating cell nuclear antigen (PCNA), was observed in all groups where LYC or tomato extract was administered (p < 0.05), indicating that LYC could inhibit cell proliferation induced by NASH. Since oxidative stress contributes to the onset of liver cancer, the levels of certain cellular components involved in oxidative stress were analyzed. In particular, it was noted that the HFD and LYC group showed an increased expression of transcription factor 2 related to nuclear factor E2 (Nrf2), a key regulator of cellular response to oxidative stress (p < 0.05). Nrf2 is the main regulator of the enzyme heme oxygenase-1 (HO-1) that acts as an antioxidant and eliminates free radicals [NASH is closely associated with hepatic fibrosis and, if not adequately treated, can evolve into HCC ,81. The  by NASH . In partradicals . The abiradicals . The purradicals . ALA seeCurrently, NAFLD presents a new challenge for scholars and physicians. A balanced and healthy diet with reduced amounts of saturated fat is the strategy that gives beneficial effects both in the prevention and in the treatment of NAFLD. Several studies are available in the literature that confirm the effectiveness of the MD in improving NAFLD owing to a great variety of nutraceutical components, among which LYC is one of the most abundant components. Several LYC biological functions in the body can be particularly effective against a multifactorial pathology, such as NAFLD. Particular attention is paid to the antioxidant activity, which is already known to act efficiently in NAFLD. In fact, many studies have reported an increase in the activity of SOD and CAT as a final result of the administration of LYC. The anti-inflammatory activity of LYC has also been confirmed in several studies performed for the reduction in TNF-\u03b1 and CYP1E2 expressions. Further, the reduction in serum levels of lipids and hepatic levels of cholesterol by LYC was confirmed, indicating a possible action on the lipid metabolism. The mechanism for these results has not yet been understood fully. Many preclinical studies are available in the literature with the aim to clarify the mechanism of LYC action. However, there is a great lack of clinical studies. In conclusion, we can state that LYC currently presents a very promising natural compound in the prevention and treatment of NAFLD, but more studies by the scientific communities are certainly indispensable to investigate some of its effects in a more useful way. A large multicenter clinical study is needed to confirm the efficacy of LYC in the treatment of NAFLD."}
{"text": "FLS in each point were calculated. A level of p < 0.05 was accepted as statistically significant. As a whole, FLS was significantly higher in FEs vs. ERMEs (p < 0.001); FS in both SCP and DCP was not significantly different between ERMEs and FEs . Correlation in focal retinal areas between FLS and FS in ERMEs was significant in SCP (p = 0.002) and not significant in DCP (p = 0.205); in FEs was significant in both SCP (p < 0.001) and DCP (p = 0.022). As previously described, these defective areas were located mainly in sites of distortion of retinal layers; therefore, it can be hypothesized that a focal change in FS, occurring mostly in SCP, could be involved in the onset of the functional defect.Inner macular layers are the most involved in the retinal distortion caused by idiopathic epiretinal membrane (iERM). They represent the anatomical structures in which the superficial (SCP) and deep (DCP) capillary plexus are embedded. We quantified flow signal (FS) in these capillary plexuses using Swept Source OCT angiography to identify possible markers for postoperative outcome. The software ImageJ was used to quantify the FS in a 150 \u00b5m radius area around each point analyzed by MAIA microperimeter. In 16 patients with unilateral iERM, focal light sensitivity (FLS) in the para- and perimacular areas was measured to evaluate macular function in 24 points overlapping macular plexuses and compared with normal fellow eyes (FEs).  Idiopathic epiretinal membranes (iERM) are a frequent macular pathology with a prevalence of 7\u201311.8% of the population. Epiretinal membranes are made of glial cells, retinal pigment epithelial (RPE) cells, macrophages, fibrocytes and collagen in varying proportions. IERM can stretch the underlying retina, depending on the cell type present in the ERM , causingPatients with ERMs usually show visual symptoms as the conditions worsen, including reduced visual acuity and metamorphopsia. A thickening of inner nuclear layer (INL) has been reported more in patients with metamorphopsia than in those without . A furthPrevious works identified preoperative markers correlated with postoperative visual function in order to predict visual recovery after surgical treatment . Recent Inner retinal layers are the anatomical structures in which the superficial (SCP) and deep (DCP) capillary plexus of the macula are located; therefore, we quantified flow signal (FS) in these retinal capillary plexuses using both Swept Source-OCT (SS-OCT) and Swept Source SS-OCT angiography (OCTA) to assess the preoperative anatomical status and identify possible markers for postoperative outcome in patients affected by iERM.The aim of this study is, therefore, to quantify by means of OCTA the FS in the SCP and DCP in eyes affected by iERM and evaluate a possible correlation with FLS.The study was conducted in a consecutive series of patients with unilateral idiopathic ERMs; 31 eyes of 16 patients, 16 eyes with ERM (ERMEs) and 15 normative fellow eyes (FEs), 9 women and 7 men, mean age 63.21 \u00b1 5.34 years, were studied.All procedures in this study adhered to the tenets of the Declaration of Helsinki and were approved by the investigational review board of Central Ethic Committee IRCCS Lazio. All subjects gave their informed consent after the aim of the study had been fully explained.Before imaging, all patients underwent ophthalmic examination, including best-corrected visual acuity and fundus examination, performed by a retina specialist using a +90 diopters (D) lens. Inclusion criteria were the presence of iERM in one eye and normal morphology without any retinal changes in the fellow eye. Exclusion criteria were the presence of any retinal or choroidal disease such as retinal detachment, retino-vascular disease, AMD, diabetic retinopathy, glaucoma or ocular hypertension, a history of previous ocular laser or surgery, eyes with refractive errors > 3D, media opacities that prevented good visualization of the fundus, any associated systematic disorders  or vascular diseases without retinopathy, which would affect the FS.\u00ae Elite 9000 . It is a swept-source OCT (SS-OCT) and OCT angiography (SS-OCTA) that provides automated segmented enface OCT with analysis of different plexus: Superficial Capillary Plexus (SCP), Deep Capillary Plexus (DCP), Avascular Retina, Choriocapillaris (CC) and Choroid (Ch). OCTA on the PLEX\u00ae Elite 9000 is generated with the OMAG\u00ae algorithm  [The macula was assessed using the split-spectrum amplitude-decorrelation angiography with PLEXography) ,9, whichography) ,11. The \u00ae Elite 9000 analyzes retinal layers using infrared (IR) light produced by a swept source laser. This IR laser ray penetrates in deeper retinal layers with less bias than light used in Spectral Domain OCT. The instrument further reduces the segmentation errors related to the projection artifacts on DCP using a software, that subtracts the artifacts related to the SCP flow signal from the enface image of the DCP.The PLEXThe software ImageJ , was used to quantify the FS in the focal area around each point analyzed by MAIA microperimeter . The MAIA microperimeter checks the fixation stability and gives an output of 3 possible fixation stability: stable, relatively stable and unstable; we repeated the microperimetry if necessary to analyze only the examinations with stable fixation and to have valid sensitivity values in each patient. To evaluate macular functional status, we measured the focal light sensitivity (FLS) in the para- and perimacular areas, as reported in previous papers ,13,14, bWe decided to assess the visual function outside the central fovea, assessed in previous studies ,16, as tAn ImageJ circle with a radius of 150 micron was used to select the focal area around each one of the 24 points analyzed by microperimeter MAIA. Thereafter, in each binarized focal area, we calculated the density of white pixels, representing the focal FS, and density was then correlated with the FLS at each point. As the aim of the study is to evaluate the correlation between morphological and functional parameters in each focal areas of the retina, the N of focal areas examined in every one of the 16 patients are 24; therefore, 24 \u00d7 16 = 384 for each capillary plexus were analyzed.The eccentricity of the 24 points analyzed in each eye was between 2.5\u00b0 and 5\u00b0, the 1\u00b0 grid was not included for its location on the border of the FAZ. The 12 points with eccentricity 2.5\u00b0 represent inner grid, 12 points at 5\u00b0 outer grid .Images with visible eye motion or blinking artefacts and with poor image quality were excluded . OCTs were visually assessed by the same retina specialist (A.M.C.) to ensure proper segmentation of SCP and DCP.By measuring macular capillary network with OCTA, an association was found between the presence of epiretinal membranes in pediatric age and the reduction in FS at the level SCP and DCP . In ordet-Test for independent samples iERMEs vs. FEs and the Pearson correlation coefficient (R2) of FS (density) vs. FSL (dB) in each point was calculated, comparing the same focal areas of the retina according to the grid between iERMEs and FEs. A level of p < 0.05 was accepted as statistically significant. The statistical analyses were performed using SPSS software version 15.0 .The p < 0.001), with a statistically significant difference, as displayed in FS and FLS values of each 24 focal areas from anyone of the 16 patients were evaluated and compared as a whole between affected and fellow eyes; in iERMEs, mean FLS was 24.39 \u00b1 4.19 dB, in FEs 25.57 \u00b1 4.18 dB   .A significant mean reduction in light sensitivity was present in affected eyes .p = 0.002) and not significant in DCP (p = 0.205); in FEs, was significant in both SCP (p < 0.001) and DCP (p = 0.022). The correlation in iERMEs is negative in both SCP (R2 = \u22120.164) and DCP (R2 = \u22120.69), whereas in FEs, it is positive in SCP (R2 = 0.279) and negative in DCP (R2 = \u22120.121), as reported in Interesting findings were standard deviations (SD) differences, for both SCP and DCP, between ERMEs and FEs, probably associated with focal changes in the retinal structure related to ERM contraction. In iERMEs, the correlation between FLS and FS was significant in SCP  and fellow eyes (FEs).Differences are present for FLS and SCP FS between inner and outer grids: inner vs. outer grid in FEs FLS Comparison between affected and fellow eyes:p < 0.004). Mean FS in iERMEs was 106.57 \u00b1 21.91 for SCP and 71.85 \u00b1 16.34 for DCP; in FEs, 107.41 \u00b1 15.03 for SCP and 70.55 \u00b1 11.01 for DCP without reaching a statistically significant difference between the two groups . Correlation between FLS and FS is significant in SCP in both iERMEs (p = 0.047) and FEs (p < 0.001), and not significant in DCP, iERMEs (p = 0.182) and FEs (p = 0.195). Correlation is positive only in SCP of FEs (R2 = 0.371) and negative in DCP of FEs (R2 = \u22120.097), SCP (R2 = \u22120.153) and DCP (R2 = \u22120.103) of iERMEs.Inner grid: in iERMEs, mean FLS was 24.94 \u00b1 3.71 dB; in FEs, 26.12 \u00b1 3.95 dB, statistically significant between fellow and affected eyes (p = 0.006), with a statistically significant difference between fellow and affected eyes. Mean FS in iERMEs were 115.43 \u00b1 21.21 for SCP and 71.21 \u00b1 16.74 for DCP, in FEs, 115.24 \u00b1 18.12 for SCP and 73.05 \u00b1 10.58 for DCP without a statistically significant difference . Correlation between FLS and FS is statistically significant in FEs only in SCP (p = 0.0001) and not significant in DCP (p = 0.115), whereas it is not significant in both capillary plexuses of iERMEs: SCP (p = 0.081) and DCP (p = 0.535). Correlation is positive only in SCP of FEs (R2 = 0.283) and negative in DCP of FEs (R2 = \u22120.118), SCP (R2 = \u22120.135) and DCP (R2 = \u22120.048) of iERMEs. Based on these data, results showed that, except for the change in the means values of FLS and FS, there is no significant difference between fellow and affected eyes related to the eccentricity of focal retinal areas  of affected eyes were analyzed to evaluate if a higher correlation between FLS and FS was present.p = 0.003 R2 = \u22120.370 and in DCP (N = 45) p = 0.006 R2 = \u22120.402 were statistically significant. In these focal areas, therefore, negative correlation is significant in both capillary plexuses  plexuses .The data of this study are consistent with the hypothesis that FS could be a marker to assess the anatomical status and predict the functional status of focal retinal areas in eyes affected by iERM. ERM contraction causes focal functional defects, as shown by the mean FLS which is statistically lower in affected vs. fellow eyes. On the contrary, the mean FS were not significantly different between the two groups in both SCP and DCP; however, this apparent discrepancy can be explained by the fact that in eyes with ERM, the irregular contraction of the ERM causes focal displacement of blood flow, thus not affecting the mean values, mainly in SCP, and in DCP. Additionally, retinal blood flow in SCP and light sensitivity are significantly different in relation to eccentricity of the focal retinal areas.Analysis of the correlation between morphological and functional parameters shows that focal functional alterations in the inner retina can occur as a consequence of focal structural change. The correlation between FLS and FS is significant in SCP in both affected and fellow eyes. These data validate a relation between the blood flow density and the functional status in focal retinal areas. Focal blood flow could be related to the conformation of the retinal structure, which can be modified by inner retinal layer shrinkage due to the entity of ERM contraction. An increase in blood flow, mainly in the SCP, is present in focal areas in which the structure of inner retina, such as in eyes affected by ERM, is more altered. We found a significant and negative correlation between FS and FLS in SCP of iERMs, whereas in FEs, the correlation between these parameters is significant and positive, showing, therefore, a different relation between FS and FLS in healthy and affected areas of the retina, as shown in 2 is higher when the correlation is calculated in groups of focal areas in which the values of FS differ more (\u00b12SD) from the mean  layer, and subsequently affecting bipolar and amacrine cells and both inner and outer plexiform layers, fed by DCP. The GC layer is more injured by the mechanical damage and a previous study of ours reported that the GC layer can also be affected by an alteration occurring before the mechanical damage related to the contraction on the iERM . MoreoveThe correlation between FS and FLS is more significant in patients with a higher difference from mean value of FS; therefore, it can be hypothesized that the severity of the functional retinal cell damage is correlated to the amount of FS change .Furthermore, the FS change and the statistically significant correlation found between SCP and FLS in affected eyes, support the hypothesis that FS focal alterations could be helpful to assess the anatomical integrity and the corresponding functional status of inner retinal layers. Consequently, FS and FLS parameters could be considered predictive markers to evaluate when patients should undergo surgical treatment and to predict post-operative outcome.\u00ae Elite 9000, infrared light SS-OCT, is more accurate with respect to SD-OCT. Additionally, the more significant capillary plexus in the affected eyes is the superficial, in which lower projection artifacts are involved, related just to the presence of epiretinal (clear) tissue. Even if the presence of projection artefacts can bias the evaluation of the FS in the DCP, in the PLEX\u00ae Elite 9000, the new software added to the infrared light of the SS-OCT significantly reduces the interference in the evaluation of the DCP. A limitation of the study is related to the series with N = 16; however, it should be considered that the number of focal retinal areas is 24 in each patient; therefore, as previously reported, 384 is the total number of focal areas examined in the study. Each focal area is an example of what happens in the retinal tissues as a consequence of ERM contraction. Following this model, we considered the N of 384 focal areas could be statistically significant to support a hypothesis reported in this pilot study. On the basis of these data, a further study in larger series is recommended to confirm that SCP could represent an interesting biomarker to assess focal retinal defects in eyes affected by iERM.It has been previously reported that the segmentation of SCP and DCP  in patieThe evaluation of FS with new generation SS-OCTA, could represent a reliable technique to analyze the macular function in eyes affected by iERM, as demonstrated by the findings of correlation between FLS and FS in macular capillary network. These results allowed us to suggest that FS, mainly in SCP, can be a biomarker to detect morpho-functional worsening in patients affected by iERM and to decide when it is the optimal time, with best benefit-risk ratio, to perform pars plana vitrectomy and peeling of the macular epiretinal membrane."}
{"text": "Cardiovascular diseases represent a significant public health issue, and the promotion of healthy lifestyles plays a major role in their prevention. Good adherence to the Mediterranean diet has a protective effect on cardiovascular health and may reduce the risk of developing cardiovascular disease. Our prospective study aimed to evaluate the impact of educational lifestyle interventions on cardiovascular risk parameters and the improvement in adherence to the Mediterranean diet of the involved population.Participants have been recruited by General Practitioners in Torresina neighborhood in Rome. From December 2018 to June 2020, 41 patients were involved in nutritional, psychological, and physical activity meetings by a multidisciplinary team of healthcare professionals. In particular, a nutritionist provided information to patients on balanced nutrition, considering the Mediterranean diet as a dietary model. Information on lifestyle, dietary habits and physical activity, anthropometric data and laboratory measurements were collected at baseline and after 12 months. The variations of the evaluated parameters were analyzed by paired t-test e Wilcoxon signed-rank test.The analysis showed statistically significant decreases in weight (p = 0.03) and BMI (p = 0.02), as well as in systolic (p < 0.001) and diastolic (p = 0.001) blood pressure and in total (p = 0.02) and LDL (p = 0.01) cholesterol level. Results also showed an improvement in the adherence to the Mediterranean diet (p = 0.001): the frequency of consumption of fruits and vegetables, legumes, cereals and fish has increased significantly, while the consumption of meat, milk and dairy products and alcohol decreased.This study highlights that a multidisciplinary educational program can be effective in improving healthy habits and in reducing cardiovascular risk factors, supporting its implementation in primary prevention at the community level.\u2022\u2002Promoting healthy lifestyle through primary prevention and health promotion actions is critical to reduce the onset of cardiovascular diseases.\u2022\u2002A population-based multidisciplinary educational intervention may be effective in improving adherence to a healthy, balanced diet and decreasing cardiovascular risk factors."}
{"text": "Thrombopoietin receptor agonists (TPO-RAs) are approved for immune thrombocytopenia (ITP), but their impact on health-related quality of life (HRQoL) remains poorly investigated in clinical practice. This observational study aimed to gain insight into real-world patient-reported experiences of the burden of ITP and TPO-RAs.An online questionnaire of closed questions was used to collect views of patients with primary ITP from Switzerland, Austria, and Belgium, between September 2018 and April 2020.Of 46 patients who completed the questionnaire , 41% were receiving TPO-RAs. A numerically higher proportion of patients reported being free from symptoms at the time of the questionnaire (54%) than at diagnosis (24%), irrespective of treatment type. Bleeding, the most frequently reported symptom at diagnosis (59%), was reduced at the time of the questionnaire (7%). Conversely, fatigue was reported by approximately 40% of patients at both diagnosis and the time of the questionnaire. Having a normal life and their disease under control was reported by 83% and 76%, respectively, but 41% were worried/anxious about their condition. Nearly 50% reported that ITP impaired their engagement in hobbies/sport or energy levels and 63% reported no impact on employment. When stratified by TPO-RA use, bleeding was better controlled in those receiving TPO-RAs than not (0% vs 11%). A numerically lower proportion receiving TPO-RAs than not reported worry/anxiety about their condition (16% vs 59%) and shifting from full-time to part-time employment (11% vs 22%). Similar proportions were satisfied with their therapy whether they were receiving TPO-RAs or not (89% vs 85%).Many factors affect HRQoL in patients with ITP. Of patients receiving TPO-RAs, none experienced bleeding at the time of the questionnaire; they also showed a more positive perspective for some outcomes than those not using TPO-RAs. However, fatigue was not reduced by any treatment. There were also some numerical differences between the cohorts for treatments currently received: a numerically lower proportion of patients using than those not using TPO-RAs were receiving steroids (37% [n = 7/19] vs 52% [n = 14/27]) and intravenous immunoglobulin (5% [n = 1/19] vs 19% [n = 5/27]), and a numerically higher proportion of patients using than those not using TPO-RAs were receiving rituximab (16% [n = 3/19] vs 7% [n = 2/27]). Furthermore, 26% (n = 7/27) of patients in the cohort not receiving TPO-RAs were not receiving any other therapy.A numerically higher proportion of patients in the total cohort reported being free from symptoms at the time of the questionnaire (54% [n = 25/46]) than at diagnosis , dizziness (0% [n = 0/19] vs 11% [n = 3/27]), and sleep deprivation  and that their disease was under control  and that their condition was under control (84% [n = 16/19] vs 70% [n = 19/27]). However, there was no marked difference between the cohorts regarding how informed they were about their disease (95% [n = 18/19] vs 89% [n = 24/27]), how well their doctor understood their situation (100% [n = 19/19] vs 92% [n = 25/27]), or the support they received from their doctor (100% [n = 19/19] vs 96% [n = 26/27]). For negative statements, a numerically lower proportion of patients receiving than those not receiving TPO-RAs felt worried/anxious about their condition (16% [n = 3/19] vs 59% [n = 16/27]). There was no marked difference for the other negative statements between patients using and those not using TPO-RAs.When asked about the impact of ITP on daily activities, engagement in hobbies/ability to play sport and energy levels were most reported to be affected equally in both cohorts, with almost half of patients  reporting that these activities were affected all of the time or often .When patients were stratified according to TPO-RA use, responses to questions about the impact of ITP on daily activities were generally aligned with the total cohort. There were some small numerical differences whether patients were receiving TPO-RAs or not for the proportion of patients for whom ITP had an impact all the time/often on sleeping (37% [n = 7/19] vs 26% [n = 7/27]), social life , or hobbies/ability to play sport (42% [n = 8/19] vs 52% [n = 14/27]).In the total cohort, 72% of patients reported that they were employed. Of the total cohort, the largest proportion of patients reported that their condition had not affected their employment (63% [n = 29/46]), while 24% (n = 11/46) had considered terminating their job (but had not), 17% (n = 8/46) had changed from full-time to part-time employment, 9% (n = 4/46) had been involuntarily terminated from their job, 4% (n = 2/46) had taken early retirement, and 2% (n = 1/46) had voluntarily terminated their job owing to ITP. When asked to describe their last working week, patients from the total cohort reported that their productivity had been affected to some degree .When stratified by TPO-RA use, the impact of ITP on employment and productivity was largely aligned with the total cohort. Almost equal proportions of patients who used TPO-RA and those who did not reported that ITP had not had an impact on their employment (68% [n = 13/19] vs 59% [n = 16/27]). Shifting to part-time employment was the outcome for which there was the clearest difference according to TPO-RA use, being numerically lower in those who used TPO-RA than those who did not (11% [n = 2/19] vs 22% [n = 6/27]). For productivity, there was no difference between responses according to whether patients were using TPO-RAs or not (mean productivity scores 66.3/100 vs 66.7/100).Similarly high proportions of patients were satisfied with their therapy over the previous 7 days whether they were receiving TPO-RAs (89% [n = 17/19]) or not (85% [n = 23/27]). Among patients receiving TPO-RAs, the majority were satisfied whether they were receiving TPO-RAs as a monotherapy (100% [n = 9/9]) or in combination with other therapies (80% [n = 8/10]).When asked about their level of agreement with negative statements that relate to treatment satisfaction, a numerically higher proportion of patients receiving than those not receiving TPO-RAs agreed with the statement regarding worry about the costs of treatment . HoweverThis real-world study, which was conducted across Switzerland, Austria, and Belgium, used an online questionnaire to assess the perspective of 46 patients with primary ITP on their symptomatic burden and the impact of the disease on their lives. Importantly, whilst other studies have explored these topics using patient-reported outcomes instruments, to the best of our knowledge, none has assessed all of these topics in a single questionnaire, or assessed patient views and opinions in a similar way to the present study. This study therefore adds to the growing body of real-world evidence by providing further insight into the multifactorial burden of this rare disease on patients\u2019 HRQoL, which goes beyond bleeding, and in some patients includes fatigue, anxiety about their condition, and limitations on their ability to engage in daily life activities, relationships, and employment . To the In the present study, over 75% of patients in the total cohort experienced symptoms at diagnosis, of which the highest proportion reported bleeding. Following diagnosis, the proportion of patients experiencing symptoms was reduced to 46%, with the proportion reporting bleeding showing the greatest reduction (59% to 7%). This is encouraging and may reflect that patients received appropriate disease management following diagnosis to address their bleeding symptoms; in line with this, at the time of the questionnaire, 85% of patients were receiving treatment.In contrast to bleeding symptoms, non-bleeding symptoms persisted at the time of the questionnaire. Fatigue in particular was reported by approximately 40% of patients both at diagnosis and at the time of the questionnaire. Results for fatigue are aligned with those of the literature that report that chronic fatigue is a commonly reported symptom in patients with ITP , 39. TheAt the time of the questionnaire, approximately 40% of patients in the total cohort felt worry/anxiety about their condition and also reported some impact of ITP on their daily life activities or employment. However, the majority of patients felt that they had a normal life, that their condition was under control, and that they were being supported by their doctor. Furthermore, at least 60% rarely/never experienced impairments in their ability to engage in daily life activities or employment. Results for impact of ITP on daily activities in the present study are largely aligned with those of the I-WISh , 39. HowWhen comparing disease and treatment burden between patients receiving and those not receiving TPO-RAs, several outcomes did not differ according to TPO-RA use. Because the study was not powered to detect statistically significant differences, it is important not to over-interpret numerical differences between groups. However, at the time of the questionnaire, a numerically lower proportion of patients using TPO-RAs reported bleeding as a symptom, feeling worried/anxious about their condition, or shifting to part-time employment than those not using TPO-RAs, and a numerically higher proportion agreed that they had a normal life and that their condition was under control. Furthermore, although overall treatment satisfaction was similar between those using and those not using TPO-RAs, a numerically lower proportion of patients receiving TPO-RAs were worried about getting infections or wanted to stop their treatment/take a different treatment. A lower proportion of patients receiving than those not receiving TPO-RAs were also worried about dietary restrictions. Dietary restrictions are a consideration only for patients receiving certain treatments, including eltrombopag. For eltrombopag, patients need to follow strict guidelines with regard to amounts and timings of calcium intake during meals. This may indicate that a majority of patients in the cohort that received TPO-RAs were receiving romiplostim, although it was not possible from the present data to identify the specific treatments that patients were receiving. Finally, as expected, owing to the price of TPO-RAs versus some other treatments , worry about cost of treatment was the only concern reported by a numerically higher proportion of patients using than those not using TPO-RAs. Further investigation is needed into the economic burden of ITP to patients from a broader perspective, including indirect costs to the patients as a result of ITP , and their reliance on social support or other financial dependencies.Overall, data stratified by TPO-RA use suggest that patients using TPO-RAs in the present study had a more positive perception of their clinical outcomes with regard to bleeding symptoms, and of their general psychosocial state, than those not using TPO-RAs. Results of the present study are aligned with those of clinical trials and extension studies, which have shown that TPO-RA treatment has a positive impact on patients\u2019 HRQoL versus no TPO-RA treatment , 29, 41.The effectiveness and tolerability of TPO-RAs may account for their positive impact on HRQoL  comparedOne of the main limitations of this study is that patient recruitment was not monitored. During the invitation to participate, it was explicitly emphasized to the patient that there would be no way to identify whether they had participated or not to ensure that those who did respond could provide their opinions anonymously, so that they could respond honestly about sensitive topics such as medical care satisfaction and about their personal lives. It was therefore not possible to investigate how many patients were contacted for participation. Furthermore, barriers to completing the questionnaire, which may have resulted in non-participation, were also not investigated. Finally, there was no requirement or incentive for physicians to meet patient recruitment quotas; it was entirely up to physicians whether they wanted to hand out envelopes with unique access codes to the questionnaire to their patients. These factors, in combination with ITP being a rare disease, may account for the rather low number of participants (N = 46) recruited during the active periods of the study (3 periods over 3 months), over 2 years. Nevertheless, this study provides important insight into the views and feelings of a cohort of patients with ITP, and the impact of ITP on their lives, including in those receiving TPO-RAs.The questionnaire used closed questions and all responses were anonymous. It was therefore not possible to investigate outcomes further, or to correlate them with clinical characteristics that were not collected as part of the questionnaire . Because responses also relied on patients\u2019 recollection of symptoms and treatment received at diagnosis, it is possible that they were not an accurate reflection of patients\u2019 experiences at that time, particularly for those who were diagnosed several years before completing the questionnaire. The online nature of the questionnaire may have also precluded some patients from participating in the study, and may account for the numerically lower numbers of patients recruited who were over 60 years of age (24%) than who were in the younger age groups.Another limitation of the questionnaire is that it was not validated. However, the topics included in this questionnaire were selected by a panel of haematologists with extensive experience in, and dedication to, caring for patients with ITP. Therefore, the questionnaire is likely to reflect issues that impact patients in their daily lives and are of importance to them. Furthermore, questions were phrased in a way that was consistent with the language used by haematologists when discussing with patients about their physical and emotional condition and their HRQoL. Therefore, it was agreed by the panel of haematologists that the questionnaire was suitable for use in the present study.Finally, patients recruited into the study were being treated by haematologists. They may therefore have had more severe disease or required treatment, compared with patients managed by general practitioners or who were not receiving or in need of treatment or management by a healthcare professional, who would not have been represented in the study. Furthermore, physicians who sought to recruit patients to participate in the study may have also actively engaged in enquiring about their patients\u2019 experiences and keeping patients informed of their condition; this would have had a positive impact on their patients\u2019 lives and may have produced biased questionnaire results. Based on these limitations, the results of this study are not generalizable to the general population of patients with primary ITP, but they do provide valuable data from the patient\u2019s perspective in a selected group of patients with primary ITP. To overcome some of these limitations, a future study could recruit patients with ITP from other sources, such as patient registries , that may include a broad population of patients, including those who are not under the care of haematologists or any healthcare professional.There is growing recognition of the importance of evaluating patients\u2019 perspectives of the impact of their disease and treatment on their lives. The current study, which was conducted across Switzerland, Austria, and Belgium, provides further insight into the burden of ITP based on the feelings and opinions expressed by a cohort of patients in clinical practice. This is particularly relevant with regard to fatigue, the impact of which is often underestimated, and which is not generally responsive to available treatments. Importantly, to the best of our knowledge, this is also among the first studies assessing the patient-reported experience in those receiving versus those not receiving TPO-RAs: patients receiving TPO-RAs had better outcomes with regard to bleeding, but also for other aspects of the disease, such as anxiety.S1 File(DOCX)Click here for additional data file."}
{"text": "The p53 tumor suppressor is a central protein in the fight against cancer. Mutations that inactivate at least one p53 allele occur in more than half of all cancer cells. Finding p53 nuclear and cytoplasmic amyloid inclusions in many tumor cell lines and cancer biopsies suggests, but does not prove, that amyloid formation inactivates p53 and that conformationally-converted p53 can be transmitted in a prion-like fashion . We rWhile the initial prion hypothesis addressed the unusual features of the mammalian PrP protein, much of what we know about prions comes from studies in yeast, where prions control several endogenous traits ,5,6. PriConcerning p53 prion formation in yeast, Billant et al. , it takeA limited number of proteins can form prions in living cells. Even for these proteins, the de novo appearance of prion seeds in the absence of infection is rare. While certain mutations or the overexpression of the protein or its prion domain can enhance the de novo appearance of prion seeds, the frequency of new seed formation is low. However, once a protein forms a prion, it is fairly stable.We showed that either wild-type or p53-R175H mutant human p53 expressed in yeast can indeed propagate stably in either a bona fide prion or non-prion form . FurtherBillant et al.  showed tHowever, we emphasize that Billant et al.\u2019s finding that mutant p53 does not inevitably take on a prion form or always exist as a prion does not lead to the interpretation that mutant or wild-type p53 cannot sometimes propagate as prions. Indeed, by definition, prion-forming proteins can take on and stably exist in either non-prion or prion forms.PSI+] prion in the presence of the [PIN+] prion at a rate of ~7 \u00d7 10\u22127 [PIN+]  .Although we have not quantified the frequency with which p53 forms a prion in yeast, we easily found p53 prions following transient p53-YFP overexpression in cells constitutively expressing untagged functional p53. We screened for cells that lost p53 activity using a color assay. When we turned p53-YFP synthesis back on in cells that retained their p53 activity, p53-YFP was diffused in the cell nuclei. This is what we see when we express p53-YFP in yeast lacking the p53 prion. However, in many of the isolates that lost p53 activity, when we turned the p53-YFP synthesis back on, p53-YFP was in nuclear and cytoplasmic aggregates, indicative of prion formation. Furthermore, cells with inactive p53 only appeared following p53-YFP overexpression, but not in the control samples expressing an empty vector. We characterized one such isolate extensively showing that the prion phenotypes of p53 inactivation and aggregation could be transferred to non-prion cells by infecting them with prion cells\u2019 cytoplasm via either cytoduction or transfection.Importantly, we showed that the prion was maintained if just untagged p53 synthesis was retained. Indeed, p53 aggregates could be seen in prion cells lacking p53-YFP expression by staining them with the amyloid-binding dye thioflavin T , p53 inactivation is therapeutic . Thus, tIn conclusion, rather than being a victim of prion fashion, p53 is a sterling example of the likely relevance of prion formation to human health."}
{"text": "Background: Neurogenic lower urinary tract dysfunction requires lifelong surveillance and management for the perseveration of patients\u2019 quality of life and the prevention of significant morbidity and mortality. Urine biomarkers are an attractive noninvasive method of surveillance for these patients. The aim of this systematic review is to search for and critically appraise studies that investigate the clinical usefulness of urine biomarkers in the management of neurogenic lower urinary tract dysfunction (NLUTD) in adults. Methods: This review was conducted according to PRISMA and MOOSE guidelines. Search strategy included PubMed, CENTRAL, and Scopus (until October 2022). Studies investigating potential urine biomarkers for the management of adults with NLUTD were included. Results: Fifteen studies fulfilled the criteria. To date, a variety of different urine molecules have been investigated for the diagnosis and management of neurogenic overactive bladder and detrusor overactivity , diagnosis of vesicoureteral reflux , urinary tract infection  and bladder cancer screening  in neurological patients. Conclusion: Further studies are needed to specify the utility of each molecule in the management algorithm of adult NLUTD. Aphorisms he associated the bubbles on the surface of fresh urine with chronic kidney disease and linked the sediment of urine with fever [A biomarker is traditionally defined as any biological parameter that can be measured objectively and evaluated as an indicator of a physiological or pathological process or a response to a therapeutic intervention . Biomarkth fever . In receth fever ,9,10,11.The lower urinary tract (LUT) consists of the urinary bladder and the bladder outlet  and serves the functions of storage of urine at low pressures and periodic urine expulsion . The actAdult neurogenic lower urinary tract dysfunction (NLUTD) according to the International Continence Society (ICS) refers to the abnormal or difficult function of the bladder and urethra (and/or prostate in men) in adults, in the context of a clinically confirmed relevant neurologic disorder . NLUTD iCurrently, the available studies on NLUTD and urine biomarkers have three main areas of research focus. The main research interest is the evaluation of the role of different molecules as potential biomarkers either in the diagnosis or in the assessment of the response to treatment and in the prediction of complications of neurogenic OAB ,25,26,27This systematic review aims to summarize and critically appraise the existing evidence on the use of urine biomarkers in the management of adult NLUTD.This systematic review followed the reporting recommendations of the PRISMA statement and the guidelines of the meta-analysis of observational studies in epidemiology (MOOSE) . We did We conducted a systematic literature search until October 2022 on three major electronic databases, including PubMed (Medline), CENTRAL (Cochrane Library) and Scopus looking for articles published in English without any publication date criteria. Additionally, so as to ensure literature saturation the reference lists from the included studies or reviews were hand-searched in order to retrieve additional studies that could have escaped the search. For the search strategy we used a combination of free text and medical subject headings (MeSH) of the terms \u201cneurogenic bladder\u201d or \u201cneuropathic bladder\u201d or \u201cneurogenic lower urinary tract dysfunction\u201d and \u201cbiomarkers\u201d or \u201curine biomarkers\u201d or \u201cnoninvasive biomarkers\u201d or \u201ccancer biomarkers\u201d. After the Medline strategy was finalized, the search string was \u201ctranslated\u201d and used in other databases. The search was conducted independently by two authors  in a blinded manner. Any discrepancies were solved after consultation with a third investigator (AA). Retrospective or prospective studies or randomized controlled trials (RCTs) of adults who have been diagnosed with NLUTD, in whom urine biomarkers were assessed with the aim of identifying their potential use in the management of NLUTD, were eligible for this systematic review. Only English language studies were included. Case reports, animal studies and conference abstracts were excluded.All relevant studies were imported into the Mendeley reference manager . Eligibility assessment was performed independently by three investigators  who screened the studies based on the predefined criteria. Disagreements were solved based on discussion. In the end, studies that were found to be eligible were screened as full text. A risk of bias assessment was performed by two reviewers , as previously described in a blinded manner. As all the eligible studies were observational studies (prospective or retrospective cohort studies), they were evaluated using the Newcastle\u2013Ottawa Scale (NOS) . Based oData from each study was extracted in an excel sheet that was designed a priori. The following details were collected: study design, biomarker evaluated, participants\u2019 characteristics, and outcome analysis. Due to the great heterogeneity of the results, a meta-analysis was not possible. The systematic search yielded 160 results following the removal of duplicates . After screening titles and abstracts, 128 were excluded as irrelevant. From the remaining 32 full-text studies, one could not be retrieved and 16 were excluded due to eligibility criteria. In the end, 15 studies were included in the qualitative synthesis and are illustrated in p = 0.0001). No difference was found between patients with DO due to CVD and normal controls .Yokoyama et al. : a prospp = 0.000). Levels of uNGF/Cr were significantly higher in patients with high voiding detrusor pressure (>40 cm H2O) compared to patients with low voiding detrusor pressure . A significant decrease in uNGF/Cr levels was observed after therapy in responders (n = 14) to BoNT-A injections  but not in non-responders (n = 5) to BoNT-A injections (0.76 pg/mL vs. 1.64 pg/mL p = 0.129) at 3 months.Liu et al. : a prospn = 93) and controls (n = 40). The median uNGF/Cr level was significantly higher in all patients with CVA than in controls (0.13 pg/mL vs. not detectable), with wide variations in uNGF/Cr levels. No significant difference in uNGF/Cr levels was found between patients with different urological conditions (urgency urinary incontinence (UI), impaired awareness of UI, OAB or no OAB, p = 0.805) and with different urodynamic findings . The increasing level of the severity of neurological impairment according to the American Heart Association Stroke Outcome Classification was correlated with significantly higher uNGF/Cr levels .Liu et al. : a prosp2 (PGE2), prostaglandin F2\u03b1 (PGF2a), NGF and substance p in patients with suprapontine brain diseases (n = 114) and controls (n = 27). The NLUTD cohort was classified into four groups: patients without LUT symptoms (n = 19); the increased bladder sensation group (IBS) (n = 37); the OAB-dry group (n = 22); and the OAB-wet group (n = 36). Urinary PGE2 levels were significantly higher in all four groups of patients compared with controls. Patients in the OAB-dry and OAB-wet groups had significantly increased levels of urinary PGE2 compared to the patient group without LUTS . In terms of urinary PGF2a levels, patients in the OAB-wet group had significantly higher levels compared to patients without LUTS (p < 0.001). Urinary levels of NGF and substance P were not significantly different between the four groups of patients and between NLUTD patients and controls. Yamauchi et al. : a prospn = 37) and controls (n = 10). The uNGF concentration was <10 pg/mL  in all investigated samples.Krebs et al. : a prosp2, transforming growth factor beta-1 (TGF\u03b2-1) and the tissue inhibitor of metallopeptidase 2 (TIMP-2) between NLUTD patients of different neurogenic etiology . Patients suffering from multiple sclerosis had significantly higher levels of urinary NGF/Cr and BDNF/Cr compared to the other two groups . Patients suffering from SCI had significantly lower urinary levels of TGF\u03b2-1 compared to spina bifida patients . Urodynamic parameters were not significantly associated with any of the urine biomarkers levels. Richard et al. : a singl2, BDNF, matrix metalloproteinase 2 (MMP-2), TIMP-2 and TGF-\u03b21 for adverse urodynamic parameters and for upper urinary tract damage in NLUTD patients due to spina bifida (n = 40). In terms of urodynamic findings, the urinary levels of TIMP-2 and MMP-2 were significantly associated with poor bladder compliance (<20 mL/cmH2O) . TIMP-2 was significantly associated with upper urinary tract damage upon contrast-enhanced computed tomography scanning .Peyronnet et al. : a prospn = 60). The levels of the urinary exosomal vitronectin were significantly higher in VUR patients (n = 25) than in no-VUR patients (n = 35) (p < 0.0001), the sensitivity and the specificity of the biomarker was 80% and 82.9%, respectively. Li et al. : a prospn = 16), NLUTD (n = 12) and controls (n = 12). The levels of urinary microRNAs miR-10a-5p and miR-363-3p were significantly higher in the NLUTD group compared to BLUTD and controls . Urinary microRNA miR-301b-3p levels were significantly higher in BLUTD patients compared to NLUTD patients and controls (p = 0.0071).Von Siebenthal et al. : a prospn = 27) participating in a prospective clinical trial, assessing the correlation of urine neutrophil gelatinase-associated lipocalin levels (uNGAL) and likelihood of UTI. Patients of the likely-UTI group had significantly higher levels of uNGAL compared to the patients in the no-UTI group and the patients in the unlikely-UTI group . A difference in uNGAL levels was also found between the no-UTI and unlikely-UTI groups (p < 0.01).Forster et al. : A seconn = 12) and spinal lesions (n = 11). Urine levels of IL-6 were found to be significantly increased during UTI in pooled and intra-individual comparisons to ABU . A threshold value of 25 ng/L in the urinary levels of IL-6 had a specificity of 93% and sensitivity of 77% in the diagnosis of UTI episodes with high-scoring symptoms.Sunden et al. : a longin = 208) had undergone a bladder biopsy due to a long-term indwelling catheter, gross or persistent microscopic hematuria, bladder stone, progressive urethral stricture or suspicious cytology. The sensitivity and specificity of the suspicious cytology results for BCa diagnosis were 71% and 97%, respectively. Stonehill et al. : a retron = 16). Eight from the eleven available cytologies were positive for bladder cancer diagnosis.Hess et al. : a retron = 457). Three patients were diagnosed with non-invasive BCa during the study period, all three tests were negative before BCa diagnosis.Davies et al. : a prospn = 79). The sensitivity and specificity of cytology for BCa diagnosis were 83.3% and 43.7%, respectively.Pannek et al. : a retroQuality assessment was conducted using the NOS, as mentioned previously. Four of them were high-quality studies, seven medium- and four low-quality. Results of the quality evaluation are illustrated on This is the first systematic review to evaluate studies that investigate the use of urine biomarkers in patients diagnosed with NLUTD. The fact that urine biomarkers can be easily collected by non-invasive methods makes them attractive to be used not only as a tool for diagnosis but also for the surveillance of various conditions in this specific group of patients. 2, PGF2a, substance P and TGF\u03b2-1) as potential biomarkers for the assessment of neurogenic DO.A common trait of most patients with NLUTD is neurogenic DO causing OAB symptoms, such as urinary frequency, urgency and incontinence, and carrying the risk of upper urinary tract deterioration . A plethNGF is undoubtedly the most studied urine biomarker in the NLUTD population. NGF is a small signaling protein that regulates the growth and survival of sensory and sympathetic fibers, which both have a vital role in the micturition pathways . In the 2, PGF2a and substance P) only PGE2 correlated with the severity of OAB symptoms [Concerning NLUTD due to suprapontine brain diseases, uNGF levels were not elevated in two studies ,22. In o2, TGF\u03b2-1, MMP-2 and TIMP-2) and urodynamic findings. In the study by Richard et al., none of the biomarkers tested were associated with urodynamic parameters. The levels of urine NGF/Cr and BDNF/Cr were associated with the etiology of NLUTD, and were found to be elevated in multiple sclerosis patients when compared to SCI and spina bifida patients [Two studies examined the association between multiple urine biomarkers  in patients with NLUTD due to spina bifida. This was the only study which assessed the diagnostic performance of the biomarkers; the area under the ROC curve (AUC) for the diagnosis of poor bladder compliance was 0.70 for MMP-2 and 0.69 for TIMP-2. In the same study, TIMP-2 was associated with upper urinary tract damage diagnosed by CT scans with an AUC of 0.72; a threshold of 405.9 pg/mL had sensitivity of 72.7% and specificity of 72.6% for diagnosing upper urinary tract damage [2O [Peyronnet et al. described positive associations between elevated urinary TIMP-2 and MMP-2 and poor bladder compliance , HOXC6 and DLX1 miRNAs or TMPRSS2:ERG, which are used in the diagnosis or surveillance of men with prostate cancer ,56,57,58Another major issue for patients with NLUTD is the difficulty to accurately diagnose urinary tract infections (UTI) from asymptomatic bacteriuria (ABU). Impaired storage and voiding function results in elevated post-void residual urine that predisposes them to the risk of UTI . DiagnosAnother biomarker that has been investigated as a potential tool in diagnosing infection in this population is uNGAL. In the adult population it has been explored as a marker for identifying acute kidney injury, while in the paediatric population with NLUTD, it has been thoroughly studied as a tool that could aid in differentiating UTI from ABU ,69,70,71BCa in the NLUTD population is a major concern. Although its incidence is not as high as thought in previous years, the aggressiveness of the disease together with a younger age at diagnosis, higher percentage of squamous cell carcinoma and a higher mortality compared to the general population highlights the need for early diagnosis and treatment ,74,75,76Regarding urine cytology, three retrospective studies have reported a sensitivity ranging between 71 and 83.3% and two studies reported a specificity of 43.7\u201397% in SCI patients with long-term indwelling catheters or symptoms of BCa ,31,33. OIn our systematic review we tried to be as comprehensive as possible, adhering to the PRISMA and MOOSE guidelines. However, this study has some limitations. Most studies were of evidence level 2 or 3  and no randomized controlled trial compared the urine biomarkers to invasive methods of management. There was great heterogeneity regarding the biomarkers studied and the study designs, thus a meta-analysis was not possible. Furthermore, some of the studies were retrospective, which could possibly introduce confounding errors that could affect their results. Moreover, many of the included studies have a small sample size thus it is difficult to interpret their results with safety. Finally, our review did not include \u201cgrey literature\u201d, thus some information related to our subject may have been missed.This systematic review summarized the available evidence for urine biomarkers in the management of adults diagnosed with NLUTD. During the last two decades, numerous urine molecules have gained interest, which are involved in the diagnosis and surveillance of neurogenic overactive bladder and detrusor overactivity, the diagnosis of vesicoureteral reflux and urinary tract infection, and bladder cancer screening in this specific population. However, the paucity of evidence and conflicting results do not allow the application of any of the urine biomarkers in every day clinical practice. Further studies providing a high level of evidence are needed to specify the utility of each molecule in the management algorithm of adult NLUTD."}
{"text": "Cancer is largely caused by genetic alterations such as mutations in a group of genes known as cancer driver genes. Many of the key advances in cancer treatment in recent years have involved blocking these driver genes using a new generation of anti-cancer drugs. Although p53 is the most frequently mutated gene in human cancers, historically, it has proved difficult to develop drugs against it. However, recently, several new drugs have become available for neutralizing the cancer-promoting effects of mutant p53. The aim of this article is to discuss the most promising of these drugs, especially those that are being investigated in clinical trials.TP53 myelodysplastic syndrome. Although no data on clinical efficacy are currently available for eprenetapopt, preliminary results suggest that the drug is relatively well tolerated. Other strategies for targeting mutant p53 that have progressed to clinical trials involve the use of drugs promoting degradation of the mutant protein and exploiting the mutant protein for the development of anti-cancer vaccines. With all of these ongoing trials, we should soon know if targeting mutant p53 can be used for cancer treatment. If any of these trials show clinical efficacy, it may be a transformative development for the treatment of patients with cancer since mutant p53 is so prevalent in this disease.Mutant p53 is one of the most attractive targets for new anti-cancer drugs. Although traditionally regarded as difficult to drug, several new strategies have recently become available for targeting the mutant protein. One of the most promising of these involves the use of low molecular weight compounds that promote refolding and reactivation of mutant p53 to its wild-type form. Several such reactivating drugs are currently undergoing evaluation in clinical trials, including eprenetapopt (APR-246), COTI-2, arsenic trioxide and PC14586. Of these, the most clinically advanced for targeting mutant p53 is eprenetapopt which has completed phase I, II and III clinical trials, the latter in patients with mutant  TP53 which encodes the p53 tumor suppressor protein.Molecularly targeted therapy has had a major beneficial impact on the outcome of patients with several different cancer types in recent years. Thus, the use of drugs targeting HER2 amplification/overexpression in breast cancer , mutant TP53 (p53) is the most widely studied gene in the human genome [TP53 mutations in most types of cancer. Indeed, across all human cancers, TP53 is the most frequently altered gene with mutations occurring in approximately 50% of cases [TP53 is the most frequently mutated gene in most types of solid tumors. Most of these mutations belong to the missense type (70\u201380%) and are located in the DNA-binding region. Functionally, these mutations may result in loss of function, exertion of a dominant-negative effect or acquisition of a new ability [n genome , with ovof cases ,7. Furth ability ,7. DespiAlthough there are currently no approved drugs for targeting mutant p53, several are currently undergoing clinical trials. Most of these investigational drugs mediate their action by either reactivating mutant p53 back to a form with at least some wild-type properties or by promoting degradation of the mutant protein ,9 Figur. The aimTP53 mutations are present in approximately 50% of all cancers. Importantly, however, mutations occur at a particularly high prevalence (>80%) in some of the currently most aggressive tumors such as high grade serous ovarian cancer [As mentioned above, n cancer , triple n cancer , esophagn cancer  and smaln cancer . Thus, iMutant p53 is believed to promote cancer development via two main mechanisms, i.e., cell intrinsic (direct effect on tumor cells) and cell extrinsic (direct effect on tumor microenvironment) ,15,16. CAvailable data suggest that several anti-cancer drugs act at least in part by promoting apoptosis mediated via wild-type p53 . ConsistClonal mutations, i.e., mutations that are present in all or most of the malignant cells within a tumor are believed to be superior targets for drug treatment than non-clonal mutations (mutations present in a subset of tumor cells). Based on available evidence, mutations in p53 appear to occur early and are clonal in many different cancer types .Mutant forms of p53 possessing missense mutations tend to accumulate in malignant cells. The accumulation may partly relate to the inability of the mutant protein to induce expression of the E3 ubiquitin ligase MDM2, which targets p53 for proteasomal-mediated catalyzed degradation ,26. In cWhile theoretically, mutant p53 is a highly attractive target for cancer therapy, there are several difficulties in designing drugs to block its functioning. Indeed, it is considerably more challenging to restore normal activity to a defective tumor suppressor protein such as mutant p53 than to block the actions of a driver oncoprotein . The difUnlike several oncogenes which are usually mutated at a small number of hot spot areas, p53 has been shown to undergo at least 2000 different mutations (giving rise to >2000 mutant/variant proteins) . Indeed,Further complicating the impact of the different mutations is that the same mutation might have different effects in different cell types, i.e., it is cell-context dependent. For example, p53 has been reported to induce the transcription of different sets of genes in different tissue types ,34. IndeAdding yet further diversity is that the mutant p53 protein can be post-translationally modified at multiple amino acid residue sites. These alterations, which include phosphorylation, acetylation, methylation, ubiquitylation and glycosylation , may alsDeveloping a specific inhibitor against all of the different forms of mutant p53 might be expected to be highly difficult, if not impossible.With the exceptions of Y220C, Y220S and Y220N, most mutant forms of p53 lack an obvious deep pocket into which low molecular compounds can bind with high affinity and specificity . DespiteMonoclonal antibodies are amongst the most specific drugs used to treat cancer, but their current use is largely confined to targeting transmembrane proteins. However, as mutant p53 is primarily located in the cell nucleus, it cannot be readily reached with the current generation of these antibodies. A possible strategy for overcoming this problem, however, is to target processed mutant p53 bound to MHC proteins on the cancer cell membranes with so-called bispecific T-cell receptor mimic antibodies ,43.By far, the most widely investigated mutant p53 reactivating drug is eprenetapopt (previously known as APR-246). As the mode of action of this compound has been previously discussed in detail ,45,46, iAs might be expected from a small molecule that binds to thiol groups, eprenetapopt/MQ can also attach to other intracellular molecules containing these structures. In particular, it can also attach to the tripeptide, glutathione (GSH) and the redox modulating enzymes, thioredoxin reductase thioredoxin and glutaredoxin ,48,49,50n = 7) or prostate cancer (n = 7). Overall, eprenetapopt was found to be well tolerated, the most frequent adverse effects were fatigue, dizziness, headache and confusion, all of which were reported to be reversible. Evidence for reactivation of mutant p53 was the finding of cell cycle arrest, induction of apoptosis and upregulation of p53 target genes in tumor cells from several treated patients.Based on several preclinical studies showing anti-cancer activity ,44, epreTP53 mutant myelodysplastic syndromes (MDS) (n = 40) or AML (n = 11) [TP53 mutations exhibited a significantly higher complete response rate than those lacking mutations . Furthermore, patients achieving a response had a reduction in both TP53 mutation levels  and p53 protein levels. The most frequent adverse events (grade \u2265 3) in this trial were febrile neutropenia (33%), leukopenia (29%) and neutropenia (29%). Essentially similar findings were reported with eprenetapopt plus azacytidine in a second phase II trial involving patients with MDS or AML [Subsequently, eprenetapopt in combination with azacytidine was investigated in a phase Ib/II trial in patients with (n = 11) . The oveS or AML .TP53 mutant MDS. Although results from this trial do not appear to have been published to date in the peer-reviewed literature, according to the Aprea website , it failed to meet the primary statistical endpoint of complete remission. Complete response rates however, tended to be higher in the combination arm, i.e., 33.3% of patients receiving eprenetapopt with azacytidine achieved a CR versus 22.4% in the azacitidine alone arm. However, the press release stated that with further follow-up, secondary endpoints such as overall response rate and duration of response might favor the combination arm.The above findings led to a phase III trial of eprenetapopt plus azacitidine versus azacitidine for the frontline treatment of patients with Despite this negative finding, eprenetapopt has received Breakthrough Therapy, Orphan Drug and Fast Track designations from the US FDA for MDS, and Orphan Drug designation from the European Commission for patients with MDS, AML and ovarian cancer.COTI-2 is described as a third generation thiosemicarbazone drug with broad anti-cancer activity ,57,58. COther actions of COTI-2 include activation of AMP-activated protein kinase (AMPK) and inhibition of mTOR signaling . These eClinicalTrial.gov website , this trial \u201cis designed primarily to assess the safety and tolerability of COTI-2 monotherapy or combination therapy in patients with advanced and recurrent malignancies to establish a recommended phase II dose for future studies\u201d. Preliminary data suggest that COTI-2 is well tolerated, the most frequent adverse effects being nausea, vomiting, fatigue and abdominal pain. Of the 24 patients treated, only 2 (8%) were reported to have to undergo a decrease in the dose of COTI2 administered [COTI-2 as a monotherapy or in combination with standard therapies has undergone evaluation for the treatment of several different types of recurrent cancers in a phase I clinical trial NCT02433626). According to the US National Library of Medicine, 6. AccordArsenic trioxide (ATO), a drug which has been used for several years to treat acute promyelocytic leukemia, was recently shown to reactivate mutant forms of p53 possessing structural mutations . This acIn addition, a reactivating p53, ATO, has also been reported to degrade the mutant protein . Thus, AClinicalTrials.gov Identifier: NCT04695223).A potential advantage of ATO over other mutant p53-reactivating drugs such as eprenetapopt and COTI-2 is that its pharmaceutical and toxicological properties are well established . It shouIn contrast to the mutant p53 reactivating drugs discussed above, PC14586 specifically reactivates mutant p53 proteins containing the Y220C mutation . As mentBAX, PUMA, MDM2 and CDKN1A (encodes p21) as well as induction of cell cycle arrest. Consistent with the ability of mutant p53 to induce immunosuppression (see above), administration of PC14586 was found to enhance immunity by increasing influx of immune cell such as CD4+ T cells, CD8+ T cells, T-regulatory cells and natural killer T cells into tumors [In mutant cell lines, PC14586 was found to reactivate and stabilize the p53 Y220C mutant protein . In turno tumors . In miceo tumors .PC14586 monotherapy is currently being investigated in a phase I/II clinical trial in patients with advanced cancers harboring the p53 Y220C mutation (NCT study identifier NCT04585750). The aim of this multicenter dose escalation study is to evaluate PC14586 safety, pharmacokinetics, pharmacodynamics and possibly efficacy in patients with advanced solid tumors possessing a p53 Y220C mutation. Preliminary findings presented at the 2022 American Society of Clinical Oncology conference suggest that PC14586 is generally well-tolerated with only grade I/II adverse events which were found in 79% of the treated patients . The mosTP53 Y220C mutation.Recently, the US FDA granted Fast Track designation to PC14586 for the treatment of cancer patients with locally advanced or metastatic solid tumors possessing a The advantage of mutant-specific drugs such as PC14586 over other mutant p53 reactivators such as eprenetapopt is that they are unlikely to bind to wild-type p53 or indeed to other intracellular proteins. The main disadvantage, however, is that the p53 Y220C mutation is relatively rare in cancer, being only the 9th most frequent mutation. Despite this low frequency, the Y220C is estimated to be responsible for in excess of 100,000 cases of cancer per year, worldwide .Another strategy for targeting \u201cdifficult to drug\u201d driver oncoproteins such as mutant p53 is to treat with compounds that promote their degradation and thus their elimination from tumor cells. Such drugs in everyday clinical use include fulvestrant which acts by degrading the estrogen receptor and is used for the treatment of patients with estrogen receptor-positive metastatic breast cancer  and lenaProof of principle that the presence of mutant p53 was necessary for the maintenance of malignancy emerged from knockdown studies which showed that depletion of the mutant protein reduced oncogenicity . AmongstThese observations led to the testing of low molecular weight HSP inhibitors as potential drugs for cancer treatment. Early work using preclinical models confirmed that treatment with low molecular weight HSP90 inhibitors such as ganetespib, 17-allylamino-17-demethoxygeldanamycin or geldanamycin did indeed result in the degradation of specific forms of mutant p53 and inhibition of tumor cell growth . In contMore recently, treatment with a group of widely used drugs known as statins, was also shown to result in degradation of mutant p53 ,81,82,83Several studies have recently shown that decreased production of MVP leads to the degradation of mutant p53 ,81,82,83p53 with conformational mutations [Consistent with their ability to degrade mutant p53, several different statins when used alone have been shown to inhibit the in vitro and in vivo growth of cancer cells in model systems ,82,83,85utations , other sClinical evidence supporting an anti-cancer role for statins are multiple epidemiological studies showing that patients with a range of different cancer types undergoing treatment with statins have a superior outcome compared with those not receiving these drugs ,86,87,88TP53 mutation and are at risk of developing large intestinal cancer . A third trial  is investigation the benefit of neoadjuvant atorvastatin plus zoledronate in patients with triple negative breast cancer . In this study, response is being related to the level of cellular p53. In addition to these trials, several studies are investigating if statins have anti-cancer activity irrespective of p53 status  (accessed on 1 June 2022).One of these is a window-of-opportunity trial to determine if administration of atorvastatin decreases levels of p53 with conformational mutations in patients with different solid tumor or AML (NCT03560882). Another is a phase II trial investigating the effect of atorvastatin for treating patients with ulcerative colitis who have a dominant-negative missense Should any of these trials produce positive findings, statins might be expected to rapidly enter clinical use for the treatment of cancer. As with ATO (see above), statins have been in clinical use for several years. Thus, their pharmaceutical and toxicological properties are also well established. Since they are relatively safe, inexpensive and off-patent, they would be ideally suitable for repurposing for the treatment of cancer.Before concluding this section, it is also important to add that in addition to degrading mutant p53 via downregulation of the mevalonate pathway, there are several other mechanisms by which statins could exert anti-cancer activity . IndeedTP53 is mutated in a large proportion of human cancers, it would be expected to act as a neoantigen and promote an immune response, thereby opening up the potential of exploiting it as an anti-cancer vaccine. Evidence from mice models and patients with cancer showed that administration of certain p53 neo-epitopes containing hot-spot mutations did indeed provoke the generation of specific T cell (CD8+ and CD4+ cells) responses against p53 peptides containing the hot-spot mutations [25\u201335, p53110\u2013124, p53149\u2013157 and p53264\u2013272 have been observed in experimental systems [A further strategy for targeting mutant p53 that has progressed to clinical trial investigations is vaccination against the mutant protein. The aim of therapeutic cancer vaccination is to stimulate a patient\u2019s adaptive immune system against tumor (neo)antigens and thus block tumor growth. As utations ,94. Thus systems ,94. Thes systems ,94. Sinc systems . Althoug systems .Before concluding this section, it should be stated that these negative clinical findings are not unique to mutant p53 as a cancer vaccine . DespiteFuture research on anti-cancer vaccines including p53 vaccines should therefore focus on optimizing delivery of the vaccine to the appropriate cellular location, selecting the optimum adjuvant to be co-administered with the vaccine, identifying the optimum dose and treating patients with early cancer (where immunosuppression would be expected to be limited) rather that patients with advanced cancer (where immunosuppression would be expected to be strong).Several other strategies for targeting mutant p53 are currently undergoing investigations, most of which are still at a preclinical level . Gene thGoing forward, one of the most promising approaches for targeting mutant proteins such as mutant p53 is likely to be the use of bispecific T-cell receptor mimic antibodies, i.e., antibodies in which one end binds to a mutant epitope on a tumor cell and the other end to a T-cell. It was mentioned above that the present generation of monoclonal antibodies cannot readily access nuclear proteins such as mutant p53. However, peptides derived from such mutant proteins can be processed by the proteasome system and converted into small peptides. These peptides can attach to HLA proteins located on the cell membrane and hence be recognized by T-cell via their T-cell receptors.TP53 gene, i.e., the R175H mutation. The bispecific single chain antibody, named H2scD, bound to both the specific mutant p53 peptide attached to an HLA allele (A*02:01) and the T-cell receptor. By binding to both sites, the bispecific antibody was able to activate T cells and consequently eliminate cancer cells possessing the specific mutant peptide. Theoretically, this strategy could be used to target other mutant forms of p53 as well as other mutant proteins such as KRAS [To target such peptides, Hsiue et al.  develope as KRAS . AlthougTP53 mutation, R273H. Evidence of gene correction was the induction of several canonical p53 target proteins such as p21, MDM4, PUMA and GADD45A. Further work by Sayed et al. [TP53 R175H mutation in a cell line derived from a colorectal cancer organoid. The challenge will be to translate this technology to patients with cancer.Another potential strategy for targeting cancer-specific mutations is the use of CRISPR/Cas9 gene editing. This technology uses genetic engineering to alter the cells\u2019 genome . In one d et al.  showed tTP53 mutations [Other potential strategies for targeting muting mutant p53 include the use of PROTACs/molecular glues which can lead to degradation of specific mutant proteins , inductiutations  and explutations . AlthougWith the multiplicity of ongoing clinical trials, we should soon know whether targeting mutant p53 has clinical efficacy in cancer. The recent demonstration of response to PC14586 provides some optimism that mutant p53 may eventually be targeted for cancer treatment. However, irrespective of the outcome of the ongoing trials, mutant p53 remains a highly attractive target for cancer treatment (see above). We, therefore, encourage academic researchers and pharma companies to intensify their research into exploiting the most frequently mutated gene in cancer for therapeutic potential. The ultimate reward could be a drug with wide application across a broad range of cancer types."}
{"text": "The purpose of this study is to explore cohort differences in the trajectories of cognition and functional limitations among oldest-old adults. Using 1992-2018 data from the Health and Retirement Study, participants included those who survived to at least 98 years (N=494). The study included three cohorts  predicting intercept and slope of cognition and functional limitations. Two separate latent growth curve models were computed, and models of cognition, \u03c72(5)=6.935, p=.226 and functional limitation, \u03c72(7)=7.743, p=.35 fit the data well. There was a significant decline in cognition levels and a significant increase in functional limitation over time. When adding cohort, results indicated that older cohorts were more likely to have a decrease in cognition and increase in functional limitations compared with the younger cohort. Future research should investigate trajectories of other health variables for a better understanding of health changes of centenarians."}
{"text": "Our understanding of Alzheimer\u2019s disease (AD) pathogenesis has developed with several hypotheses over the last 40 years, including the Amyloid and Tau hypotheses. More recently, the p53 protein, well-known as a genome guardian, has gained attention for its potential role in the early evolution of AD. This is due to the central involvement of p53\u2019s in the control of oxidative stress and potential involvement in the Amyloid and Tau pathways. p53 is commonly regulated by post-translational modifications (PTMs), which affect its conformation, increasing its capacity to adopt multiple structural and functional states, including those that can affect brain processes, thus contributing to AD development. The following review will explore the impact of p53 PTMs on its function and consequential involvement in AD pathogenesis. The greater understanding of the role of p53 in the pathogenesis of AD could result in more targeted therapies benefiting the many patients of this debilitating disease. Alzheimer\u2019s disease (AD) is a pernicious condition of the aging population that leads to progressive degeneration of brain function, whose characteristic anatomopathological hallmarks, firstly described by Alois Alzheimer, remain the senile plaques and neurofibrillary tangles which consist of beta-amyloid peptides and hyperphosphorylated Tau proteins . The forThe amyloid hypothesis of AD pathology suggests that the increased presence of amyloid-beta (A\u03b2) is the triggering factor, which then links directly to Tau protein hyper-phosphorylation, synapse loss, and cell death .However, the classical amyloid hypothesis has been questioned  and otheGSK-3\u03b2 is a critical cell cycle regulation factor and has an important function in neurons for Tau regulation. Increased GSK-3\u03b2 activity has been shown to be one of the first events in AD pathology, leading to increased A\u03b2 production and Tau hyper-phosphorylation . MoreoveMitochondrial dysfunction has also been implicated in AD pathology, Oxidative stress, damage by free radicals in parallel with changes in the expression of superoxide dismutase (SOD) and catalase, both important anti-oxidant enzymes, have been shown in tissue from AD patients .Interestingly, In the AD brain, fatty acid oxidation has been linked to Tau pathology, and p38 MAPK has been linked as a potential candidate gene within the relevant MAP Kinase pathway . FurtherThe development of clinical symptoms of AD requires many decades for pathological changes to develop . HoweverAs seen in cancer, cellular response mechanisms involve multiple pathways. The role of one of these genes, TP53, is now being revealed as central to the cellular response to cancer and other diseases, including obesity and aging-associated neurodegenerative disorders . Given tEver since the discovery of p53  it has bTumorigenesis of p53 has been shown in numerous mouse experiments where p53 loss-of-function predisposes cells to permanent damage and tumor transformation . Tumorigp53 has been shown to have further roles including the control of cell metabolism, pluripotency, epigenetic control, and aging . As p53 The role of p53 is now realized to be much greater than simply dealing with DNA damage. Thus, p53 should now be regarded not only as a \u201cgenome guardian\u201d but as a \u201cstress guardian.\u201dThere are several cellular mechanisms that control p53 function, and post-translational modifications (PTMs) are the most common and effective type of regulation .O-GlcNAcylation, ADP-ribosylation, and \u03b2-hydroxybutyrylation  are induced by cytotoxic stimuli, which results in p53 activation and stabilization . p53 aceMethylation of lysine and arginine residues is an important control mechanism of p53 function as it\u2019s a reversible process . InteresMDM2 ubiquitination is critical for maintaining p53 levels in the cell. This has been postulated to occur by polyubiquitination of p53 leading to proteasome pathway mediated protein degradation, which then inhibits p53-mediated transactivation . The majDifferent types of p53 ubiquitination are known to cause changes in p53 function as well as stabilization, by regulating its cytosolic location and nuclear export. p53 ubiquitination is also implicated in the disruption of its binding to promoters of target genes, similar to the action of transcription factors in the nucleus. This results in halting the cell cycle and triggering apoptosis . In unstThese include neddylation and SUMOylation .These modifications all have several common features: (i) Multiple sites: on many different amino acids. (ii) Multiple functions: that are site-specific. (iii) Reversibility: such that there are functionally modifying and de-modifying mechanisms. (iv) Cross talk: modifications at certain sites can affect modifications at other sites .Aerobic metabolism has several by products including ROS and reactive nitrogen species (RNS). RNS can initiate signaling that is transduced through redox-based PTMs of proteins. It is known that redox signaling is a critical process in cell physiology and is an important regulator of redox-sensitive proteins . The brain vitro model of amyloid precursor protein (APP) overexpression-induced A\u03b2 load. Concomitantly, they observed an associated increase in the expression of unfolded p53, and neuronal dysfunction. Restoring the native conformational state of p53 with Zinc chloride prevented neuronal dysfunction, supporting the role of p53 in maintaining neuronal functionality  and the TP53 induced glycolysis and apoptosis regulator (TIGAR) . An impaIn vitro experiments on BACE, the rate-limiting enzyme in the generation of A\u03b2, showed its transcription is repressed by p53, thus affecting potential A\u03b2 accumulation. During oxidative damage exposure, p53 expression is upregulated. This leads to a decrease in the level of BACE1 expression and potentially contributes to AD pathogenesis , is known to regulate cellular metabolic activity through the process of deacetylation/acetylation. Sirtuins are NAD+-reliant deacetylases. Sirtuin deficiency disrupts mitophagy in numerous diseases, including AD .A recent publication has shown another potential p53 regulated pathway involved in AD development involving sirtuin 6 (SIRT6). The SIRT6 pathway promotes cell longevity by controlling several different processes including energy metabolism, genome integrity and inflammation, along with DNA repair. Interestingly a reduction in the level of the SIRT6 protein has been observed in human AD brain samples . The conUpregulation of p53 protein by Nutlin-3 avoids SIRT6 decline, and DNA impairment induced by A\u03b2 .Sirtuin family is both a stimulatory and inhibitory factor linked to p53 and, in postnatal cortical neurons, the p53 related apoptosis required PUMA transcriptional activation. PUMA, along with the Bax-mediated permeabilization of the mitochondrial outer membrane, induces cytochrome C release, caspase activation, triggering cell death. SIRT1 inhibits the apoptosis pathway by deacetylation of p53 in several senescence models .Interestingly, a recent study showed the exposure of neurons to fragments 25\u201335 of A\u03b2 peptide activates Cdk5, which promotes p53 phosphorylation and stabilization . Mitocho2O2), there is no activation of p53 dependent cell cycle regulators p21, GADD45, and BAX1 genes   . The con(GAP-43) . As a co(GAP-43) . Using i(GAP-43) . StudiesThe main AD signaling pathways implicated with p53 conformational change are summarized in The relationship between Tau, A\u03b2, and p53 has been explored in many studies and has been considered as a potential therapeutic target for AD . The rec2+-chelating activity, metal ions are sequestered from the p53 DNA binding domain, inducing conformational changes in p53, inhibiting its activity. This directly links to When A\u03b2 is present at nanomolar levels, through the HIPK2 inhibition, it induces metallothionein 2A expression and, with Znin vitro study  from AD patients . InteresAs previously discussed, the expression of the unfolded conformational variant of p53 was investigated in other dementias, and other brain disorders such as Parkinson\u2019s disease, where the data indicated that p53 is differentially expressed when compared to AD patients, thus suggesting specificity for AD .To further investigate the relationship of unfolded p53 isoforms in AD, The recent study by AZ), was further validated as a potential prognostic biomarker in AD in a set of plasma samples from the longitudinal and retrospective biobank, AIBL .The conformational variant of p53, detected by 2D3A8 and then quantified by mass spectroscopy (identified as U-p53nk, AIBL . In detaThe exact pathology of AD is controversial and remains to be fully elucidated. The validity of the amyloid hypothesis is still being challenged due to the failure of several high-profile drug trials. A significant amount of evidence supports the potential role of p53 in AD pathogenesis, particularly due to the protein\u2019s functional dysregulation and involvement in many AD pathways. PTMs are an important part of the normal regulation of p53 cellular function, and the presence of a conformational change in p53 induced by redox dysfunction in AD, leading to a loss of function in many cellular response pathways, is an indication of a central role in AD. Further studies on the conformational unfolding of p53 showed that the conformational change of the protein impacts on its role, directly increasing or decreasing the activation of specific pathways involved in AD. A recent study has also shown that p53 is expressed in the AD brain in a phosphorylated isoform in correlation with p-Tau . This stFurther studies will be necessary to further elucidate the mechanism of actions for the specific PTMs of p53 and their involvement in AD pathways.JC, SP, and PK defined the review focus. JC and SP wrote the manuscript. GA, PK, DU, and RK provided approval for publication of the content. All authors contributed to manuscript revision, read, and approved the submitted version.JC, PK, and SP were employed by Diadem srl. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
{"text": "Keratins are a group of proteins that can constitute intermediate fibers. It is a component of the cytoskeleton and plays an important role in cell protection and structural support. Keratin 17, a Type I keratin, is a multifunctional protein that regulates a variety of biological processes, including cell growth, proliferation, migration, apoptosis and signal transduction. Abnormal expression of KRT17 is associated with a variety of diseases, such as skin diseases. In recent years, studies have shown that KRT17 is abnormally expressed in a variety of malignant tumours, such as lung cancer, cervical cancer, oral squamous cell carcinoma and sarcoma. These abnormal expressions are related to the occurrence, development and prognosis of malignant tumors. In this review, we summarized the expression patterns of KRT17 in a variety of malignant tumours, the role of KRT17 in the development and prognosis of different malignant tumors and its molecular mechanisms. We also discuss the potential clinical application of KRT17 as a valuable therapeutic target. Malignant tumours are considered a serious threat and one of the leading causes of death worldwide. In addition, with the increase of the global population and unhealthy lifestyles, the situation will become more serious . AlthougKeratin is an important part of the cytoskeleton and a member of the intermediate filament superfamily. The intermediate filament (IF) cytoskeleton of mammalian epithelia is generated from pairs of type I and type II keratins, which are encoded by two large gene families and consist of 54 genes in humans and the mice . AccordiKRT17 is a type I keratin that is commonly expressed in epithelial cells . KRT17 iKeratin 17 (KRT17) belongs to the family of Type I intermediate keratins. Intermediate filaments (IFs) are a new cytoskeletal filament assembly between actin microfilaments (\u223c6\u20138\u00a0nm) and microtubules (\u223c25\u00a0nm). The diameter is \u223c10\u00a0nm . Like alin vitro and in vivo , degree of differentiation (p = 0.011), sex (p < 0.001), age (p = 0.024), and lymph node metastasis (p = 0.04). Subgroup analysis showed that the high expression of KRT17 was only associated with sex (p = 0.002) in lung squamous cell carcinoma, while the high expression of KRT17 was associated with degree of differentiation (p = 0.036), advanced TNM stage (p = 0.005), and lymph node metastasis (p = 0.011) in lung adenocarcinoma  is a disease that can be caused by many factors . It is tColorectal cancer (CRC) is one of the major causes of cancer-related morbidity and mortality in the world . MetastaGallbladder cancer (GBC) is the sixth most common gastrointestinal malignancy and the most common bile duct cancer in the world, with a 5-years survival rate of 5\u201310% in most cases . TherefoPancreatic cancer is a highly fatal disease. The 5-years survival rate is only 9%, and the global incidence has increased significantly over the past few decades . SurgicaKidney cancer makes up a small percentage of all cancer types; only 2.2% . Renal cUrothelial carcinoma (UC) is a common urinary tract tumor , and aboCervical cancer ranks fourth in the incidence of malignancies and cancer-related deaths among women , accountEndometrial carcinoma (EC) is a common malignancy in women . The maiOvarian cancer is a common gynecological cancer and even the main cause of death of gynecological cancers. Although the prognosis is good at early diagnosis, the vast majority of patients are diagnosed with advanced stage, with a 5-years survival rate of about 30\u201340% . Therefop-mTOR, HIF1\u03b1 and HIF1\u03b1 glucose transporter 1 target gene expression also decreased. The re-expression of p-Akt, p-mTOR or HIF1\u03b1 inhibited the inhibitory effect of KRT17 on cell proliferation and glycolysis. The results showed that KRT17 knockout inhibited the proliferation and glycolysis of osteosarcoma cells by inhibiting the Akt/mTOR/HIF1\u03b1 pathway (Sarcoma is a mesenchymal malignancy with high heterogeneity. Although it accounts for only 1% of all malignancies, it is the second most common malignancy in children and adolescents . When th pathway . In Ewin pathway . AlthougSkin cancer is the most common cancer in humans. The most common types are keratinocyte derived basal cell carcinoma and SCC , but theKRT17 is highly expressed in a variety of human malignancies and may be considered a valuable gene for research. However, KRT17 is underexpressed in some tumors, and it is considered that KRT17 may play a role as a tumor suppressor gene in these tumors. The high expression of KRT17 is associated with poor clinicopathological parameters of various malignant tumours, such as tumour size, depth of invasion, lymph node metastasis, tumour differentiation, distant metastasis and survival rate. Hence, KRT17 can be used as a potential prognostic biomarker for malignant tumours. However, the prognostic significance of KRT17 in malignant tumours needs to be confirmed by a large number of clinical case studies. Similarly, KRT17 also shows the high expression levels in malignant tumour cell lines. Knockout or silenced KRT17 can inhibit the proliferation, migration, adhesion and invasion of tumour cells, and promote the apoptosis of tumour cells. It may regulate a wide variety of tumour-related molecules or pathways that play a role in tumour formation. Hence, the upstream and downstream regulatory pathways of KRT17 are of significance, but further validation is needed, especially in unreported tumours or more precise mechanism studies. In addition, KRT17 knockout can increase the chemotherapeutic sensitivity for paclitaxel and cisplatin, suggesting that KRT17 is also a feasible and valuable therapeutic target, but the exact mechanism of action remains unclear and needs further elucidation. However, whether KRT17 can improve the sensitivity of radiotherapy and the efficacy of immunotherapy and biotherapy may be a new research direction of KRT17 in the future . In addiIn conclusion, KRT17 is an important, but really understudied gene. It plays a key role in promoting the growth and metastasis of tumour cells, highlighting its role in the pathogenesis of tumour. However, KRT17 as a biomarker and therapeutic target for tumour prognosis remain a major challenge, as the exact molecular mechanisms of KRT17 action remain unclear. At present, there is no unified and standardized detection method for clinical screening of KRT17 expression."}
{"text": "TP73 gene belongs to the p53 family comprised by p53, p63, and p73. In response to physiological and pathological signals these transcription factors regulate multiple molecular pathways which merge in an ensemble of interconnected networks, in which the control of cell proliferation and cell death occupies a prominent position. However, the complex phenotype of the Trp73 deficient mice has revealed that the biological relevance of this gene does not exclusively rely on its growth suppression effects, but it is also intertwined with other fundamental roles governing different aspects of tissue physiology. p73 function is essential for the organization and homeostasis of different complex microenvironments, like the neurogenic niche, which supports the neural progenitor cells and the ependyma, the male and female reproductive organs, the respiratory epithelium or the vascular network. We propose that all these, apparently unrelated, developmental roles, have a common denominator: p73 function as a tissue architect. Tissue architecture is defined by the nature and the integrity of its cellular and extracellular compartments, and it is based on proper adhesive cell-cell and cell-extracellular matrix interactions as well as the establishment of cellular polarity. In this work, we will review the current understanding of p73 role as a neurogenic niche architect through the regulation of cell adhesion, cytoskeleton dynamics and Planar Cell Polarity, and give a general overview of TAp73 as a hub modulator of these functions, whose alteration could impinge in many of the Trp73\u2013/\u2013 phenotypes.The  TP73 gene belongs to an evolutionary conserved family of transcription factors, the p53 family, with key functions to vertebrate\u2019s biology. The genes that constitute this family, TP53, TP63, and TP73, have evolved from a common ancestor and, consequently, share a similar modular structure which consists of an amino-terminal transactivation domain (TAD), a central DNA binding domain (DBD) and a carboxy-terminal oligomerization domain (OD)  domain. This C-terminal region, involved in protein\u2013protein interaction, might give p63 and p73 their unique signaling network of regulators and transcriptional targets  . Althougscovered , TP63 anhomologs  and diff targets . In addiM domain .Trp73\u2013/\u2013  and in the dentate gyrus, and loss of Cajal-Retzius (CR) neurons , has shed new light on the role of p73 isoforms in the development of the murine brain   and has in vitro . In accoin vitro . On the in vitro . This lene brain . These mhenotype . The authenotype . Intereshenotype . In the henotype . HoweverNeurogenesis in the mammalian brain is complex and requires specialized microenvironments called \u201cniches\u201d . In the Trp73\u2013/\u2013 mice is the complete lack of cytoarchitecture in the SVZ neurogenic niche  10 and 12, when the tight junctions that couple neuroepithelial cells convert into adherens junctions, and the cells acquire features associated with glial cells . It is nto E16.5 . This isto E16.5 . By E16 to E16.5 . Moreoveto E16.5 . Neverthto E16.5 . Thus, cto E16.5 , should Trp73 is necessary for the efficient establishment of both types of PCP , even ahead of p73 role in controlling cell death/cell proliferation . Also coZonula occludens protein ZO2, a scaffold protein that physically links transmembrane tight junction proteins to the apical cytoskeleton of actomyosin  or Scd4 (DEG in Saos-TetOn and E14TG2\u03b1). FSCN1 is an actin binding-protein involved in the formation of essential cell structures for migration, cell-to-cell interactions and cell-matrix adhesion (A similar situation occurred for genes related to actin cytoskeleton regulation. In this case, we draw our attention to the functional annotation terms \u201cGO:0030029\u223cactin filament-based process,\u201d \u201cGO:0032970\u223cregulation of actin filament-based process\u201d and \u201cGO:0007015\u223cactin filament organization.\u201d Among the DEGs shared within cell models, we could find some genes playing relevant roles for cytoskeleton dynamics, like adhesion ; therefoadhesion . Syndecaadhesion . Loss ofadhesion . FindingTrp73 knockout scenario are further supported by the recent study of Trp73 gene isoforms. Altogether, the analyzed studies indicate that the integration of -omics data could be a very valuable strategy to provide a more comprehensive dissection of p73 regulated molecular networks and, overall, they place p73 as a central hub in the regulation of cell adhesion and cytoskeleton dynamics, two cornerstones for tissue architecture.The DEG analysis of cell adhesion and cytoskeleton dynamics clusters for the individual models led as to ask whether we could define a more global transcriptional profile by comparing the whole list of DEGs assigned to these clusters in Saos2-TetOn, MGCs, and mESCs . For botTP73 belongs to one of the most intensively studied gene families in molecular oncology. The considerable interest stems from the fact that most human tumors have subverted the function of the founding member of this family, the p53 protein. Thus, since its serendipitous discovery (TP73 gene is a very rare event in cancers involving chromosome 1p (iscovery , p73 tumiscovery . Howeverosome 1p . Moreoveosome 1p .Trp73 deficient mice have revealed that p73 function is essential for the organization and homeostasis of different complex microenvironments governing various aspects of tissue physiology. Altogether, this has raised the idea that TAp73 was not evolved for tumor suppression, but rather to perform unique functions in regulating developmental processes through p53-independent mechanisms (Trp73\u2013/\u2013 mice are the reflection of the p73 requirement for the establishment and/or maintenance of tissue organization (The discovery of the TA- and DN-p73 isoforms with antagonist anti- and pro-oncogenic functions, and the TAp73KO mice predisposition to spontaneous tumorigenesis, demonstrated TAp73 role as a tumor suppressor gene . Howeverchanisms . We propnization . This funization . Thus, iIn this model, p73 regulates distinct transcriptional nodes in a hierarchical manner that would functionally interact with each other in a cell context and time dependent manner. Cell adhesion mechanisms are responsible for assembling cells together and, along with their connections to the internal cytoskeleton, determine the overall architecture of the tissue . In thisMCM and MMM conceived the review and took the lead in writing. LM-A performed data analysis and crafted the figures. LL-F performed a critical revision of the article. All authors provided critical feedback and contributed to the final 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
{"text": "FANCM protein truncating variants (PTVs) are emerging as risk factors for ER-negative and triple negative breast cancer. Here, we discuss evidence that greatest risk associates with PTVs, such as p.Arg658*, that extensively truncate the 2048 amino acid FANCM protein. Moreover, risks associated with other less-truncating FANCM PTVs such as p.Gln1701* and p.Gly1906Alafs12* may be amplified by additional gene variants acting as modifiers. Further studies need to be conducted taking into considerations these aspects. BRCA1 and BRCA2 genes are associated with a high-risk of breast and ovarian cancers. These genes are now widely screened to assess the risk of developing these diseases in variant carriers and their relatives. Over the last 15 years, protein-truncating variants (PTVs) in several other genes have been described or proposed to cause moderate- to high-risk for breast cancer but insufficient data hamper a conclusive annotation. Early this year, two large case-control studies were published in the New England Journal of Medicine, which aimed to clarify which genes should be used clinically and to estimates the breast cancer risk magnitude associated with PTVs in each gene. These studies were based on the sequencing of 34 and 28 known or candidate breast cancer risk genes in 113,000 women from 25 countries, and 64,000 women from the US, respectively2. Both studies found that PTVs in the 8 genes BRCA1, BRCA2, PALB2, BARD1, RAD51C, RAD51D, ATM, and CHEK2 were significantly associated with breast cancer risk. In particular, PTVs of ATM and CHEK2 were associated with risk of estrogen receptor (ER) positive breast cancer, and PTVs of BARD1, RAD51C, and RAD51D with a risk of ER-negative breast cancer. Moreover, among the genes previously proposed to be associated with an increased risk of breast cancer, the majority including NBN and RECQL, were found not associated. The unparalleled statistical power of such a large dataset appears to make this a definitive designation4. But for one of the candidate genes, FANCM, the role in breast cancer predisposition appears more nuanced. In the Dorling et al. study, FANCM PTVs were not found to be associated with overall breast cancer risk nor to have an effect on age at diagnosis, but were statistically associated with risk in ER-negative breast cancer cases  disease subtypes  change expected according to the genetic code. Instead, as we showed in cell-based experiments, the variant creates a consensus sequence for a splicing silencer triggering the skipping of exon 22. This causes a frameshift starting at aa position 1906 forming a stop codon 12 codons later6. Therefore, while the c.5791\u2009C\u2009>T variant is universally known as p.Arg1931*, the correct protein annotation of this variant would be p.Gly1906Alafs12*. FANCM:c.5101\u2009C\u2009>T (p.Gln1701*) is another PTV identified in a Finnish population study, by exome sequencing of 11 breast cancer families. Further genotyping of this allele found it to be significantly associated with breast cancer risk with higher ORs detected in ER-negative and TNBC cases8. In addition to single PTVs genotyping studies, two burden analyses of all FANCM PTVs discovered by gene sequencing were published10. Altogether, these data indicate that, while the association with risk for breast cancer was inconclusive, FANCM PTVs might be risk factors for familial breast cancer or for ER-negative and TNBC disease subtypes , which is the third most common FANCM PTV found in Europeans, in a large study including breast cancer cases and controls from 19 European countries. Surprisingly, we only found p.Arg658* to be associated with risk of ER-negative and TNBC subtypes11. We also functionally tested these three PTVs for their capacity to rescue survival and chromosome fragility in a FANCM\u2212/\u2212 patient-derived cell line exposed to the DNA damaging agent diepoxy butane. We observed that p.Arg658* was unable to rescue the cell survival and chromosome fragility while p.Gln1701* and p.Gly1906Alafs12* showed an intermediate effect11. This is consistent with the fact that the p.Gln1701* and p.Gly1906Alafs12*, located in the C-terminus of the protein, are both expected to just disrupt the binding domain of the partner protein FAAP24; while p.Arg658*, located much upstream in the gene, is expected to cause the loss of greater protein portions. Based on these genetic and functional data, it is possible to speculate that the effects on breast cancer risk of the different FANCM PTVs might be due to the extent of protein truncation of the PTV. In support of this hypothesis, two women with early-onset breast cancer (at age 29 and 32); were genotyped as homozygotes for p.Arg658*. In addition, one developed several cancers, and the other demonstrated chromosomal fragility12. In the same study, two women genotyped as homozygotes for p.Gln1701*, and one homozygous for p.Gly1906Alafs12*, developed breast cancer at age 52 or later, and their cells did not demonstrate chromosome fragility.More recently, we tested 13 which are lost in the Arg658* variant protein. Previous in vitro work has shown that the ability to recruit Fanconi and Bloom syndrome complexes is more important to the function of FANCM protein than the association with FAAP2413. Furthermore, although yet to be experimentally validated, Arg658* can be predicted to act in a dominant-negative fashion. Because the Arg658* protein variant retains the N-terminal DNA binding and ATPase domain, it could potentially block the recruitment of DNA repair complexes by the full-length protein in heterozygotes and reduce DNA repair capacity. In most other breast cancer genes, including BRCA1 and BRCA2, it is the loss of DNA repair capacity that associates with variant severity.As FANCM protein is a large, multi-domain anchor that binds both DNA and other protein complexes at DNA damage sites, different effects may arise relating to breast cancer predisposition, depending upon the protein domains deleted. For example, p.Gln1701* and p.Gly1906Alafs12* delete the C-terminal DNA binding domain and FAAP24 interaction site but retain interaction with two complexes necessary for DNA repair (the Fanconi Anemia core complex and the Bloom syndrome complex). These protein complexes associate at distinct regions located between aa 960\u20131210BRCA1, BRCA2, and other genes associated with breast cancer, there is also an impact of population ancestry and potential modifier alleles. Could this explain the differences between studies investigating FANCM variants and their impact on breast cancer? First, the Finnish studies were based on the p.Gln1701* and p.Gly1906Alafs12* genotyping in geographically, ethnically, and genetically matched cases and controls. In addition, the p.Gln1701* and p.Gly1906Alafs12* carrier frequencies are high in Finns being 1.62% and 0.92%, vs. 0.21% and 0.21% in non-Finnish Europeans (https://gnomad.broadinstitute.org/), respectively. This has allowed the authors of these studies to derive robust associations with ER-negative and TNBC risk in a relatively small sample size. Second, the multicentric study was based on greater case-control numbers allowing to reach a statistical power sufficiently large to show that p.Arg658*, despite a low carrier frequency of 0.033% in non-Finnish Europeans (https://gnomad.broadinstitute.org/), was associated with ER-negative and TNBC risks11. It is hence puzzling that in this study no association was found for p.Gln1701* and for p.Gly1906Alafs12* and risk for breast cancer or the ER-negative and TNBC disease subtypes. One potential explanation is the relative genetic isolation of the Finnish population. Finns have recurrent BRCA1/2 founder PVs and at least 12 other PVs in the moderate risk genes ATM, CHEK2, FANCM, PALB2, RAD51C, and RAD51D have known that account for the majority of Finnish breast cancer cases with a genetic component14. In addition, an excess of double heterozygosity for variants in the moderate penetrance genes has also been documented in Finns14. It can therefore be speculated that some other modifier allele exists within this population which amplifies the p.Gln1701* and p.Gly1906Alafs12* risk effects, although the studies to locate such a modifier have yet to be conducted.For FANCM PTVs could be moderate risk factors for ER-negative and TNBC. It is possible that deletions that remove more of the FANCM protein, such as p.Arg658*, are likely to have a stronger effect on both the DNA repair defect and the risk for ER-negative or TNBC. Conversely, the risk effects of FANCM:p.Gln1701* and p.Gly1906Alafs12* on these breast cancer subtypes are probably lower but may be amplified by additional variants acting as modifiers. Importantly, the two new very large studies from Dorling et al. and Hu et al. have not analyzed each FANCM PTVs singularly nor grouped based on their location within the gene. More clear results about which of the FANCM PTVs are moderate risk factors for ER-negative or TNBC and which are not may arise if this were to be done.In conclusion, all these observations indicate that certain"}
{"text": "As 87Sr/86Sr ratio, and furthermore, the 87Sr/86Sr ratio is far more influenced by the type of bedrock  than the age of bedrock.Generally, the geological age of the bedrock does not necessarily indicate a certain 87Sr/86Sr ratio does not seem to have been successful, as this isn\u2019t even shown. Such an evaluation was done only in combination with further isotopic parameters analyzed.Based on the geological situation of Slovenia, as shown by Gregorcic et al. 2021) [ [1], a f87Sr/86Sr ratio of soil  or also enriched (acidic-magmatic and metamorphic rocks)   , (Fi, (Fi87Sr87Sr/86Sr ratio for determination and control of geographic origin can be a very potent tool, depending on the exact question intended to be answered. Areas with homogenous bedrock geology and variations in 87Sr/86Sr ratio are very well suited, whereas heterogeneous bedrock geological settings are a challenge for this method, as outliers and heterogeneous values within individual areas are to be expected. One must keep in mind that the highest 87Sr/86Sr ratio for Slovenian truffles [Application of truffles ,7 was re"}
{"text": "The tumor suppressor p53 is a complex cell signaling hub encompassing multiple transcription programs and governs a vast repertoire of biological responses. However, despite several decades of research, how p53 selects one program over another is still elusive. Recent attempts have used meta-analyses of p53 ChIP-seq data to determine the core p53 transcriptional program, conserved across different models and stimuli. This review highlights the complexity of the multiple layers of p53 regulation and the context specificity of p53 target genes. More specifically, we discuss the controversy over the mechanisms of p53-dependent transcriptional repression and its potential role in the flexibility of p53 response.p53 is a major tumor suppressor that integrates diverse types of signaling in mammalian cells. In response to a broad range of intra- or extra-cellular stimuli, p53 controls the expression of multiple target genes and elicits a vast repertoire of biological responses. The exact code by which p53 integrates the various stresses and translates them into an appropriate transcriptional response is still obscure. p53 is tightly regulated at multiple levels, leading to a wide diversity in p53 complexes on its target promoters and providing adaptability to its transcriptional program. As p53-targeted therapies are making their way into clinics, we need to understand how to direct p53 towards the desired outcome  selectively in cancer cells without affecting normal tissues or the immune system. While the core p53 transcriptional program has been proposed, the mechanisms conferring a cell type- and stimuli-dependent transcriptional outcome by p53 require further investigations. The mechanism by which p53 localizes to repressed promoters and manages its co-repressor interactions is controversial and remains an important gap in our understanding of the p53 cistrome. We hope that our review of the recent literature will help to stimulate the appreciation and investigation of largely unexplored p53-mediated repression. The transcription factor p53 is the most frequently mutated gene in cancer and arguably the most critical barrier against tumorigenesis. Because of its crucial importance in most types of cancer, it has become one of the most studied genes . The higWhile transcription-independent functions of p53 that participate in its pro-apoptotic properties have been described , the priRecently, several studies tried to simplify the complexity of the p53 response toward its core target genes by performing a meta-analysis of p53 cistrome and transcriptome in multiple cell lines upon different stimuli. The authors of these studies suggest that the direct p53 core genes involve a certain number of activated target genes, while p53-dependent repression is only indirect, occurring via induction of p21 followed by activation of the downstream DREAM repressive complex  ,7. In thp53 induces a remarkable variety of biological responses, ranging from transient or permanent growth arrest (senescence), induction of pro-oxidant response and apoptotic cell death , modulatReactivation of the p53 killer function  in established cancers is the ultimate goal of p53-based therapies. However, the induction of p53-mediated apoptosis in normal tissues is the cause of toxicity upon chemotherapy and the pathological loss of cells in neurodegenerative diseases, ischemia and stroke. Induction of senescence by p53 might also contribute to aging . On the p53 is a transcription factor that binds to its response elements (RE) in target genes and facilitates or prevents the formation of active transcription complexes, thus activating or repressing gene expression. p53 transcriptional activity is tightly regulated at multiple levels .p53 is heavily regulated by more than 300 different posttranslational modifications (PTMs), such as phosphorylation, acetylation, ubiquitination, neddylation, SUMOylation and methylation (reviewed in ). PTMs hOn top of PTM complexity, the TP53 gene can give rise to twelve different protein isoforms by a combination of alternative splicing, usage of IRES and alternative promoter . All p53Another level of regulation is the persistence of p53 on the promoter. Several studies have highlighted the importance of p53 dynamics for its transcriptional response ,37,38. AOverall, a network of multiple factors affects p53 transcriptional response, but our understanding of p53 life or death decisions constitutes only the tip of the iceberg. More systematic studies are required to address these urgent questions of p53 biology and provide new ideas for combination therapies to direct p53 response to the desired outcome.ca 100 p53 target genes have been recently defined by comparing multiple complementary datasets. These are induced irrespective of PTMs and biological outcomes (growth arrest/senescence) upon nutlin treatment in three cancer cell lines of different origins [Ccng1, Eda2r, Gtse1, Mdm2, Polk, Psrc1, Zfp365), while the canonical p53 target gene p21 was not among them. This extreme context specificity of the p53 response illustrates how the definition of the core p53 program is challenging and may be biologically irrelevant in a broader picture.Our analysis of genome-wide chromatin occupancy by p53 using ChIP-seq revealed the \u201cp53 default program\u201d , irrespective of the activating compound  and regardless of distinct transcriptional and biological responses triggered by these compounds . The cor origins . The autOverall, considerable evidence supports the notion that the diverse p53-mediated transcriptional programs are governed by the epigenetic state of the chromatin and thus the availability of p53 RE, its PTMs and the repertoire of co-regulators, which greatly vary among cell types and upon different stresses ,42.Thus, genes involved in \u2018non-core functions\u2019, including p53-repressed genes, most probably determine the extraordinary flexibility of the context-specific p53 response and drive its tumor suppressor properties in different tissues.p21). The direct repression by p53 involves binding to the gene and recruitment of co-repressors/HDACs or competition with/sequestration of transcriptional activators (reviewed in [The molecular mechanism of p53-mediated gene repression is a subject of active debate . p53 caniewed in ). Despitiewed in ,44). Antiewed in ,46.Another interesting possibility is that p53-dependent repression could be mediated by epigenetic silencing. Indeed, p53 has been shown to regulate the transcription of the DNA methyltransferases DNMT1, DNMT3a and DNMT3b, and the TET enzymes Tet1 and Tet2; hence, p53 can regulate DNA methylation homeostasis . FurtherDrosophila and zebrafish models, demonstrating its importance during evolution [A gene is regarded as a direct p53 target if it matches at least three of the four criteria: (1) p53-dependent differential expression; (2) the presence of p53 response element (RE) in the promoter; (3) p53 binding to its RE; (4) p53-dependent regulation of the Luc reporter containing p53 RE from the gene. Based on these strict criteria, at least two dozen p53-repressed genes have been defined as direct targets . A few evolution .However, in recent years, several studies have suggested that p53 represses genes solely indirectly through the induction of CDK inhibitor p21, which prevents phosphorylation of Rb and Rb-like proteins, thus transmitting the signal from p53 to a repressor DREAM complex ,59 Figuc. Among The major limitation of meta-analysis is that it considers only those peaks and genes that are reproduced in all data sets, disregarding lineage- and stress type-specific gene regulation by p53\u2014since these, by definition, are not common for all datasets, and therefore appear to have lower p53 signal in meta-analysis. Thus, by no means does this meta-analysis of different datasets identify the whole p53 cistrome and transcriptome. These studies have identified a core, or default, p53 chromatin occupancy pattern and transcriptional program, which are triggered in all types of cells irrespective of cellular chromatin state, the nature of activating signal, p53 PTMs and diverse biological outcome. As mentioned above, all these factors create the diversification of context-dependent p53 transcriptional regulation, which needs to be investigated to enable us to manipulate p53 for a long cancer-free life.We have previously analyzed p53 occupancy at repressed genes and noted that p53 peaks are usually smaller on repressed genes in comparison to activated ones . We suggA comprehensive meta-analysis of ChIP-seq data from different experiments remains a challenge; as such, most computational methods consider only the top few hundred highest peaks with the highest affinity RE, whereas low peaks/weaker RE are completely ignored. Nevertheless, several studies have highlighted the role of low-affinity REs for robustness, specificity and precision of gene regulation (reviewed in ).Previous studies (reviewed in ), as welNotably, analysis of the p53 REs sequence in repressed genes showed more flexible dinucleotide combinations in the core CWWG of p53 RE compared to p53-activated genes . ImportaInterestingly, the lower affinity binding sites might require a higher level transcription factor to achieve a productive binding, as shown for another transcription factor\u2014c-Myc, which can activate and repress genes. c-Myc occupancy at repressed genes is lower than that at activated genes and requires higher concentrations of c-Myc for transcriptional repression . This imRecently, a thorough meta-analysis of 46 p53 ChIP-seq datasets has found that only 14% of genes bound and repressed by p53 are DREAM targets . By consIn summary, to identify genes involved in the diversification of the p53 transcriptional response in a signal- and lineage-dependent manner, we need to consider lower affinity/lower occupancy p53 binding sites, including those in p53-repressed genes. More systematic studies are required to elucidate the difference between the p53 RE in activated versus repressed genes. It is possible, for example, that the presence of another transcription factor at the promoter due to a composite binding site  can switch p53 activity towards transcriptional repression. Interestingly, our bioinformatics search for transcription factors with composite binding sites in p53-repressed promoters has identified several of those, including E2F family members (unpublished data). We speculate that activated p53 and E2F bound to hypophosphorylated Rb might cooperate to repress genes. Further studies will hopefully help clarify these issues and identify cellular cofactors and small molecules that can switch p53 activity from gene activation to repression in a controlled fashion."}
{"text": "Numerous studies indicate that zinc and the new zinc-related adipokine, zinc-\u03b12-glycoprotein (ZAG), are involved in lipid metabolism. Excess body fat lowers blood concentrations of Zn and ZAG, leading not only to the development of obesity but also to other components of the metabolic syndrome. Zinc homeostasis disorders in the body negatively affect the lipid profile and cytokine secretion. Zinc appears to be a very important ZAG homeostasis regulator. The physiological effects of ZAG are related to lipid metabolism, but studies show that ZAG also affects glucose metabolism and is linked to insulin resistance. ZAG has a zinc binding site in its structure, which may indicate that ZAG mediates the effect of zinc on lipid metabolism. The review aimed to verify the available studies on the effects of zinc and ZAG on lipid metabolism. A literature review within the scope of this research area was conducted using articles available in PubMed (including MEDLINE), Web of Science and Cochrane Library databases. An analysis of available studies has shown that zinc improves hepatic lipid metabolism and has an impact on the lipid profile. Numerous studies have found that zinc supplementation in overweight individuals significantly reduced blood levels of total cholesterol, LDL (Low-density lipoprotein)cholesterol and triglycerides, potentially reducing cardiovascular morbidity and mortality. Some results also indicate that it increases HDL-C (High-density lipoprotein) cholesterol levels. ZAG has been shown to play a significant role in reducing obesity and improving insulin sensitivity, both in experimental animal model studies and in human studies. Furthermore, ZAG at physiologically relevant concentrations increases the release of adiponectin from human adipocytes. In addition, ZAG has been shown to inhibit in vitro leptin production. Further studies are needed to provide more data on the role of zinc and zinc-\u03b12-glycoprotein. Zinc, as a trace element, plays an important role in human life. It is involved in the growth and development of the body and, as a catalyst for enzymes, participates in the metabolism of lipids, proteins and carbohydrates. It regulates the expression and activation of many molecules and builds over three hundred metalloenzymes. This metal is involved in the regulation of inflammations by affecting pro-inflammatory cytokines and plays a role in reducing oxidative stress. Zinc deficiency impairs growth, reproduction, metabolism and immunity in both humans and animals and may predispose those affected to the development of obesity and other metabolic diseases ,2,3,4. AThis paper constitutes a literature review of 196 articles available in PubMed (including MEDLINE), Web of Science and Cochrane Library databases. The inclusion criterion for papers was information on the role of zinc and/or ZAG in lipid metabolism. Keywords used to search for papers were \u201czinc\u201d, \u201czag\u201d, \u201czinc-\u03b12-glycoprotein\u201d, \u201clipids\u201d \u201clipid metabolism\u201d, \u201cadipose tissue\u201d and \u201cobesity\u201d . ExperimZinc (Zn) is currently one of the most important micronutrients in the human body. It is also an essential part of life processes, bone development and body growth.Zinc plays a major role in the metabolism of carbohydrates, fats and proteins. It is a component of more than three hundred metalloenzymes and exhibits antioxidant activity, thus participating in the reduction of oxidative stress. Zinc is also involved in the synthesis, storage and transport of insulin ,6,8. TheZinc deficiency in the body means that the energy production process is disrupted. This is due to the abnormal behaviour of metalloenzymes that include zinc, such as carbonic anhydrase. It is involved in energy production reactions in the body. When this process is disrupted, fat tissue is formed instead of energy, which promotes the development of overweight and obesity. Studies also show that at low blood levels of this element, lipid management is disrupted leading to an increase in total cholesterol, triglycerides and LDL cholesterol ,10.Zinc is a very important regulator of ZAG homeostasis, which plays a role in lipid metabolism and glucose homeostasis . In addiZAG contains trace elements such as zinc. ZAG has been shown to have 2 strong and 15 weak zinc binding sites, and the attachment of zinc at these sites enables ZAG to bind to fatty acids and \u03b2-adrenergic receptors. Correct blood zinc concentrations are also essential to maintain adequate ZAG activity, as Zn facilitates the binding of adipokine to substrates. In vitro research has shown zinc binding to play a key role as it induces oligomerisation of the zinc-\u03b12 glycoprotein, allowing ZAG to bind to fatty acids ,13,14,15Zinc-\u03b12 glycoprotein is a polypeptide with a molecular weight of 43 kDa. It was first isolated from human plasma in 1961 . ZAG is The structure of ZAG is similar to major histocompatibility complex (MHC) class 1 proteins. Similar to MHC class I molecules, ZAG has an open groove between its A1 and A2 helix domains. The ZAG groove contains polyethylene glycol (PEG). Calorimetry and fluorescence spectroscopy have shown that the ZAG groove can bind hydrophobic ligands, especially polyunsaturated fatty acids ,22,23.ZAG has been classified as a novel adipokine because it is a lipid mobilising factor . StudiesZAG also affects glucose metabolism and is linked to insulin resistance (IR) . IntraveZAG is also used as a tumour marker as its overexpression is found in several types of malignant cancers ,29,30. OThe ZAG impact mechanism on lipid metabolism has not yet been clearly defined, but the overwhelming majority of studies indicate that ZAG may affect this process in multiple ways .ZAG increases lipolysis in white adipose tissue (WAT) by acting through the classical cyclic AMP pathway . It stimPhysiologically, ZAG plasma levels range from a few mg/dL in a foetus, 8\u201312 mg/dL in young people, and 18\u201330 mg/dL in healthy adults and older men . ZAG is The expression of ZAG in adipose tissue changes depending on various factors. Increased expression is influenced by PPAR\u03b3, glucocorticoids, some \u03b23-adrenergic receptor agonists, thyroid hormones and growth hormone (GH), among others. On the other hand, chronic inflammation and increased serum leptin levels may reduce ZAG secretion in the adipose tissue ,27,39.Animal studies by Tinkov et al.  showed tLi et al.  in theirp < 0.01) and alanine transaminase (p < 0.05). Oral zinc supplementation reversed the effects of the HFD diet. Zinc reduced triglyceride levels and raised HDL-C cholesterol levels, but did not affect changes in total cholesterol and LDL-C cholesterol. In addition, it decreased the expression of high-sensitivity C-reactive protein (hs-CRP) and interleukin-6. The results of these studies suggest that zinc may protect the liver and the cardiovascular function subject to an unhealthy, high-fat diet.A study by Xu et al.  suggestsStudies show that people with excessive body fat have reduced levels of zinc and ZAG in their blood ,54,55,56Studies by Cayir et al.  found thAs shown by Voruganti et al. , even sh2, p = 0.030 and 90.4 \u00b1 15.4 vs. 88.7 \u00b1 15 kg, p = 0.020, respectively) and highly significant reductions in blood triglyceride levels . On the other hand, in a study by Kelishadi et al. [Findings indicate that impaired Zn homeostasis in obese individuals also affects circulating lipid concentrations in the blood . Numeroui et al. , supplemZinc is also involved in regulating the expression of pro-inflammatory cytokines produced by adipocytes . Zinc adZinc-\u03b12 glycoprotein plays a huge role in the regulation of adipose tissue mass. ZAG acts multidirectionally, playing a role in stimulating lipolysis, inhibiting lipid accumulation in adipose tissue, regulating serum lipid values and influencing the secretion of other adipokines .The results of ongoing studies suggest that ZAG expression in adipocytes is inversely related to fat mass. In a study by Bing et al. , it was Adipocytes and liver cells are major AZGP1 expression sites in rodents . AdminisResearch by Gong et al.  clearly Fan et al. reached similar conclusions . They foFurthermore, evidence from Gao et al.  indicateRussell et al. also observed ZAG impact on adipose tissue browning . Ob/ob mIn contrast, Rolli et al.  showed tStudies by Mracek et al.  and CepeWhite adipose tissue is found in excess in obese and overweight people. Studies indicate that ZAG has the potential to induce WAT browning in 3T3-L1 adipocytes. Xiao et al.  observedIt turns out that ZAG is also found in cord blood and influences the development of body fat as early as infancy. In the study by N\u00e4f et al.  ZAG in cp < 0.001) and that they correlated positively with age, waist circumference and BMI, fasting insulin, indices of insulin resistance, adipocyte and fatty acid binding protein (A-FABP), serum TG, and CRP. That research provided the first evidence that the excessive elevation of ZAG in individuals with metabolic disorders and obesity may be a compensatory regulation of the body to the oxidative stress present. Elevated ZAG levels may also indicate ZAG resistance, similar in mechanism to hyperinsulinaemia and hyperleptinaemia caused by the body\u2019s resistance to insulin or leptin. These studies suggest that the serum ZAG concentration value can be considered as a circulating biomarker of obesity and metabolic syndrome. However, further research is needed to determine whether the theses are correct and to fully understand the pathophysiological functions of ZAG.A study by Yang et al.  in a groIn contrast, the results obtained by Morse et al.  were incLiu et al.  in theirp < 0.05). Reduced ZAG levels were also associated with an increased risk of developing metabolic syndrome . The results of this study suggest that the ratio of serum ZAG to fat mass, may be a future diagnostic biomarker for the diagnosis of metabolic syndrome. Lei et al. reached similar conclusions [In addition, Wang et al.  examinedclusions .2 and 9 obese men (BMI = 34.7 \u00b1 1.2 kg/m2) with a similar lifestyle. Using RT-PCR, they found that the expression of the ZAG gene was reduced by 70% in the subcutaneous tissue of obese individuals compared to slim subjects. Additionally, analysis of the data showed that gene expression positively correlated with serum adiponectin and negatively correlated with plasma leptin levels and waist circumference in obese subjects.In contrast, in the study by Marrades et al. , 18 younp < 0.01). Women with excessive body weight and women with elevated blood glucose levels also exhibited lower blood ZAG levels. It also demonstrated that there was an increase in circulating ZAG after 12 weeks of treatment with either exenatide or metformin (p < 0.01). The study shows that ZAG can be used as an observational indicator in the treatment of PCOS.Overweight and obesity accompany many metabolic diseases, including the polycystic ovary syndrome (PCOS). Studies indicate that ZAG levels are significantly lower in women with PCOS than in healthy women ,96,97. ZUsing genetic profiling, it was found that ZAG transcripts in adipose tissue are reduced in obese women . It has p = 0.00088) and during diet-induced weight loss (p = 0.059). Furthermore, in healthy subjects, ZAG levels were correlated with blood triglyceride levels (p = 0.035), and in obese subjects on a VLCD diet also with LDL-C (p = 0.035 and HDL-C (p = 0.055), but not with TG. The ZAG gene was associated with circulating blood cholesterol levels, which may indicate that ZAG plays a role in its metabolism. This correlation may be due to the role ZAG plays in the body, namely in lipolysis. The researchers suggest that the correlation between ZAG and total cholesterol is the result of increased TG lipolysis, caused by the effects of ZAG.ZAG also potentially affects cholesterol metabolism. A study by Olofsson et al.  showed tEvidence that ZAG is involved in the development of cancer cachexia was presented by Mracek et al. . In cancBoth in vivo and in vitro results show that there is increased hormone-induced lipolysis in patients with cancer cachexia . Ryd\u00e9n eThere are still too few studies to determine the exact pathway through which zinc and ZAG affect lipid metabolism. Both zinc and ZAG are reduced in overweight and obese individuals, which may indicate that a deficiency of these factors indirectly impacts further development of obesity and associated diseases. Zn and ZAG show a negative correlation with body weight, BMI and adipose tissue. Research also indicates that both zinc and ZAG can regulate the lipid profile, thus preventing the development of cardiovascular diseases. In addition, zinc and ZAG have also been shown to affect other adipokines secreted by the adipose tissue. ZAG expression in human adipose tissue was positively associated with adiponectin expression. Serum zinc levels show a negative correlation with inflammatory markers.Summarising the currently available studies, it may be concluded that ZAG and Zn should be more widely used as markers for several biochemical reactions occurring in the human body, both in therapeutic and pathogenetic aspects."}
{"text": "This review aims to investigate the different levels of vitamin D and its role in muscle strength in healthy children and non-athletes. A search conducted in three databases  resulted in 655 articles, which were systematically analyzed and selected based on the following criteria: (a) original cross-sectional studies and clinical trials; (b) healthy children aged 5-11 years; (c) no language restriction or year of publication; and (d) studies that assessed the possible relationship between vitamin D levels and muscle strength. Six studies were included because they met all the inclusion criteria. According to the findings of this review, factors such as sex, skin color, and vitamin D supplementation early in life modulate the levels of vitamin D in the body, and there is a relationship between muscle strength and vitamin D levels. Interestingly, vitamin D supplementation is not always significantly associated with increased muscle grip strength. However, there is a scarcity of studies that aim to analyze the possible effects of different levels of vitamin D on muscle function and neuromuscular variables in physically inactive children and non-athletes without previously diagnosed disease. Further studies are warranted in the future to address the gap in the literature. Serum levels of vitamin D may suffer variability due to genetic and environmental factors related to general metabolism, resulting in changes in its integumentary synthesis and bioavailability ,2. In ad2D) (Vitamin D can increase the capacity to produce strength through the readiness of type II muscle fibers  and favo2D) , the bio2D)  and intr2D) . Thus, vPalacios and Gonzales  reportedThis systematic review adopted the criteria recommended by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) method , which wThe following databases were used to perform our searches: PubMed (MEDLINE), Scopus, and Psycinfo with the following combination of terms contained in the Medical Subject Headings: \u201cVitamin D\u201d AND \u201cMuscle Strength\u201d OR \u201cStrength, Muscle\u201d and \u201cChild\u201d.Two independent researchers (ABJ and TS) read the study titles and abstracts and, if necessary, read the entire text. In case of disagreement, a third researcher was consulted to establish a consensus. The following inclusion criteria were established: (a) original cross-sectional studies and clinical trials; (b) healthy children aged 5-11 years; (c) no language restriction or year of publication; and (d) studies that assessed the possible relationship between vitamin D levels and muscle strength. Studies that included child athletes, carriers of some diseases, or children receiving maintenance drug therapy were excluded. In addition, the Population Intervention Comparator Outcome strategy was used to select studies with greater specificity.ABJ and TS independently reviewed the selected articles to extract relevant data for the preparation of this review. Conflicts of opinion between the authors were mitigated by a third reviewer. It is important to note that searches performed using electronic databases were performed without the aid of any software.The Joanna Briggs Institute (JBI) tool  was usedA total of 655 titles and 88 abstracts were analyzed. Eighty-two articles were excluded because they did not meet the eligibility criteria or because they were duplicates. A total of 262 studies were excluded because they included unhealthy individuals, which constituted the main cause of exclusion. Four cross-sectional studies and two clinical trials were included. The flowchart for the selection and inclusion of articles is shown in The study participants included in this review were located in different geographic locations, including North America, Europe, and Asia. The age range was 5-11 years. Three studies were identified with insufficient classification of vitamin D levels -24, and It is important to note that vitamin D3 undergoes enzymatic conversion in the liver by vitamin D-25-hydroxylase to produce 25-hydroxyvitamin D (25OHD). 25OHD is the main circulating form of vitamin D in the body.Possible relationships between handgrip strength (HGS) and vitamin D levels were analyzed in all studies, as shown in p=0.96)Filteau et al.  analyzedTrilok-Kumar et al.  studied p=0.005). After supplementation, all children had vitamin D levels \u226550 nmol/L and muscle strength did not differ between groups. Before supplementation, the HGS was 92.18\u00b14.9 N (placebo group), 107.87\u00b15.88 N (10 \u03bcg/day) and 106.89\u00b14.90 (20 \u03bcg/day). After supplementation, the HGS was 98.06\u00b15.88 N (placebo group), 112.78\u00b15.88 N (10 \u03bcg/day) and 109.83\u00b14.90 N (20 \u03bcg/day). In this study, there was no difference between the sexes in terms of muscle strength.To determine whether muscle strength is associated with the plasma levels of 25OHD, Mortensen et al.  analyzedp=0.013). However, in girls, there was a significant association , demonstrating an increase in serum 25OHD of 25 nmol/L resulted in an increase in HGS of 2.25 N. It is worth mentioning that the mean HGS value for these children was 82.76\u00b116.96 N.Al-jwadi et al.  found thp=0.010) and Taql genotype \u201cCC\u201d (p=0.038). In addition, three VDR haplotypes were significantly associated with HGS in the dominant and non-dominant hands .Bozsodi et al.  determinThis review investigated scientific literature concerning the relationship between serum vitamin D levels and muscle strength in healthy children. In general, the studies included in this review observed a relationship between muscle strength, measured through HGS, and the serum levels of 25OHD. Variables such as sex, age, and ethnicity can be intervening factors in this relationship. Furthermore, it was found that vitamin D supplementation was not always significantly associated with an increase in HGS, suggesting the need for further studies to consolidate results on this association.It is important to note that none of the studies included in this review (selected according to our inclusion criteria) were executed in countries with a low Human Development Index nor in countries with a tropical climate. Both are important sociogeographic factors that directly influence an individual's serum vitamin D levels, environmentally and with regard to vitamin D synthesis .p=0.256).HGS was used as a predictor of motor performance in healthy children in all studies included in this review. HGS is favorable among muscle function tests because it is simple, non-invasive , and ineA systematic review conducted in 2019 . Humans One of the studies included in this review  presentep<0.001). However, it is necessary to better explain the differences between the sexes to determine the possible relationship between HGS and vitamin D levels from this perspective.Regarding the influence of sex, changes in HGS related to 25OHD levels were observed in children of both sexes. However, only female individuals showed a positive and significant association ,31. In cAccording to our results, supplementation with vitamin D can increase serum 25OHD levels, although this change does not appear to be related to increases in HGS. Studies conducted in children with health impairments have reported the effects of vitamin D supplementation on muscle strength -40 and cAlthough there are discrepancies regarding the association between vitamin D and muscle strength, studies conducted in a population with some motor limitations or in individuals with deficient or insufficient 25OHD levels showed significant positive effects of vitamin D supplementation on neuromuscular abilities ,38,41. IVitamin D deficits in childhood can result in physical and mental health problems in adulthood and old age. One of the many variables that influence serum levels of vitamin D is ethnicity. Some reviews point out that cultural effects may cause vitamin D deficiency, often severe, due to factors such as clothing, beliefs, eating habits, and even the levels of physical activity -47. The Silva ABJ and Carmo TS contributed to the research conception, data collection, interpretation of results, and critical review of the manuscript. Souza APS, Silva MRM, and Fernandes MSS contributed to data analysis and interpretation, manuscript drafting and critical review. Souza VON and Barros WMA contributed to the data collection and critical review of the manuscript."}
{"text": "Buffy coat rLTL and mtDNA content were quantified by monochrome multiplex quantitative polymerase chain reaction. Generalized linear mixed-effects models were used to predict the absolute and percent change in rLTL and mtDNA content after 12 weeks. Iron supplementation was not associated with an absolute or percent change in rLTL after 12 weeks compared with placebo . However, iron supplementation was associated with a smaller absolute and percent increase in mtDNA content after 12 weeks compared with placebo . Thus, daily oral iron supplementation for 12 weeks was associated with altered mitochondrial homeostasis in our study sample. More research is needed to understand the risk of iron exposure and the biological consequences of altered mitochondrial homeostasis in order to inform the safety of the current global supplementation policy.There is limited evidence regarding the potential risk of untargeted iron supplementation, especially among individuals who are iron-replete or have genetic hemoglobinopathies. Excess iron exposure can increase the production of reactive oxygen species, which can lead to cellular damage. We evaluated the effect of daily oral supplementation on relative leukocyte telomere length (rLTL) and blood mitochondrial DNA (mtDNA) content in non-pregnant Cambodian women (18\u201345 years) who received 60 mg of elemental iron as ferrous sulfate ( Anemia is defined as a hemoglobin concentration <120 g/L in non-pregnant women of reproductive age . In 2011In Cambodia, ~43% of non-pregnant women of reproductive age have anemia . AlthougThere is strong evidence that iron supplementation is effective in treating women who are iron-deplete  . HoweverGlutathione is an abundant antioxidant that plays a critical role in protecting cells against oxidative damage . The ratn = 69 randomly selected women who received iron versus placebo. The results from this pilot study provided the impetus to further investigate markers of cellular damage in the larger study sample (n = 376). Thus, we evaluated the effect of daily oral supplementation with 60 mg of elemental iron for 12 weeks, compared with placebo, on two biomarkers of cellular damage: relative LTL (rLTL) and absolute mtDNA content in n = 376 Cambodian women.First, we conducted a pilot study to measure the GSH/GSSG ratio, an indicator of oxidative stress, in clinicaltrials.gov (NCT-02481375). The complete design, protocol, and methodology have been previously published , p = 0.02) and only adherent women , such that older age was associated with a smaller increase or larger decrease in LTL change. Similarly, baseline rLTL was significantly associated with the adjusted change in rLTL for the models that included all women and only adherent women (p < 0.001). While mtDNA content increased in both groups after 12 weeks, the magnitude of the increase associated with iron supplementation was significantly smaller, compared with placebo, among all women and among only adherent women.p = 1.00 and p = 0.99) or mtDNA content (p = 0.84 and p = 0.71) after 12 weeks. Similarly, the presence of any genetic hemoglobinopathy did not modify the effect of iron supplementation on the adjusted change in rLTL (p = 0.97 and p = 0.95) or mtDNA content (p = 0.09 and p = 0.19) after 12 weeks.Baseline serum ferritin concentration did not significantly modify the effect of iron supplementation on the adjusted change in rLTL (n = 81/191) in the iron group and 58% (n = 108/186) in the placebo group, and iron deficiency based on low ferritin concentrations was 1% (n = 2/190) in the iron group and 26% (n = 48/184) in the placebo group.At 12 weeks, anemia prevalence was 42%  in a model that was adjusted for multiple factors, including age [Similar to the findings described above, some cross-sectional studies have demonstrated a positive association between iron status or exposure and mtDNA content ,52. Datading age . In a reding age  reportedding age .n = 17 had an elevated serum NTBI (\u22650.1 \u03bcmol/L) after 12 weeks of supplementation, but this did not appear to be associated with iron supplementation (n = 9 were in the Fe group and n = 8 were in the placebo group) [Despite previous cross-sectional studies demonstrating a negative association between iron status or iron supplementation and rLTL ,58,59,60o group) . Howevero group) . We alsoo group) . HoweverHigher doses, durations, and adherence increase iron exposure and can cause more oxidative stress, which may explain why Xu et al.  observedAs our study sample contained a mix of both iron-deplete and iron-replete women at baseline, we assessed the interaction effect between baseline serum ferritin concentration and iron supplementation on both rLTL and mtDNA content at 12 weeks. No interaction was observed, suggesting that the effect of iron supplementation on these outcomes was independent of the presence or absence of iron deficiency at baseline. We similarly observed no interaction effect between the presence of a genetic hemoglobin disorder and iron supplementation on rLTL and mtDNA content. Future studies should also consider these factors as they may impact policy decisions regarding untargeted supplementation regimes.There are no known published studies that have investigated the effect of 60 mg of elemental iron for 12 weeks on the change in rLTL or mtDNA content. The dose and duration of exposure to iron supplements that may lead to the damage of telomeric DNA are not yet clear. The rigorous study design of our randomized controlled trial is better suited to measure changes in rLTL and mtDNA content that are a result of iron supplementation, as compared with cross-sectional studies that can only infer association. Further, the MMqPCR assay used in our study for the rLTL and mtDNA content analyses is a robust high-throughput method that has been validated against other methods of telomere length and mtDNA content measurement . Some liIn conclusion, it is likely that 60 mg/d of elemental iron as ferrous sulfate for 12 weeks does not affect rLTL, but is associated with altered mitochondrial homeostasis. More research is needed to understand the effect of iron supplementation on mitochondrial homeostasis and potential associated health consequences."}
{"text": "Functional neurological disorders (FNDs) are commonly encountered in practice; however, there is a paucity of data in Africa.To identify and describe the clinical profile of patients presenting with FNDs, underlying medical and psychiatric diagnoses and review the investigation and management of these patients.Inkosi Albert Luthuli Central Hospital (IALCH), a tertiary-level hospital in Durban, South Africa.A retrospective chart review and descriptive analysis were performed over a 14-year period (2003\u20132017) on cases meeting the study criteria.Of 158 subjects, the majority were female (72.8%), had a mean age of 32.8 years, were single (63.3%), unemployed (56.3%) and of black African ethnicity (64.6%). The most common clinical presentation was sensory impairment (57%) followed by weakness (53.2%) and seizures (38.6%). Inconsistency was the most frequent examination finding (16.5%). Medical conditions were identified in half of the study population (51.3%), with hypertension (22.2%) and human immunodeficiency virus (HIV) (17.2%) being most common. Of patients with a psychiatric diagnosis (55.1%), 25.3% had depression. Magnetic resonance imaging (MRI) was the most frequently performed investigation (36.1%). The majority of patients received psychotherapy (72%) and most had not shown improvement (55.3%) at a median follow-up of 2 months, whilst 17% had deteriorated.Functional neurological disorders were most frequently diagnosed in young unmarried females, of black African ethnicity. Family history, personal exposure to a neurological illness and certain socioeconomic factors may be potential risk factors. Sensory impairment was the most common clinical phenotype. Further studies are needed to better understand and manage FNDs in the South African context. Psychiatric terms include conversion disorder, somatisation, dissociative motor disorder, hypochondriasis, factitious disorder, Munchausen syndrome and malingering. All carry different connotations with regard to causation and may affect the patient\u2019s perception of their illness.7\u2018Functional neurological symptom disorder\u2019 was added to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V) criteria in 2013 and distinguished functional disorders from intentional disorders such as malingering or factitious disorder, although this distinction is debated.8\u2018Functional neurological symptom disorder\u2019 is a broad term implying a change in function rather than structure and has become a preferred term, as it avoids the insinuation that \u2018everything is in the patient\u2019s mind\u2019 and uncertainty as to whether the problem is neurological or psychological.10Functional neurological disorders are commonly encountered in neurology clinics and may cause levels of disability and impaired quality of life similar to multiple sclerosis and Parkinson\u2019s disease.11 and approximately 50% have functional symptoms even if it is not the main complaint.8One-third of neurological outpatients experience symptoms regarded as \u2018not at all\u2019 or \u2018only somewhat\u2019 explained by disease,Neurologists and non-neurologists alike are often faced with the challenge of inconsistent examination findings, uncertainty as to whether there is co-existent organic pathology and the need to investigate these patients, sometimes at unnecessary cost. In resource-limited settings with high burdens of disease such as South Africa, the need to practise evidence-based, cost-effective medicine is essential.13Although a number of international studies have described the spectrum of FNDs, there is a paucity of data from South Africa and recent literature from Africa. Studies that describe the spectrum of FNDs were either conducted over 50 years ago or if more recent have focussed primarily on psychogenic non-epileptic seizures (PNES) and included relatively small sample sizes.15The aim of this study was to document the experience at a tertiary-level referral centre in Durban, South Africa, and identify the clinical profile, investigation and management of patients with FND presenting to the Inkosi Albert Luthuli Central Hospital (IALCH) over a 14-year period. Inkosi Albert Luthuli Central Hospital serves a population of approximately 18 million people. Patients attending public health facilities in Kwazulu-Natal (KZN) (approximately 79.5% of the population) are mostly of black African ethnicity (90.3%) reflecting local demographics.Addressing the relative lack of local data on this subject may lay the foundation for future research in this important yet neglected field in Southern Africa.A retrospective chart review was conducted on patients presenting to IALCH with a diagnosis of FND between 2003 and 2017. The tenth revision of the International Statistical Classification of Diseases and Related Health Problems (ICD) coding system was used to identify cases. ICD codes F44 \u2013 dissociative (conversion disorders), F45 \u2013 somatoform disorders, F51 \u2013 non-organic sleep disorders and F99 \u2013 mental and behavioural disorders were included. Data were extracted by using the Clickview software program, a data extraction program that is able to select patient charts via specific patient numbers according to ICD-10 codes entered. Charts were accessed via the Meditech software system. Patients with an organic neurological disease that may have explained their symptoms and those with insufficient clinical information were excluded. A total of 158 patients were analysed.A data collection tool created by the authors was used to document demographic data, clinical presentation, examination findings, organic disease, psychiatric illness if known or diagnosed during admission, investigations performed, management and follow-up.Descriptive analysis was used to address all the objectives. Continuous variables were summarised by using mean, standard deviation and range, whilst categorical variables were tabulated as frequency and percentage. Categorical variables were analysed by using the Chi-square test, and non-parametric testing was performed for continuous variables. IBM SPSS version 25 was used to analyse the data.The Ethics committee of the Faculty of Health Sciences at the University of Kwazulu-Natal, Durban, South Africa, granted the ethical approval for the study, reference number: BE042/18.During the 14-year study period, 158 subjects with FND were analysed. Patient demographics and characteristics are listed in n = 90/158 (57%), was the most common clinical complaint followed by weakness, n = 84/158 (53.2%), and seizures, n = 61/158 , that reported sensory disturbances. When noted, the sensation was reported to split the midline including vibration sense in six patients. Other sensory complaints were paraesthesias (n = 6), facial numbness (n = 1) and hyperalgesia (n = 1).Hemisensory loss was found in a third of subjects, n = 31/84 (36.9%) of presentations with weakness, followed by hemiparesis, n = 28/84 (33%) (n = 15/28 (53.6%), and when handedness was documented in these patients, all were noted to be right-handed. Thirty-three per cent (n = 3) of the nine patients who presented with quadriparesis reported associated incontinence. This was higher than the percentage of patients who had incontinence in association with other forms of weakness  patients of the total study population complained of weakness. Paraparesis accounted for 84 (33%) . In patin = 33/61 (54%), were more common than focal seizures, n = 22/61 , long duration (n = 5), no postictal phase (n = 1), hyperventilation (n = 1) and high numbers seizures (n = 2).There were 61 patients in whom a diagnosis of psychogenic non-epileptic seizures was documented. Generalised seizures, /61 36%; . Other dn = 12/61) of patients who presented with seizures themselves, a significantly higher proportion than patients without seizures and a positive family history, n = 2/97 . One patient with seizures was noted to live with a known epileptic patient, although not related. Seven patients were admitted for long-term electroencephalographic (EEG) monitoring of seizures, and all studies were normal apart from one documenting a single epileptic seizure amongst many other non-epileptic seizures. Anti-epileptic drugs were stopped in three patients and continued in three patients. Data on treatment discontinuation were not captured for the rest of the sample.A family history of seizures was present in 19.7% , hesitancy (n = 2), appear anxious (n = 2), uncooperative (n = 2), resistant (n = 2) and tearful (n = 1) during examinations.Inconsistency was the commonest clinical finding supportive of FND, 8 16.5%; . Patientn = 87/158). Depression was the commonest psychiatric diagnosis documented overall (n = 18/87) were diagnosed before admission. Of those diagnosed pre-admission, n = 7/18 (38.9%) had depression, n = 6/18 (33.3%) had both anxiety and depression and n = 4/18 (22%) had other diagnoses. When psychiatric diagnoses were made after admission (n = 66), 15 (22.7%) were diagnosed with depression, 1 with anxiety and 14 (21%) with other diagnoses.A psychiatric diagnosis was present in 55% of patients , asthma (n = 5), stroke (n = 4), hypertension (n = 2), multiple sclerosis (n = 1), spinal pathology not specified (n = 1), brain tumour (n = 1), depression (n = 1), visual loss (n = 1) and hearing loss (n = 1)  . Twelve n = 4), work-related problems (n = 1) and sexual orientation (n = 1). Domestic abuse or rape was reported as events preceding the onset of the FND in five patients. Bereavement (n = 2), alcohol abuse (n = 1), incarceration (n = 1) and school failure (n = 1) were noted in others. Trauma related to a motor vehicle accident (n = 3), surgery (n = 1) and a snake bite (n = 1) were also identified as preceding events (Stressors documented included domestic (g events .n = 86/238 (36%), computed tomography scan of the brain (CTB), n = 68/238 (29%), EEG, n = 57/238 (24%), and lumbar punctures (LPs), n = 27/238 (11.3%) (Investigations performed included magnetic resonance imaging (MRI),  (11.3%) . AbnormaElectroencephalographic findings were abnormal in three patients, of which two did not correlate with the patient\u2019s clinical seizure semiology. The other showed an epileptic seizure captured in association with multiple non-epileptic seizures.n = 2/27 (7.4%). One was explained by the patient\u2019s HIV status, and the other had an isolated raised protein of 0.61g/dL with 150 red blood cells noted.Cerebrospinal fluid (CSF) results were abnormal in n = 132/184 (72%), either received or were referred for psychotherapy. The use of antidepressants, n = 32/184 (17.4%), and physiotherapy, n = 20/184 (10.9%), was less common , attended a follow-up appointment. Symptoms had improved in n = 16/47 (34%) of subjects at a median follow-up time of 2 months and had worsened in n = 8/47 (17%), but remained static in the majority of patients 17 but older than in the 2018 Tanzanian study in which almost 50% of patients with FND were below the age of 20.12In keeping with international studies and other African studies, a female predominance was noted.16 reported that most patients with FND were married and well-educated. In that study no association was found between FND and employment status. Our investigation found a predominance of unmarried individuals who were unemployed and of black African ethnicity. Two South African studies reported that most patients (77% and 60%) who presented with PNES were of the white ethnic background; however, these studies analysed patients presenting with PNES only, with one of them limited to patients in a private healthcare setting, which is not accessible to the majority of the local population.19Jacob et al.5 Multiple social stressors including physical and sexual abuse, alcoholism, scholastic failure and bereavement were reported amongst subjects over the study period, which is similar to international data.16As suggested in our study, social circumstances contribute to the development of FNDs and their role is thought to be causative in many cases.20A South African study of pseudoseizures in patients with neurocysticercosis found 17/29 (58%) female patients had experienced sexual or physical abuse and problems related to alcoholism and other socio-economic problems.21 Contributing and causative factors stem from the influences of culture, tradition and religion but the socioeconomic impacts of poverty, alcohol abuse, gun-violence and poor access to legal services are also major role players.22 In addition to the physical and behavioural consequences of GBV, post-traumatic stress disorder (PTSD), major depression and generalised anxiety disorders are documented in victims.22 Domestic abuse or rape was reported in five of our subjects, and psychiatric diagnoses of PTSD, depression and anxiety were also documented. This may aid in understanding why FNDs are more prevalent in the female population in South Africa.Gender-based violence (GBV) in South Africa is amongst the highest in the world.23 Such beliefs are commonly observed in the local population, and many patients may seek the help of traditional healers or \u2018sangoma\u2019.24 We identified only two subjects whose family members believed that symptoms were because of witchcraft; however, it is possible that many were not directly interrogated on their beliefs or accessing traditional medicine.Older studies in Africa have documented the influence of cultural beliefs on mental health presentations.27 This corresponds to our finding that a range of psychiatric conditions are prevalent amongst patients with FND, with depression being most common.A high rate of psychiatric comorbidities including depression, anxiety, post-traumatic stress and personality disorders have been documented locally and internationally in patients presenting with PNES.Our study also examined the overlap with organic disease, with hypertension being most commonly followed by HIV, asthma and diabetes mellitus; however, no significant correlations were found. Given the high prevalence of HIV in Southern Africa, this may reflect an under-recognised aspect of the neuro-psychiatric consequences of the HIV epidemic in sub-Saharan Africa. It is possible that the stress of being diagnosed with HIV or HIV-related neurocognitive impairment may contribute to the development of FNDs, but an incidental correlation between the two is also likely because of the high burden of HIV in South Africa.16 Our study did not support the predominance of the PNES phenotype as sensory disturbance was the most common neurological complaint, followed by weakness, seizures and gait dysfunction.In the 2018 Tanzanian case series and 1-year long retrospective review in the United States, the most common FND phenotypes reported were PNES and gait impairment, respectively.28We identified a predominance of paraparesis when patients were weak, hemisensory loss when patients had sensory symptoms and tremor when patients had a movement disorder. Tremor, being one of the most common forms of functional movement disorder, has been documented previously.29 Over half of the hemiparetic subjects in our study had a left-sided weakness.Previous reviews have reported a predilection for the left in up to 60% of patients presenting with functional weakness and sensory symptoms.30 Many of these symptoms were found in our patient sample, with headache being the most commonly documented. Specific diagnoses of IBS, chronic fatigue syndrome and fibromyalgia were not documented; however, pain was a frequently reported symptom suggesting that such functional pain syndromes may have been present.Multiple somatic symptoms such as headache, pain and fatigue are found in patients with FND. Irritable bowel syndrome (IBS), chronic fatigue syndrome, fibromyalgia and chronic pain syndromes are also documented.5 was corroborated by our findings and in particular those who presented with PNES.The impact of family members\u2019 illnesses on phenotypic presentations of FND, or \u2018modelling\u2019 as described by Stone et al.,5 A patient may be able to walk but, when examined supine, is unable to lift his or her leg off the bed. Intermittency was documented in 15.8% of our subjects and describes the power that \u2018comes and goes\u2019 but is normal with encouragement.5 Variability, whether of frequency, amplitude or distribution was the third most common clinical finding in our study, and not only in relation to movement disorders as is often observed.5 Hoover\u2019s sign, which demonstrates the discrepancy between voluntary and involuntary hip extension, is the only sign found in controlled studies to have a high specificity .32 It was only documented in 5.7% of our subjects. Distractibility, or improvement in abnormal movements when performing tests of mental concentration,5 was also documented infrequently. The \u2018hemisensory syndrome\u2019, recognised for over a century, refers to the splitting of sensation down the midline.33 However, in subjects with FND, vibration sense is also discrepant on either side of the sternum or frontal bone despite each vibrating as single units, a distinguishing feature.5 This was observed in six subjects. Not all \u2018positive functional signs\u2019 that have been documented in previous studies were assessed in our patients.5 Examining the sensitivity and specificity of particular signs prospectively are a consideration for future studies in South Africa.To distinguish FNDs from organic pathology, \u2018positive functional signs\u2019 may be found on examination. Some of these signs include inconsistency, intermittency, variability, Hoover\u2019s sign, distractibility and the \u2018hemisensory syndrome\u2019. The commonest signs found in FNDs have not been documented locally. Inconsistency, the most frequently documented sign in our study, refers to discrepancies in findings but does not differentiate whether this is conscious or unconscious.34 A recent systematic review highlighted that individuals with FND and somatic symptom disorders (SSD) might exhibit sensorimotor, prefrontal, striatal-thalamic, paralimbic and limbic structural alterations and therefore may have both a \u2018software\u2019 and \u2018hardware\u2019 problem.34A number of considerations arise when investigating functional disorders. Investigations are performed to exclude organic pathology but some studies propose neuroimaging to support the diagnosis of FND. This includes the emerging theory of a \u2018software problem\u2019, which describes abnormal neurobiological mechanisms (\u2018software\u2019) despite an intact macroscopic brain structure (\u2018hardware\u2019).12 As our study reviewed patients attending a tertiary-level hospital with most investigations being readily available, this may not be representative of most hospitals in South Africa where access to specialist care, neuroimaging and neurophysiological studies is very limited, if available at all. This emphasises the need for clinical education on FNDs to ensure that resources are utilised cost-effectively, without compromising the quality of patient care.In resource-limited settings, however, the exclusion of organic pathology overrides proving a pathophysiological basis of FND. For example, the 2018 study in northern Tanzania had access to a single CT scanner and EEG machine with the closest MRI facility being approximately 80 km away.17 the number of investigations carried out and resource utilisation contributed to high costs incurred as a result of FNDs. The authors found that the EEG was the commonest study requested. This is in contrast to the present study, in which MRI was the most frequently performed investigation.In an audit conducted in the United Kingdom by Parry et al.,Cerebrospinal fluid analysis was the least frequently performed investigation analysed in our study and did not aid in diagnosis. Besides the attendant cost, LPs are invasive tests with procedural complications, and in patients with underlying psychiatric illnesses they may precipitate further somatic symptoms. This is another reason, besides cost, why limiting unnecessary investigations in patients with FNDs is crucial.35 These modalities were not analysed in our study as apart from being rarely performed (n = 4/158), their use in the context of FND in South Africa has not been studied.Neurophysiological studies other than the EEG, such as motor-evoked potentials (MEPs) and Bereitschaft potentials, may assist in further defining pathophysiology in FNDs.37Management of FNDs remains challenging, as demonstrated by the high proportion of subjects in our study who showed little or no improvement on follow-up. Whilst data on optimal management strategies are limited, there is evidence that a multidisciplinary team (MDT) approach is effective. This was shown in studies of inpatients with severe and chronic FNDs.38 has been underutilised locally, with only 13.3% of subjects in our study having received physiotherapy. This may have been because of the fact that sensory disturbances were the most common functional symptom documented in our study and clinicians may have been unaware of the benefit of physiotherapy even in these patients. Another consideration is that clinicians themselves did not believe that their patients\u2019 symptoms were \u2018real\u2019 and therefore did not warrant physiotherapy.The benefit of rehabilitative physiotherapy, demonstrated in a randomised control study,17The majority of subjects were referred for or received psychotherapy (72%) and a smaller proportion were commenced on antidepressants (17.4%). This represents a higher proportion of patients with FND referred to psychiatric services than in a United Kingdom audit.39Psychotherapy, in particular, cognitive behavioural therapy (CBT) has shown benefit in the management of PNES compared with standard medical care.18 whilst a local public sector study on PNES reported that only 30% of patients received psychotherapy.19 The establishment of similar networks in the public sector would be a major advance in the management of FND and encourage a multidisciplinary approach.Access to psychological support services may vary between the private and public sectors in South Africa. Patients attending an epilepsy-monitoring unit at a private hospital facility were noted to have access to an FDN integrating the work of psychologists, psychiatrists and neurologists,1 An increasing awareness of \u2018mind\u2013body\u2019 connections has prompted renewed interest in this area, and relaxation techniques such as \u2018mindfulness\u2019 and meditation may hold promise in the future management of functional disorders.Emerging data on the links between emotional states and FNDs have implicated autonomic dysregulation, abnormal limbic motor interactions and abnormal bodily awareness.40Therapeutic sedation has been used with some benefit in a case series although it remains a contentious form of treatment raising ethical concerns.41Newer treatment modalities such as transcutaneous electrical nerve stimulation (TENS) and transmagnetic stimulation (TMS) have not been described in the present study or other local studies, and whether such data from the developed world can be extrapolated to the South African context remains unknown.42The prognosis of patients with FNDs on long-term follow-up appears to be guarded. We found that of the subjects who were followed up in our study, the majority had no improvement in their symptoms and 17% reported deterioration at follow-up. This is in keeping with international data on the outcomes of functional motor disorders and PNES; however, these studies were limited by a lack of validated outcome measures.43 addressed the inconsistency of outcome measures and concluded that few are well validated such as the Functional Movement Disorder Rating Scale (S-FMDRS)44 and the Conversion Disorder Scale (CDS).45 Future studies that apply validated outcome measures will be of benefit to patients and clinicians.A recent review by Pick et al.19 Our study presents the spectrum of FNDs seen at an academic centre in South Africa, and we anticipate that future studies will continue to expand our knowledge and proficiency in this area. As recent studies have suggested, there is a need to eradicate the stigma attached to the diagnosis, to patients and their clinicians and reform the approach taken in managing this condition.7Interest in the complex field of FNDs in South Africa is growing, and as more data on this subject becomes available, current practice will continue to evolve. Lack of training in the identification and management of patients with FNDs is noted to be a barrier to patient care; therefore, raising awareness amongst clinicians is an area of particular concern.13 this study is the first in South Africa to assess the full clinical spectrum of FND, rather than a subset of patients. This is also the largest study conducted on FND in Africa to date. The electronic database available at the hospital provided a reliable, secure data source. The study was conducted in the public healthcare setting, which is accessed by the majority of patients in South Africa and is, therefore, likely to be representative of the general population.Although there have been reports of FND in Africa since the 1950s,This was a retrospective study that did not employ specific outcomes measures, and some patient data were missing. Our study was also limited to a specific geographical area and to a single tertiary referral centre although patients were referred to this facility from throughout the province of KwaZulu-Natal.5 The majority of subjects were female, unmarried, unemployed and of black African ethnicity and the impact of cultural and socioeconomic factors requires further study. Comorbid psychiatric disease and clinical signs such as inconsistency may be useful in establishing the diagnosis of FND. Neuroimaging and neurophysiological studies play a role in the exclusion of organic pathology but should be individualised in a resource-limited setting. The prognosis of patients in this context was poor, but evidence suggests a multidisciplinary approach may offer better outcomes. Further research in Africa is required to expand current knowledge and expertise in this area, reduce stigma and improve both clinician awareness and patient care.Whilst the spectrum of FND is broad and heterogeneous, our study found the most common phenotype to be sensory impairment. A significant proportion of those with PNES had a family history of epilepsy, supporting the theory of \u2018modelling\u2019.Abnormal MRI findings included cerebral atrophy, cerebellar atrophy, small vessel disease, sinusitis, periventricular non-specific lesion, chronic lacunas, degenerative spine disease, disc protrusions, old infarcts and an incidental pineal cyst. Only one patient had mesial temporal sclerosis reported on MRI; however, this patient was known with epilepsy and the reason for presenting was a hemiparesis rather than seizures.Computed tomography brain findings included hemiatrophy, cerebellar atrophy, lacunar infarcts, incidental granulomas, calcification, arachnoid cyst, prominent pituitary gland and bilateral basal ganglia hyperintensities."}
{"text": "No gene has garnered more interest than p53 since its discovery over 40 years ago. In the last two decades, thanks to seminal work from Uri Alon and Ghalit Lahav, p53 has defined a truly synergistic topic in the field of mathematical biology, with a rich body of research connecting mathematic endeavour with experimental design and data. In this review we survey and distill the extensive literature of mathematical models of p53. Specifically, we focus on models which seek to reproduce the oscillatory dynamics of p53 in response to DNA damage. We review the standard modelling approaches used in the field categorising them into three types: time delay models, spatial models and coupled negative-positive feedback models, providing sample model equations and simulation results which show clear oscillatory dynamics. We discuss the interplay between mathematics and biology and show how one informs the other; the deep connections between the two disciplines has helped to develop our understanding of this complex gene and paint a picture of its dynamical response. Although yet more is to be elucidated, we offer the current state-of-the-art understanding of p53 response to DNA damage. TP53 gene in humans provide many regulatory functions. Specifically, its role in healthy cells is to suppress changes to a cancerous phenotype by gatekeeping key cellular processes such as apoptosis and cell cycle arrest 92], a co complex , or a hy complex . Bifurcas, as in , appear Spatial models similar to those of Sturrock et al.  and DimiAn alternative approach when developing a p53-Mdm2 network model is to incorporate additional steps or intermediaries, which add additional upregulating pathways. The simplest way in which to couple a positive feedback loop to the negative p53-Mdm2 system is to include a positive feedback loop on p53, a type of model coined as a relaxation oscillator by . This sip53-Mdm2 negative feedback loop with a positive feedbackIf we assume that a physiological delay in the p53-Mdm2 auto-regulatory network is attained through auto-catalytic positive feedback, as shown in This system yields an oscillatory response, for a suitable choice of parameters given in . In FiguZhang et al. study, and compare four different positive feedback mechanisms incorporated into the negative p53-Mdm2 system: Mdm2 as a promoter as well as an antagonist of p53; autocatalytic p53; autocatalytic Mdm2; p53 as a downregulator of Mdm2 . Recall,Other models have incorporated a positive feedback loop on ATM (in this case without Wip1) ,104,105.Another frequent approach is to couple the p53-Mdm2 negative feedback loop to the more complex p53-PTEN-Akt-Mdm2 pathway, which is \u201cglobally\u201d positive, see As discussed in The mathematical model proposed by Alon and coauthors has only a few components but it is complicated in the sense that many different relationships are taken into consideration. Moreover, a necessary time lag between p53 activation and p53-dependent induction of Mdm2 is incorporated in the form of a hypothetical intermediary . Neverthbout 6 h , see Figbout 6 h  remains bout 6 h ,112,113.bout 6 h . It is nbout 6 h  and mathbout 6 h ), ratherbout 6 h . Moreovebout 6 h . In Figubout 6 h .As reviewed in the previous sections, most mathematical studies of p53 dynamics in individual cells focus on the underlying mechanisms behind the p53 oscillatory response to DNA damage. They may depend solely on mathematical modelling, or combine mathematical modelling with qualitative/quantitative experimentation. As we have seen, early mathematical models such as ,100,102,Mathematical models may be used to further reveal, so far unknown aspects of p53 signalling, as well as crosstalk between the p53 pathway and pathways of other regulators, activated in response to genotoxic stress. Batchelor et al. investigated p53 signalling in response to UV . They diresponse  without adiation  with thaKonrath and coworkers  combine borators , successNo paper on p53 would be complete without mentioning cancer. Certainly a frequent feature of p53 experiments and mathematical models has been to identify suitable targets for cancer therapy. Indeed, several protocols and p53-based anti-cancer therapies have been developed and already used in clinical trials. These include retrovirus- or adenovirus-mediated gene therapy to restore p53 function lost due to mutations; targeting p53-deficient cells with modified adenoviruses; pharmacological modulation of p53 protein functions; identification and design of small-molecule inhibitors of the Mdm2-p53 interactions such as nutlins or selective Mdm2 inhibitors CGM097 and HDM201 ,118. HowOptimisation of drug administration is perhaps one of the biggest challenges in developing anticancer therapies, for example, prolonged daily administration of some drugs over several days may result in poor tolerability . To deteExtensive PK/PD modelling is used in  to improIn almost all attempts to model p53 intracellular dynamics, many simplifications have to be made, and much biological information has to be excluded. Biomathematicians and mathematical modellers often face the question of what is the \u201cright\u201d information to include in a model? For example, as we have seen in Another contradiction can be found, for example, in the interpretation of ATM-dependent phosphorylation of Mdm2 at Ser395. Experimental and computational works of Batchelor et al. ,82 and MThese two examples and apparent inconsistencies in experimental data point to the value that mathematical modelling can offer this area of research, but pose an important modelling question: simplification or clarification? Which biological processes should be included in the modelling? One can consider all the events hitherto described, even those which appear to oppose each other, and construct a large model (large in the number of parameters). Such an approach is likely to lead to \u201coverfitting\u201d, one can fit whatever experimental data is available with a sufficiently large parameter model, which may lead to false model interpretations and wrong conclusions. Or, one can focus on model reduction and simplification; parameter sensitivity analysis can perhaps point to obvious extraneous model terms, while common generic network motifs can be incorporated . Indeed Toettcher et al. lead by example, by introducing what they call a \u201csynthetic-natural oscillator\u201d; a model in which core aspects of the network are modelled in full while periphery interactions are distilled into artificial terms . The issIt is perhaps fair to say that the technologies to study oscillations in single cells remain quite limited and this simple fact is why we still do not have the full picture of the dynamical behaviour of p53 under normal versus stressed conditions. Since there are several ways one can view oscillations, mathematics can certainly help to fill some of the gap. It is perhaps important to identify as many oscillators as possible in a particular pathway, to try to understand, from both experimental and mathematical points of view, if and how these different factors interact with each other, what their individual functions in the oscillations are, and how they communicate. Do they have the same target, or do they act on several targets in the same pathway? It is conjectured that there must be some limitations to the cellular repertoire, in terms of which mechanisms are in place to generate an oscillating feedback system. How many of these are required as a minimum to achieve a tightly regulated cell biological response?\u201cThe Functional Landscape of p53\u201d was to offer a snapshot of the different mathematical mechanisms, which provoke oscillatory p53 dynamics, as well as commenting on the interplay between mathematical modelling and experiments in this lively field. Although every effort has been made to provide a comprehensive survey of the literature, the topic of p53 has garnered much interest from experimentalists and mathematicians alike. For additional reviews of the extensive literature please see [Over the last 20 years a wealth of study into the dynamics of p53 has permeated the literature. The purpose of this review for this special edition ease see ,132,133."}
{"text": "Skin cancers are the most common types of cancer worldwide, and their incidence is increasing. Epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, and it is associated with poor prognosis in the advanced stage. The most important risk factor for cSCC is long-term exposure to solar ultraviolet radiation, which induces oncogenic mutations in epidermal keratinocytes. The most common mutations are inactivating mutations in tumor suppressor p53, which result in accumulation of additional mutations. Recently, the role of p53 in the progression and invasion of cSCC has also been elucidated. In this review we will discuss the role of p53 in development of cSCC and as a potential new therapeutic target in advanced cSCC.TP53 gene and inactivation of the tumor suppressor function of the tumor protein 53 gene (TP53) in epidermal keratinocytes, which then leads to accumulation of additional oncogenic mutations. Additional genomic and proteomic alterations are required for the progression of premalignant lesion, actinic keratosis, to invasive and metastatic cSCC. Recently, the role of p53 in the invasion of cSCC has also been elucidated. In this review, the role of p53 in the progression of cSCC and as potential new therapeutic target for cSCC will be discussed. Skin cancers are the most common types of cancer worldwide, and their incidence is increasing. Melanoma, basal cell carcinoma (BCC), and cutaneous squamous cell carcinoma (cSCC) are the three major types of skin cancer. Melanoma originates from melanocytes, whereas BCC and cSCC originate from epidermal keratinocytes and are therefore called keratinocyte carcinomas. Chronic exposure to ultraviolet radiation (UVR) is a common risk factor for skin cancers, but they differ with respect to oncogenic mutational profiles and alterations in cellular signaling pathways. cSCC is the most common metastatic skin cancer, and it is associated with poor prognosis in the advanced stage. An important early event in cSCC development is mutation of the  Skin cancers are the most common types of cancer worldwide, and their incidence is increasing. Melanoma, basal cell carcinoma (BCC), and cutaneous squamous cell carcinoma (cSCC) are the three major types of skin cancer. Melanoma originates from melanocytes, whereas BCC and cSCC originate from epidermal keratinocytes. BCC is derived from basal cells of the interfollicular epidermal layer, and cSCC is derived from keratinocytes of the interfollicular epidermal layer and hair follicle stem cells . ChronicNOTCH1 . Cli. Cli1]. Only a portion of AKs will progress to invasive cSCC ,28. The TP53 in epidermal keratinocytes is an early event in cSCC development  and p14(ARF), are also common in cSCC [CDKN2A occurs at later stage in the progression from AK to cSCC, since it is not mutated in the sun-exposed normal skin  genes in cSCC include nd ERBB4 ,48. Sustnd ERBB4   can grow over wild-type cells to colonize normal epidermis, but the epidermis can adapt to the mutant clones and revert the expansion of the mutant cells over time [It has been shown that p53 mutations arise very early in mouse skin after UVB exposure, and that the growth of the p53-mutant keratinocytes is driven by UVB ,89. Intever time . Short-tver time . What dever time . In addiver time . TP53 and NOTCH1/2 [TP53 is mutated three times more frequently in the normal skin of patients with a high skin cancer burden compared to the low-burden group, indicating that elevated accumulation of UV-signature mutations is associated with an increased burden of cSCC [Normal sun-exposed skin adjacent to a cSCC harbors a high mutation burden, including the UV-targeted genes NOTCH1/2 . The oveNOTCH1/2 . Particu of cSCC .+/\u2212;p73+/\u2212) drives spontaneous cSCC development in mice, indicating that p73 may play a role in cSCC development [p53 shares a high degree of structural and functional homology with two of its ancestral genes, p63 and p73 . They arelopment . The \u0394Npelopment .TP63 gene has been reported in cSCCs, and given the substantially higher expression of the \u0394Np63 compared to TAp63 in normal epidermis, the oncogenic function of p63 is primarily dependent on the overexpressed \u0394Np63 isoform in the skin [A1AT), which is upregulated through the mut53\u2013p63 complex, promotes tumor invasion by inducing the expression of several epithelial\u2013mesenchymal transition (EMT) markers [p63 and p73 are rarely mutated in cancer, but the balance between their different isoforms can be critical for the cell fate and cancer development ,95. For the skin . The \u0394Npthe skin . Moreovethe skin . The strthe skin ,95. In athe skin . The co-the skin . One suc markers .TP53 gene are early events in skin carcinogenesis, and they predispose skin cells to further genomic instability. In accordance with this, several studies show remarkably high overall mutational burden in human cSCCs, which occur mostly at older age [Mutations in the lder age ,5,6,7. TTP53 gene in cSCC tumors is very high, ranging from about 50% in primary cSCCs to nearly 95% in aggressive tumors [TP53 gene, but not all mutations are equal. As in many other human cancers, also in cSCCs the missense mutations in TP53 gene are frequently found in the conserved p53 DNA-binding domain  oncogenic activities, such as increased pro-growth signaling, invasiveness, and tumor metastasis B 104,10,106. A dR248W contact mutant is commonly detected in cSCCs  [R175H exhibits GOF activity in many cancer types. For example, in HNSCC cells, p53R175H drives the expression of oncogenic transcription factor FOXM1, which is upregulated in human oral premalignant and HNSCC tissues [R175H has been shown to interact with other p53 family members, p63 and p73, and inhibit their transcriptional program to promote cell invasion [R175H drives cSCC development in mice, when specifically expressed in the epidermis [In addition to the contact mutant p53 , missense mutations in the (HNSCCs) , and it (HNSCCs) , althoug tissues . Mutant invasion ,115,116.pidermis . This p5pidermis ,119. TP53 gene, a significant fraction of human cancers carry TP53 nonsense mutations, which introduce a premature stop-codon [In addition to the missense mutations in the op-codon . This wiop-codon . These nop-codon . Expecteop-codon ,125,126.op-codon . Not allop-codon ,129. Intop-codon . In addiop-codon . In accoIn summary, distinct p53 mutants possess a plethora of functional mechanisms in which they can regulate several hallmarks of cancer, from activation of cell proliferation to promotion of invasion and metastasis. The biological consequence of distinct p53 mutants is very context dependent. Moreover, the recent evidence of the cancer-associated p53 isoforms, particularly the truncated ones which can escape degradation, indicates that the loss of function or nonsense p53 mutants should be regarded as an additional subgroup of GOF p53 mutants. Yet, the existence and potential function of different p53 isoforms in the process of skin carcinogenesis is not known.TP53 plays a significant role in cSCC development. Mutant p53 is stable and accumulates in the nucleus, and a strong p53 immunopositivity is a widely used characteristic to detect mutated p53 in tumor tissues [TP53 mutations are frequently detected in normal sun-exposed skin and AKs, increased p53 immunopositivity in AKs is associated with increasing severity of dysplasia, and with progression of AK to cSCC [TP53 mutations present an early indication of UV damage, and increased TP53 mutational frequency is associated with potential cSCC development. However, there is also evidence that UV-induced mutation and inactivation of NOTCH1 may precede mutation of TP53 [Mutated  tissues ,130. Con tissues ,102. The tissues . While p to cSCC ,89,131.  of TP53 .The protective role of tumor suppressor p53 in skin cancer has been shown in several studies. Loss of p53 accelerates skin tumor formation after UV irradiation , and losCDKN2A gene encodes two important tumor suppressors, p16(INK4a) and p14(ARF), which are frequently inactivated in cSCC , which constitute an essential layer of gene regulation ,138. MicNORAD (noncoding RNA activated by DNA damage), are direct transcriptional targets of p53 [MiRNAs are evolutionarily conserved, single-stranded ncRNAs ~22 nucleotides in length. They inhibit gene expression specifically by binding to the 3\u2019-untranslated region of the target mRNA in the cytoplasm, resulting in translational repression or degradation of target mRNA . Compares of p53 ,143.MALAT1 (metastasis associated lung adenocarcinoma transcript 1), which is commonly upregulated in cancer, has been shown to interact with mutated p53 in breast cancer [MALAT1 to mutant p53-ID4 protein complex, delocalizing MALAT1 from nuclear speckles and favoring its association with chromatin [As discussed above, the tumor-suppressive function of p53 is frequently lost in cancer, particularly in skin cancers, and mutated p53 can acquire oncogenic GOF activity. In addition to the proteins that cooperate with mutated p53 to transactivate specific target genes in cancer, also ncRNAs have been shown to participate in the pathogenic signaling via p53 mutants . As an et cancer . Here, ohromatin . This wihromatin .TPX2 (TPX2 microtubule nucleation factor) is upregulated [TP53 mutation and aggressive clinical behavior in breast cancer, supporting a TPX2-p53 regulatory circuit [While the role of ncRNAs in cSCC progression is emerging ,146, onlegulated . High nu circuit . In cSCC circuit . Another circuit .PRECSIT (p53 regulated carcinoma-associated STAT3 activating long intergenic non-protein coding transcript) is a recently characterized lncRNA with a tumorigenic function in cSCC [TP53 can cause premature p53 translation termination, leading to undetected p53 protein expression. Here, PRECSIT was shown to be upregulated in cSCC, and its high expression was specifically associated with loss of p53 in cSCC in vivo [PRECSIT expression in cSCC progression. In accordance with this, delivery of a wild-type p53 into cSCC cells, which harbor mutated p53 led to downregulation of PRECSIT expression, indicating that its expression is suppressed by p53. PRECSIT was further shown to activate STAT3-signalling and expression of MMPs and invasion of cSCC cells [MiRNA regulation is very context-dependent, and certain miRNAs are specifically regulated by mutant p53. For example, miR-1246 is induced by mutant p53 in colon cancer, and miR-34 is suppressed by mutant p53 in lung cancer . Thus, i in cSCC   has been shown to restore the proper folding and transcriptional activity of p53 mutant inducing apoptosis in the p53ft model . In addiell line . Howeverr cSCCIS ,113.Delivery of the p53 gene directly into tumor cells is one option to target p53 mutations, and wt-p53 can be transferred to cells by viral vector in combination with cancer treatments. Combining chemotherapy, immunotherapy, or radiation therapy with the p53 gene therapy may increase the efficacy of tumor cell targeted therapies. For example, in HNSCCs, intra-tumoral p53 gene delivery is being studied. In phase 2 multi-center open label clinical trial adenoviral p53 (Ad-p53) gene therapy is administered intratumorally in combination with immune checkpoint PD-1 and PD-L1 inhibitors in patients with recurrent or metastatic HNSCC and other solid tumors approved for anti-PD-1 or anti-PD-L1 therapy . AnotherIn summary, p53 targeting is an interesting area in drug development, but the efficacy of p53 targeted therapies has not yet been tested in advanced and metastatic cSCCs. It is conceivable that drugs targeting several mutants of p53 are needed, and that p53-targeting drugs would be used in combination with surgical excision, immunomodulators, radiation therapy, or chemotherapies. However, given the important role of p53 mutations in the progression of cSCC, it is expected that p53-targeted therapies could be useful already at an earlier stage of cSCC development, in addition to treatment of advanced and metastatic cSCC.Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, and it is associated with poor prognosis in the advanced stage. The mutation rate of cSCC is one of the highest among the malignant tumors, and the majority of mutations found in cSCC are UV-induced. An important early event in cSCC development is mutation and inhibition of the wild-type function of tumor suppressor p53. This leads to the accumulation of additional oncogenic mutations. It has become evident that additional alterations, for example in non-coding RNAs, are required for the progression of premalignant lesion and actinic keratosis to invasive and metastatic cSCC. In addition, the role of p53 in the invasion of cSCC has also been elucidated. Together, these observations suggest mutant p53 as a putative target in both at the early stage of cSCC progression, as well as in advanced and metastatic stages. It is possible that multiple p53 targeted therapeutic approaches under development may be feasible in the treatment of cSCC at different stages of tumor progression."}
{"text": "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+/\u2212 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 \u201cpseudoorgans\u201d . 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 TNF\u03b1 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 IFN\u03b2, 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 IFN\u03b2 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 \u201cwinner\u201d 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 \u201csuperfit\u201d 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 . IFN\u03b2, 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"}
{"text": "SNORD118 gene in humans, is an atypical C/D box snoRNA as it promotes rRNA cleavage rather than 2\u2032\u2013O\u2013methylation 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\u2013step 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\u2032\u2013O\u2013methylation 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"}
{"text": "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\u2009cm.For the EMSA shown in Fig. Supporting Informationreproducibility checklist"}
{"text": "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\u2019s 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\u201310 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 \u221280 \u00b0C. 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 \u00b1 2 \u00b0C), light/dark cycles (12 h/12 h) and humidity (50 \u00b1 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 \u221280 \u00b0C. 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 \u221280 \u00b0C 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 \u03b2-ACTIN were used as housekeeping genes in the mouse and human study respectively. Relative expression of the specific genes was determined using the 2\u2212\u2206\u2206Ct method [RNA from mouse and human samples was extracted with TRIzol\u2122 reagent , according to the manufacturer\u2019s instructions. RNA quality and concentrations were measured by Nanodrop Spectrophotometer ND1000 . RNA (1\u20135 \u00b5g) was then incubated with DNase I  for 30 min at 37 \u00b0C and reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit  according to the manufacturer\u2019s 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\u2013Wallis test followed by post hoc test for multiple groups-comparisons after testing the normality with Shapiro\u2013Wilk tests. Student\u2019s 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."}
{"text": "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\u201310 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\u201310 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\u201313 (n = 50), and 14\u201317 years (n = 93). The experimental design was approved by the Hospital\u2019s 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\u201317 years . Volunteers were recruited from children and adolescents attending the Department of Paediatrics at the Cl\u00ednica 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 \u2265 85th and < 95th percentile), or with obesity (BMI \u2265 95th percentile)  based on\u00ae, 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) .\u00ae, 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\u00ae 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\u00ae CRP (Latex) ultrasensitive assay . Thyroid-stimulating hormone (TSH) concentrations were measured by an electro-chemiluminescence immunoassay (ECLIA) using Roche Elecsys\u00ae 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 \u00b1 standard error of the mean (SEM). Differences between groups were analyzed by ANOVA followed by Fisher\u2019s 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\u2019s 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\u201317 years (p < 0.001) children and adolescents as compared to the 8\u201310 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\u201310 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\u201310 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\u201310 years age range; and that (vi) serum leptin levels correlate with REE/FFM in the 8\u201310 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\u201310 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\u201310 years, which is the only age group in which serum leptin levels are positively correlated with REE/FFM."}
{"text": "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. Sandstr\u00f6m 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\u22126 vs. 0.32 g/100 mL), a finding that was also observed in concentrations of zinc ions in solution (1.6 \u00d7 10\u22127 vs. 0.017 mol/L). Dietary zinc can interact with dietary phytate in the intestinal lumen  [From H 6\u20137.4) , formingH 6\u20137.4) . The preH 6\u20137.4) . In otheH 6\u20137.4)  a phytat6 L/mol (site 1) and 8 \u00d7 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."}
{"text": "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-HIF1\u03b1 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\u00a0A 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\u00a0A 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, HIF1\u03b1, NF-\u03baB, or mTOR likely contributes to restraining anabolic metabolism , p53-med"}
{"text": "Our data support \u0394Np73 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, \u0394Np73, TAp73, and \u0394133p53, in healthy subjects (n = 29), individuals with premalignant lesions (n = 49), and colorectal cancer patients (n = 42). Extracellular vesicles\u2019 \u0394Np73 levels were already significantly high in subjects with premalignant lesions. \u0394133p53 levels were statistically increased in colorectal cancer patients compared to the other two groups and were associated with patients\u2019 survival. Remarkably, TAp73 mRNA was not detected in any of the individuals. The evaluation of \u0394Np73, \u0394133p53 and CEA sensitivity, specificity and AUC values supports \u0394Np73 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 \u0394Np73 may identify in screening programs those individuals at higher risk of presenting a premalignant lesion. In addition, \u0394133p53 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 \u0394Np73, TAp73, and \u0394133p53 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 \u0394TAp73  [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 \u0394N\u2019p73) . In pati \u0394N\u2019p73) ,16,17 an \u0394N\u2019p73) . Finallyor types ,20,21,22gression ,24,25.TP53 variant \u0394133p53 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 \u0394Np73, the N-terminal truncated ffectors ,27,28. Affectors ,30 and fffectors  and moduffectors . In addiffectors . \u0394133p53The deregulated expression of \u0394Np73 and \u0394133p53 in different tumor types and its association with cancer patients\u2019 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\u2019 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 \u0394Np73, TAp73 and \u0394133p53 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\u20135 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 \u00b0C. 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\u2019s 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\u2019s J index. For \u0394Np73, we selected 0.76 and \u22120.83 as the cut-off values for disease-free survival (DFS) and overall survival (OS), respectively. For \u0394133p53, we selected 2.93 and 1.99 as the cut-off values for DFS and OS, respectively. \u0394Np73 and \u0394133p53 mRNA levels were classified in low and high levels accordingly. We used the log-rank test to compare DFS and OS according to \u0394Np73 and \u0394133p53 mRNA levels. Kaplan\u2013Meier 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 \u2264 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\u2019Agostino\u2013Pearson and Shapiro\u2013Wilk tests were used to assess the normality of the data distribution. Subsequently, the Kruskal\u2013Wallis and U-Mann\u2013Whitney 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 \u0394Np73 and \u0394133p53 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\u2019 mRNA content of these isoforms at the different stages of the disease. Non statistical significative differences were observed for \u0394133p53. However, \u0394Np73 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 \u0394133p53 group. In the whole cohort of CRC, patients with low \u0394133p53 levels did not reach the median OS and patients with high \u0394133p53 levels had a median OS of 50 months , with a HR of 0.23 .Survival analysis did not reveal a correlation between \u0394Np73 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 \u0394Np73 and \u0394133p53 EV content was evaluated. We have observed a negative correlation trend in \u0394Np73\u03b1 seroreactivity and \u0394Np73 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, \u0394Np73 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, \u0394133p53, has attracted during the last few years the interest of the scientific community due to its putative oncogenic properties. \u0394133p53 may act as a survival factor [The overexpression of different arcinoma , neuroblarcinoma , lung caarcinoma , gastricarcinoma , acute parcinoma , and colarcinoma . \u0394Np73 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 \u0394Np73 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 \u0394Np73 in EVs and CEA levels in plasma in the three groups of study clearly revealed a much greater potential of \u0394Np73 to identify individuals with PL. Our findings highlight the plausible usefulness of \u0394Np73 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\u2019 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 \u0394133p53 in EVs in CRC. Here, we observed higher levels of \u0394133p53 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 \u0394133p53 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 \u0394Np73. These findings are similar to those previously reported  regardinNo correlation was observed between \u0394Np73 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 \u0394Np73 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 \u0394Np73, TAp73 and \u0394133p53 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\u2019 \u0394Np73 content to detect premalignant lesions as the earliest stage of the disease. Interestingly, although \u0394133p53 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 \u0394Np73 in EVs as we describe here."}
{"text": "Due to the lack of an etiologic treatment for SARS-CoV-2 and the difficulties involved in developing new drugs, some drugs already approved for other diseases or with efficacy against SARS and MERS, have been used in patients with COVID-19. This systematic review aims to summarize evidence on the efficacy and safety of five antivirals applied to patients with COVID-19, that have proven to be effective either in vitro studies or in studies on SARS-CoV and MERS.; An intensive search of different databases  has been carried out until the end of April 2021. This systematic review has been conducted according to the PRISMA statement. From each of the included studies, the characteristics of the intervention and comparison groups, demographic data and results were extracted independently; Remdesivir is well tolerated and helps to accelerate clinical improvement but is ineffective in reducing mortality. Favipiravir is safe and shows promising results regarding symptom resolution but does not improve viral clearance. The use of lopinavir/ritonavir has been associated with an increased risk of gastrointestinal adverse events and it has not proven to be effective. No significant differences were observed between patients treated with ribavirin or umifenovir and their respective control groups; Remdesivir and favipiravir are well tolerated and effective in accelerating clinical improvement. This systematic review does not support the use of lopinavir/ritonavir, ribavirin and umifenovir in hospitalized patients with COVID-19. Members of the family Coronaviridae are widely spread among mammals, usually triggering respiratory infections . Until 2However, this perception changed in 2002 as a result of an outbreak of cases in China with severe respiratory symptoms, currently known as Severe Acute Respiratory Syndrome (SARS) . A similThe latest coronavirus with pathogenicity for humans that has been identified to date is SARS-CoV-2 . At the SARS-CoV-2 is responsible for the disease known as COVID-19, whose dominant route of transmission is respiratory  and patiSARS-CoV-2 is a positive-sense single-stranded RNA virus (+ssRNA)  and encoThe entry of SARS-CoV-2 into the host cell occurs thanks to the binding of the viral spike (S) proteins to its host target cell receptor, which is the angiotensin-2 converting enzyme (ACE-2) . This peAs the virus replicates inside the host cell, the production of type I interferon (IFN) begins. It will interact with other cells of the immune system such as macrophages and neutrophils, considered major sources of pro-inflammatory cytokines and chemokines such as IL-1\u03b2, IFN\u03b3, tumor necrosis factor alpha (TNF\u03b1), inducible protein-10 (IP-10) and monocyte chemoattractant protein- 1 (MCP-1), which can lead to the activation of T-helper-1 cells (Th1) . In addiThe most frequent symptoms are fever, cough, asthenia, dyspnoea, myalgia or arthralgia, ageusia and anosmia ,24. OtheIf the immune response is effective, approximately 80% of infected patients have a mild or even an asymptomatic clinical course . HoweverUp to the present day we lack an etiological treatment against COVID-19, which is why the therapeutic strategies used are being oriented in three main directions .One way is trying to reduce or eliminate the SARS-CoV-2 viral load using molecules capable of interfering with viral replication. Another way is using immune therapies that allow regulating the inflammatory response and finally, the other therapeutic strategy used is symptomatic treatment, for example, providing oxygen . AntivirSome of the antivirals used against SARS-CoV-2, among other non-antiviral molecules are shown in Since the beginning of the pandemic, a major scientific effort has been made to develop a vaccine that will make it possible to immunize the population and thus reduce the incidence of this virus. As of 14 July 2021, a total of 3,400,884,367 vaccine doses have been administered around the world [Currently there are three main types of vaccines against SARS-CoV-2 virus. On the one hand, the messenger-RNA (mRNA) vaccines use manufactured nucleoside-modified, single stranded mRNA, that contain the genetic code for the synthesis of the SARS-CoV-2 S protein antigen ,38. GiveIt should be noted that this type of vaccine is very vulnerable at room temperature, which is why it must be stored at \u221270 \u00b0C or \u221220 \u00b0C. This can be a problem in developing countries, where the continued supply of electricity to large industrial freezers is unreliable .Nowadays there are two COVID-19 mRNA vaccines approved for market: mRNA-1273 developed by Moderna and BNT162b2 developed by Pfizer-BioNTech . On the We can also find inactivated coronavirus vaccines, created by using radiation, heat or chemical stress . This tyGiven the urgent need to reduce the cost, time and risks derived from the development of new medicines, drugs already approved for other indications have been \u2018\u2019repurposed\u2019\u2019 and used to treat COVID-19 patients.Nowadays, not too many studies regarding the use of antiviral drugs on patients with SARS-CoV-2 infection have been carried out. The present study aims to summarize evidence on the efficacy and safety of five antivirals applied to patients with COVID-19, that have proven to be effective either in in vitro studies or in studies on SARS-CoV and MERS ,43.The search was made using PubMed, Scopus, Web of Science, MEDLINE, and Cochrane COVID-19 Study Register, which also includes trials published in ClinicalTrials.gov-COVID-19 subset and WHO International Clinical Trials Registry Platform (ICTRP).The studies have been identified by combining the name \u201ccoronavirus\u201d, \u201cCOVID-19\u201d and \u201cSARS-CoV-2\u201d with the following keywords: lopinavir/ritonavir, remdesivir, favipiravir, umifenovir, ribavirin or their respective trade names. MeSH terms  and the Boolean operators \u201cAND\u2019\u2019 and \u2018\u2019OR\u2019\u2019 have also been used in the search.The study was conducted according to the PRISMA statement . ArticleRegarding type of studies, both randomized controlled clinical trials (RCTs) and observational studies were included. The rest of the inclusion criteria have been proposed according to the PICO algorithm .The proposed exclusion criteria for this systematic review have been: (a) studies with insufficient data, (b) in vitro, in silico, in vivo animal studies, (c) comments, expert opinions, case reports, letters to the editor, reviews, protocols and trial registry records, (d) studies including patients with other coronaviruses, SARS, or MERS, (e) studies that do not include as an intervention at least one of the five antivirals evaluated in this systematic review, (f) studies using medicinal plants.The following information has been extracted from each of the included studies: (a) clinical trial registration number, (b) author, (c) publication date, (d) trial design, (f) participant characteristics (severity of the disease), (g) country, (h) interventions carried out with their respective dosage regimen, (i) comparison group dosage regimen (i) number of participants recruited/allocated/evaluated, (j) age and gender of participants, (k) outcomes.We included nine studies about remdesivir, which is the first FDA-approved drug for COVID-19 . Six of p = 0.02) [p = 0.18) [p > 0.05) [One of the RCTs carried out in multiple centres in the USA, Europe and Asia simultaneously, compared a 10-day course of remdesivir and a 5-day course with standard of care . Patient = 0.02) . In cont = 0.18) . Other o > 0.05) .p = 0.14), length of hospital stay, discharge rate, time to recovery, and mortality [Goldman et al. compared a 5-day course of remdesivir with a 10-day course in patients with severe COVID-19. Despite an apparent trend towards better outcomes in patients treated with remdesivir for 5 days than those receiving it for 10 days, the differences were not statistically significant. After adjusting for baseline imbalances in severity disease, similar results were obtained between both groups in terms of clinical status at day 14  as well as higher odds of improvement in the eight-category ordinal scale on day 15, where \u201c1\u201d corresponds to not having any limitations and \u201c8\u201d means death  [The first phase of the \u201cAdaptive COVID-19 treatment trial or ATT-1\u201d constitutes a RCT where 541 patients allocated to remdesivir therapy were compared with 521 patients randomized to placebo . The dat1.2\u20131.9) . Additional secondary end points that support the use of remdesivir are: a shorter time to improvement of one and of two categories on the 8-category ordinal scale, a shorter time to discharge, and a shorter length of hospital stay (12 days versus 17 days in the remdesivir and placebo groups respectively). Moreover, mortality by day 29 was 11.4% in the remdesivir group versus 15.2% in the placebo group  .p = 0.0672) did not show a significant improvement in the intervention group either [Wang et al. recruited a total of 237 severe COVID-19 patients (158 randomized to remdesivir therapy and 78 to placebo) in China at the start of the pandemic . Althougp either . It is ip either .p = 0.50) nor the percentage of patients requiring mechanical ventilation after randomization [These results are in line with those of the \u2018\u2019WHO Solidarity Trial Consortium\u201d, in which several intervention arms were compared with standard of care. The use of remdesivir as monotherapy did not significantly reduce 28-day mortality  [One of the observational studies included in this systematic review retrospectively compared 25 critically ill patients undergoing mechanical ventilation, who had received remdesivir with 26 patients treated with standard of care . In this <0.001) . It shou <0.001) .p < 0.001). In addition, remdesivir was associated with greater clinical improvement, assessed using a 7-category ordinal scale, as well as, with 62% lower odds of all-cause death compared with the standard of care cohort  [Olender et al. used data from a phase 3 remdesivir RCT and compared it with a longitudinal retrospective cohort of patients treated with standard of care . By day = 0.001) . p > 0.05). No significant differences could be found in terms of time to clinical recovery and time to discharge  [In line with the results seen so far, there are also those observed in another retrospective study where a group of 99 patients treated with remdesivir was compared with 125 patients who had received supportive care . Despitectively) .p = 0.03) and in increasing improvement in clinical status by day 15  [Finally, there is a double-blind RCT that has analysed the potential efficacy of remdesivir in combination with another drug. More specifically, it compares the use of remdesivir associated with barticinib versus remdesivir + placebo . This st1.0\u20131.6) .p = 0.02). Adverse events that were more common in the remdesivir group than in the standard of care group were: nausea, hypokalaemia, and headache. With regard to severe adverse events, they were numerically lower in both remdesivir intervention arms than in the control group, albeit not statistically significant [The RCT by Spinner et al. showed that those randomized to a 10-day course of remdesivir had significantly higher rates of adverse events compared to those receiving standard of care  and those allocated to a long course of remdesivir (10 days) .Data from the ACTT-1 trial suggest that remdesivir treatment may have prevented the progression to more severe respiratory disease. This can be evidenced by the lower proportion of serious adverse events due to respiratory failure among patients in the remdesivir group as well as a lower incidence of new use of oxygen among patients who were not receiving oxygen at the time of recruitment  .In the RCT proposed by Wang et al. remdesivir was well tolerated and no new safety concerns were identified. The overall proportion of patients with serious adverse events tended to be lower in remdesivir-treated patients (18%) than in placebo-treated patients (26%) .Nevertheless, there was a higher rate of premature discontinuation of treatment in the intervention group (12%) compared to the placebo group (5%) because of adverse events such as gastrointestinal symptoms , increases in aminotransferase or bilirubin levels and worsening of cardiopulmonary status .p > 0.05) [Another observational study found that the therapy with remdesivir did not increase liver test abnormalities and was not associated with a higher risk of acute kidney injury  .p = 0.03) [In the case of the study that has compared the combination of remdesivir with barticinib versus remdesivir alone, those receiving dual therapy have presented significantly fewer serious adverse reactions  .Five RCTs and ten observational studies regarding lopinavir/ritonavir have been included .In an RCT carried out in China, 99 patients with severe COVID-19 randomized to lopinavir/ritonavir were compared with those allocated to standard of care. This study found that treatment with lopinavir/ritonavir was not associated with significant clinical improvement, mortality reduction or reduction in viral RNA detectability, since SARS-CoV-2 RNA was still detectable in 40.7% of the patients treated with lopinavir/ritonavir at the end of the study (day 28) . It shoup = 0.60), in length of hospital stay, as well as in risk of progressing and needing invasive mechanical ventilation , which is why they conclude that lopinavir/ritonavir monotherapy is not an effective treatment for hospitalized COVID-19 patients [The results of the RECOVERY trial carried out in the United Kingdom, which enrolled 5040 patients, indicate that compared to standard of care, lopinavir/ritonavir is not associated with significant reduction in mortality , as well as in a faster resolution of symptoms assessed by the SOFA score  [In the RCT by Hung et al., the patients allocated to the triple therapy of lopinavir/ritonavir + ribavirin + IFN-\u00df1b were compared with those in the control group receiving lopinavir/ritonavir monotherapy. The results show that this triple therapy applied within 7 days of symptom onset is effective in suppressing SARS-CoV-2 shedding . This po= 0.041) . The DisCoVery trial is an RCT carried out in multiple French healthcare centres simultaneously, in which several lines of treatment have been investigated . While iNone of these investigational treatments resulted in significant clinical improvement at day 15 and at day 29, as measured on the WHO-7-point ordinal scale, where \u201c1\u201d implies the absence of activity limitations or need for hospitalization and \u201c7\u201d equals death .p = 0.39), while at day 29 it was OR = 0.93  [p = 0.08) and at day 29 it was OR = 0.76  [In more detail, the odds for clinical status improvement at day 15 in the lopinavir/ritonavir monotherapy group versus the standard of care group was OR = 0.83  . In the  = 0.19) . Likewis = 0.19) .p = 0.97) and the percentage of patients requiring mechanical ventilation after randomization [These results are in line with those of the WHO Solidarity Trial Consortium, in which several lines of treatment were compared with standard of care internationally. One of them was lopinavir/ritonavir in monotherapy, but it failed to reduce mortality , the time to viral clearance , nor prevents the progression to severe cases  [Regarding the observational studies included, Gao et al. have found no evidence that lopinavir/ritonavir monotherapy accelerates the remission of symptoms like fever  .Following this line of results, Grimaldi et al. compared a total of 57 patients receiving lopinavir/ritonavir with a cohort of 85 patients treated with standard of care . Here, tp < 0.0001) [The same occurs in the retrospective cohort study by Choi et al., where the median viral shedding duration was longer in patients treated with lopinavir/ritonavir (23 days) compared to those who only received standard of care (18 days) ( 0.0001) .p = 0.0219), as well as time to defervescence (p = 0.0364) when compared to standard of care [On the contrary, Ye et al., observed that patients treated with lopinavir/ritonavir monotherapy accelerated time to viral clearance , ventilator free days, 14-day mortality and 28-day mortality and change in viral load between admission and day 7, was the retrospective study by Lecronier et al., where 20 patients treated with lopinavir/ritonavir were compared with 22 receiving standard of care .p = 0.001) and median duration of hospital stay  [Lu et al. studied the use of lopinavir/ritonavir monotherapy in paediatric patients with mild COVID-19. They could observe that the intervention group had a disadvantage compared with those receiving standard of care, in terms of time to viral clearance  .p = 0.213) and 30-day mortality (p = 0.271) [Other authors have tried to evaluate the differences in clinical efficacy between an early treatment (begin within 5 days of symptom onset) of lopinavir/ritonavir + hydroxychloroquine and a delayed treatment . The res= 0.271) .p < 0.01). Additionally, the duration of positive RNA test was shorter in the arbidol group (p < 0.01) [Lopinavir/ritonavir has also been compared with other antivirals. In a retrospective study carried out in China 36 patients who received lopinavir/ritonavir were compared with 16 patients treated with arbidol (trade name of umifenovir) . Arbidol < 0.01) .p = 0.319) or in-hospital mortality (p = 0.639) [Vernaz et al. propose in their retrospective single-centre study several intervention arms containing lopinavir/ritonavir. In one of them lopinavir/ritonavir monotherapy is compared with standard of care and no statistically significant differences can be found between both groups in terms of length of hospital stay (= 0.639) .p = 0.697) and it was significantly associated with an increase in length of stay (p < 0.001) [In another of the intervention arms, a group of patients treated with hydroxychloroquine + lopinavir/ritonavir was compared with a cohort that had received standard of care. This combination therapy failed to reduce mortality (< 0.001) .p = 0.02). However, subgroup analysis revealed that this shorter duration was only statistically significant in those patients treated within 10 days from symptom onset (log-rank p < 0.0001), unlike those who received it eleven or more days later (log-rank p = 0.51) [Finally, the last observational study included in this systematic review coincides with the results proposed by Yan et al., since the group of patients treated with lopinavir/ritonavir had a significant shorter duration of viral shedding compared to the control group ( = 0.51) . It is t = 0.51) .Cao et al. conclude that gastrointestinal adverse events were more common in lopinavir/ritonavir group than in those who received standard of care. However, the number of patients with severe complications  was higher in the control group. In total, 13 patients had to stop prematurely the use of lopinavir/ritonavir because of adverse events .On the contrary, the DisCoVery trial recorded a significantly higher number of serious adverse events in those treated with lopinavir/ritonavir, either in monotherapy or associated with IFN-\u03b21a. The most frequently reported ones were: acute respiratory failure (11%), acute kidney injury (8.2%), acute respiratory distress syndrome (8%), arrhythmia (7%), pulmonary embolism (5%) and sepsis (4%) .p > 0.05), being those side effects generally mild and self-limiting [Hung et al. reported no statistically significant differences between intervention and control group regarding adverse events , which is why this study raises concerns about the safety profile of this antiviral [Grimaldi et al. registered a statistically significant higher incidence of cases of acute kidney injury (AKI) and need for renal replacement therapy (RRT) at day 28 in the lopinavir/ritonavir group compared to the control group  [In the study by Lu et al. the use of lopinavir/ritonavir in paediatric patients was associated with significantly higher adverse reactions compared to those who received conventional treatment, mainly because of a longer nasopharyngeal swab negativitation time  .Finally, coinciding with Cao et al., a retrospective study also observed that the most frequent adverse events in patients treated with lopinavir/ritonavir were gastrointestinal symptoms including nausea, diarrhoea and elevated liver enzymes, in fact 8.1 % of the patients developed grade 2\u20133 gastrointestinal disorders and had to discontinue the treatment prematurely .After literature search and careful review of full-text articles, a total of six RCTs and two observational studies on favipiravir have been included .p > 0.05) [p = 0.048) [One of the RCTs carried out in Japan, compared the early administration of favipiravir in patients with asymptomatic or mild COVID-19 with its late administration (starting on day 6 of study) . Early f > 0.05) . What wa= 0.048) .Another RCT was carried out in India and it compared the use of favipiravir in adults with mild to moderate COVID-19 with supportive care .p = 0.098) and of time to hospital discharge  could not be reached, significant results were found regarding a reduction in time to clinical cure in those treated with favipiravir  [While a statistically significant difference in terms of time to cessation of viral shedding  . This le= 0.019) .The results obtained in another RCT carried out in Russia, in which 17 patients receiving favipiravir monotherapy were compared with 22 patients receiving standard of care, have been encouraging . It shoup < 0.05 respectively) [Favipiravir turned out to be more effective than the standard etiotropic therapy used as control group in various aspects such as: in reducing the time to defervescence, in having lower lactate levels in blood at the end of the treatment and in reducing the area of pulmonary parenchyma lesion according to the CT data (ctively) .p = 0.06), lower mortality rates (p = 1.00) and none of them required mechanical ventilation (p = 0.118) [In Egypt, the authors Dabbous et al. found in their RCT promising results regarding the use of favipiravir when compared to chloroquine. Although the differences were not statistically significant, those receiving favipiravir had a shorter duration of hospital stay (= 0.118) .p = 0.018) [p = 0.007) [In a phase II/III RCT, Ivashchenko et al. studied two possible favipiravir dosing regimens and compared each of them with a standard of care. In this study the patients were randomized 1:1:1 to receive either 1600 mg favipiravir, twice the first day, followed by 600 mg twice a day for 14 days, or 1800 mg favipiravir, twice the first day followed by 800 mg twice a day for 14 days or standard of care . On both= 0.018) . Likewis= 0.007) .p = 0.019) and in reducing mortality [In the last included RCT, three possible intervention arms were compared: favipiravir + tocilizumab; favipiravir monotherapy and tocilizumab monotherapy . The simortality . The comortality .p < 0.001), as well as to improve chest CT inflammatory changes on day 14 after treatment (p = 0.004) [Combinations of favipiravir with other drugs were studied in the following observational studies. Cai et al. proposed a comparative study in which the intervention group received favipiravir and IFN-\u03b11b, while the control group received lopinavir/ritonavir associated with IFN-\u03b11b. Those treated with favipiravir appeared to accelerate viral clearance (= 0.004) .p = 0.237), which could be due to the small sample size of the study. On the other hand, favipiravir was superior to lopinavir/ritonavir in shortening the length of ICU stay (p = 0.01) [In a retrospective study carried out in Turkey favipiravir was also compared with lopinavir/ritonavir, however, in this case both were simultaneously given with hydroxychloroquine . On the  = 0.01) .The most frequent adverse event recorded in 84.1% of the patients after favipiravir administration was hyperuricemia. Other reported adverse events were: elevation of serum triglycerides (11%) and serum alanine aminotransferase elevation (8.5%) .These observed adverse events coincide with those of Udwadia et al., where most of them were mild and transient. Hyperuricemia and elevated liver enzymes were the most common ones detected in the favipiravir group. The reported gastrointestinal disorders were minimal and similar between intervention and supportive care groups .In another RCT, it was found that, in terms of QTc prolongation, the use of favipiravir was safer than standard therapy, represented mostly by hydroxychloroquine and azithromycin. The rest of the recorded side effects were mild in form and associated with hepatotoxicity .Following this line of results, the undesirable events reported by a study with two dosing regimens for favipiravir were mild to moderate in severity and consistent with those reported so far for this antiviral. In addition, those who received higher doses of favipiravir did not experience any increasing toxicity .p < 0.001), being the most frequent one gastrointestinal disorders including diarrhoea, liver injury, nausea and vomiting [In one of the studies comparing the efficacy of favipiravir versus lopinavir/ritonavir, higher rates of adverse events in lopinavir/ritonavir group (55.56%) were noticed compared to favipiravir group (11.43%) , time to fever resolution (p=0.55), proportion of patients with SARS-CoV-2 nucleic acid negativity at day 14 and day 28, CT-changes improvement (p = 0.76), length of hospital stay (p = 0.56) or mortality [The other RCT included in this systematic review compared the efficacy of ribavirin in combination with other drugs, specifically, 33 patients were randomized to treatment with ribavirin and IFN-\u03b1, 36 received lopinavir/ritonavir + IFN-\u03b1 and 32 patients were assigned to a triple therapy of ribavirin, IFN-\u03b1 and lopinavir/ritonavir . The resortality .p < 0.001) [Among the studies where ribavirin was given concomitantly with other drugs, in the one by Elalfy et al. the combination treatment of ribavirin + nitazoxanide + ivermectin + zinc supplement proved to be effective in clearing SARS-CoV-2 in a shorter time than those treated with standard of care (< 0.001) .p = 0.475) or accelerate time to viral clearance (p = 0.314) [Finally, one of the observational studies compared the use of ribavirin in patients with severe COVID-19 with a cohort of patients who had received supportive care and found that ribavirin failed to significantly reduce mortality (= 0.314) .Huang et al. found that the combination of ribavirin with lopinavir/ritonavir was associated with a significant increase in gastrointestinal disorders, thus suggesting that both antivirals should not be administered simultaneously in patients with COVID-19 .Eslami et al., also observed that the most frequent adverse events in the ribavirin group were gastrointestinal in nature such as nausea, vomiting, diarrhoea, abdominal pain and bleeding .In the study by Tong et al., ribavirin was well tolerated, with anaemia being the most frequent side effect. To this must be added, that there was no premature discontinuation of treatment as a consequence . In the This systematic review has included one RCT and five observational studies regarding umifenovir or better known as its trade name, arbidol .p = 0.02) and by presenting a higher peripheral oxygen saturation on the seventh day of treatment (p = 0.02) and milder involvement of the chest-CT scan after 30 days (p = 0.004) [Starting with the Iranian RCT that enrolled 100 patients, 50 of them were assigned to a double therapy of arbidol + hydroxychloroquine, while the other half received hydroxychloroquine and lopinavir/ritonavir . In this= 0.004) . p = 0.2). Other parameters like need of intubation (p = 0.6), need of mechanical ventilation (p = 0.6) or mortality (p = 0.5) did not show significant differences either [Regarding time to defervescence, despite being numerically lower in the arbidol group, the difference was not statistically significant . However, in terms of length of hospital stay, the differences between arbidol and standard therapy were not statistically significant [Another retrospective study found that arbidol compared to standard therapy could shorten time to viral clearance, as well as accelerate clinical cure by relieving fever and dry cough faster (nificant .p < 0.05) [Deng et al. compared in their retrospective study a cohort of patients who had received arbidol and lopinavir/ritonavir versus a control group only treated with lopinavir/ritonavir. They observed that the combination group presented higher viral negative conversion rates ( < 0.05) .p = 0.057) or reducing length of hospital stay (p = 0.056). However, it did significantly accelerate pneumonia absorption observed through CT (p = 0.037) [In another retrospective cohort where arbidol associated with IFN-\u03b12b was compared with IFN-\u03b12b monotherapy, dual therapy was not effective in terms of accelerating viral clearance (= 0.037) . The dat= 0.037) .p = 0.002) and shorter length of hospital stay [Arbidol given to a group of 90 patients revealed beneficial effects in comparison to 45 patients who received standard of care. These benefits were mainly observed in aspects such as higher viral clearance rates , and the dual therapy lopinavir/ritonavir, in terms of their efficacy and safety when applied to SARS-CoV-2 infected patients.Regarding remdesivir efficacy, none of the randomized controlled clinical trials have shown statistically significant benefits in terms of mortality ,47,49,50When it comes to the question of how long remdesivir should be given, there are discrepancies between studies. In the study by Spinner et al. remdesivir therapy for 5 days was more likely to have a better clinical status on day 11 compared to standard of care .Parallelly, Goldman et al. compared a 5-day regimen of remdesivir versus a 10-day course and despite noticing a trend toward better outcomes in patients receiving it for 5 days, the differences were not statistically significant . These rOn the one hand, this meta-analysis reveals that those who received a 5-day remdesivir therapy had greater clinical improvement compared to those in the control group . On the other hand, no statistically significant differences were observed between those with a 10-day remdesivir regimen and those receiving placebo  .Contrary to the results recorded in the RCTs, several of the included observational studies, do detect a significant reduction in mortality in remdesivir group ,52. However, it must be taken into account that the lack of randomization, the small sample size in the case of Pasquini et al.  or the uRegarding safety, no significant association has been found between an increased risk of suffering an adverse event and the use of remdesivir ,48,49,53Large randomized controlled clinical trials on the use of lopinavir/ritonavir applied to COVID-19 patients, have revealed a lack of efficacy in terms of clinical improvement, mortality reduction, viral clearance, length of hospital stay, oxygenation-free days and mechanical ventilation-free days ,55,56,58These results are consistent with those of a meta-analysis published online in April 2021 where the differences between lopinavir/ritonavir group and those receiving standard therapy were not statistically significant in terms of mortality, viral clearance  and radiological improvement  .The majority of the observational studies included in this systematic review coincide with the results proposed by the RCTs and neither did they find benefits regarding the use of lopinavir/ritonavir compared to standard of care ,63,64,67Other authors have compared the efficacy of lopinavir/ritonavir administered simultaneously with other drugs such as IFN-\u00df1a  or hydroRegarding safety, gastrointestinal adverse events like nausea, vomiting and diarrhoea have been more common in those who received lopinavir/ritonavir ,57,65.While it is true that most of the studies included in this review recorded mild and self-limited adverse events, in some studies like the one by Grimaldi et al. there was a higher incidence of acute renal injury and need for renal replacement therapy in those receiving lopinavir/ritonavir, thus raising doubts about its safety profile . Likewise, lopinavir/ritonavir proved to be inadequate to treat paediatric population since the number of adverse events was significantly higher in this group compared to those who received standard therapy .Treatment with favipiravir has shown efficacy in accelerating clinical cure, specifically time to defervescence ,71,73. HIt should be noted that the benefits recorded in the study by Balykova et al. regarding the use of favipiravir versus standard of care are difficult to quantify since the standard therapy consisted of various combinations of drugs, mainly hydroxychloroquine, azithromycin and lopinavir/ritonavir .In a meta-analysis published in September 2020, several of the studies included in this systematic review were analysed. The group of patients treated with favipiravir showed a significant clinical improvement by day 14  compared to the control group. In contrast, viral clearance by 14 day  and need for oxygen or non-invasive mechanical ventilation  did not show statistically significant differences between groups .The combination of favipiravir with tocilizumab seems to have promising results in terms of reducing lung parenchyma inflammation, as well as in recovering clinical symptoms compared to favipiravir monotherapy . FinallyIn terms of safety, patients treated with favipiravir experienced fewer adverse events than their respective control groups, being the most common symptom hyperuricemia. The results obtained in the just mentioned meta-analysis, revealed no significant differences between favipiravir and control group  .Ribavirin has not proven to be effective in reducing mortality, time to viral clearance, length of hospital stay, or in alleviating symptoms in any of the studies included in this review ,78,79,81Some of the studies assessed the efficacy of ribavirin combined with other drugs ,78,80, bIn terms of safety ribavirin was well tolerated, however, its concomitant use with lopinavir/ritonavir seems to potentiate gastrointestinal disorders, therefore this combination is not recommended in patients with COVID-19 .We have only been able to include one RCT on arbidol in this systematic review. Here, the association of arbidol with hydroxychloroquine significantly reduced the length of hospital stay, as well as improved radiological lung parenchyma changes compared to the group treated with lopinavir/ritonavir .Likewise, one of the included observational studies also verified the superiority of arbidol against lopinavir/ritonavir and registered significantly higher viral clearance in the group treated with arbidol . This apFinally, three studies compared the efficacy of arbidol versus standard therapy ,84,87. TNone of the studies detected serious adverse events leading to discontinuation of treatment because of the use of arbidol. A meta-analysis published in January 2021 supports some of the results just presented, such as the safety of arbidol  or the lack of efficacy of arbidol in reducing the length of hospital stay [Some of the antivirals of the current paper have either been administered concomitantly or compared with hydroxychloroquine. This drug is a 4-aminoquinoline compound that has been used as an antimalarial for years . Due to In fact, given its lack of benefit in terms of mortality, the hydroxychloroquine arms were discontinued in both SOLIDARITY  and RECOThese findings have subsequently been confirmed by several meta-analyses. In one published in September 2020, those treated with hydroxychloroquine failed to achieve a significant reduction in mortality  as well as to shorten time to defervescence compared to those treated with standard of care . The useOnce summarized evidence on the efficacy and safety of some antivirals that have proven to be effective, there is no doubt that the SARS-CoV-2 pandemic implies a major threat to human health. However, while it is essential to find a vaccine to immunize the population, this is a long-term solution. In the meantime, we must continue to invest in and support pharmacological research. Firstly, to be able to treat already infected patients. Secondly, to ensure that in regions where access to vaccines is limited, there are means to counteract this disease. Thirdly, since it is yet unclear whether the authorized vaccines are effective against the new viral strains of concern, which are the result of mutations accumulation .As a reflection, given the similarity of this new virus to the coronaviruses responsible for the SARS and MERS epidemics in 2002 and 2012 respectively, we could think that, almost a decade later, effective antivirals would be available to treat them. However, this is not the case. This leads us to believe that, by affecting only a limited geographical region, once the incidence started to decrease and the disease was controlled locally, it lost the interest of large institutions to continue researching on a suitable pharmacological treatment. Hence, it is very important to keep researching, even when effective vaccines are already available or the incidence is starting to decline. New variants of the virus may emerge in the future or a new wave of cases may arise, for which having an effective drug would be very helpful.The main limitations found in the studies included in this systematic review are the following. On the one hand, most of them had an open design, so knowing the drug being administered may have influenced the clinical outcomes. On the other hand, many of the studies did not collect detailed information about adverse events or reasons for abandoning treatment. Therefore, the conclusions obtained in terms of safety are reduced, which limits their interpretation.The absence of placebo as a comparative method in most of the included studies makes it difficult to determine whether the observed results are due to the evolution of the curative process of the disease itself, to the antiviral treatment used, or to a combination of both. Another limitation would be the small sample size used in some of the studies.In the studies where patients with COVID-19 of mild to moderate severity were recruited, it was more difficult to assess the symptoms, thus contributing to a possible underestimation of potential clinical benefits of the antivirals applied. That is why, the results of these studies cannot be extrapolated to severe or critically ill COVID-19 patients.Many of the antiviral agents were used in combination with other medications, implying that the observed results cannot be exclusively attributed to the antiviral treatment.Finally, it should be noted that even though systematic reviews constitute the \u201cgold standard\u201d when it comes to synthesizing the evidence to answer a scientific question, these types of articles require a great investment of time, which makes their information quickly obsolete, especially in an emergency health situation like the current one, in which new studies are constantly being published.Remdesivir may help accelerate clinical improvement in hospitalized COVID-19 patients, but it is not effective in reducing mortality. Regarding the duration of the treatment, a 5-day course seems to be sufficient. In terms of safety, this drug has proven to be as tolerable as placebo or other drugs that it has been compared to.Lopinavir/ritonavir has not proven to be an effective treatment for patients with SARS-CoV-2 infection in improving clinical status, reducing mortality, or increasing viral clearance. In fact, it has been associated with a higher risk of gastrointestinal adverse events.Treatment with favipiravir is associated with significant clinical improvement, mainly in reducing time to fever resolution, and is also safe and well-tolerated. Likewise, the combination of favipiravir with tocilizumab seems to have promising results in terms of reducing lung parenchyma inflammation and symptom resolution. However, the use of favipiravir has failed to increase viral clearance.Ribavirin has not proven to be an effective treatment for COVID-19 patients, and its concomitant use with lopinavir/ritonavir is not recommended, due to an increase in the risk of adverse events.Arbidol has proven to be a safe and well-tolerated treatment for COVID-19 patients. However, it is not associated with a significant reduction in the length of hospital stay. Due to the small number of studies included in this systematic review regarding arbidol, we do not have sufficient evidence to support the use of this drug in patients with COVID-19."}
{"text": "Introduction: It is unknown if the Fontan circulation has an impact on sexual health in men. This study assessed self-reported sexual health and fertility in men with a Fontan circulation.Aims: In this prospective, cross-sectional study, Australian men \u226518 years enrolled in the Fontan Registry of Australia and New Zealand were invited to complete the International Index of Erectile Function (IIEF), alongside questions assessing fertility. These data were compared to historical, age-matched controls.Results: Of 227 eligible men, 54 completed the survey; of those 37 were sexually active and included in the final analysis. Mean age was 28 \u00b1 3 years, age at Fontan was 5 \u00b1 3 years. Fontan type was extra-cardiac conduit in 15 (41%), lateral tunnel in 12 (32%), and atriopulmonary connection (APC) in 10 (27%). Ventricular function was normal in 24 (83%), and all were New York Heart Association Class I  and II . Nine participants (24%) had erectile dysfunction (IIEF-EF score \u226425). The severity was mild (IIEF 22\u201324) in six (16%), mild\u2013moderate (IIEF 17\u201321) in two (5%), and moderate (IIEF 11\u201316) in one (3%). Baseline characteristics and current medication usage were similar in those with and without erectile dysfunction. Compared with historical control values, erectile function was not significantly impaired in the Fontan population (p =0.76). Men with a Fontan circulation had decreased levels of sexual desire and overall satisfaction (p < 0.001). There was no correlation between the presence of erectile dysfunction and any assessed parameter. Eleven (30%) of the cohort reported a pregnancy with a prior partner.Conclusion: In our cohort, overall erectile function was comparable between men with a Fontan circulation and historical controls, however sexual desire and overall satisfaction were reduced. There was no correlation between study parameters and the presence of erectile dysfunction. The proportion of the cohort who had a prior pregnancy was congruent with population data. Since its initial development almost 50 years ago, the Fontan procedure has been increasingly implemented for children who have complex single ventricle cardiac anatomy \u20134. Over There are numerous long-term morbidities which are well documented complications of a Fontan circulation due to the altered physiological state characterized by systemic venous hypertension, reduced cardiac output, endothelial dysfunction, neurohormonal activation and chronic cyanosis in many . Late covia the Fontan Registry of Australia and New Zealand. A total of 227 patients across all Australian states were identified and contacted between March 2019 and July 2020. A mailed questionnaire was sent to eligible men. If no response was received, they were contacted via telephone and given the opportunity to complete the questionnaire online. The study was approved by the Human Research Ethics Committee at all involved institutions (HREC2019.042).For this prospective, cross-sectional study, men over the age of 18 years who had consented to involvement in research were contacted The survey utilized the International Index of Erectile Function (IIEF), an internationally validated sexual health questionnaire. The IIEF examines the domains of erectile function, orgasmic function, sexual desire, intercourse satisfaction and overall satisfaction. Questions were also included to provide a qualitative assessment of fertility. Patients self-reported appropriate scores for each domain based on their experience in the preceding 4 weeks. The IIEF defines a diagnosis of erectile dysfunction (ED) as an erectile function score of \u226426 (out of a possible 30). Mild ED is defined as an IIEF-EF score of 22\u201325, mild-moderate 17\u201321, moderate 12\u201316, and severe <11. These data were compared with historical control data with mean age of 35 years . Correlations were completed using Spearman rank correlation and Pearson correlation coefficient. All statistical analysis was completed via SPSS v26 for Windows. Statistical significance was based on a Of 227 eligible men, 135 were unable to be contacted or did not complete the survey, 22 were excluded due to a history of mental disorder, 17 refused to complete the survey upon contact, 13 did not have sexual activity during the study period, two declined to have their registry data accessed, and one person died during the study period, leaving 37 men included in the final analysis.p = 0.23). Fourteen patients in total had a history of arrhythmia, eight in the cohort without ED and six in the cohort with ED (p = 0.75). All arrhythmias were atrial in origin.Baseline characteristics are shown in p < 0.001) and overall satisfaction at 8.3 \u00b1 1.9 compared to 9.5 \u00b1 0.8 in controls (both out of 10) (p < 0.001).Patients with a Fontan circulation had significantly decreased levels of sexual desire at 8.1 \u00b1 1.2 compared to 9.1 \u00b1 1.0 (p = 0.76). p < 0.001). Patients with ED also had significantly decreased scores in the domains of orgasmic function (p = 0.05), intercourse satisfaction (p = 0.01), and overall satisfaction (p = 0.01). There was no difference between groups in sexual desire (p = 0.29).As demonstrated in The range of IIEF-EF scores are demonstrated in p = 0.44) and intercourse satisfaction (p = 0.14). There was no association between clinical indices and IIEF-EF . There was no difference in IIEF-EF score between those with APC compared to those with total cavopulmonary connection .There was no difference in orgasmic function (p = 0.85). Of those who did not report a pregnancy, 19 (51%) reported they had only ever used contraception. Four men (11%) reported they had been trying to conceive with a partner for greater than 6 months. There was no difference in IIEF-EF score between the patients who had and those who had not reported a prior pregnancy (p = 0.41). Patients who had an APC Fontan procedure were more likely to report a pregnancy compared to those who underwent a TCPC procedure (p < 0.001).A total of 11 men (30%) in the cohort reported a pregnancy in a prior sexual partner. There was no significant difference in the proportion of those with and without ED who reported a prior pregnancy  literature that ED is typically of mild severity . Prior sMost studies that have examined sexuality in the setting of CHD have included heterogenous CHD types without distinction as to the underlying pathology or anatomy; this is an important consideration for the Fontan population who are affected by unique physiological problems including chronic venous hypertension and reduced cardiac output, especially during exertion. Elevated systemic vascular resistance, chronic cyanosis and cardiovascular drugs including anti-arrhythmics and some diuretics (spironolactone in particular) may also contribute to dysfunction , 22.n = 7) with a Fontan circulation. This study included a questionnaire and interview assessing sexual satisfaction and fertility  were either unable to be contacted or did not complete the survey. A number of these had outdated contact details. Others never returned the survey despite repeated contact. Thus, selection bias is an important consideration. It is feasible that due to the personal nature of the questioning, men with erectile dysfunction may have been less inclined to complete the survey. Men with more significant erectile dysfunction may also have a propensity to avoid sexual intercourse, and thus have been excluded from final analysis once the survey was completed. The overall small number of patients within both cohorts makes it important to consider that the study was underpowered to detect true differences in baseline characteristics between those with and without erectile dysfunction. It is theorized however, that chronic venous hypertension may be implicated in the pathogenesis of sexual dysfunction in men with a Fontan circulation. As our study did not incorporate invasive haemodynamic assessment, we have been unable to assess whether this is contributory.A further limitation to research in sexual health is the reliance on self-reported questionnaires. It is also noted that several different research tools have been used in this area. We utilized the IIEF, as it has a broad base of supporting literature and has been used previously in the field of adult congenital heart disease , 16. OthIn our cohort, erectile function was comparable in men with a Fontan circulation compared with age-matched historical controls, though they did report decreased levels of sexual desire and overall satisfaction. Overall, men with a Fontan circulation reported levels of fertility congruent with that of the general population. Clinicians should be aware that erectile dysfunction may occur in this population and consider asking appropriate questions to ensure that concerns regarding sexual health and fertility are not overlooked.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by HREC Royal Children's Hospital, Parkville, Victoria. The patients/participants provided their written informed consent to participate in this study.RC: concept design, project supervision, and data analysis. IR: assistance with concept design, survey implementation, and data analysis. DT, AB, VW, DB, Yd'U, KP, DK, ML, DZ, and DC: assistance with project design, data interpretation, and manuscript preparation. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
{"text": "The occurrence and prevalence of heart failure remain high in the United States as well as globally. One person dies every 30 s from heart disease. Recognizing the importance of heart failure, clinicians and scientists have sought better therapeutic strategies and even cures for end-stage heart failure. This exploration has resulted in many failed clinical trials testing novel classes of pharmaceutical drugs and even gene therapy. As a result, along the way, there have been paradigm shifts toward and away from differing therapeutic approaches. The continued prevalence of death from heart failure, however, clearly demonstrates that the heart is not simply a pump and instead forces us to consider the complexity of simplicity in the pathophysiology of heart failure and reinforces the need to discover new therapeutic approaches. We will show that despite many approaches having targeted individual/single components of E\u2013C coupling to date, all have shown no benefit and/or differing levels of promise. This suggests a complexity to the current simplicity of thought regarding the treatment of heart failure.This review article will cover the origins of several schools of thought regarding E\u2013C coupling in the failing heart and the treatment of heart failure . We will2+ indicator that can be used in multicellular preparations i, markers of myocardial energy supply, and connective tissue content were quantified. Although weanling animals with POH showed a similar degree of hypertrophy to that found in juvenile animals with POH, there were differences in peak twitch force despite there being no difference in peak [Ca2+]i. Connective tissue content, however, showed significant differences. In weanling animals with POH, connective tissue content was 10 \u00b1 2% compared to 24.0 \u00b1 6.8% in juvenile animals with POH (p < 0.001), which might have contributed to the lower peak twitch force in juvenile animals. Despite a lack of change in connective tissue content or resting [Ca2+]i in weanling animals, passive stiffness in muscles was increased. Lactate dehydrogenase was significantly higher (38%) in weanling animals. However, total creatine kinase activity and total creatine content were significantly less (22%) in hearts from juvenile animals. This study demonstrated that despite having similar levels of hypertrophy, there can be differences in the remodeling of the extracellular matrix (increased fibrosis) as well as molecular remodeling (decrease in the creatine kinase system)  and a prolonged diastolic relaxation. These abnormalities are more pronounced at higher frequencies of stimulation and may be caused by an altered Ca2+ resequestration into the SR. SERCA activity has been reported to be positively correlated with diastolic force. Diastolic force has an inverse relationship with the SERCA activity in failing myocardium. These findings suggested that a reduction in SERCA activity could contribute to both an impairment in systolic as well as diastolic function in failing human hearts [Two of the most significant characteristics of failing human myocardium that have been reported are an increase in diastolic [Can hearts .2+]i availability and myofilament contractile activation is cross-bridge cycling dynamics  might be beneficial in the treatment of heart failure. Studies on isolated muscles from failing human hearts demonstrated a decrease in inotropic response to milrinone . In ordeholidays .2+]i and enhancing inotropy/myocardial contractility in heart failure patients. A simple increase in intracellular Ca2+ was being shown to be clinically detrimental , which may explain in part the failure of the gene therapy trial (CUPID) using SERCA2a in heart failure patients i and SR Ca2+ release were not the sole contributors to heart failure. A novel mutation in TnC was also reported that further strengthened this school of thought  resulted in a further rightward shift in non-failing human myocardium, but did not further shift the Ca2+-force relationship in fibers from failing hearts. Importantly, cyclic guanosine monophosphate (cGMP) resulted in a greater decrease in myofilament sensitivity in fibers from failing hearts. From these studies, it was suggested that not only changes at the level of the thick myofilaments, as previously reported by Leinwand and Bristow i, and there was a change in the Hill coefficient (myofilament cooperativity). These findings indicated that there was a decrease in myofilament Ca2+ sensitivity and a change in cooperativity among thin myofilament proteins most likely reflecting phosphorylation of thin filament regulatory proteins by PKC. Both TnT and TnI have at least two phosphorylation sites. In heart failure tissue, there are two TnT isoforms. These findings might reflect the effect on relative states of phosphorylation of two TnT isoforms. PKC activation, which decreased SR Ca2+ release while decreasing the Ca2+ responsiveness of the myofilaments with a change in cooperativity among thin myofilament proteins, might be a future approach to the treatment of heart failure and Ca2+ regulation in the failing heart. However, the impact of PKC stimulation on vascular smooth muscle remains unclear and must be considered before possible future clinical development  and SR Ca2+ release are key contributors to the pathophysiology of heart failure and possible targets for therapeutic intervention, there is a dynamic state that occurs in the failing heart, especially when blood circulation is impaired and when increases in heart rate can induce a hypoxic milieu [Despite indications that changes at the level of the thick and thin myofilaments and diastolic [Cac milieu ,190,191.c milieu ,193,194.c milieu ,195. Carc milieu ,199,200)O-linked N-acetylglucosamine modification [2+ current, and RyR Ca2+ leak, which facilitate the progression of the above-mentioned diseases [Calcium/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase that has emerged as a key regulator of cardiac physiology and pathology ,203,204.fication ,206. Excfication ,210,211.diseases ,212. Thediseases ,214. Bendiseases ,216,217.diseases . One readiseases . Future A main upstream regulator of CaMKII is the oxidation at M281/282 by reactive oxygen species (ROS) ,220,221.2+ cycling protein SERCA2a [Despite progress in the clinical management of heart failure, it continues to be associated with a high rate of mortality and morbidity worldwide ,227,228. SERCA2a ,234,235, SERCA2a ,239,240."}
{"text": "The key tumor suppressor protein p53, additionally known as p53, represents an attractive target for the development and management of anti-cancer therapies. p53 has been implicated as a tumor suppressor protein that has multiple aspects of biological function comprising energy metabolism, cell cycle arrest, apoptosis, growth and differentiation, senescence, oxidative stress, angiogenesis, and cancer biology. Autophagy, a cellular self-defense system, is an evolutionarily conserved catabolic process involved in various physiological processes that maintain cellular homeostasis. Numerous studies have found that p53 modulates autophagy, although the relationship between p53 and autophagy is relatively complex and not well understood. Recently, several experimental studies have been reported that p53 can act both an inhibitor and an activator of autophagy which depend on its cellular localization as well as its mode of action. Emerging evidences have been suggested that the dual role of p53 which suppresses and stimulates autophagy in various cencer cells. It has been found that p53 suppression and activation are important to modulate autophagy for tumor promotion and cancer treatment. On the other hand, activation of autophagy by p53 has been recommended as a protective function of p53. Therefore, elucidation of the new functions of p53 and autophagy could contribute to the development of novel therapeutic approaches in cancer biology. However, the underlying molecular mechanisms of p53 and autophagy shows reciprocal functional interaction that is a major importance for cancer treatment and manegement. Additionally, several synthetic drugs and phytochemicals have been targeted to modulate p53 signaling via regulation of autophagy pathway in cancer cells. This review emphasizes the current perspectives and the role of p53 as the main regulator of autophagy-mediated novel therapeutic approaches against cancer treatment and managements. Autophagy, a self-degradative intracellular process, is an essential mechanism of the cell that facilitates renewal or removal of cellular molecules, thereby balancing the cell\u2019s energy consumption and maintaining homeostasis . HoweverTumor suppressor p53 has been implicated in a wide variety of cellular processes, including genomic stability, cell-cycle arrest, DNA repair, apoptosis, cellular senescence, and autophagy . GeneralLiterature-based online databases, Google Scholar, Web of Science, PubMed, Google, and Scopus were accessed to collect information on the published articles that reported molecular mechanism of p53 and autophagy modulation in cancer prevention. Several keywords were used in the search, such as p53, autophagy, cancer, phytochemicals, natural compounds, solid tumors, and lymphomas perspectives role of p53 and autophagy in cancer therapy. Figures were created with the Adobe Illustrator software.The p53 is a central transcription factor that has the capacity to induce diverse cellular responses likely DNA damage repair, cell cycle arrest, apoptosis, and senescence followed by various stress signals  Subbura. The masin vitro and in vivo have signified that the oncogenic activities of p53 mutant variants have heterogeneous which can vary with tissue type in addition to genetic background of the cells  which signify lysosomal-mediated degradation process to facilitate cell survival . It has Accumulating evidence indicated that autophagy could decide whether cancer cells are promoted or suppressed in certain conditions . In thatCell death regulation is a complicated process of maintaining cellular homeostasis by preventing oncogenic growth and recycling damaged cell debris . DysreguThe research to date has found several tumor suppressor proteins and oncogenes to be essential regulators of autophagy. The loss or mutation of these proteins contributes to tumor formation. In addition to being one of these tumor suppressors, the mammalian cell \u201cjanitor\u201d p53 may be one of the most frequently mutated genes in human tumors. Most human cancers exhibit p53 mutation, which is found in approximately half of all tumors . P53 actThere are many different cell stressors that might activate p53 . It is p\u2212/\u2212 cells, only the cytoplasmic p53 can inhibit autophagy through suppressing AMPK and inducing mTOR, resulting in the hyperphosphorylation of AMPK, TSC2, and acetyl CoA carboxylase (ACC) and hypophosphorylation of mTOR substrate, p70S6K   . microRNant P53) .p-mTOR and increase of p-AMPK , has been described to possess anti-cancer potential through excessive ROS generation and induce p53-dependent apoptotic cell death by autophagy inhibition in cervical cancer (HeLa) and colon cancer (HCT116) cells  cells . Sulfora6) cells . A sterot of p53 .The role of p53 in autophagy regulation in cancer progression has established into a strongly knit, exciting, and rapidly changing disciple in biological science. However, the study of the ability of p53 to modulate autophagy in addition how this modulation of regulation of cancer metabolism raises numerous issues. The basic process of autophagy is important for normal cellular function as its dysregulation is generally encountered during human tumor development . HoweverThe role and impact of modulation of p53 in regulation of autophagy is complex and far from fully clarified. Emerging evidence and rapidly developed omics as well as genome editing techniques have likely been to revolutionized a new p53 roles in autophagic activities of different p53 proteins may vary along with changes in tumor microenvironment. Therefore, novel technologies may shed a new perceptions for a knowledge-based insights to recognize gaps-existing knowledge in addition to analyze scenarios which involve a reconsideration for the function of p53 modulation in autophagy signaling in cancer. Recently, autophagy has been established as a dual role in tumor suppression process likely involved in human cancer research. p53 might be an essential player in the modulation of autophagy pathway, although the exact molecular mechanisms and cellular function in cytoplasmic and nuclear p53-mediated autophagy regulation have not been well studied. However, cellular function and role of p53-mediated autophagy, as well as molecular metabolism in cancer progression, require a strongly related and rapidly altering field. The regulation of cancer metabolism by p53 target genes can diverge according to the stress signal, cell type, and other conditions. Additional, it is evidently established that p53 stabilization is a tumor-specific vulnerability, approaches to indorse the degradation of p53 through autophagy which represents an attractive anti-cancer method. Nevertheless, our augmented understanding of the function of p53 and autophagy will hopefully offer a prospective approach to cancer treatment. Therefore, this review revealed that p53 could be targeted as an important implication of cancer therapy via modulation of autophagy signaling. Hitherto the actual therapeutic use of p53-mediated autophagy induction needs detailed knowledge of how the autophagy-lysosomal pathway may affect in cancer progression."}
{"text": "According to these results, we conclude that there is no evidence to recommend ES in egg-donor IVF cycles, regardless of the number of previous failed cycles.Endometrial scratching (ES) has been proposed as a useful technique to improve outcomes in in vitro fertilization (IVF) cycles, particularly in patients with previous implantation failures. Our objective was to determine if patients undergoing egg-donor IVF cycles had better live birth rates after ES, according to their previous implantation failures. Secondary outcomes were pregnancy rate, clinical pregnancy rate, ongoing pregnancy rate, miscarriage rate, and multiple pregnancy rate. We analysed the results of 352 patients included in the Endoscratch Trial (NCT03108157). A total of 209 were patients with one or no previous implantation failures , and 143 were patients with at least two previous failed implantations . We found an improvement in pregnancy rates (62.9% in group A1 vs. 55.8% in group B1 vs. 70.4% in group A2 vs. 76.4% in group B2,  In 2003, Barash et al. published for the first time their results on patients undergoing endometrial scratching (ES) before an in vitro fertilization (IVF) cycle. They aimed to enhance endometrial receptivity, as this had been seen in guinea pigs when an intentional injury was caused before embryo implantation . They peIn 2019, Vitagliano et al. published a meta-analysis on the impact of ES in infertile women undergoing a first embryo transfer, and they could not find a difference between groups in terms of clinical pregnancy rates (CPR), miscarriage rate (MR), ongoing pregnancy rate (OPR), LBR, multiple pregnancy rate (MulPR), and ectopic pregnancy rate (EPR) in fresh or frozen embryo transfers . The samThe underlying mechanism that would lead to benefits of ES remains uncertain. Some authors have suggested that ES would lead to a better synchronization between embryo and endometrium through the endometrial maturation retardation , while op = 0.823), CPR  and LBR  between intervention and control groups in intention-to-treat (ITT) analysis.It is known that ovarian stimulation may induce an asynchrony between the embryo and the endometrium , due to The definition of RIF has been subject of debate during the last years as it is difficult to determine if a negative result is due to implantation deficient phenomena or to impaired embryo quality. This has led to many different definitions among authors, who may consider recurrent implantation failure from two to six previous failed embryo transfers, with fresh or frozen embryos . In 2014Thus, we performed a subgroup analysis of the outcomes of patients included in the Endoscratch Trial (NCT03108157), dividing our patients into RIF patients (with two or more previous failed implantations) and non-RIF patients (with a maximum of one previous failed embryo transfer) to determine the efficacy of ES in improving live birth rates in egg-donor IVF cycles according to previous implantation failures.The overall number of patients included in the Endoscratch Trial (NCT03108157) was 352, of whom 176 were allocated to receive an ES in the luteal phase of the cycle preceding the embryo transfer cycle (group A), and another 176 to receive no intervention (group B). Sample size was calculated upon egg-donor IVF average pregnancy rates (60%) in order to detect a difference of 15% between the two groups.We performed an analysis according to the number of previous failed embryo transfers, including own egg and donor egg, and fresh and frozen embryo transfers. This secondary analysis of our RCT was not included in the original design of the trial and thus sample size according to the number of implantation failure patients to be included in each arm was not calculated. Out of the 352 patients, 209 had a maximum of 1 previous failed embryo transfer (non-RIF patients), and 143 had at least two previous failed transfers (RIF patients). Among the 209 non-RIF patients, 105 belonged to group A (group A1) and received an ES, and 104 patients belonged to group B (group B1) and did not receive any intervention. Similarly, among the 143 RIF patients, 71 were included in group A (group A2) and 72 in group B (group B2).n = 27 patients excluded).ES was performed as described in the literature, with a biopsy catheter  under abdominal ultrasound guidance . No anaeAll egg donors underwent a conventional antagonist IVF protocol. Eggs were fertilized by ICSI (intracytoplasmic sperm injection) and embryos were kept in culture media until day 5 (blastocyst stage), when embryo transfer was performed. All recipients underwent endometrial preparation with oral  or transdermal oestrogens (oestradiol 100\u2013200 mcg), and vaginal progesterone (400 mcg micronized progesterone every 12 h) was added 5 days before transfer. Treatment efficacy was determined by a transvaginal scan to check the endometrial pattern and thickness, and a blood test to determine oestradiol and progesterone levels on the day of the egg fertilization. Those patients with ovarian activity were prescribed an oral contraceptive pill to help synchronization with their donor, as well as GNRH analogues to induce ovarian quiescence.A positive pregnancy test was considered if \u03b2-hCG levels were over 10 mUI/mL 12 days after embryo transfer. CPR was diagnosed if a vaginal ultrasound confirmed an intrauterine gestational sac 4 weeks after the embryo transfer (6th week of pregnancy). IR was calculated by the ratio between the number of gestational sacs observed and the number of replaced embryos. The birth of a living baby beyond the 24th week of pregnancy confirmed a live birth and all pregnancies lost before that moment were considered as miscarriages.Statistical analysis was done using Stata 16 for Windows . We use mean and standard deviation to describe qualitative variables, frequency distribution to summarize qualitative variables and median and 25% and 75% centiles for non-normal variables. Outcomes were analysed with a chi-squared test or Fisher\u2019s exact test.The ES effect is described by absolute and relative frequencies, and the association strength is determined by raw risk ratio (RR) with 95% confidence intervals. A general linear model with a log link and binomial distribution was used to estimate the strength of association between outcomes, adjusted by independent variables. Results are presented as RR and 95% confidence intervals. Statistical significance was considered 0.05 (5% both sides \u03b1 error) for all comparisons.All groups had similar baseline characteristics in terms of patient age, partner age, smoking habits, body mass index (BMI), race, and previous live births, as is shown in p = 0.164), nor in endometrial preparation duration . Hormonal parameters on the day of egg fertilization were also similar , and a comparable number of extra embryos were frozen in each group after the embryo transfer   .Embryo quality was also similar in the four groups, as is detailed in p = 0.028), but we could not find this difference when we performed a subgroup analysis of the results in the control and intervention groups in non-RIF patients  and RIF patients . In addition, we found no differences when we compared CPR among groups , which confirms that the increase in PR was not clinically relevant. Finally, a total of 55/105 patients in group A and 45/104 patients in group B in non-RIF patients, and 41/71 patients in group A and 40/72 patients in group B in RIF patients had a live birth , which meant no differences in terms of LBR among these populations either, as is detailed in We performed an ITT analysis and we found a lower PR in non-RIF patients  versus non-RIF patients (group A1 and B1), but this difference was not detected when we compared subgroups in the RIF-group (group A2 vs. group B2), which means that this increase is not associated with the ES. Accordingly, our study reveals that there is no difference in CPR and LBR when comparing patients according to whether they have had previous implantation failures or not, both in group A and B. These results are consistent with those of the largest study published in 2019 by Lensen et al. that included own-egg IVF cycles in RIF and non-RIF patients, and subgroup analysis did not find any differences between these groups regardless of whether ES was performed or not . These rIn addition, our results show that patients with RIF have a higher previous miscarriage rate than those who have not suffered previous implantation failures, which may mean that the former had an unknown underlying additional reason for these pregnancy failures.p = 0.00011), PR , and LBR  in those patients who underwent four endometrial injuries during the cycle prior to the embryo transfer [Nevertheless, the possible benefits of ES remain a controversial issue as studies published to date on this subject are very heterogeneous, which entails a real problem when we try to estimate an overall effect. The first study by Barash et al. included RIF and non-RIF patients and found a two-fold increase in IR (27.7% vs. 14.2%, transfer . On the transfer ,23,24. Rtransfer ,26, and transfer ,13,23, otransfer . Most sttransfer , and othtransfer , obtainitransfer ,30,31. IThe main strength of our analysis is that it refers to an RCT including only egg recipients who had homogeneous endometrial preparations and who received good quality embryos. On the contrary, the main limitation is that this subgroup analysis was not considered at the initial study design and thus no randomized stratification was performed and no sample calculation for these subgroups was considered, which may lead to a limited power to establish robust evidence.According to our results, based on the post-hoc analysis of an RCT, ES cannot be recommended as a strategy to improve clinical outcomes in patients undergoing egg donor IVF cycles, regardless of their previous failed treatments. Nevertheless, since our analysis concerns a low number of patients, these conclusions should be taken cautiously and larger studies in egg donor recipients targeting RIF patients should be performed."}
{"text": "TP53 gene, including 19 retrogenes , which are partially active, whereas humans possess only one copy of TP53 and have an estimated cancer mortality rate of 11\u201325%. The mechanism through which p53 contributes to the resolution of the Peto\u2019s paradox in Animalia remains vague. Thus, in this work, we took advantage of the available datasets and inspected the p53 amino acid sequence of phylogenetically related organisms that show variations in their lifespans. We discovered new correlations between specific amino acid deviations in p53 and the lifespans across different animal species. We found that species with extended lifespans have certain characteristic amino acid substitutions in the p53 DNA-binding domain that alter its function, as depicted from the Phenotypic Annotation of p53 Mutations, using the PROVEAN tool or SWISS-MODEL workflow. In addition, the loop 2 region of the human p53 DNA-binding domain was identified as the longest region that was associated with longevity. The 3D model revealed variations in the loop 2 structure in long-lived species when compared with human p53. Our findings show a direct association between specific amino acid residues in p53 protein, changes in p53 functionality, and the extended animal lifespan, and further highlight the importance of p53 protein in aging.Recently, the quest for the mythical fountain of youth has produced extensive research programs that aim to extend the healthy lifespan of humans. Despite advances in our understanding of the aging process, the surprisingly extended lifespan and cancer resistance of some animal species remain unexplained. The p53 protein plays a crucial role in tumor suppression, tissue homeostasis, and aging. Long-lived, cancer-free African elephants have 20 copies of the  The promise of eternal life has inspired research into this topic across many civilizations and through the millennia, dating back to Herodotus and his writings 2500 years ago. Although the average human lifespan is increasing, our health span appears to be lagging. Several studies argue that the human lifespan is physiologically and genetically limited , yet recTP53 gene and/or in its regulatory pathways [Given the high cancer susceptibility in humans and the role of p53 in regulating cell fate, p53 is regarded as the key regulator of humans\u2019 healthy lifespan ,14. Whenpathways . As revipathways , cancer-pathways . Multiplpathways ,18,19. Fpathways , cell cypathways ,22, apoppathways , ferroptpathways , DNA reppathways , embryonpathways . In the pathways ,28,29. Opathways ,31.TP53 gene is specific for the Holozoa branch, where its ancestral p63/p73-like genes emerged approximately one billion years ago [TP53, TP73 and TP63 genes are rarely mutated in cancers. Yet, the tumor suppressor function of p73 (tp73) is often attenuated in human cancers. The mechanism of suppression is via the hypermethylation of CpG islands at promoter 1, the binding to the overexpressed dominant-negative p73 isoform, dNp73 [From the evolutionary point of view, the ears ago ,33. The ears ago . Unlike m, dNp73 , or to Em, dNp73 . Notablym, dNp73 ,25.CDKN1A (p21) and promyelocytic leukemia protein, PML. The study by Tyner et al. showed that heterozygous mice having one TP53 allele with the deletion of the first six exons  aged prematurely. These mutant mice exhibited enhanced resistance to spontaneous tumors, yet displayed accelerated aging compared to Tp53+/+ mice [TP53 gene. Thus, over-activated p53 per se might not be a critical driver of accelerated aging. Yet, the role of p53\u2032s hyperactivity in aging appears to be conflicting. Fibroblasts derived from hereditary segmental progeroid syndrome patients with the homozygous antiterminating mutation c.1492T > C in the MDM2 gene showed p53 hyperstability and accelerated aging [Mdm2\u2212/\u2212 mice show embryonic lethality in a wtp53 background, which indicates that p53 regulation by MDM2 is critical for development. Yet, the conditional deletion of Mdm2 in the epidermis induced p53-mediated senescence and accelerated aging [p53 is a major factor that regulates cellular senescence, and the mechanism is via the activation of cyclin-dependent kinase inhibitor 1 +/+ mice . A study+/+ mice . Based o+/+ mice , as the ed aging . This sted aging . Thus, aCDKN1A. Yet, several other factors contribute to aging, such as the activation of E2F and mTOR, as described elsewhere [Gradual DNA damage and mitochondrial decline are hallmarks of physiological aging. DNA damage that is activated by telomere attrition in an aging cell induces p53 and mitochondrial dysfunction through the repression of the PPAR\u03b3 co-activator 1\u03b1 (PGC1\u03b1). This induces senescence . Furtherlsewhere . In prinlsewhere .CDKN1A [In addition to full-length p53, p53 isoforms may also play an important role in the modulation of longevity. The expression of certain short and long forms of p53 protein might contribute to a balance between tumor suppression and tissue regeneration . For exaCDKN1A .Considering the critical role of p53 in maintaining tissue homeostasis, high frequency of gain of function (GOF) mutations in cancer, and the limited and conflicting information on p53 role in organismal aging in Animalia, in the present work, we employed currently available datasets and tools to analyze p53 protein sequences in species possessing an extended lifespan. Our thorough analysis depicted a surprising correlation between the changes in the p53 protein sequence and the organismal lifespan, both in short- and long-lived species. Many of the identified changes occurred in the DNA-binding domain and might have a detrimental effect on p53 DNA-binding activity. Overall, we found that, when compared to the majority of closely related organisms within their phylogenetic groups, animals with unusually long lifespans share atypical p53 protein sequence features when compared to human p53 in the position corresponding to the human 180\u2013192 p53 region, which points to the important contribution of loop 2 in the p53 core domain regarding life expectancy.The growing evidence implies that p53 activity might play a pivotal role in aging in humans and little is known about the molecular signatures of extended lifespan in animals including humans; thus, we inspected all currently available sequence data of long-lived animals to explore a link between longevity  and p53 protein sequences. For this, we used the longevity data from the AnAge Database . We mergBalaena mysticetus) from Artiodactyla (subgroup Cetacea), with a maximal lifespan of 211 \u00b1 35 years [Balaena mysticetus had a unique leucine substitution in the proline-rich region, corresponding to amino acid residue 77 in human p53 , which is the only exclusively cave-dwelling chordate, has a maximal documented lifespan of 102 years. A comparison of the p53 protein sequences in amphibians showed a previously unrecognized insertion in Proteus anguinus. The p53 protein from this species had additional serine and arginine residues in the core domain (corresponding to an insertion after amino acid L188 in human p53), which had a deleterious effect on p53 functionality according to the PROVEAN tool  is a long-lived, large, flightless, nocturnal, ground-dwelling parrot that is endemic to New Zealand with a lifespan of around 95 years (TP53 Mutations (PHANTM) classifier, the N131H mutation decreases p53 transcriptional activity by 47.19% [The kakapo (95 years B. Interey 47.19% . In addiMyotis brandtii) is an extremely long-lived bat with a documented lifespan of 41 years [Myotis lucifugus, they had significantly longer lifespans than other bats  and provide a significant exception from Max Kleiber\u2019s law (mouse-to-elephant curve) since their lifespan is extremely long in relation to their small body size [Next, our analysis identified alterations in p53 in species having a long lifespan in the Chiroptera order. The Brandt\u2019s bat ), followed by the goldfish (Carassius auratus (41 years)). The Siamese fighting fish (Betta splendens) had the shortest lifespan in the group (2 years). The correlation analyses show that fifteen amino acid residues in the p53 core domain were significantly associated with a prolonged lifespan (The organisms with the longest lifespan in the Neopterygii dataset were the carp (lifespan A. We fouTerrapene carolina triunguis (138 years)) and the kakapo (Strigops habroptila (95 years)). The green anole (Anolis carolinensis) had the shortest lifespan in the group (7.2 years). The correlation analyses showed that, similar to Neopterygii, a specific fifteen-amino-acid-residue fragment in the p53 core domain was associated with a prolonged lifespan (The lifespans of species in Sauria were significantly variable. The organisms with the longest lifespan in this group were the three-toed box turtle (lifespan A. The molifespan A. SimilaHomo sapiens (122 years)) and the western gorilla (Gorilla gorilla (60 years)). Tarsier (Carlito syrichta) had the shortest lifespan in the group (16 years). The correlation analyses showed that the specific amino acid triad\u2014Q104, S106, L289\u2014was significantly associated with a prolonged lifespan  or histidine (H) at position 104, asparagine (N) at position 106, and phenylalanine (F), serine (S), or tyrosine (Y) at position 289 were associated with short-living primates. While studying human longevity, one needs to consider that the prolonged lifespan of Heterocephalus glaber (31 years) and Castor canadensis (23). The shortest lifespan in the group was Rattus norvegicus (3.8 years). The correlation analyses showed that ten amino acid residues were significantly associated with a prolonged lifespan (The dataset of Glires contained seventeen species with lifespans ranging from 3.8 to 31 years. The organisms with the longest lifespan in this group were lifespan A. Two thMyotis brandtii (41 years)) and the little brown bat (Myotis lucifugus (29 years)). The pale spear-nosed bat (Phyllostomus discolour) had the shortest lifespan (9 years). The correlation analyses showed that nine-amino-acid-long motif was associated with a significantly prolonged lifespan (The organisms with the longest lifespan in the dataset of Chiroptera were the brandt bat (lifespan A.Ursus maritimus) and the panda (Ailuropoda melanoleuca). The shortest lifespan in the group was that of the ferret (Mustela putorius furo). The correlation analyses showed that two amino acids in the p53 core domain, position 148 and 232, were significantly associated with lifespan ) followed by the orca (Orcinus orca (90 years)). Correlation analyses showed that twelve amino acid residues in the p53 core domain were significantly associated with prolonged lifespan  [The changes at the molecular level are often a result of the adaptation of species to environmental forces. To evaluate whether the amino acid residues in the p53 core domains  share some relevant features in relation to the convergent evolution, we constructed a sequential circular representation of the multiple sequence alignments and the mutual information it contains A. The fiturtles) .2+. The loss of Zn2+ triggers the aggregation of L2 and the loss of DNA-binding specificity. The 3D structures of DNA binding domain (DBD) in selected long-lived species revealed intrinsic variations in L2 structure when compared to human p53 (The longest region in p53 that was associated with longevity spanned amino acids in loop 2 (L2) of human p53  and insertions , a complete lack of p53 mRNA expression was found in Turritopsis sp.+-dependent manner and inhibits p53 transcription activity [Cebus imitator, a model organism for studying extreme longevity in primates, where the amino acid sequence is different from all other primate SIRT1s. 3D modeling revealed that the predicted structure of Cebus imitator\u2019s SIRT1 was significantly different from the structures of SIRT1 from Homo sapiens and Sapajus apella (a close relative of Cebus imitator)  .Cebus imitator gained new functions, which might result in the decreased activity of p53 when compared to other primates and slow down the aging processes, most likely via the transient inhibition of p53. Yet, it remains to be elucidated which factors might be affecting the altered target recognition by SIRT1.We hypothesized that SIRT1 from Myotis brandtii (long-living bat described above), in addition to p53, had two atypical protein sequences, one in UFM1  and the other in the p73 .In addition to SIRT1, we investigated other key factors in the p53 pathway. Surprisingly, we found that Recently, it was reported that UFM1 covalently modifies p53; this phenomenon is called UFMylation . UFMylatMyotis brandtii   and in the rest of mammals, including humans, no such extension occurs. We hypothesize that the extended UFM1 protein might contribute to the extreme longevity in myotis bats through the loss of function and consequent changes in p53 protein degradation patterns. Yet, more experimental evidence is needed to draw a clear conclusion.In UFM1, we found an approximately 20-amino-acid-long extension of the C-terminal end in tis) see . In contMyotis brandtii bat (XP_014401672). There were multiple large deletions (>10 amino acid residues) in critical p73 regions, which were found exclusively in this extremely long-lived bat. p73 is the transcription factor that undergoes similar cellular regulation as p53 protein and its role in aging is attributed to the induction of senescence through the upregulation of CDKN1A [Lastly, we found unique changes in the p73 protein sequence in the f CDKN1A .Taken together, our analyses revealed the unexpected correlation between p53 sequence variations and longevity in the animal kingdom. The changes may affect p53 functionality and, thus, influence the activation of replicative senescence, a hallmark of molecular aging. In long-lived species, with no changes in p53, the upstream regulatory proteins, including SIRT1 and UFM1, displayed amino acid changes that may affect their functionality and in consequence alter p53 activity. Yet, further studies are needed to fully comprehend the role of amino acid changes in p53 and its role in the long-lived species described in our work.TP53 is the most often mutated gene in human cancers. On the cellular level, in humans, increased p53 activity protects from cellular stress and enables genome stability, whereas altered mutant p53 protein functionality is essential for cells\u2019 immortalization and neoplastic transformation [Turritopsis jellyfish is immortal and can repeatedly rejuvenate, reverse its life cycle, and thus, was the first and only known \u201cimmortal\u201d animal on Earth [Turritopsis sp. [Turritopsis might be directly related to its unique ability of life cycle reversal and \u201cimmortality.\u201dThe p53 protein is a well-known tumor suppressor and ormation . Howeverormation . The semon Earth . Here, wpsis sp.  and surpTelomere shortening in humans induces replicative senescence, which is a process that is regulated by p53. In the absence of p53, the replicative lifespan of human cells is extended and the concurrent loss of retinoblastoma protein (RB) extends the replicative lifespan to a greater extend (reviewed in ).Intriguingly, our results obtained using Protein Variation Effect Analyzer  show tha2+. We speculate that in long-lived species, L2 affects the p53 binding to DNA and/or other transcription factors and, consequently, affects the replicative senescence program. On other hand, in humans, the L1 region is responsible for p53 binding to the major groove and was reported to undergo the most dynamic changes among the DNA contacting loops (L1\u2013L3) when located on a non-target or target DNA sequence [We identified the 180\u2013192 region, corresponding to the loop 2 (L2) region of human p53, as the longest region that is associated with longevity. The 3D model revealed variations in L2 structure in long-lived species when compared to human p53. Loop 2 is responsible for binding to the minor groove and its structure depends on the presence of Znsequence . Our finCDKN2B-AS1 and IGF2R. The TP53 gene was not among the singled-out variants, which, in accordance with our observations, indicates that no changes in human p53 might be attributing to longevity in humans [Based on what is known about the processes underlying aging, we anticipate that the altered gene expression programs would enable the following changes : (1) morn humans . Our anaSomniosus microcephalus) are available. Compared to other sharks (with a life expectancy of up to seventy years), its lifespan is exceptional. It will thus be very interesting to know the sequence of their p53 protein. A recent study suggests that certain animals may have evolved to have longer lifespans compared to other species belonging to the same taxa [The maximal lifespan according to the AnAge database is attributed to the Greenland shark, with an estimated maximal life span of 300\u2013500 years. Unfortunately, no transcriptomic nor genomic data for the Greenland shark  could act on species with an extended lifespan. In the naked mole rat , which is the longest-living small rodent and weighs only around 35 g, a unique hyperstabilization and nuclear accumulation of the p53 protein were recently reported [Experimental data support our hypothesis that specific p53 variations are associated with longevity. For example, it was found that the reduced expression of the ongevity . It was lifespan . The samlifespan  and a melifespan . In a relifespan . Anotherp53 gene . In thiso stress . In conto stress  in cetacreported . The nakreported . The hypStrigops habroptila, Myotis brandtii, Myotis lucifugus, Proteus anguinus, Balaena mysticetus, and Heterocephalus glaber. It is thus likely that low or no exposure to the UV light promotes the evolutionary changes in the p53 protein structure that alter the p53\u2032s pro-senescence activity. In addition, we speculate that in those species, the endogenous levels of reactive oxygen species might be lower when compared to animals from other, less extreme habitats. This might be a consequence of the changes in the metabolism rates that might affect the overall rate of oxidative phosphorylation and, consequently, slow down the generation of free radical species through the electron transport chain. This hypothesis agrees with the recent study showing that rapamycin, which is a widely studied inhibitor of mTOR, prevents UV-induced skin aging through the inhibition of p53, reversal of UVA-induced cellular senescence, and induction of autophagy [Intriguingly, the animals with extreme longevity that we have identified are mostly nocturnal or live in absolute darkness. These include utophagy .Despite the high complexity of the p53 proteins family, modern methods of comparative genomics provide useful tools for exploring protein variations in closely related species and correlating the extracted molecular information with lifespan . Accordihttps://genomics.senescence.info/species/, accessed on 4 May 2020); AnAge currently contains data on the longevity of more than four thousand animals [To access data on longevity and maximal lifespan, we used the AnAge Database  and merged them with the AnAge Database. We received the p53 sequence information of 118 species with information about their lifespan and sorted them according to their phylogenetic group  [Proteus anguinus (SRX2382497) and Sphenodon punctatus (SRX4014663); the resulting assemblies are enclosed in Transcriptomic data for the bowhead whale was obtained from uly 2020 . When thuly 2020  from theer 2020)  with defhttp://www.phylogeny.fr/alacarte.cgi, accessed on 15 October 2020) [https://phylot.biobyte.de/, accessed on 4 September 2020) and visualized in iTOL (https://itol.embl.de/) [The protein tree was built using the Phylogeny.fr platform (er 2020) ,81 and cer 2020) , which wer 2020)  using ther 2020) ,84. The er 2020) . The reambl.de/) .http://provean.jcvi.org/index.php, accessed on 20 May 2021) [The effect of the p53 variations in long-lived organisms was predicted and statistically evaluated using the Protein Variation Effect Analyzer web-based tool  ,87. PROVay 2021) . All inshttps://www.swissmodel.expasy.org/interactive, accessed on 3 March 2021) [Homo sapiens with bound DNA [We used the SWISS-MODEL template-based approach (ch 2021)  to prediound DNA . The resound DNA . Effectsound DNA .http://www.cbs.dtu.dk/services/SigniSite/, accessed on 22 October 2020) [p-value of \u22640.05. A Bonferroni single-step correction for multiple testing was applied for the global correlation of all sequences, no correction was applied for smaller groups of taxonomically related animals. The manually curated set of 118 high-quality p53 protein sequences obtained from the NCBI  was used as the input file. These sequences were taken from the RefSeq database and the canonical isoform corresponding to the human full-length p53 isoform (NP_001119584.1) was manually filtered for each vertebrate species. The resulting set of these 118 p53 sequences was aligned within the UGENE workflow [http://genomics.senescence.info/species/, accessed on 5 September 2020) [Residue level genotype/phenotype correlations in p53 multiple sequence alignment were performed using SigniSite 2.1 (workflow  and the workflow  with defer 2020) .http://mistic.leloir.org.ar/index.php, accessed on 18 November 2020), with PDB 2ocj (A) as the reference and using default parameters [Multiple sequence alignments of the p53 core domains from 118 species were uploaded to the MISTIC webserver (rameters .TP53 gene gain or losses were inspected using Ensembl Comparative Genomics toolshed [TP53 gene query ENSG00000141510: https://www.ensembl.org/Homo_sapiens/Gene/SpciesTree?db=core;g=ENSG00000141510;r=17:7661779-7687550, accessed on 8 December 2020.toolshed  via EnseMyotis brandtii, Myotis lucifugus, Balaena mysticetus, Heterocephalus glaber, Strigops habroptila, and Proteus anguinus displayed unique p53 protein sequence properties that were not shared with their close relatives that have a shorter lifespan. Altogether, our data show the convergent evolution of p53 sequences supporting a higher insensitivity to p53-mediated senescence under prolonged stress conditions in long-lived vertebrates. Our observations that specific variations of p53 protein are correlated with lifespan provide important grounds for the further exploration of p53 sequences in species displaying extreme longevity. Most importantly, our data implies a general mechanism at work in all vertebrates that leads to extended lifespan, which might be translated to studies on the extension of the health span in humans.This study revealed a previously overlooked correlation between longevity and changes in p53 function due to the amino acid variations in the animal kingdom. Strikingly, several long-lived species, including"}
{"text": "TP53 gene is mutated in approximately half of all human malignancies, including those of the breast, colon, lung, liver, prostate, bladder, and skin. When DNA damage occurs, the TP53 gene on human chromosome 17 stops the cell cycle. If p53 protein is mutated, the cell cycle is unrestricted and the damaged DNA is replicated, resulting in uncontrolled cell proliferation and cancer tumours. Tumor-associated p53 mutations are usually associated with phenotypes distinct from those caused by the loss of the tumor-suppressing function exerted by wild-type p53protein. Many of these mutant p53 proteins have oncogenic characteristics, and therefore modulate the ability of cancer cells to proliferate, escape apoptosis, invade and metastasize. Because p53 deficiency is so common in human cancer, this protein is an excellent option for cancer treatment. In this review, we will discuss some of the molecular pathways by which mutant p53 proteins might perform their oncogenic activities, as well as prospective treatment methods based on restoring tumor suppressive p53 functions.The p53 protein is a transcription factor known as the \"guardian of the genome\" because of its critical function in preserving genomic integrity. The  TP53 gene on human chromosome 17. The p53 protein is posited to inhibit the phenotypic and genomic alterations associated with cancer development through a complex interplay with several signaling pathways known to play critical roles in essential cellular processes such as cell division, maintenance of genomic stability, apoptosis, autophagy, immune response, and regulation of tumor microenvironment (TME) . T. T126]. TLR) gene expression in T-lymphocytes and to a lesser extent in macrophages in a way that is dependent on genetic stress and the host genetic background [Wild-type p53 controls Toll-like receptor  induce illness. Targeting senescent cells through SASP regulation, or cellular reprogramming could be a new therapeutic path for cancer and age-related illnesses like neurodegeneration, pulmonary fibrosis, and renal failure. The A repair .TP53 and RB2/P130 [P53 and RB2/P130 were identified as the major regulators of senescence in human MSCs [ELK1 could be targeting the promoters of RB2/P130 . This fiman MSCs \u2013146.In the context of senescence, p53 plays a critical role in deciding the fate of cells, and its activation can be DDR-dependent or DDR-independent . In the The role of DDR activation as a necessary and causal element in p53 activation and senescence induction has lately been questioned. Many recent investigations have shown that many OIS routes can really activate p53 without going through the DDR as in fact they might occur throughAKT via downregulation of MnSOD, through the onco-suppressor PTEN depletion, prompting mTORC1 and mTORC2 to bind to p53 instead of MDM2, and MAPK p38 by direct phosphorylation of p53 . These fBecause wild-type p53 is an efficient promoter of apoptosis and senescence  in tumorSeveral studies have shown that transfection of cancer cells with wild-type p53 expressing plasmids can induce apoptosis and/or growth arrest, implying that a gene therapy method for cancer treatment could be based on restoring normal p53 expression and function. Several clinical research investigations using viral and non-viral vectors delivering p53 genes, alone or combined with other therapeutic agents, have been completed to far .Some tumor-derived mutations that cause wild-type p53 loss-of-function can be restored by other point mutations that help stabilize the p53 protein, indicating that the structural change is reversible . PhiKan0When zinc is added to the structural mutants G245C and G245D, the wild-type structure is for the most part restored  As a conCompounds like APR-246, PK11007, and COTI-2 are promising treatments for patients with trible negative (TN) breast tumours because p53 is mutated in the great majority of them. However,mutant p53 can work as a biomarker in breast cancer, is not clearly defined .Critical Outcome Techonologies Inc.'s (COTI-2) third-generation thiosemicarbazone, as well as particular peptides, have recently been shown to convert reactive mutant p53 protein to a form with wild-type characteristics. These drugs have been demonstrated to have anticancer action in preclinical models expressing mutant p53, which is consistent with the reactivation of mutant p53. Two of these drugs, APR-246 and COTI-2, have made it to clinical trials so far. APR-246 had no major side effects in a phase I/IIa clinical trial. APR-246 is now being tested in patients with advanced serous ovarian cancer in a phase Ib/II trial, while COTI-2 is being tested in patients with advanced gynaecological tumours in a phase I trial. However, whether any mutant p53 reactivating chemical is effective in the therapy of human cancer remains to be seen .Shenzhen SiBiono GeneTech Co. Ltd. produced Gendicine (recombinant human p53 adenovirus), which was approved by the China Food and Drug Administration (CFDA) in 2003 as a first-in-class gene therapy treatment to treat head and neck cancer and went on the market in 2004. Gendicine is a biological therapy that can be administered via intratumoral injection, intracavity infusion, or intravascular infusion. Depending on the cellular stress circumstances, the wild-type (wt) p53 protein expressed by Gendicine-transduced cells is a tumour suppressor that is triggered by cellular stress and promotes cell-cycle arrest and DNA repair, or produces apoptosis, senescence, and/or autophagy. Based on more than 30 published clinical trials and 12\u00a0years of commercial use in over 30,000 patients, Gendicine has a proven track record of safety, and when combined with chemotherapy and radiotherapy, it has shown to produce much higher response rates than traditional therapies alone. In addition to head and neck cancer, Gendicine has been used to successfully treat a variety of other cancer kinds and stages. Thirteen published trials with long-term survival data found that Gendicine combination regimens result in considerably longer progression-free survival periods than standard treatments alone. Despite the fact that the p53 gene is mutated in more than half of all human malignancies, the presence of a p53 mutation had no effect on efficacy or long-term survival in Ad-p53-treated patients .The growing understanding of mutant p53 actions has contributed to the identification of a number of compounds with promising therapeutic potential. However, further experiments are required to fully characterize mutant p53 function in cancer. The fact that mutant p53 might play a role in promoting metastasis \u2013 the primary cause of cancer-related mortality \u2013 is particularly attractive in terms of possible therapeutic applications. Although many tumors express mutant p53, it is unclear if the many mutations present on this protein have similar activity, and we might have to personalize therapy depending on the presence of a particular mutation rather than only consider whether a cancer express a wild-type vs a mutant p53 mutant."}
{"text": "The transcription factor p73 is a structural and functional homolog of TP53, the most famous and frequently mutated tumor-suppressor gene. The TP73 gene can synthesize an overwhelming number of isoforms via splicing events in 5\u2032 and 3\u2032 ends and alternative promoter usage. Although it originally came into the spotlight due to the potential of several of these isoforms to mimic p53 functions, it is now clear that TP73 has its own unique identity as a master regulator of multifaceted processes in embryonic development, tissue homeostasis, and cancer. This remarkable functional pleiotropy is supported by a high degree of mechanistic heterogeneity, which extends far-beyond the typical mode of action by transactivation and largely relies on the ability of p73 isoforms to form protein\u2013protein interactions (PPIs) with a variety of nuclear and cytoplasmic proteins. Importantly, each p73 isoform carries a unique combination of functional domains and residues that facilitates the establishment of PPIs in a highly selective manner. Herein, we summarize the expanding functional repertoire of TP73 in physiological and oncogenic processes. We emphasize how TP73\u2019s ability to control neurodevelopment and neurodifferentiation is co-opted in cancer cells toward neoneurogenesis, an emerging cancer hallmark, whereby tumors promote their own innervation. By further exploring the canonical and non-canonical mechanistic patterns of p73, we apprehend its functional diversity as the result of a sophisticated and coordinated interplay of: (a) the type of p73 isoforms (b) the presence of p73 interaction partners in the cell milieu, and (c) the architecture of target gene promoters. We suppose that dysregulation of one or more of these parameters in tumors may lead to cancer initiation and progression by reactivating p73 isoforms and/or p73-regulated differentiation programs thereof in a spatiotemporally inappropriate manner. A thorough understanding of the mechanisms supporting p73 functional diversity is of paramount importance for the efficient and precise p73 targeting not only in cancer, but also in other pathological conditions where TP73 dysregulation is causally involved. In bony fishes, the p63/p73 and the p53 genes are separated into distinct entities and undergo positive selection to control different processes: p63 got specialized in the production of epithelial cells and p73 in neuronal development, whereas p53 is better adapted as a tumor suppressor , the core DNA-binding domain (DBD) and the oligomerization domain (OD), with DBD being the most conserved domain. They have an extra SAM  domain in their C-terminus, which confers protein stability . Althougppressor . These frganisms . These cN classes of isoforms, (b) alternative splicing in the 5\u2032 end, resulting in amino-truncated \u0394TA isoforms  that partially or entirely lack the transactivation domain and, together with \u0394N, constitute the so called DN isoforms, (c) alternative splicing in the 3\u2032 end, putting forth several C-terminal splice variants   use of an extrinsic (P1) and alternative intrinsic promoter (P2) in the 5\u2032 end, generating TA and \u0394, and \u03b8)   . We also, and \u03b8) . In the , and \u03b8) , while D, and \u03b8) . In cont, and \u03b8) . Using k, and \u03b8) . The \u0394TA, and \u03b8)  and, anaitiation  and cancitiation . For insitiation . Consistitiation . For a dper se. Furthermore, the p53/p63 cell content often influences p73 functions, because p73 can form stable hetero-oligomers with p63 or mutant p53  cells in the developing brain. Hippocampal dysgenesis appears to be a consequence of deprivation of CR cells that are physiologically deputed to direct brain architecture during embryonic development. These effects appear to be highly isoform-dependent , which are vital for respiration, neurogenesis and fertility. Moreover, TAp73 shows a significant ability to regulate cellular metabolism. The abovementioned physiological functions of p73 in these organ systems have been extensively reviewed elsewhere . BesidesLast but not least, several lines of evidence imply a key role of TP73 protein products in the differentiation and homeostasis in several types of muscle tissues. In skeletal muscles, TAp73\u03b1 but not TAp73\u03b2 isoform suppresses myogenic differentiation , while \u0394Oncogenic transformation occurs through progressive acquisition of key adaptations, the so-called cancer hallmarks, which include sustaining proliferative signaling, resisting cell death, evading growth suppressors, activating invasion, enabling replicative immortality, inducing angiogenesis, reprogramming energy metabolism and evading immune destruction. Transformation is further facilitated by tumor-promoting inflammation and genome instability. Strikingly, TAp73 isoforms inhibit all these hallmarks [reviewed in detail in It is noteworthy that in some cancer-related processes and cell-contents, TAp73 isoforms show an effect inconsistent with their traditional tumor suppressive function. For example, TAp73 activates anabolic pathways compatible with proliferation and cancer promotion by regulating glucose metabolism to control cellular biosynthetic pathways and antioxidant capacity [reviewed in Using our previous work on melanoma as a representative example of p73\u2019s regulation of cancer outcomes via orchestrating molecular networks, we further made the striking observation that TAp73/DNp73-controlled pathways can co-ordinate extracellular changes in the TME and intracellular gene regulation by intercepting with major cellular signaling cascades which respond to growth factors in the TME. These intersections suggest that p73 isoforms internalize the information from extracellular signals that are received by cell surface receptors and convey them to the nucleus, leading to global changes in p73-transcriptome. In particular, we used the melanoma paradigm and constructed a comprehensive regulatory map by exploiting own high-throughput and experimental data from melanoma tissue culture, mouse metastasis models and patient tumor samples. These results were integrated with data from the literature, previous mathematical models describing sections of the map , and parOf particular interest is that neurodevelopmental defects and cancer-related phenotypes co-exist in TAp73 knockout and, to a much lesser extent, in \u0394Np73 KO mice. The ability of p73 isoforms to modulate tumor initiation and progression may be relevant to their neurological functions. In particular, it is becoming increasingly evident that cancer and neuronal cells develop reciprocal interactions via mutual production and secretion of neuronal growth factors, neurothrophins and/or axon guidance molecules in the TME. Intriguingly, tumors can stimulate their own innervation during cancer progression, and this phenomenon is termed neoneurogenesis. Tumors produce and excrete neurogenic factors that modulate the TME and induce formation of new nerves that eventually infiltrate tumors . RecentlIt is possible that the ability of p73 isoforms to regulate several cancer hallmarks may not be independent from their neurological and/or immunomodulatory functions, but instead might imply co-options of relevant p73-governed pathways in a cancer cell context. Looking at the processes regulated by p73 isoforms in normal and cancerous tissues more closely, analogies can be found between some physiological processes and cancer hallmarks. It is well-accepted that \u0394Np73 overexpression becomes a positive advantage for tumor progression due to co-option of its pro-angiogenic capacity in tumors that trigger neoangiogenesis . In an aIn view of these data, our results suggest that p73 isoforms co-regulate stemness and neurodifferentiation to control tumor progression. The tumor-specific p73\u0394Ex2/3 isoforms, which are established metastasis inducers and CSC regulators, have the ability to activate key neurodifferentiation players. Increased cancer stemness, together with the loss of original cell identity (de-differentiation), and the acquisition of characteristics of neuronal cell types upon p73 isoform expression, are overall indicative for their ability to switch from a melanocyte to a neuronal-like cell phenotype which would be theoretically able to foster a newly-emerged dangerous liaison between melanomas and the nervous system .In light of the overwhelming functional diversity of the products of TP73, it is reasonable to assume that the typical mode of direct transactivation/transrepression that has been described for TAp73 and DNp73\u2019s, on its own, is not sufficient to support such high level of functional pleiotropy across several organ systems. Besides the TA/DN ratio, isoforms synthesized by other members of the p53 family, like p53 and p63, also influence p73 activity . HoweverIn this section we provide compelling evidence to support that p73-regulated functions are a result of a sophisticated combination of at least three parameters: (a) the type of p73 isoforms, (b) the presence of p73 interacting partners in the cellular milieu, and (c) the intrinsic properties of promoters of the target genes. Tissues are characterized by distinct proteomes, hence the expression of the same p73 isoform in one tissue content could lead to functional diversification, since this isoform is able to form protein\u2013protein interactions (PPIs) with a particular set of factors that is contained in one tissue but not in another. In addition, the C-terminus of p73 determines whether PPI occurs, because even if the interactor is present in the cell, the expressed p73 splice variant may not interact with it. Furthermore, p73 isoforms form protein complexes with several other key transcriptional regulators on target gene promoters to fine-tune their transcriptional regulation. Besides, some p73 isoforms appear to have non-transcriptional functions by interacting with proteins other than transcriptional regulators outside the nucleus , suggestThe p73 protein interactome is a decisive parameter of the tissue- and/or cell context-specific p73 activity. Several proteins have been described to physically associate with p73 isoforms, by recognizing the TA domain, the DBD domain, the OD or the C-terminus. These interactions usually take place in the nucleus and regulate the transactivation activity of p73 either positively or negatively. However, in some cases the interactions occur in the cytoplasm . AnotherSeveral p73 protein binding partners induce post-translational modification, proteolytic degradation, phosphorylation-dependent activation or inhibition, acetylation or gene target co-regulation, often in a p73 C-terminus-dependent manner . Other pd13/d13 mice, in which a switch from the \u03b1 to \u03b2 form through knockout of exon 13 results in distinct phenotypic abnormalities that do not overlap with those of other p73 knockout mice  the yes-associated protein YAP, a phosphoprotein that interacts with a non-receptor Src tyrosine kinase encoded by the c-yes protooncogene, (b) NEDD4-like ubiquitin protein ligase 2 (NEDL2), (c) cytoplasmic tumor suppressor oxidoreductase (Wwox), and (d) E3 ubiquitin ligase Itch. YAP, NEDL2, and Wwox enhance the transcriptional activity of p73, while Itch is a YAP antagonist, that leads to p73 ubiquitination and degradation and impairs transcriptional activity  nalysis:  candidatnalysis:  candidatnalysis:  the likenalysis: .b) Tethering scenario: we checked ORTFBS in DNp73 genes that are highly increased  or decreased  and found non-overlapping groups of ORTFBS between both groups (A2 vs. B2) (\u2217) in a non-overlapping manner.( vs. B2) . In A2, In summary, the interplay between p73 isoforms with different C-termini, their interacting partners, and the architecture of the target gene promoter supports a high degree of heterogeneity in the mechanisms underlying tissue-specific p73-driven functions in a variety of organ systems including immunity, neurodevelopment and reproduction. Such sophisticated mechanistical patterns may explain, at least partially, the different and sometimes opposing results across different cell contents and experimental settings. Dysregulation of one or more of the above parameters in tumors could lead to tumor initiation and progression by, at least in some cases, reactivating p73-regulated differentiation programs in a spatiotemporally inappropriate manner.The main body of research on TP73 has been initially performed in the cancer setting, due to its functional and structural similarity with the tumor-suppressor TP53. In cancer, the networks controlled by the typically anti-oncogenic TAp73 isoforms offer functional redundancy to the intricate circuitries regulated by p53, through activating fully or partially overlapping pathways, which can circumvent blocks attributed to mutations in TP53 or its downstream effectors . Neverthde novo combinatorial activation of multiple distinct and developmentally distant transcriptional modules (In view of recent findings, the potential of p73 isoforms to modulate cancer initiation and progression might not be independent from the physiological functions of TP73. Instead, it could reflect the recapitulation of the same p73-regulated developmental/tissue homeostasis pathways within the cancer cell content. Specifically, a new idea proposes that metastatic transcriptional programs arise from  modules . Althoug modules . This pr modules .The functional pleiotropy of TP73 assumes existence of mechanistic heterogeneity that extends beyond its typical transactivation mode of action. The PPI-mode of action of TP73 can provide a basis for its multifunctionality and tissue specificity. We propose that multiple p73 isoforms can establish PPIs with many proteins via several motifs in the N-terminus, core domain, and C-terminus, with the C-terminus being particularly enriched in protein binding motifs and residues. In different tissues, p73 isoforms with different C-termini may have different binding partners that can be recognized by the appropriate interacting regions of 73, causing the formation of a variety of p73 coregulator complexes. The localization of these complexes can also lead to functional diversification. Thus, p73-containing complexes within the nucleus select direct and indirect p73 target genes and regulate them via composite and tethering mechanisms based on a specified promoter architecture. In addition, p73 isoforms may be able to bind to other proteins as transcriptional regulators, such as the miRNA processing complex and SAC. p73 isoforms also physically associate outside the nucleus with proteins localized in a number of subcellular organelles. The ER- and Golgi-related interactome reflects to some extent the well-established tendency of TP73 gene products to undergo post-translational modifications. However, their potential to interact directly with proteins in intracellular compartments, such as the cell membrane, lysosomes, endosomes, and mitochondria might indicate novel, non-transcription-mediated functions of p73 isoforms that are worth unveiling in the future. A comprehensive scheme of the heterogeneity of mechanisms supporting p73 functional pleiotropy and diversity is shown in From the therapeutic perspective, these new insights provide a roadmap for efficient and selective manipulation of p73 isoforms toward precision medicine. On the shoulders of structural systems pharmacology , these mSL and BP conceived the review and took the lead in writing. SM developed bioinformatics pipelines and analyzed high-throughput data. CR conducted CoIP-MS and data analysis. NM, CR, and SL performed literature search. AS, SM, and SL crafted the illustrations. All authors contributed to and approved the final 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
{"text": "TP53) plays a central role in response to various cellular stresses. A variety of biological processes are regulated by p53 such as cell cycle arrest, apoptosis, senescence and metabolism. Besides these well-known roles of p53, accumulating evidence show that p53 also regulates innate immune and adaptive immune responses. p53 influences the innate immune system by secreted factors that modulate macrophage function to suppress tumourigenesis. Dysfunction of p53 in cancer affects the activity and recruitment of T and myeloid cells, resulting in immune evasion. p53 can also activate key regulators in immune signaling pathways which support or impede tumor development. Hence, it seems that the tumor suppressor p53 exerts its tumor suppressive effect to a considerable extent by modulating the immune response. In this review, we concisely discuss the emerging connections between p53 and immune responses, and their impact on tumor progression. Understanding the role of p53 in regulation of immunity will help to developing more effective anti-tumor immunotherapies for patients with TP53 mutation or depletion.As a key transcription factor, the evolutionarily conserved tumor suppressor p53 (encoded by  As an intensively studied protein, the fame of p53 mainly stemming from its role as a tumor suppressor which is activated when responding to stress signals such as genotoxic damage, or nutrient deprivation . MutatioBesides the capability of governing cellular homeostasis to curb tumourigenesis, accumulating observations suggest that p53 also plays the role in inflammatory reactions . ChronicChronic inflammation enhances the risk of cancer. As the crucial transcription factor, NF-\u03baB is constitutively activated in most tumors. p53 and NF-\u03baB pathways play crucial roles in response to various stresses and the NF-\u03baB activity usually shows an antagonistic relationship with that of p53 . In contIntriguingly, the reciprocal activation of p53 and NF-\u03baB has been also found in certain cases . It has As the most frequently genetic alterations in cancer, p53 mutations exist in over half of human cancers. However, many p53 mutants (mutp53) gain new activities to augment pro-inflammatory and survival properties, termed gain-of-function (GOF). Several studies have shown that GOF mutp53 can activate some of the NF-\u03baB target genes . For exavia IKK, which is required for nuclear localization. At the transcriptional level, r-Roscovitine inhibits the transcription of NF-\u03baB-regulated genes such as MCP-1, ICAM-1, COX2, FLIP, and IL-8  . The TMEIn the TME, cancer-associated fibroblasts (CAFs) play an important role in modulating tumor progression and metastasis . In CAFsOne of the most important components in TME is the extracellular matrix (ECM), which are comprised of various macromolecules that regulate cellular functions in tumors. Tumor cells manipulate the arrangement and orientation of ECM to enhance tumor progression and create a positive tumourigenic feedback loop . PreviouImmune cells are important cellular compartments in TME that are heterogeneous across tumor types and are associated with cancer progression and prognosis , b. Prod\u2013/\u2013 mice show more susceptibility to inflammation and auto-immunity which favors tumor establishment and progression , including retinoic-acid- inducible gene I (RIG-I)-like receptors (RLRs), stimulator of IFN genes (STING) protein, and Toll-like receptors (TLRs) . The rolThe TLRs are membrane glycoproteins and previous studies reveal that p53 transcriptionally regulate several TLRs, constituting a crucial bridge between cellular stresses and TLR-induced innate immune response . Notablyin vivo via up-regulation of type I IFNs  that enhance NK cell-mediated cytotoxicity . However+ T cells, and the direct role of p53 in DC antigen presentation requires further exploration.The development of effective immunotherapies for oncology patients is now becoming a clinical reality. Notably, the interaction between T cells and DCs is developing as one of the key targets for immunotherapy. As an important sensor to activate adaptive immune responses, p53-mediated activation of innate immune cells, particularly DC, is expected to promote adaptive immunity. Although the direct effect of p53 on the function of DCs has not been clarified, many results suggest that p53 activation is necessary for DC function. Treatment with Nutlin 3, an MDM2 inhibitor that activates wild-type p53, has been reported to increase the ability of DCs to stimulate T-cell proliferation, suggesting that p53 is involved in the activation of DCs . It has via PD-L1  and cytotoxic T lymphocyte antigen 4 (CTLA4), which are important for T cell tolerance to evade immune attack . Links bia PD-L1 . Consistia PD-L1 .During the resolution of injury and infection, normal cell turnover and clearance is an important process in preventing autoimmunity and triggering immune recognition of antigens by dying cells . FailureAs mentioned above, the regulation of p53 in the tumor immune response exhibits a yin-yang balance. On the one hand, p53 counteracts pro-inflammatory factors, such as NF-\u03baB and STAT3, to promote tissue homeostasis and avoid excessive immune responses. On the other hand, p53 contributes to the recognition of non-self antigens and thus activates anti-tumor immunity through multiple pathways. All these p53 features will allow us to develop more effective tumor therapies in combination with current immunotherapies.Cancer cells are always accompanied by unstable genetic changes and produce new antigens that distinguish cancer cells from normal cells. The accumulation of p53 hotspot mutations in cancer has been considered as immunologically active neoantigens for immunotherapy. However, progress in this field has been limited by the lack of efficiency of recognition of mutant p53 antigens in cells . A recenAlthough significant advances have been made in antitumour immune checkpoint inhibitor (ICI) therapy, only a minority of cancer patients respond well to immune checkpoint inhibitors . An effeAs a tumor suppressor, the cell-autonomous function of p53 in suppressing malignant tumors has been extensively studied. More recently, growing evidence suggest a potential link between p53 and immune function, and dysfunction of p53 is also associated with inflammatory diseases. Dysfunction of p53 in tumors is shown to regulate not only immune recognition but also affect the stromal compartment, which plays an important role in controlling tumor progression. Thus, as a \u201cguardian of genomic integrity,\u201d p53 also functions in response to homeostatic stress, including innate and adaptive immunity as described above. There are still many uncharacterized issues that presumably have a broad impact on immunity and inflammation, which may ultimately lead to tumor development. For instance, how exactly p53 dysregulation affects the immune response to various external or internal stimuli, and what is the role of p53 in immune cell development. Moreover, depletion or mutation of p53 is likely to reprogram the microenvironment, especially the extracellular components in tumors, but the molecular regulatory mechanisms involved remain still largely unknown. p53 mutations can promote tumor cell metastasis. How the immune regulation and response are changed during this process, and in particular which immune cells\u2019 functions are altered. In addition, the role of p53 in the remote regulation and communication between different tissues or organs will also be a highly anticipated research direction. There is no doubt that, understanding these issues will significantly improve our knowledge of both biologic and pathologic functions of p53, allowing for the development of targeted therapeutic approaches in the future.DS and PJ conceived the manuscript. DS reviewed the literature, drafted the manuscript, and drew the figures. PJ revised the manuscript. Both authors read and approved the final 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
{"text": "Efficient proteostasis is crucial for somatic maintenance, and its decline during aging leads to cellular dysfunction and disease. Selective autophagy is a form of autophagy mediated by receptors that target specific cargoes for degradation and is an essential process to maintain proteostasis. The protein Sequestosome 1 (p62/SQSTM1) is a classical selective autophagy receptor, but it also has roles in the ubiquitin-proteasome system, cellular metabolism, signaling, and apoptosis. p62 is best known for its role in clearing protein aggregates via aggrephagy, but it has recently emerged as a receptor for other forms of selective autophagy such as mitophagy and lipophagy. Notably, p62 has context-dependent impacts on organismal aging and turnover of p62 usually reflects active proteostasis. In this review, we highlight recent advances in understanding the role of p62 in coordinating the ubiquitin-proteasome system and autophagy. We also discuss positive and negative effects of p62 on proteostatic status and their implications on aging and neurodegeneration. Finally, we relate the link between defective p62 and diseases of aging and examine the utility of targeting this multifaceted protein to achieve proteostatic benefits. The health and survival of an organism is reliant on efficient proteostasis, and breakdown of this process results in accumulation of toxic protein aggregates that contribute to aging and age-related diseases . The maivia the nuclear UPS machinery  are more apparent in p62 cluster formation than K48-linked cargoes, suggesting the K63-linked chains specifically triggered clustering, while K48-linked chains needed higher concentrations for clustering, which might occur during proteasomal inhibition  accounts for nearly 80% of protein degradation in the cell . p62 colThe UPS and autophagy are two major intracellular degradation routes and p62 is a key mediator of crosstalk between these pathways . ProteasLike the UPS, inhibiting autophagy also causes accumulation of p62, but this accumulation delays delivery of ubiquitinated proteins to the proteasome and thus reduces flux through the UPS . AutophaToxoplasma gondii by encapsulating them in vesicles that do not fuse with lysosomes  . p62 expression is age- and disease-dependent, characterized by a decline of expression with age and senescence in mice  and flie(ALS) ; however(ALS) .Drosophila melanogaster, but only in females that have the overexpression initiated at middle-age. This follows the endogenous expression pattern: transcript levels of p62 are increased in early adulthood, but the sharp decrease in expression after midlife can be rescued by p62 overexpression only at that stage  causes SQST-1/p62 to accumulate, leading to a decrease in lifespan  is a progressive neurodegenerative disease that destroys memory and cognitive functions and is characterized by accumulation of amyloid-\u03b2 (A\u03b2) and hyperphosphorylated tau, causing amyloid plaques and tau tangles, respectively, in the brain. Low expression of p62 has been observed in the frontal cortex of AD patients as well as in transgenic AD mouse models; however, the remaining p62 is associated with tangles, and is believed to play an important role in tau degradation . In addiParkinson\u2019s Disease (PD) is characterized by intracellular accumulation of Lewy bodies and Lewy neurites, which consist of p62-associated aggregated proteins, including \u03b1-synuclein, parkin, and ubiquitinated proteins . DisruptC. elegans model of ALS showed defective autophagy and increased levels of p62; however the removal of p62 alleviated the locomotion defect without restoring the autophagy defects  is characterized by ubiquitin-p62 positive intraneuronal inclusions, with increased levels of p62 in the spinal cord and motor neurons. Some cases of ALS are associated with p62 mutations in the UBA, LIR, or KIR domains. UBA domain mutations prevent interactions with ubiquitinated proteins tagged for degradation , and LIR defects , suggestFrontotemporal Lobar Degeneration (FTLD) is characterized by neuronal cytoplasmic inclusions of TDP-43, which are correlated with neurodegeneration. Overexpression of p62 leads to the mislocalization of TDP-43 to the cytoplasm, causing aggregates and neuronal death . p62 hasHuntington\u2019s Disease (HD) is characterized by neuronal degeneration associated with a CAG repeat expansion (polyQ) in the huntingtin gene (mHTT). Early work investigating mTOR inhibition by rapamycin treatment in a HD mouse model demonstrated reduce HD pathology ; however2O2 leads to inhibition of proteasome activity and an increase in p62 expression in RPE cells. Silencing of p62 increases the accumulation of protein aggregates in RPE cells that showed decreased autophagy and Nrf2-mediated antioxidant response , leading to the accumulation of cellular waste such as lipofuscin. Oxidative stress induced by Hresponse . RPE provia autophagy induction.p62 is commonly upregulated in human tumors , and theMost degenerative diseases are characterized by protein aggregation, so naturally it would seem that targeting autophagy would be the best option to clear large aggregates. But what is the best way to induce this process when it requires coordination of multiple steps for autophagy to work? Since p62 has a multitude of roles beyond protein turnover and is also not essential for autophagy , specifiLoss of p62 leads to accelerated aging due to a decline in proteostasis, dysregulation of signaling pathways, and inability to sufficiently respond to oxidative stress. However, overexpression of p62 has potential detrimental effects, including accumulation of cellular p62-aggregates , tumorig"}
{"text": "APOB gene. Haplotype analysis showed that these patients presented the same mutant haplotype. Moreover, there was a decrease in plasma levels of PCSK9 in affected individuals compared to the non-affected and a significant positive correlation between circulating PCSK9 and ApoB levels in all studied probands and their family members. Some of the p.(Arg490Trp) carriers suffered from diabetes, hepatic steatosis or neurological problems. In conclusion, the p.(Arg490Trp) pathogenic variant seems a cause of FHBL in patients from Lebanese origin, accounting for approximately 70% of the probands with FHBL presumably as a result of a founder mutation in Lebanon. This study is crucial to guide the early diagnosis, management and prevention of the associated complications of this disease.Familial hypobetalipoproteinemia (FHBL) is a codominant genetic disorder characterized by reduced plasma levels of low-density lipoprotein cholesterol and apolipoprotein B. To our knowledge, no study on FHBL in Lebanon and the Middle East region has been reported. Therefore, we conducted genetic studies in unrelated families and probands of Lebanese origin presenting with FHBL, in order to identify the causes of this disease. We found that 71% of the recruited probands and their affected relatives were heterozygous for the p.(Arg490Trp) variant in the  APOB gene which encodes the apolipoprotein B [PCSK9 gene [APOB gene is located on the short arm of chromosome 2 at position 2p24.1 [Familial hypobetalipoproteinemia  is the most common monogenic form of primary hypobetalipoproteinemia . It is arotein B ,7. Less SK9 gene ,6, a pron 2p24.1 . This gen 2p24.1 ,12,13. Mn 2p24.1 ,15. Thesn 2p24.1 . The firn 2p24.1 ,17.APOB gene are generally asymptomatic but present an increased risk of developing liver disease [MTP) gene [Patients who are heterozygous for mutations in the  disease . Truncat disease , and thu disease . PatientTP) gene ,11. ThesTP) gene ,11,12. LPL gene causing type I hyperlipoproteinemia [ABCA1 gene causing Tangier disease [APOB gene responsible for this phenotype, presumably as a result of a founder effect.To our knowledge, no genetic study on FHBL has been conducted in the Middle East (ME) region. In fact, the ME region is characterized by a high prevalence of consanguineous marriages, metabolic syndrome, dyslipidemia characterized by low high-density lipoprotein cholesterol (HDL-C) levels and high triglycerides levels, familial hypercholesterolemia (FH), diabetes , and nonteinemia , and mor disease . In thisIn the first family , the proIn the second family , the proIn the third family , the proIn addition to these families, four unrelated probands were also recruited. Proband 4 was a 49-year-old male who presented low LDL-C levels (0.45 mmol/L)  and was APOB gene, in which the pathogenic c.1468 C>T variation (rs771541567) has been previously reported by Burnett et al. in an extended Lebanese family living in Australia [We sequenced exon 11 of the ustralia ,17. It wustralia  considerIn the first family, sequencing of exon 11 revealed the presence of the p.(Arg490Trp) variant at the heterozygous state in the proband (II.1) and the affected relatives  and its absence in non-affected individuals (I.2 and II.3), highlighting a segregation with the FHBL phenotype in the family . In the \u22126 in the general population according to GnomAD.Interestingly, the amino acid arginine at position 490 is well conserved among species. The p.(Arg490Trp) variant was predicted to be disease causing on Mutation Taster (Grantham Matrix score of 101), deleterious on PROVEAN (with a score of \u22125.204), with probably damaging consequences on the functionality of the protein according to Polyphen-2 , and presented a CADD score of 34 suggesting that this variant is predicted to be among the top 0.1% of the most deleterious substitutions in the human genome. The allele frequency of this variant is estimated to be 7.96 \u00d7 10PCSK9 gene in which loss-of-function mutations have been associated with hypocholesterolemia [We also sequenced all the exons of the erolemia . The seqerolemia   while others presented the E3/E3 isoforms (I.1 and II.2)  . The seqnd II.2) .The relatively high frequency of the p.(Arg490Trp) variant in the present FHBL cohort and its occurrence in Lebanese families living abroad raised questions about its origin and the presence of a founder effect. Therefore, we conducted a haplotype analysis to determine whether the chromosomal background of the variation detected in some individuals is identical. The latter showed that in families 1 , 2 and 3For the HincII and PvuII markers, all heterozygous carriers presented at both sites a nucleotide substitution which is predominant compared to the wild type nucleotide, with a prevalence of 86% and 93% respectively. For the AluI marker, all heterozygous individuals did not present a polymorphism at this site, the wild type amino acid Ala was conserved, with a prevalence of 47%. For the XbaI marker, all heterozygous individuals had a nucleotide substitution, with a prevalence of almost 50%. For the MspI and EcoR1 markers, all heterozygotes had the wild type amino acid conserved. The prevalence of the wild type amino acid is respectively around 90% and 81%. These prevalences are reported in Non-Finnish European according to GnomAD and in an Austrian population reported in previous studies .p = 0.004) as well as in FHBL patients carrying the p.(Arg490Trp) variant .We measured plasma and serum PCSK9 levels in family 1 and in some of the other individuals of the present cohort, for whom plasma was available. The results are presented in APOB.This is the first study conducted to investigate the genetic causes of hypobetalipoproteinemia in Lebanon, and to measure PCSK9 levels in familial hypobetalipoproteinemia caused by a missense mutation. We searched for the p.(Arg490Trp) variant which was described for the first time in 2003 by Burnett et al. in an extended family with FHBL of Lebanese origin living in Australia ,17. ThisAPOE gene and two others for the p.Leu21dup in exon 1 of the PCSK9 gene. The presence of a single E2 allele is thought to be associated with a \u2248 10 mg/dL reduction in TC compared to people with other APOE allele combinations [PCSK9 gene was detected, we observed that there was a significant decrease in PCSK9 levels in affected individuals of family 1 compared to the unaffected. These results are consistent with the work of Fazio et al., who studied the association between plasma PCSK9 levels and low LDL-C levels in subjects with FHBL due to truncations in the ApoB protein and found that PCSK9 levels were decreased in subjects harboring a truncating mutation [Moreover, two carriers of the p.(Arg490Trp) were heterozygous for the E2 allele in the inations , and theinations . While nmutation .APOB gene are at increased risk to develop fatty liver because of a defective synthesis and export of VLDL from the liver, which causes an accumulation of TG in the hepatocytes [APOB gene variants does not seem to cause an increase in the risk of diabetes mellitus (DM) [The pathogenicity of this variant has been proven in both in vitro and in vivo functional studies ,26. It matocytes . In the atocytes ,33,34, watocytes . Relatiotus (DM) . Moreovetus (DM) ,39,40. Itus (DM) . Therefotus (DM) .Another interesting observation is that three individuals from the present cohort carrying the p.(Arg490Trp) suffered from a neurological manifestation ranging from neuropathies to Parkinson\u2019s disease and multiple system atrophy. It is not clear whether the neurological disorders developed by these individuals are caused by FHBL or by other factors. In fact, Parkinson\u2019s disease  and a caThree unrelated families and four unrelated probands from different regions in Lebanon were recruited in collaboration with endocrinologists and gastroenterologists at the Endocrinology and Gastroenterology Clinics of H\u00f4tel Dieu de France hospital in Beirut upon perceiving permanent low LDL-C levels . Inclusi\u00ae analyzer .Blood samples were obtained after the subjects had fasted overnight, plasma and serum were prepared. Lipid measurements were determined on a COBAS INTEGRA\u00ae DNA Kit from Qiagen  according to the manufacturer\u2019s instructions. The exon 11 of the APOB and all exons of the PCSK9 genes and their flanking exon-intron boundaries, as well as the region containing the two SNPs for genotyping the APOE gene [\u00ae Software.Genomic DNAs of all the participants were extracted from peripheral blood leukocytes using FlexigenePOE gene  were amphttp://gnomad.broadinstitute.org/, accessed on 7 April 2021), and for pathogenicity prediction we used Polymorphism Phenotyping version 2 , Protein Variation Effect Analyzer , Mutation Taster , and the Combined Annotation Dependent Depletion score .For frequency determination of the variant, we used the Genome Aggregation database  that modify a recognition site by a specific restriction enzyme: p.Ala618Val (AluI), p.Thr2515 (XbaI), p.Arg3638Gln (MspI), p.Glu4181Lys (EcoRI), and two occurring in intron 4 (HincII and PvuII) [These diallelic markers resulted from the following substitutions in d PvuII) . We measured plasma and serum PCSK9 levels using a commercial ELISA kit  and the Bio-Plex Pro assay technology  as previously described .APOB gene, in several Lebanese families and probands presenting with FHBL (in approximately 70% of the probands), presumably because of a founder mutation. Therefore, in the case of a patient presenting with primary hypobetalipoproteinemia or with a hepatic steatosis without any risk factors and low LDL-C levels, family investigation should be conducted in order to identify a familial aspect of the hypobetalipoproteinemia. Molecular diagnosis should be implemented by searching firstly for the p.(Arg490Trp) variant in exon 11 of the APOB gene. Moreover, these patients and their families should be advised on the risk of consanguineous marriage, especially that its prevalence in Lebanon was of 35.5% [In this article, we highlight the presence of a pathogenic variant, p.(Arg490Trp), in exon 11 of the of 35.5% , knowingof 35.5% . In conc"}
{"text": "Importance: The protein p53 is an unequivocal tumor suppressor that is altered in half of all cancers. The immune system produces systemic p53 autoantibodies (p53 Abs) in many cancer patients. Objective: This systemic review and meta-analysis focuses on the prognostic value of p53 Abs expressed in the serum of patients with solid tumors. Data Sources: All the clinical investigations were searched on PubMed from the first study dated 1993 until May 2021 (date of submission of the manuscript). Study Selection: Studies were included that met the following criteria: (1) participants with cancer; (2) outcome results expressed in relation to the presence of a p53 antibody; (3) a primary outcome  expressed as hazard ratio (HR). The following exclusion criteria were used: (1) insufficient data available to evaluate outcomes; (2) animal studies; (3) studies with less than 10 participants. As a result, 12 studies were included in the analysis. Data Extraction and Synthesis: PRISMA guidelines were used for abstracting and assessing data quality and validity by three independent observers. The summary estimates were generated using a fixed-effect model (Mantel\u2013Haenszel method) or a random-effect model (DerSimonian\u2013Laird method), depending on the absence or presence of heterogeneity (I2). Main Outcome(s) and Measure(s): The primary study outcome was to determine the prognostic value of p53 Abs from a large population of patients with solid tumors, as determined before data collection. Results: In total, 12 clinical studies involving 2094 patients were included in the meta-analysis, and it was determined that p53 Abs expression in the serum significantly correlated with poorer survival outcomes of cancer patients . Conclusions and Relevance: This is the first meta-analysis proving the diagnostic utility of p53-Abs for cancer patients in predicting poorer outcomes. The serum-p53 value  may be useful for future theranostics. The P53 protein is an unequivocal tumor suppressor mutated in almost half of human cancers ,2,3,4. IMice lacking MDM2 show embryonic lethality, while the dual presence of p53 and MDM2 can rescue lethality . The p53Prognostic biomarkers have a crucial role in measuring the progression of diseases from samples of patients, such as metastasis in cancer, and they can aid clinicians in intervening with more precise medical interventions. In addition to the common theory that in humans the loss of p53 increases genomic instability, this loss has been linked to the proliferation of the stem-cell characteristic that ultimately leads to highly aggressive cancers with invasive and metastatic properties. p53 antibodies (s-p53-Abs) are stably expressed in the sera of cancer patients, and could have an important prognostic application. Many clinical studies have assessed in cancer patients the correlation between the expression of s-p53-Abs with tumor invasiveness grades, metastasis and prognosis .In our review, we performed a meta-analysis of the current literature, investigating the prognostic role of serum p53-Abs in cancer patients.p < 0.0001, 2 = 19%).After screening the article according to flow chart in The funnel plot  of the iThe meta-analysis showed that high levels of p53 antibodies significantly correlated with worse clinical outcomes. However, our study had some limitations. First, the retrospective nature of the study was intrinsically susceptible to biases. Second, different forms of solid tumors were included pre- or post-treatment with various types of therapies, as the typology requirements were at different stages. Consequently, in our analysis patients were observed independently of treatment and tumor type because of the relatively low number of randomized studies at our disposal. Third, there was a lack of follow-up with patients from different clinical trials. Thus, differences in survival probability may have been influenced by the durations of the studies. This may have given rise to different age populations, which could ultimately have affected the data. All these variables may ultimately have influenced the results.As medicine advances, studies involving greater numbers of patients could help to evaluate the impact of our findings and treatment response.In summary, p53 is a well-established tumor suppressor, and its absence is commonly found in patients diagnosed with cancer. The p53 protein has been demonstrated to be absent or mutated in approximately one out of two malignancies. It is known that p53-wt cancers have a better prognosis compared to p53-mut cancers. Our data are not in contradiction with this notion. Although both mutated and wild type p53 antibodies can be detected in cancer patients, their role is still controversial and a matter for debate. Recently, a few studies have reported that these antibodies are statistically associated with the survival of patients diagnosed with different malignancies. To the best of our knowledge, our meta-analysis is original and is the first study gathering p53 (wild type/mutated) antibody data generated from 1993 thus far. Overall, the investigation includes 12 studies and a total of 2094 patients.The studies were identified according to the following inclusion criteria: (1) participants with cancer; (2) outcome results expressed in relation to the presence of a p53 antibody; (3) a primary outcome  expressed as hazard ratio (HR). The following exclusion criteria were used: (1) insufficient data available to evaluate outcomes; (2) animal studies; (3) studies with less than 10 participants.Two independent researchers revised the included studies, and all potential disputes that could have arisen were evaluated with the corresponding author.2). A subgroup analysis was performed to highlight any differences between studies in terms of Overall Survival (OS), Disease-Free Survival (DFS), Progression-Free Survival (PFS), as summarized in The summary estimates were generated using a fixed-effect model (Mantel\u2013Haenszel method)  or a ranWhen we used the keywords \u201cp53 antibodies in early cancer\u201c, p53 antibodies in metastatic cancer\u201d, \u201cp53 antibodies impact on cancer progression\u201d, the PubMed search yielded 1375 potentially relevant articles. Studies as duplicates, animal studies, cellular studies, or letters to the editor or reviews were excluded. After viewing the titles and abstracts, the full texts of 34 studies were retrieved and 12 studies ,19,20,21We observed that serum antibodies generated in the blood of cancer patients against p53 (and mostly p53-wt) were deleterious. Given the straightforward detection in blood of p53 antibodies as a biomarker for cancer survival, as summarized in a simple workflow in The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in, or financial conflict with, the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript."}
{"text": "LDLR, APOB, PCSK9, LDLRAP1 and STAP1) has been performed by next-generation sequencing (NGS). In cases where a copy number variation (CNV) has been detected by NGS, confirmation by multiplex ligation-dependent probe amplification (MLPA) has also been performed. We identified 47 causal or potentially causal (including variants of uncertain significance) LDLR and APOB variants in 44 index patients. The most common variant in the APOB gene was the c.10580G>A p.(Arg3527Gln) missense alteration, this being in accordance with literature data. Several missense variants in the LDLR gene were detected in more than one index patient. LDLR variants in the Hungarian population largely overlap with variants detected in neighboring countries.Familial hypercholesterolemia (FH) is one of the most common autosomal, dominantly inherited diseases affecting cholesterol metabolism, which, in the absence of treatment, leads to the development of cardiovascular complications. The disease is still underdiagnosed, even though an early diagnosis would be of great importance for the patient to receive proper treatment and to prevent further complications. No studies are available describing the genetic background of Hungarian FH patients. In this work, we present the clinical and molecular data of 44 unrelated individuals with suspected FH. Sequencing of five FH-causing genes ( LDLR and APOB genes are responsible for familial hypercholesterolemia, and sometimes alterations in the PCSK9 gene, but more FH-related genes have also been described, such as the LDLRAP1 and STAP1 genes. Variants in these genes have a negative effect on the proper elimination of low-density lipoproteins (LDL) from the blood, which results in an elevated LDL cholesterol (LDL-C) and total cholesterol (TC) level.Familial hypercholesterolemia FH, MIM#143890) is one of the most common autosomal, dominantly inherited diseases; it is a disorder of the cholesterol metabolism and one of the major risk factors for early arteriosclerosis and cardiovascular complications. Disease prevalence is 1:200\u2013250. The estimated FH prevalence in Hungary was found to be 1:340 [43890 is In the case of a heterozygous alteration, the FH patient has an LDL-C level higher than 5 mmol/L, whereas homozygous or compound heterozygous alterations generally result in an LDL-C level higher than 13 mmol/L [LDLR and 250 APOB pathogenic variants have been described in the literature . No mutational hotspots have been identified in the LDLR gene; the genetic variants are spread from the promoter region to the very last exon of the gene. According to literature data, pathogenic variants occur more frequently in some regions of the gene, such as the ligand-binding domain and the epidermal growth factor-like domain.To date, over 2100 APOB gene is located on chromosome 2, consists of 29 exons and encodes 4563 amino acids. Apolipoproteins play an important role in binding the lipoprotein particle to lipoprotein receptors. One of the main roles of lipoproteins is supplying the cells with lipids and transporting the unnecessary lipids. ApoB has two forms, ApoB 100 and ApoB 48. Both forms are the product of the APOB gene, but the ApoB 48 is a truncated version of ApoB 100. ApoB 100 is produced in the liver, whereas ApoB 48 is produced in the proximal small intestines.The LDLR gene has five functional domains: an N-terminal ligand-binding region, an epidermal growth factor (EGF)-precursor homology region, a region containing O-linked sugars, a transmembrane domain, and a C-terminal cytosolic domain.The human LDLR is encoded by a gene of ~44.5 kb located on chromosome 19p13.3. The 18 exons of the gene are translated into an 860 amino acid-long type I transmembrane protein. The LDLR variants have been found in patients with familial hypercholesterolemia (FH) that can impair LDLR activity at different levels and therefore are classified according to their phenotypic behavior as follows: class 1 (no protein synthesis), class 2  and class 5 (diminished LDLR recycling capacity) [The LDLR mediates the clearance of cholesterol and cholesteryl ester-containing low-density lipoprotein (LDL) particles from blood . LDLR plOur goal was to examine the genetic background of patients with familial hypercholesterolemia in Hungary to map variants in the Hungarian population, as no such data have been available to date. Here, we describe the clinical and molecular data of 44 unrelated suspected FH patients.Patients clinically diagnosed with FH were referred to our laboratory for genetic testing from all around Hungary. Family members of index patients who tested positive were also examined when available. All participants or their legal representatives gave informed consent to genetic testing according to national regulations. Relevant clinical data were available in the case of most patients.Clinical diagnosis, whenever possible, was established by a clinical geneticist or internist based on the patient\u2019s laboratory findings and possible phenotypic symptoms. Among the patients with a pathogenic alteration, there were 31 female and 13 male patients. The mean age of the patients was 48 years (14\u201378 years). The average LDL-cholesterol level was 7.2 (mmol/L) and the average total cholesterol level was 10.4 (mmol/L) in those who had at least one FH-causing variant.Genomic DNA was isolated from peripheral blood leukocytes using the QIAamp Blood Mini kit .APOB: NM_000384.3, LDLR: NM_000527.5, PCSK9: NM_174936.4, LDLRAP1: NM_015627.3, STAP1: NM_012108.4.Genes related to familial hypercholesterolemia were analyzed using the Devyser FH v2 kit . This amplicon-based library preparation kit can examine the coding region and exon/intron boundaries (minimum +/\u2212 10 base pairs) of the following genes: Sequencing was performed on Illumina MiSeq with 300 cycles according to the manufacturer\u2019s protocol. Raw data analysis was carried out with the NextGENe  software, with a minimum coverage requirement of 40x. Copy number variation detection was performed based on coverage data.https://primer3.ut.ee/) (accessed on 15 October 2021). Primers are listed in The detected pathogenic variants were confirmed by Sanger sequencing. Primers were designed using Primer3 software  according to the manufacturer\u2019s protocol.All detected variants with an MAF > 0.01  in the gnomAD population database were filtered. The remaining variants were classified according to the ACMG standards and guidelines . A web-bLDLR gene surrounding the deletion to determine the suspected breakpoints  precursor homology domain, one in the O-linked sugar region, and a further 3 variants were found in the cytoplasmic domain  [We detected a pathogenic alteration in the In two index patients, we detected two missense variants , c.10438A>G, p.(Lys3480Glu)) where pathogenicity is unclear, but they are missing from population databases, therefore not present in the normal population. Furthermore, we did not detect any other alteration in the genes examined in either of the patients. According to the ACMG classification criteria, they are classified as variants of uncertain significance (VUS), so further testing is needed to decide their pathogenicity.APOB gene, the c.13242delG frameshift variant was detected. This deletion results in a premature stop codon at the 4415 amino acid position, leading to a truncated protein. As the variant is located in the last exon, it is unlikely that the consequence of it would be nonsense-mediated mRNA decay, rather, the truncated protein likely lacks 148 amino acids from its C-terminal. The variant is not present in the population databases, but ClinVar describes it as a variant of uncertain significance with a single submission of a clinical test result (ClinVar: SCV001357780). The prediction algorithms classify it as VUS/Likely Pathogenic. Downstream to this variant, other pathogenic truncating alterations have been described, which also suggests that this variant is a pathogenic one [APOB variants in details.In exon 29 of the enic one . Table 1LDLR gene in a total of 37, patients of which three patients carried variants in the compound heterozygous form .We detected 40 alterations in the LDLR gene were present in more than one index patient. The most common variant, c.1618G>A, p.(Ala540Thr), was present in four families, the c.1130G>A, p.(Cys377Tyr) change in three families, and the c.2054C>T, p.(Pro685Leu) was also detected in three families. The c.662A>G, p.(Asp221Gly), c.1775G>A, p.(Gly592Glu), c.2000G>A, p.(Cys667Tyr) and c.1865A>G, p.(Asp622Gly) alterations were detected in 2-2 families  and c.337G>T, p.(Glu113*) nonsense variants were detected in 22 families, whereas the c.1048C>T, p.(Arg350*) alteration was present in one family in heterozygous form. In patient 13, the c.337G>T, p.(Glu113*) variant was detected together with another pathogenic missense change .We detected variants resulting in a splicing defect in eight patients . Two of LDLR gene in one patient (patient 34)  ..LDLR gene in a homopolymer region consisting of five guanines. As a result of the insertion, there will be six guanines instead of five, which disrupts the reading frame, leading to a premature stop codon. The variant belongs to class three as it results in a defective receptor.The c.2416dupG, p. variant was detected in two families in a heterozygous form. This is a 1 bp insertion in exon 17 of the LDLR gene disrupts the reading frame and leads to a premature stop codon. In this patient, we also detected a second heterozygous missense change, the c.862G>A, p.(Glu288Lys).We detected the c.2167delG, p.(Glu723Argfs*7) alteration in patient 28 in a heterozygous form. This small deletion in exon 15 of the An in-frame deletion ), was detected in a single patient in our cohort .We had three patients in our cohort with a compound heterozygote genotype . The clinical symptoms were severe in all three patients, as can be seen in Patient 13 had a nonsense ) and a missense ) variant in a heterozygous form. Cascade screening revealed that the patient inherited the nonsense variant from the mother. The father had previously died because of a cardiovascular complication, so the paternal DNA sample was not available for testing .In Patient 28, we detected a missense ) and a frameshift ) heterozygous variant.The c.2167delG variant was described previously, where a patient had highly elevated TC level . Our patLDLR gene. Only one of the two short 32 bp-long homologue DNA sequences before and after exon 7 was detected in the PCR product. The copy loss of this sequence indicates that deletion of exon 7 occurred ) and deletion of the whole exon 7 (c.941-190_c.1061-270del). We performed targeted testing of the available family members as well, which proved that the two variants were inherited in trans . We haveoccurred .LDLR gene in three patients, in one case in a compound heterozygous form in the above-described patient 12.We detected a copy number variation in the In patient 38, we detected the duplication of exons 4\u20138 and in patient 33 we detected the deletion of exons 3\u20138 in a heterozygous form based on coverage from NGS data .Patients clinically diagnosed with FH were examined with a next-generation sequencing method that detects not only the small-scale variants but large-scale deletions and insertions as well.LDLR alterations (c.1706-2A>G) is described here the first time, whereas the other 39 LDLR variants have already been reported in the literature. Although the deletion of exon 7 of the LDLR gene has been described previously [LDLR variants, most of them are in the ligand-binding domain and the epidermal growth factor-like domain.One of the eviously , we haveLDLR gene in the Polish population can be found in our cohort as well [To our knowledge, no study describing the variants related to familial hypercholesterolemia in the Hungarian population has been published before. Regarding the mutation spectrum, there is a large overlap between Hungary and neighboring countries, such as Poland or the Czech Republic. The most common variants in the  as well ,26.The 1618G>A, p.(Ala540Thr) missense change was detected in four index patients , and in a further two family members during cascade testing. This pathogenic variant has been described in several European and Latin American populations, and it is a commonly occurring alteration among our patients as well. The phenotypes of our patients are similar to the ones described in the literature. This variant is located in the epidermal growth factor (EGF)-precursor homology domain. Its pathogenicity is demonstrated by the fact that, as described in the literature before, patients homozygous for this variant present with severe clinical symptoms at a young age such as premature coronary heart disease, tendon xanthomas and high total cholesterol level ,30.The c.1130G>A, p.(Cys377Tyr) missense change was detected in three families. First, it was described in a study examining Swedish FH patients . Its patA missense change, c.2054C>T, p.(Pro685Leu) was detected in three families in our cohort. According to a comprehensive study describing more than 2000 FH-causing variants, this alteration is pathogenic. It should be noted that seven alterations were found in this study which have been described in several countries on every continent, and one of these is the c.2054C>T, p.(Pro685Leu) missense change . This suThe c.1775G>A, p.(Gly592Glu) missense change was detected in two patients in a heterozygous form. This variant has been previously described in several populations and was associated with a strong founder effect in a Greek region according to a previous study, because it was present in almost one-third of the patients examined and detected in a homozygous form in several cases. According to literature data, this class 5 variant has no drastic cholesterol-elevating effect associated with its homozygous form, suggesting that it results only in a small decrease in LDLR activity .The nonsense alterations have been previously described as FH-causing pathogenic variants. These alterations result in a truncated form of the LDLR protein which is nonfunctional. The uptake of LDL particles is reduced in the liver and other tissues.LDLR gene. A luciferase-based functional assay has suggested that this variant may significantly reduce LDLR promoter activity [The c.-153C>T promoter variant has been previously detected in Czech FH patients. This variant is located in the conserved sterol-dependent regulatory element 2 (SRE2) in the promoter region of the activity .The c.940_940+14del variant has been previously described in a study examining a Scandinavian population, where it segregated with the disease in the family tested . In thatInterestingly, the c.694+2 T>C variant was previously described as a founder variant in the Icelandic population that could be traced back to the 18th century. It affects a conservative splicing site and its pathogenicity has been proved by segregation studies as well .The pathogenicity of the novel c.1706-2A>G variant is suggested by the fact that variants with other nucleotide changes have been previously identified in the same position . The c.1706-2A>T variant has been detected in two Arabic families, and further testing showed that the A>T exchange at this position is pathogenic because of the large reduction in LDLR mRNA levels .The c.2416dupG, p. variant was first described in a Swedish population study, then it appeared in Pakistani and other population studies as well. Elevated TC levels of the index patient and further eight family members were typical for heterozygous FH patients (7.76\u201312.93 mmol/L), whereas the 5-year-old child of the index patient had a TC level typical for homozygous FH patients (23.25 mmol/L) and had typical xanthoma [The c.654_656delTGG, p.(Gly219del) deletion has been previously described in the Ashkenazi Jewish population as a founder variant, and identified as the FH-causing variant in more than 21% of the cases . The varLDLR gene and it belongs to class 1 as it results in a null allele. It is a known pathogenic variant and was first described by Hobbs et al. [The c.337G>T, p.Glu113*) variant is in exon 4 of the 3* varians et al. .The c.862G>A, p.(Glu288Lys) missense change was first described in German patients . In ItalAPOB testing was characteristic because of the size of the APOB gene, examining the most common FH-causing variants as well [APOB gene is available, therefore more FH and other lipid metabolism disorder-related alterations have been described [Earlier, targeted  as well . Nowadayescribed .APOB gene, the most common variant in the Hungarian population is the c.10580G>A p.(Arg3527Gln) missense change, which is in accordance with literature data. By testing the whole APOB coding region, we could detect a further three variants, of which two missense alterations have not been described before, and the third frameshift variant ) has been previously reported in the ClinVar database, and is also related to FH. This result reflects on the fact that the sequencing of the whole APOB gene instead of targeted APOB testing can greatly improve the diagnostic sensitivity.In the LDLR gene, while most of the variants that are present in the Hungarian population have already been described.Our study revealed one novel variant in the The proportion of variant types and their localization within the gene are also highly similar in population studies.Indicating the importance of establishing a genetic diagnosis, the timing of initiation of lipid-lowering therapies may greatly influence the onset and severity of expected complications. According to a large study that followed therapy in children for 20 years, early treatment greatly reduced or delayed the risk of cardiovascular complications in adulthood . To init"}
{"text": "APRT sequence variants in a diverse set of large genomic databases. A thorough search was carried out for all APRT variants that have been confirmed as pathogenic under recessive mode of inheritance, and the frequency of the identified variants examined in six population genomic databases: the deCODE genetics database, the UK Biobank, the 100,000 Genomes Project, the Genome Aggregation Database, the Human Genetic Variation Database and the Korean Variant Archive. The estimated frequency of homozygous genotypes was calculated using the Hardy-Weinberg equation. Sixty-two pathogenic APRT variants were identified, including six novel variants. Most common were the missense variants c.407T>C (p.(Met136Thr)) in Japan and c.194A>T ) in Iceland, as well as the splice-site variant c.400\u2009+\u20092dup (p.(Ala108Glufs*3)) in the European population. Twenty-nine variants were detected in at least one of the six genomic databases. The highest cumulative minor allele frequency (cMAF) of pathogenic variants outside of Japan and Iceland was observed in the Irish population (0.2%), though no APRT deficiency cases have been reported in Ireland. The large number of cases in Japan and Iceland is consistent with a founder effect in these populations. There is no evidence for widespread underdiagnosis based on the current analysis.Adenine phosphoribosyltransferase deficiency is a rare, autosomal recessive disorder of purine metabolism that causes nephrolithiasis and progressive chronic kidney disease. The small number of reported cases indicates an extremely low prevalence, although it has been suggested that missed diagnoses may play a role. We assessed the prevalence of APRT deficiency based on the frequency of causally-related  Adenine phosphoribosyltransferase (APRT) deficiency  is a rare disorder of purine metabolism that is inherited in an autosomal recessive manner [APRT gene, located on chromosome 16q24, is 2466 base pairs long and contains five exons encoding a 180 amino acid protein (NP_000476.1) [The human 00476.1) . The Hum00476.1) , a T ins00476.1) . Individ00476.1) .Although the small number of reported cases worldwide indicates that APRT deficiency is an extremely rare condition, the large number of patients identified in Japan, France and Iceland has raised concerns that the prevalence may be underestimated in many populations. Furthermore, the commonly reported occurrence of delayed or missed diagnosis suggests that lack of awareness among clinicians might contribute to the low number of reported cases , 10, 11.APRT variants confirmed as disease-causing under recessive mode of inheritance that have been reported in the literature and registered in online disease databases to date, and assess the frequency of these variants in a set of large population genomic databases containing information on individuals from diverse geographic locations and ethnic groups.As whole-genome and whole-exome sequencing have become more widely available, studies on the molecular basis of rare diseases using open-access population-based data have provided further insight into their pathogenesis and allele frequency \u201316. The The study was approved by the National Bioethics Committee of Iceland (NBC 09-072) and the Icelandic Data Protection Authority.APRT variants in reported cases of APRT deficiency using PubMed, HGMD [Medical literature and databases. We performed a web-based search and assessement of ed, HGMD , OMIM [1ed, HGMD , and Clied, HGMD .APRT variants, we also assessed the full text of published articles, conference abstracts and book chapters identified using PubMed\u00ae (https://pubmed.ncbi.nlm.nih.gov/) and Google\u00ae (http://www.google.com), with the terms \u201cAPRT deficiency\u201d, \u201cadenine phosphoribosyltransferase deficiency\u201d, \u201c2,8-dihydroxyadenine\u201d, \u201c2,8-dihydroxyadeninuria\u201d and \u201c2,8-DHA\u201d.Expanded web sources. To search for http://www.rarekidneystones.org). Information on APRT variants was obtained from the APRT Deficiency Registry and through personal communication to the research group. The RKSC was established in 2009 to study hereditary kidney stone disorders and has since maintained a private database with clinical information on participating individuals. As of September 2019, 63 patients from eight countries were enrolled in the APRT Deficiency\u00a0Registry, 56 of whom had undergone genetic testing with the identification of biallelic disease-causing APRT variants.APRT Deficiency Registry of the Rare Kidney Stone Consortium  and transcript number (ENST00000426324.6).https://varseak.bio/).As consequence at the RNA level has not been reported for the vast majority of the variants, a prediction analysis of the variant effect at the splice junction on the splicing was carried out using consensus sequence frequencies, maximum entropy modeling and the varSEAK Online splice prediction tool   browser The Human Genetic Variation Database of Japan , 25 compThe Korean Variant Archive (KOVA)  includesWe used six databases in all, containing information on sequence variation for over 300,000 individuals from various geographic locations and ethnic groups:The minor allele frequency (MAF) of each causally-related sequence variant among different ancestries was exctracted.APRT variants, we used the sum of the allele frequency of individual variants in databases of genome and exome sequences available for a given geographic area or ethnic group. We note that each of the individual variants is rare and none are reported to be present on the same haplotype. As APRT deficiency is an autosomal recessive disease, each affected individual is expected to carry two copies of a disease-causing variant, either the same (homozygous) or different variants (compound heterozygous). The Hardy-Weinberg equation (p2\u2009+\u20092pq\u2009+\u2009q2\u2009=\u20091) was used to calculate the expected genotype frequencies for heterozygous (2pq) and homozygous (p2) genotypes, using the minor and total allele counts, where p is equal to the cMAF of causally-related variants and q is (1\u2009\u2212\u2009p).To determine the cumulative minor allele frequency (cMAF) of the reported APRT gene were identified in homozygous or compound heterozygous patients with APRT deficiency ), from Australia and the c.346G>A (p.(Ala116Thr)) missense variant from China. These variants were quite common in the databases searched; in gnomAD, the p.(Ala116Thr) variant had an allele frequency of 0.23%  in the East Asian population and p.(Arg89Gln) an allele frequency of 0.41%  in the South Asian population. However, neither of the two variants have been observed in confirmed cases of APRT deficiency and, thus, were not included in our set of 62 causally-related variants.APRT variants, 56 had already been reported in the literature. The missense variants c.407T>C (p.(Met136Thr)) and c.194A>T ) and the splice-site variant c.400\u2009+\u20092dup (p.(Ala108Glufs*3)) were most commonly reported. Additionally, six novel pathogenic variants were discovered through our APRT Deficiency Research Program. A C-to-G substitution in intron 1 (c.81-3C>G) was identified in the homozygous state in two affected siblings in the US and one patient in Italy, and a C-to-T substitution in exon 1 ) was detected in a homozygous patient in the UK. Two compound heterozygous patients from the US had novel sequence variants; one of these patients had a frameshift variant in exon 1, c.23dup ), and a deletion in exon 5, c.522_524del (p.(Ser175del)), while the other had a missense variant in exon 3, c.264G>T (p.(Lys88Asn)), in addition to a variant that had already been reported. The sixth variant was identified in a patient from India who was referred to our program but had already been found to be a compound heterozygote when evaluated in his home institution, carrying a missense variant in exon 3, c.227C>T ), in addition to one previously reported variant.Of the 62 pathogenic All cases harboring the three most common variants causing APRT deficiency in a homozygous or compound heterozygous state are presented in Table\u00a0The T-to-C missense variant at codon 136 in exon 5, p.(Met136Thr), has been described in 134 patients of Japanese descent, all but one of whom were previously reported in the literature. The additional case, identified in the APRT Deficiency Registry, was a Japanese patient living in the US. This pathogenic variant was also found in the Human Genetic Variation Database in Japanese cohorts from Tohoku University  and the University of Tokyo . This geographic distribution is consistent with previous reports [APRT variant in 1299 alleles, yielding a minor allele frequency (MAF) of 1.2% . Icelanders have ancestries originating from Scandinavia and the British Islands. Out of 14,904 whole-genome sequenced Scandinavian individuals at deCODE, only two heterozygotes carrying the p. variant were detected, and one single heterozygote was identified in the UK  variant have been reported. Of these, 37 individuals\u00a0are from Iceland, all of whom are homozygous. One patient from Spain and one from the UK were homozygous for the same variant and two other patients were compound heterozygotes, one from France and one from Australia. On whole-genome sequencing of 53,964 Icelanders in the deCODE database, we observed this n\u2009=\u200929), Germany, Austria, Belgium, Italy and Poland. The variant has also been found in patients in the US (n\u2009=\u20096) and Australia (n\u2009=\u20091). The variant was consistently present in European populations in the genomic databases  and p., are clearly indicative of a founder effect in Japan and Iceland, respectively, with very low frequency in the public databases. The third variant, c.400\u2009+\u20092dup, shows a wide distribution among cases from multiple European countries and the US, and is the disease-causing variant with the highest\u00a0occurence in public databases. Besides these three variants, the most commonly reported ones were c.294G>A (p.(Trp98*)) in 26 Japanese cases and the c.521_523del (p.(Phe174del)) variant\u00a0which was observed in six cases from European countries and three in the US.APRT variants in the deCODE database (Table\u00a0Iceland is the country where the highest proportion of the population has been sequenced (around one in six Icelanders). In addition to the p. variant, we discovered five individuals heterozygous for one of the two other known pathogenic se Table\u00a0. Of thesBased on the Hardy-Weinberg principle, the cMAF of p. in the Icelandic population was 1.2%, with a predicted frequency of homozygous individuals of 1 in 6840 Table\u00a0. In the The overall frequency of the reported pathogenic variants in gnomAD was similar among the European (non-Finnish) population, with a calculated allele frequency of 0.05% . Frequencies of causally-related variants in Latinos and East Asians was similar to that observed in gnomAD for Europeans, whereas other groups had lower frequencies. Thus, there could be as many as 300 cases in the East Asian population (1.7 billion) and up to 170 cases in the Latin American population (642 million).APRT variants in a total of 311 patients undergoing molecular diagnosis. Six novel variants were discovered through the APRT Deficiency Registry. Three variants are by far most common among the APRT deficiency cases, namely the missense variants p.(Met136Thr)\u00a0and\u00a0p. and the splice variant c.400\u2009+\u20092dup, collectively\u00a0accounting for 64% of the disease-associated alleles.A comprehensive search for APRT deficiency cases, using multiple web-based resources and the APRT Deficiency Registry, identified 62 pathogenic Twenty-nine of the 62 pathogenic variants were detected in large population genomic databases and were assessed for the individual and cumulative frequencies. Evaluation of the frequency of the Icelandic missense variant p. in large datasets from ancestral populations in Scandinavia and UK revealed that the variant is approximately 100 times less common in these geographic locations. Thus, the high carrier rate of the variant of 1.2% in the Icelandic population clearly represents a founder effect. Not surprisingly, analysis of the large Icelandic genomic data disclosed a number of additional undiagnosed homozygous individuals.The high frequency of the missense variant p.Met136Thr) in the Japanese population is also consistent with a founder effect. A previous study assessing the frequency of p.(Met136Thr) observed an allele frequency of 0.13% (3/2326) among Japanese and 0.05% (1/1898) among Koreans, while this variant was absent in samples from Taiwanese individuals. In the same study, the geographic distribution in Japan was determined to be rather uniform [6Thr in tThe c.400\u2009+\u20092dup variant was observed in cases from many countries in Europe, the US and Australia, as well as in most of the public databases used in the current study. In a report of a French APRT deficiency cohort, this variant was found in 54% of the patient population, all of whom originated from metropolitan France except for one Italian family . The allWhen calculating the cMAF of reported causally-related variants to assess the expected genotypic frequency, we found that there may be as many as 200 individuals in Japan homozygous for p.(Met136Thr) and 50 in Iceland homozygous for p.. As expected, the two described founder variants in Japan and Iceland are very rare outside of their respective countries. In the European (non-Finnish) population, the cMAF of any pathogenic variant was 0.05%. The cMAF in the Irish population was higher and is consistent with approximately 30 homozygous individuals. Interestingly, no cases have been diagnosed in Ireland to the best of our knowledge, indicating that the disease may be underreported or underdiagnosed.APRT missense variants, c.266G>A (p.(Arg89Gln)) [APRT variants. It is unclear if and to what extent individuals homozygous for these variants will develop clinical manifestations of APRT deficiency. Multiple copies of both variants were present in the genomic databases and the allele frequencies were quite high in both the East Asian and South Asian populations. If proven to be disease-causing, there could be roughly 20,000 homozygotes in South Asia and 5000 in East Asia, assuming the Hardy-Weinberg equilibrium. However, as no cases of APRT deficiency caused by these two variants have been reported, a classical phenotype appears unlikely as the disease would then be seriously underdiagnosed. Hence, homozygosity for these variants would either be expected to cause a very mild clinical phenotype or no disease at all.Two g89Gln))  and c. 3g89Gln)) , have beOf the 62 pathogenic variants, 33 were not observed in any of the public genomic databases. Most were detected in one or two cases except for c.188G>A (p.(Gly63Asp)) which was found in six patients, most of whom were of Lebanese decent. The reason for this may be the high proportion of consanguineous marriages in Lebanon, previously reported to be ~35%, and/or that Middle Eastern populations might be underrepresented in the genomic databases used in this study .We also examined sequence variants that have not been detected in patients with APRT deficiency in gnomAD, which is the largest population database with the highest allele count  in the Icelandic population clearly represents a founder effect and the same appears to hold true for the missense variant p.(Met136Thr) in Japan. While there is no definite indication of extensive underdiagnosis, this may nevertheless be the case in certain areas. It is important to recognize\u00a0that genomic data are still lacking for many countries. Hence, future work should assess the frequency of pathogenic APRT variants in countries and populations where the number of expected cases is higher than currently reported. An updated list of all variants causally related to APRT deficiency will be maintained and made publicly available on the RKSC website.In conclusion, we identified 62 confirmed pathogenic Supplementary_Tables_1_To_7_Clean"}
{"text": "The coronavirus disease 2019 (COVID-19) pandemic has had a significant impact on the mental health and wellbeing of Ontario's youth. Our study investigated the psychological impacts of COVID-19 on the pediatric population of Ontario, using a survey derived from the Revised Children's Anxiety and Depression Scale (RCADS) system to identify children who may benefit from seeking professional help. Our cross-sectional study examined the potential risk factors that contributed to worsening mental health and wellbeing in children, including changes in sleep patterns, appetite, and physical activity levels, as well as the diagnosis of a family member with COVID-19. Our study found that 24%, 9.4%, and 15.5% of participants exhibited symptoms of depression, anxiety, and obsessive-compulsive disorder (OCD), respectively, according to the RCADS system. Furthermore, there were significant associations between the presence of symptoms and the diagnosis of a family member with COVID-19 or a frontline worker in the family. This suggests a need to create interventions to support the families of frontline workers and those directly affected by a COVID-19 diagnosis. The coronavirus disease 2019 (COVID-19) pandemic has had a significant impact on the mental health of people across the whole world, including Ontario's youth . For chiThis is highlighted in a recent study by McArthur et al., where 846 mothers and children participated in a COVID-19 mental health questionnaire in Calgary, Canada [Another longitudinal study by Bignardi et al. that assessed childhood depression symptoms during the pandemic found that there was a significant increase in depression symptoms as a result of the lockdown in the UK . Mental Our study aimed to investigate the psychological impacts of COVID-19 on the pediatric population of Ontario, using a survey completed by parents. Although a survey is not an appropriate diagnostic tool for mental health conditions, our survey uses the Revised Children's Anxiety and Depression Scale (RCADS) system to identify children who may benefit from seeking professional help. Our cross-sectional study examined the potential risk factors that contribute to worsening mental health and wellbeing in children, including changes in sleep patterns, appetite, and physical activity levels, as well as the diagnosis of a family member with COVID-19.Our study is a cross-sectional study in which we asked the parents of children in grades 3 to 12 of all races and genders to fill out an anonymous online survey investigating any possible development of symptoms of anxiety, depression, and obsessive-compulsive disorder during the COVID-19 pandemic. Furthermore, our survey asked questions pertaining to noticeable behavioral changes in the child concerning aspects such as physical activity, sleep, and appetite. Children were excluded from the study if they had psychological or physical comorbidities prior to the beginning of the COVID-19 pandemic. Parents were asked to provide informed consent on behalf of the child\u00a0prior to filling out the survey. Anonymity was preserved through a lack of personal identifying information. Our survey was a standardized survey that was derived from the Revised Children\u2019s Anxiety and Depression Scale\u00a0(RCADS) that is available for use at no cost, given in a Likert scale, and designed using Cognito Forms  (Appendix 1). A poster with an easily accessible link and QR code that took parents to the survey form was used to recruit participants (Appendix 2). The poster was shared on\u00a0various social media sites including Facebook and Instagram. Research Ethical Board approval was obtained through the Scarborough Health Network . The data collection period was July 8 to October 8, 2021, and we were able to recruit 246 children in total.To contextualize this time period, by early July in 2021, the province had seen continued improvement in key public health and health care indicators\u00a0such as hospitalizations, ICU occupancy, and the weekly cases incidence rates . On\u00a0JulyThe demographic characteristics of the participants are presented in Table A total of 246 children participated in this study. The majority of participants were male (52.9%), in Grade 3 (14.23%), had no personal or family COVID-19 diagnosis (83.74%), and had no frontline workers in the family (59.35%).Depression symptoms2 (n = 59) = 49.358, p = 0.014) or a frontline worker in the family (X2 (n = 59) = 45.004, p = 0.039). The difference in depressive symptoms between genders was nonsignificant (X2 (n = 59) = 36.549, p = 0.191).Twenty-four percent  of participants were found to be positive for symptoms of depression using the RCADS clinical cut-off of T-scores over 65. As shown in Table Anxiety symptoms2 (n = 23) = 52.733, p = 0.000) or a frontline worker in the family (X2 (n = 23) = 35.600, p = 0.017; see Table 2 (n = 23) = 26.403, p = 0.153)  developed symptoms of anxiety using the RCADS clinical cut-off of T-scores over 65. The presence of a personal or family diagnosis of COVID-19 (X developeOCD symptoms2 (n = 38) = 55.596, p = 0.000) and a frontline worker in the family (X2 (n = 38) = 48.453, p = 0.000; see Table 2 (n = 38) = 20.919, p = 0.191)  of participants had symptoms of anxiety. Significant associations were found between anxiety symptoms and the presence of a personal or family diagnosis of COVID-19 (XThis cross-sectional study aimed to assess the impact of the COVID-19 pandemic on Ontario\u2019s pediatric population by examining potential risk factors. We discovered that there were significant differences in the development of children\u2019s symptoms of depression, anxiety, and OCD when associated with the child having family members working on the frontline or if there was a positive COVID-19 diagnosis in the family. Gender was not a significant risk factor for the development of anxiety, depression, or OCD symptoms in children during COVID-19.Our findings are consistent with other studies that also suggest the rise of youth mental health difficulties during the COVID-19 pandemic . This maCost et al. examined COVID-19 exposure as a potential risk factor for pediatric mental health concerns . HoweverThe association found between the development of pediatric mental health disorders and the chronic and acute stressors associated with frontline work is alarming, suggesting a heightened need to create effective interventions to help frontline workers cope with and adequately support their children and families. Perhaps this is indicative of a weakness in the Canadian response to the pandemic. Efforts must be made by workplaces when implementing occupational health measures to look beyond people\u2019s function as frontline responders and to adopt a more holistic approach that takes into account their societal roles as parents, spouses, and offspring. A study among Moroccan healthcare workers explores the effects of how 40% of healthcare workers marry each other, causing a \u201cchildcare crisis\u201d that causes disruption to traditional solidarity systems, potentially exposing children of healthcare workers to additional emotional challenges caused by the absence of both parents .This prompts one to consider the state of childcare for frontline workers in Canada. As of March\u00a017, 2020, licensed child care centers were closed, aside from select locations to support health care and other frontline workers. Then, from June 12, 2020, child care centers were allowed to reopen throughout the province . It was On the other hand, the findings of our study may hint at something greater than an infrastructure issue such as parents being ill-equipped to face the novel challenges posed by parenting due to the pandemic. In a few studies, it was shown that a rise in parental stress levels is associated with harsher parenting while perceived control over stressful environments while supportive family environments act as a buffer to decrease stress -22. COVIThis study has several limitations. The study design was cross-sectional, preventing us from making causal inferences on the relationship between frontline worker families, families with a positive COVID-19 diagnosis, and symptoms of pediatric mental health disorders. Longitudinal research is required to evaluate these potential risk factors and capture their effect in light of changing COVID-19 restrictions. The RCADS scale used in this study while a validated screening tool for symptoms of depression, anxiety, and OCD\u00a0cannot ascertain cases with certainty. A clinician needs to be present to review each individual case in order to make a diagnosis.\u00a0Another limitation of the study was that the survey collected limited data regarding participant characteristics, preventing a more holistic examination of sociodemographic risk and protective factors for the development of pediatric mental health concerns. Additionally, while advertisements were shared in networks serving Ontarians and participants were required to self-identify as Ontario residents, it cannot be said with certainty that all respondents were from Ontario exclusively. While the results were consistent with the general literature, our findings cannot be broadly generalized. Finally, our survey was only administered in English, which excludes the experiences of non-English-speaking parents in Ontario. However, as of 2016, only 2.5% of Ontarians were found to be non-English speaking so perhaps these results may still be generalizable to the Ontario population .Despite these limitations, our findings illustrate the important associations of COVID-19 diagnosis exposure, frontline workers within families, and pediatric mental health disorders. Our findings have widespread implications for prevention and intervention programming such as developing the infrastructure to provide frontline workers with more flexibility and access to childcare supports. This may include increasing flexibility for workers with families and reducing barriers to accessing care for these vulnerable populations\u00a0such as subsidizing transportation costs and expanding no-cost emergency childcare programs. In addition, the findings imply that parents may benefit from public health messaging and improved family-centered healthcare that better equips them with the difficulties of raising children in the context of a global pandemic. Finally, our study suggests that there is a gap in the literature regarding the effects of occupational stress on frontline workers' and their families' well-being, warranting further research.In conclusion, this study examined the psychological impact of COVID-19 diagnosis exposure and family members who are frontline workers on a pediatric sample in Ontario. Institutions with frontline workers and parents will benefit from knowing about the potential downstream impacts of occupational stress on children and adolescents. In addition, public health and education agencies may benefit from a greater understanding of stressors affecting children, allowing integration into intervention programs. The current research provides preliminary insight into the myriad of factors affecting pediatric individuals."}
{"text": "Dear editor,Telomeres are the physical ends of eukaryotic chromosomes, and their structures are essential to maintain genome stability and limit cell proliferation . NormallCancer cells must avoid the regulatory role of the telomere-p53 axis to ensure replicative immortality. To achieve this, they need to activate a telomere-maintenance mechanism (TMM), in which 90% of the cells reactivate telomerase, and 10% activate alternative lengthening of telomeres (ALT) ,5. AnothC16ORF72, which they renamed Telomere Attrition and p53 Response 1 (TAPR1) , as well as p53-induced senescence/apoptosis. This placed this newly identified protein in a key position at the nexus of telomere integrity and p53 regulation.Both of these phenomena highlight the central role of the telomere-p53 axis in the process of carcinogenesis. Recently, Benslimane et al. uncovered the function of a previously unannotated gene, TAPR1, the authors suggest that this molecule may have a role in cancer biology by suppressing p53 activity. Therefore, we tried to gather evidence for this by performing an analysis of this gene in the 33 tumors of The Cancer Genome Atlas (TCGA) using cBioPortal, GEPIA, and UALCAN  and are absent in 10 out of 33 types of cancer. Additionally, alterations in TAPR1 do not change the overall and disease-free survival of patients (data not shown).First, we show the somatic alteration landscape of  cancers . ProportTAPR1 and TERT (telomerase protein subunit), ACD , and TP53. Interestingly, we noticed a trend of co-occurrence between mutations in TAPR1 with TERT , ACD , and TP53  in TCGA cancers. Next, we analyzed the TAPR1 gene expression and found that all tumors express this gene (data not shown).The authors who identified this gene also reported a genetic interaction between TAPR1 modulates p53, we compared the mRNA levels of this gene in relation of TP53 mutation status (when available) (p<0.05), we found that TAPR1 is more expressed in tumors  with p53 mutations. This reinforces the oncogenic role of TAPR1 when there are deficiencies in p53. The only exception to this phenomenon is in BRCA, in which TAPR1 is upregulated in cancers without mutations in p53.Considering that ailable) . Taking TAPR1 has differential expression in cancer in relation to normal tissue (only in tumors where control is available) . On the other hand, TAPR1 may have a protective role in cancer progression since its downregulation increases the risk of disease relapse (p<0.0001). In the original study, the authors suggest that TAPR1 may have a double behavior in cancer: it can suppress both apoptosis/senescence (oncogenic role) and the tumorigenesis process itself (tumor suppressive role).Next, we analyzed whether ailable) . The UALTAPR1 in cancer, so we verified its association with each cancer individually using a hazard ratio heat map. The upregulation of TAPR1 was significantly associated (p<0.05) with poor overall survival in LGG and THCA (TAPR1 in these cancers. On the other hand, in KIRC, the upregulation of TAPR1 had a protective role in terms of both overall (The previous analysis did not elucidate the general role of and THCA  and withand THCA . These f overall  and dise overall , suggestP53 is probably the most important tumor suppressor in the human genome. Among its various functions, it keeps telomere length in check, thus limiting cell proliferation . For thiTAPR1 in cancer. Our analyses reinforce the hypothesis that this newly identified gene may have an important role in oncology. Using TCGA datasets, we showed an association between TAPR1 expression and p53 mutation status in several cancers and that this gene is differentially expressed in CHOL and HNSC.When Benslimane et al.  discoverTAPR1 expression and cancer survival in some tumors. However, this association can be both positive and negative. This suggests that the role of this molecule is context-dependent and needs to be studied further. Evidently, more robust studies are needed to understand the exact role of TAPR1 in disease progression, but this gene may become an important target in oncology or a new player in cancer biology.In addition, our analysis indicates an association between"}
{"text": "Dictyostelium and mammals. These have processing pathways similar to p67, and are thought to have phospholipase B-like (PLBL) activity. The mouse PLBD2 crystal structure revealed that the PLBLs represent a subgroup of the larger N-terminal nucleophile (NTN) superfamily, all of which are hydrolases. NTNs activate by internal autocleavage mediated by a nucleophilic residue, i.e. Cys, Ser or Thr, on the upstream peptide bond to form N-terminal \u03b1 (gp32) and C-terminal \u03b2 (gp42) subunits that remain non-covalently associated. The N-terminal residue of the \u03b2 subunit is then catalytic in subsequent hydrolysis reactions. All PLBLs have a conserved Cys/Ser dipeptide at the \u03b1/\u03b2 junction (Cys241/Ser242 in p67), mutation of which renders p67 non-functional in RNAi rescue assays. p67 orthologues are found in many clades of parasitic protozoa, thus p67 is the founding member of a group of hydrolases that likely play a role broadly in the pathogenesis of parasitic infections.p67 is a type I transmembrane glycoprotein of the terminal lysosome of African trypanosomes. Its biosynthesis involves transport of an initial gp100 ER precursor to the lysosome, followed by cleavage to N-terminal (gp32) and C-terminal (gp42) subunits that remain non-covalently associated. p67 knockdown is lethal, but the only overt phenotype is an enlarged lysosome (~250 to >1000 nm). Orthologues have been characterized in  Trypanosoma brucei ssp.) are parasitic protozoa that cause human African trypanosomiasis , as well as nagana in livestock. These diseases have devastating impact throughout sub-Saharan Africa, wherever the tsetse fly vector is found. A total of >65 million people in 36 countries are at risk of transmission, and although reported human cases have fallen steeply in recent years, it remains a serious veterinary problem. Only a handful of drugs are in use for treating HAT, all of which are either toxic, expensive and/or require a difficult regimen. As vaccination is not possible, and infection is inevitably fatal, there is a critical need for new drug development. Thus, a better understanding of the basic biology of the parasite is essential, particularly of targets amenable to therapeutics. The lysosome is such a target as it impacts the host\u2013pathogen balance in multiple ways. Expression of lysosomal activities is differentially regulated through the life cycle  intermediate glycoform  trypanosome ricin-binding proteins , and containing much internal membranous material as seen by transmission electron microscopy  makes a nucleophilic attack on the upstream carbonyl group leading to peptide bond hydrolysis creating N-terminal \u03b1 and C-terminal \u03b2 subunits. The subunits remain non-covalently associated, and the upstream linker region is typically trimmed, by a second round of autocatalysis or by another protease as proposed for cephalosporin acylase  activities, but phospholipase B (acylase) activity has been claimed (see below).The NTN superfamily of hydrolases was first proposed based on the common et al., et al., Dictyostelium, mice and humans   was identified by proteomics of mannose-6-phosphate selected lysosomal proteins, and lysosomal localization was confirmed by immunofluorescence  and C-terminal (\u03b2) subunits, analogous to gp32 and gp42 of p67, respectively. The N-terminus of the 40\u00a0kDa \u03b2 subunit is C249, which is equivalent to C241 of p67  and T. brucei . Most orthologues are soluble proteins; all have conserved Cys/Ser dipeptides at the \u03b1/\u03b2 junction mapped for p67 and PLBD2  in mice, humans and other mammals, and many more orthologues throughout the Eukaryota, often with multiple paralogues in a given species, e.g. eight in ntamoeba . Intereset al., Dictyostelium and human orthologues these proteins have been annotated as PLBs. However, this designation has been challenged by the fact that most NTN enzymes are amidases, not esterases, and because the PLBD2 active site may be too small to accommodate typical phospholipids  wild type and double mutant p67 genes, each with a C-terminal HA-tag for discrimination from the native protein. The mutations are Cys241Ala/Ser242Ala spanning the known N-terminus of gp42. The wild type and mutant RNAiR genes are designated p67CS and p67AA, respectively. These have been constitutively expressed in an inducible p67 RNAi cell line targeting the endogenous gene product. Upon induction of RNAi the parental cell line ceases growth over a 24\u00a0h period . These results establish that one or both of the residues at the gp32/gp42 junction is essential for p67 function. Collectively, these data provide compelling supportive evidence that p67 is an NTN of the PLBL subgroup: (i) lysosomal localization; (ii) conserved autocatalytic residues; (iii) similar biosynthetic processing to \u03b1 (gp32) and \u03b2 (gp42) subunits; (iv) good structural modelling and (v) essentiality of C241/S242 at the gp32/gp42 junction.Is there more evidence that p67 is an NTN of the PLBL subgroup? First, the p67 sequence, despite only 28% identity, models tightly onto the PLBD2 crystal structure . Importah period , top. Gret al., et al., et al., p67 is an essential lysosomal membrane protein in African trypanosomes. For many years, it was assumed to play an ill-defined role in maintenance of lysosomal physiology \u2013 until recently when homology to the PLBL class of lysosomal hydrolases has been recognized. These enzymes are part of the NTN superfamily, but are poorly studied in any system. There is evidence that they do in fact have PLB activity , and hence are likely to have the same activity. On the contrary, the two mammalian orthologues are only 32% identical and are likely to have different substrate specificities. That said, there may be considerable redundancy. PLBD1\u2212/\u2212 and PLBD2\u2212/\u2212 knockout mice are viable, and at least in the PLBD1 knockout there are no overt phenotypes . This could explain why no lysosomal storage diseases have ever been associated with mutations in these genes. The situation in other parasitic protozoa is likely to be even more complex given the greater number of paralogous genes in the individual species. The T. brucei p67 RNAi cell line may provide a novel approach to defining these specificities. Xeno-complementation (rescue) of the RNAi phenotype by orthologous genes from other parasitic protozoa would indicate overlapping enzymatic activity with p67. Failure to complement would suggest a different activity/substrate specificity, and in this case constitutive expression in trypanosomes, in conjunction with \u2018Omics\u2019, could provide insights into enzymatic activity. Most interesting would be xeno-complementation with the human PLBD genes, which might provide insights into the possibility of specific therapeutic targeting of p67 in trypanosomes. Finally, all of these efforts would be augmented by structural studies that push beyond modelling on the known PLBD2 structure, and these efforts are also underway.The reassignment of p67 as a lysosomal hydrolase raises many questions \u2013 foremost being what is its enzymatic activity? The answer to this question will come from lipidomic and metabolomic analyses of RNAi silenced cells, and these studies are currently underway. Elevated levels of any specific metabolite or lipid will point to the likely substrate(s), and this in turn will provide opportunities for enzyme assay development, and perhaps even small molecule inhibitor screens. A broader question is what are the enzymatic activities of orthologues from other parasitic protozoa, and from mammals? The two Whatever comes of these future experiments the outlook for research on this understudied group of enzymes is bright. For too long p67 has remained a protein without a function, but it is likely soon to take its rightful place as the founding member of a novel class of important lysosomal hydrolases."}
{"text": "Selenium is an essential trace element required for human health, and selenium deficiency has been associated with many diseases. The daily recommended intake of selenium is 60 \u00b5g/day for adults, which increases to 65 \u00b5g/day for women when pregnant. Selenium is incorporated into the 21st amino acid, selenocysteine (sec), a critical component of selenoproteins that plays an important role in a variety of biological responses such as antioxidant defence, reactive oxygen species (ROS) signalling, formation of thyroid hormones, DNA synthesis and the unfolded protein response in the endoplasmic reticulum (ER). Although 25 selenoproteins have been identified, the role of many of these is yet to be fully characterised. This review summarises the current evidence demonstrating that selenium is essential for a healthy pregnancy and that poor selenium status leads to gestational disorders. In particular, we focus on the importance of the placental selenoproteome, and the role these proteins may play in a healthy start to life. Pregnancy is a condition of enhanced demand for nutrients and heightened oxidative stress. Deficiencies in micronutrients such as selenium hinder antioxidant responses and can expose the placenta to an accumulation of free radicals and oxidative insult. This can cause placental insufficiency and dysfunction, leading to complications in pregnancy such as pre-eclampsia, preterm birth, gestational diabetes (GDM), inter-uterine growth restriction (IUGR) and small for gestational age (SGA) babies ,2,3. An According to the Australian Government (NHMRC), the RDI of selenium is 60 \u00b5g/day in women, which increases to 65 \u00b5g/day in pregnant women and 75 \u00b5g/day during breastfeeding. Similarly, in the US, the recommended daily allowance according to the NIH is 55 \u00b5g/day in women, and this increases to 60 \u00b5g/day in pregnancy and 70 \u00b5g/day during lactation. When considering the daily intake of selenium, this recommended dosage increases to 95 \u00b5g/day to maximally express selenoproteins such as plasma glutathione peroxidase ,5. HowevPre-eclampsia (PE) is a hypertensive disorder of pregnancy, which is the leading cause of maternal mortality and morbidity , occurrip < 0.0001). Serum/plasma selenium levels of >95 \u00b5g/L were considered selenium sufficient, and significantly reduced pre-eclampsia incidence was reported for countries above this value (p = 0.0007) [Many studies have demonstrated that selenium concentrations in women with pre-eclampsia are significantly lower than those women experiencing a normal pregnancy, suggesting that selenium status is associated with the incidence of pre-eclampsia ,10,11,12 0.0007) . This st 0.0007)  have demMore recently a double-blind placebo-controlled pilot trial randomised 230 primiparous UK pregnant women to either 60 \u00b5g/day of selenium or placebo from 12\u201314 weeks of gestation until delivery . The priHigh levels of oxidative stress and endoplasmic reticulum stress because of reduced antioxidant capacity in the placenta are characteristic of pre-eclampsia. Selenoprotein activities in pregnancy have been investigated in numerous studies, where the activity of enzymes such as glutathione peroxidases (GPx), thioredoxin reductases (TrxR) and selenoproteins S, or selenoprotein P  has demoIUGR is defined as a foetus that has failed to reach its growth potential. It affects 10\u201315% of pregnancies and is the second most frequent cause of perinatal morbidity after pre-maturity . IUGR isp = 0.013) [1 quartile (\u226456.60 \u00b5g/L) had approximately three times higher risk of SGA  compared to women in the higher quartiles [The relationship between selenium in the aetiology of IUGR/SGA infants has been analysed due to the importance of antioxidant selenoproteins in the development of gestational disorders . Less ex= 0.013) . The sam= 0.013) . In anotuartiles . OverallPreterm birth complications are a major cause of foetal death worldwide occurring in 5\u201318% of pregnancies across 184 countries with more than 60% of these preterm births occurring in Africa and South Asia . An infat = 2.9, p = 0.001) [As detailed above, selenium has been implicated in pregnancy complications associated with poor placental function such as pre-eclampsia and IUGR, which are also a common cause of preterm birth. There are a variety of studies that measure the levels of selenium in preterm birth which have presented conflicting evidence. Studies measuring the relationships between first trimester levels of selenium in a cohort of Polish women found significantly lower levels of selenium in pregnancy-induced hypertension in women who delivered in <34 weeks compared to >34 weeks . A simil= 0.001) . Further= 0.001) . Africa GDM is one of the most common diseases that develops in pregnancy, affecting up to 14% of pregnant women . GDM devp\u2009 = \u20090.005) [Supplementary antioxidants such as selenium have been widely studied as a therapy to reverse the oxidative status in pregnancies complicated by GDM. There have been multiple studies that have determined that patients with GDM have lower selenium levels and that selenium supplementation improves glucose homeostasis in these patients ,33,34,35 \u20090.005) . In a raSelenoproteins are defined by an active selenocysteine residue and, to date, 25 have been described in the literature. Of these, many have been identified in the human placenta, where they play essential roles in maintaining cell function and viability see . Seleniu2O2 but also hydroperoxides in complex lipids [Glutathione peroxidases (GPx) play important roles in oxidative balance and are one of the first and most well characterised groups of selenoproteins. Glutathione peroxidases are part of the glutathione disulphide system that protects cells from oxidative damage by converting hydrogen peroxides into water . There ax lipids . GPx4 hax lipids ,8. GPx6 x lipids .Whilst GPx\u2019s have been identified in a variety of tissues, their roles in the placenta have not been fully characterised. Broadly, GPx was identified to be synthesised by the human placenta both extracellular (eGPx) and cellularly (cGPX) and secreted extracellularly into the maternal circulation . In the Multiple studies have identified that GPx expression is compromised in pre-eclamptic pregnancies in comparison to normotensive pregnancies ,67,68. OThe expression of GPx2 has recently been investigated in developing mouse embryos, where expression of GPx2 mRNA was higher in the extraembryonic tissues than in the embryo ,60. In tGPx3, the extracellular plasma enzyme, has been demonstrated to play a role in maternal\u2013foetal selenium transfer mechanisms, important in pregnancy and birth, in pre-eclampsia and in the growth of large healthy follicles . MaternaGPx4 is found in placental tissue and is vital for embryonic development and male fertility ,36. GPx4Thioredoxin reductases (TrxR) are selenoproteins that are part of a key antioxidant system that regulates oxidative stress. The thioredoxin system is formed by thioredoxin reductase and its associated substrate, the redox active protein thioredoxin, where thioredoxin reductases recycles oxidised thioredoxin at the expense of NADPH ,75,76. IThere are three deiodinases that have been identified across all cell types; however, only DIO2 and DIO3 are expressed in the placenta and may be responsible for the regulation of thyroid hormones that are vital for healthy foetal development . DIO2 isThyroid hormones are required for normal foetal development, and the foetus relies on maternal supply until mid-gestation. The exchange of bioactive molecules between maternal and foetal circulations occurs via transport through the plasma membrane and cytoplasm of placental trophoblast cells, and in the first trimester, foetal levels of thyroid hormone can be 100 times less than those of maternal serum . In healThere is limited evidence on the regulation of selenium in pregnancy in relation to the expression of these DIO enzymes in the human placenta. In some studies, it has been found that mild selenium deficiencies result in partial protection of the thyroid hormone axis and thyroid hormone metabolic pathways. In healthy selenosufficient people and in isolated mild selenium deficiencies, thyroid hormone concentrations were not altered and there was no evidence for impaired activity of these enzymes . It has It has been recognized for some time that SelP is produced by the placenta and with GPx3 is released into both the foetal and maternal circulation, playing an important role in transporting selenium . SelP leThere is limited evidence associating abnormal SelP production with gestational disorders. A study by Altinove et al. in 2015  observedSelenoprotein H (SelH) has a conserved CXXU thioredoxin-like motif and has been shown to have oxidoreductase activity. It is localised to the Golgi and nucleus where it has role in regulating gene expression in response the redox status, antioxidant defences and oxidative stress. The human placenta expresses SelH but little is known of its function in the placenta and whether changes in placental expression are associated with gestational disorders.In a recent series of experiments in our laboratory, we supplemented transformed trophoblast cells, BeWO, Jeg and Swan-7, with sodium selenite or selenomethionine in an attempt to protect cells from oxidative stress induced by rotenone and antimycin . The preThe endoplasmic reticulum is a critical organelle in the secretory pathway and is involved in intracellular protein synthesis and folding, glycosylation, secretion, lipid synthesis and the regulation of intracellular calcium signalling ,93. Prot2+ homeostasis, inflammation, beta cell function, thyroid hormone synthesis and muscle development [Cellular perturbations such as hypoxia, nutrient deprivation, point mutations in secreted proteins, redox changes, loss of calcium homeostasis can cause ER stress . The unfelopment . ER-resi2+) homeostasis [SEPS1 polymorphism in pre-eclamptic and healthy pregnancy controls [SEPS1-105G > A polymorphism was shown to be a significant risk factor for preeclampsia in this population, and genetic variation in SEPS1 gene was strongly associated with circulating levels of TNF-\u03b1, IL-6, and IL-1\u03b2, suggesting a crucial genetic link in influencing these proinflammatory cytokines [2 = 7.87, p = 0.005) in women with pre-eclampsia, 1.34 times more likely to have the gene in comparison to normal controls, suggesting SEPS1 has a role in protection from pre-eclampsia in that population [SEPS1 polymorphisms in 569 preterm singleton neonates and 673 term neonates, which found that compared with the GG genotype, -105A positive genotypes (GA + AA genotypes) were associated with significantly increased susceptibility to spontaneous preterm birth  [As illustrated in eostasis . Despiteeostasis ,51,97. Aeostasis . A retroytokines . There wSelF was reduced (p < 0.05) within the maternal liver, SelM was significantly reduced (p < 0.05) in all maternal tissues and SelT expression was reduced in the heart (p < 0.05). Another study measured the selenoprotein mRNA expression in the placenta across the maternal and foetal tissues. It was reported that selenoproteins TrxR1, TrxR2, TrxR3, DIO3, SelH, SelI, SelK, SelN, SelO, SelS, SelV and SelW exhibited the highest expression in the middle section of the placenta, whereas GPx1, DIO2, SelM, SelT, and Sel15, exhibited the highest expression levels in foetal-facing placental tissue and GPx4, SelP, SPS2, and DIO1 exhibited a pattern whereby expression was nearly equal in the foetal-facing and middle section, and lower in the maternal facing portion of the placenta [Other selenoproteins that reside in the ER, such as Sep15, SelK, SelM, SelN and SelT, are less studied when it comes to expression in the placenta. A recent study by Hofstee et al. analysed the expression of many of these ER selenoproteins in pregnant mice who were allocated a low-selenium diet in comparison to a control selenium diet . At embrplacenta . FurtherMore specifically, there is limited information on the roles of the other ER-resident selenoproteins. SelK has been demonstrated to participate in oxidation resistance, calcium flux regulation and the ER associated degradation (ERAD) pathway in immune cells; however, not in placenta or trophoblast cells . One stuThe placenta is a highly specialised organ of pregnancy that supports the growth and development of the foetus. It does so by connecting the maternal and foetal circulation to allow for the exchange of gases, nutrients, and waste products. The crucial role of micronutrients in the health of an individual in pregnancy is still under investigation, but there is a body of literature describing the critical role of micronutrient intake during all periods of foetal growth and development. Evidence strongly suggests that where there is an excessive increase or decrease in any nutrient, it has a direct relationship on the development of placental co-morbidities. For many years, selenium has been a micronutrient of interest in pregnancy where deficiencies have been directly linked to an increased chance of developing pregnancy complications such as pre-eclampsia, preterm birth and intrauterine growth restriction . A comprMany studies have implicated the role of oxidative stress in pregnancy pathologies. When antioxidant defences are overwhelmed by reactive oxygen and nitrogen species (RONS), this leads to oxidative insults that damage proteins, lipid and nucleic acids structures. Placental development is an intricate process that is highly dependent on oxygen status. During the first trimester, the placental intervillous space is low in oxygen as trophoblast cells plug the tips of uteroplacental spiral arteries, limiting supply of oxygenated maternal blood . At the The placenta expresses low levels of antioxidants in early pregnancy; therefore, pre-mature maternal foetal circulation and widespread oxidative stress overwhelm cellular homeostasis, leading to placental injury. This is the potential cause of spontaneous first trimester abortions, and insufficient placental perfusion and ischemia reperfusion induced oxidative stress are associated with pre-eclampsia and IUGR . Placent"}
{"text": "Anterior temporal lobectomy (ATL) is the most common surgical treatment for drug-resistant temporal lobe epilepsy (TLE). Right ATL has been reported to reduce facial memory ability in patients with TLE, as indicated by poor performance on the Warrington Recognition Memory Test for Faces (RMF), which is commonly used to evaluate visual memory in these patients. However, little is known about whether patients with TLE exhibit difficulties in identifying faces in daily life after ATL. The aim of this study was to investigate facial memory ability and self-awareness of face identification difficulties in patients with TLE after ATL. Sixteen patients with TLE after right ATL, 14 patients with TLE after left ATL, and 29 healthy controls were enrolled in this study. We developed the multiview face recognition test (MFRT), which comprises a learning phase  and a recognition phase . Facial memory abilities were examined in all participants using the MFRT and RMF, and self-awareness of difficulties in face identification was evaluated using the 20-item prosopagnosia index (PI20), which has been widely used to assess developmental prosopagnosia. The MFRT performance in patients with TLE after ATL was significantly worse than that in healthy controls regardless of the resected side, whereas the RMF scores in patients with TLE were significantly worse than those in healthy controls only after right ATL. The MFRT performance in patients with TLE after both left and right ATL was more influenced by working memory load than that in healthy controls. The PI20 scores revealed that patients with TLE after left ATL were aware of their difficulties in identifying faces. These findings suggest that patients with TLE not only after right ATL but also after left ATL might have difficulties in face identification. Epilepsy is one of the most frequent chronic neurological disorders \u20134. DespiNaturally, facial memory can be influenced by facial perception processing . PreviouIn the real world, it is rare to be confronted by so many faces. In addition, people do not always recognize faces from the front, and hairstyles and clothes can change every day. Furthermore, one previous study showed that many participants were able to identify individuals in a modified RMF that removed all internal facial feature information . AlthougSeveral previous studies have reported that patients with focal hippocampal damage due to anoxia or encephalitis have impaired higher-order visuospatial perception and recognition, including unknown faces, when working memory demand is slightly increased , 37. In The main aims of this study were to investigate facial memory ability and self-awareness of face-identifying difficulties in patients with TLE after ATL. Our hypothesis is that (1) not only right TLE patients but also left TLE patients after ATL exhibit deficits in identifying unknown faces if they see the faces at different angles or the faces are partially hidden; (2) the deficits will become more severe when working memory load increases; and (3) not only right TLE patients but also left TLE patients after ATL are aware of face identification difficulties in their daily life. For these purposes, we developed a new test to assess facial memory that not only uses frontal face images but also oblique face images and those covered with noise. In addition, the test required participants to learn only one or three unknown faces to investigate the alteration of facial memory ability due to working memory load and to better reflect situations encountered in daily life. Moreover, we assessed face identification difficulties in daily life using a validated self-assessment questionnaire. We selected the 20-item prosopagnosia index (PI20), which has been confirmed to be correlated with the Cambridge Face Memory Test scores . It is iThe ethics committees of the Tohoku University Hospital approved this study. Written informed consent was obtained from all participants after they had been given a detailed description of the study.Thirty consecutive patients with drug-resistant TLE who underwent unilateral ATL at Tohoku University Hospital or National Center of Neurology and Psychiatry Hospital were enrolled in this study. The inclusion criteria were as follows: (1) 1 year or more after surgery; (2) \u2265 16 years old; (3) a Performance Intelligence Quotient index on the Wechsler Adults Intelligence Scale-III (WAIS-III) of \u2265 70; (4) a native Japanese speaker; and (5) a best-corrected Snellen acuity \u2265 20/50. The exclusion criteria were as follows: (1) concurrent extratemporal lesions on MRI and (2) no history of neurological or psychiatric disease other than TLE-related conditions.Of the 30 patients, ATL with amygdalohippocampectomy was performed in 24 patients . The antero-inferior temporal lobe was removed by up to 35 mm on the dominant side and 45 mm on the nondominant side from the tip of the temporal lobe, and then the inferior horn of the lateral ventricle was opened. The amygdala was aspirated subpially to the level of the anterior choroidal artery or optic tract. The mesial and basal temporal structures, including the hippocampus, entorhinal and perirhinal cortex, and anterior part of the parahippocampal and fusiform gyrus, were removed en bloc and further aspirated to the level of the superior colliculus in the anterior\u2013posterior direction. ATL with amygdalectomy was performed in four patients . One of the remaining two patients underwent left ATL with multiple hippocampal transactions, and the other patient underwent left ATL preserving both the amygdala and hippocampus. One patient had a cerebral infarction adjacent to the resected brain area in the right temporal lobe immediately after right ATL. The lateral and basal temporal areas resected in the patients with ATL are demonstrated in Postoperative seizure outcome was evaluated using Engel\u2019s classification. Of the patients with right ATL (right ATL group), 11 (68.8%) had achieved seizure freedom (Class I), two had rare seizures (Class II), two had a worthwhile improvement (Class III), and one had no change (Class IV) 1 year after surgery. Of the patients with left ATL (left ATL group), 10 (71.4%) had achieved seizure freedom (Class I), two had rare seizures (Class II), one had a worthwhile improvement (Class III), and one had no change (Class IV). The right ATL and left ATL groups did not differ from each other in age at onset, disease duration, or seizure outcome. The demographic data are summarized in Twenty-nine healthy controls (HCs) matched for age and sex were recruited from the local community through advertisements. Participants with any history of neurological or psychiatric disease and/or impaired visual acuity (a best-corrected Snellen acuity that was poorer than 20/50) were excluded. Although there was no significant difference in handedness among both the ATL groups and the HC group, the averages of education years in patients after left and right ATL groups were significantly lower than those in the HC group .In the present study, all neuropsychological tests, including standardized and developed tests, were administered to the patients after ATL. There was no significant difference in the mean intervals from ATL to assessment between the right and left ATL groups . All patients underwent the following standardized neuropsychological tests: the WAIS-III for verbal capacities, visuoconstructive functions, working memory, and cognitive speed ; the WecIn addition, to evaluate retrograde facial memory and familiar face recognition ability, we developed a famous face identification task in which participants were presented with 20 faces of Japanese actors, athletes, or politicians . Participants were required to provide both the first and last names of the famous individuals. If they could not correctly name the famous person, they were asked to provide the information about the famous person. These tests yield two scores: the naming score, which is the number of total correct responses , and the recognition score, which is calculated by adding the number of total correct responses to the number of total correct but insufficient responses (a participant correctly answers either the first or last name or correctly provides one or more pieces of information about the famous person). Each score ranges from 0 to 20. The famous face identification task was administered to all participants.3 on frontal face images. Selected examples of face stimuli are shown in We collected 144 nonfamous Japanese faces  for test stimuli, of which 60 were drawn from the face database of the Advanced Telecommunications Research Institute International Inc. and the others were obtained from local volunteers. A 10-point rating of facial emotional valence was assessed in each face image by 10 healthy volunteer raters . Sixty neutral face images were selected in order from the average emotional valences closest to 5.5 points. The average emotional valence of the selected male and female face images was 4.70 (SD = 0.40) points and 4.89 (SD = 0.59) points, respectively. There was no significant difference in valence between the male and female face images (p = 0.150). Hair-removed frontal and oblique [left\u201430 degrees] views of each face were generated using FaceGen software (Singular Inversion). We removed the facial blemishes manually using Adobe Photoshop CS 6.0 and Adobe Illustrator CS 5.1. All face images were transformed into grayscale with a black background. Noise-masked face images were created by overlaying black-and-white visual noise of spatial frequencies of 1/fParticipants sat in front of a personal laptop computer . The computer was placed in an easily viewable and operable position. The participants were instructed both verbally and in written instructions. Each trial of the test consisted of a learning phase and a recognition phase. During the learning phase, participants were asked to remember frontal face images that were displayed on a screen for 5 s. This presentation time and the angle of face (oblique face image) was defined based on a pilot study in 7 healthy volunteer subjects  so that the total accuracy rate of the MFRT became approximately 80%. We varied the numbers of frontal face images displayed (one or three images) . In the We found significant differences in the scores between each time interval between the learning and recognition phases , both in the one-image and three-image blocks in the HC group . As showTo assess patients\u2019 insight into their own facial recognition abilities, we used the PI20 , which iOne-way ANOVA and post hoc Bonferroni tests were used for between-group comparisons in the Warrington Recognition Memory Test scores (words and faces), and Welch\u2019s t-test was used to compare the other neuropsychological test scores, except for the recognition score of the famous face identification task and the MFRT scores, between the left and right ATL groups. A three-way repeated measures ANOVA was used to analyze between-group differences  in the MFRT scores for the three stimulus conditions  and between the two working memory loads (Memory: one image or three images). The Kruskal\u2013Wallis test and a post hoc Mann\u2013Whitney U-test with Bonferroni correction were used to compare the recognition score of the famous face identification task and PI20 scores between the three groups.Statistical analyses were performed using IBM SPSS statistics software , and the threshold for statistical significance was set at p < 0.05.2 = 0.396). Post hoc pairwise comparisons with Bonferroni correction revealed that the mean naming score of the famous face identification task score in the left ATL group was significantly lower than that in the right ATL (p = 0.050) and HC groups (p < 0.001). In contrast, there were no significant differences in the median recognition scores of the famous face identification task among the three groups . The mean scores of the Warrington Recognition Memory Test for words  = 5.87, p = 0.005, \u03b72 = 0.173) and for faces  = 6.67, p = 0.003, \u03b72 = 0.192) were significantly different among the three groups. Post hoc pairwise comparisons with Bonferroni correction revealed that the Warrington Recognition Memory Test for words score in the left ATL group was significantly lower than that in the HC group (p = 0.003), whereas there were no such significant differences between the right ATL and HC groups (p = 0.791) or between the left and right ATL groups (p = 0.126). In contrast, the RMF score in the right ATL group was significantly lower than that in the HC group (p = 0.002), and there were no such significant differences between the left ATL and HC groups (p = 1.000) or between the left and right ATL groups (p = 0.105).The neuropsychological assessment results are summarized in 2 = 0.032) for the MFRT scores. However, the interactions between Group and Memory  = 7.54, p = 0.001, \u03b72 = 0.212) and between View and Memory  = 10.15, p < 0.001, \u03b72 = 0.270) were significant. There was no significant interaction between Group and View  = 1.17, p = 0.153, \u03b72 = 0.058). The main effects of Group  = 6.66, p = 0.003, \u03b72 = 0.192), View  = 14.66, p < 0.001, \u03b72 = 0.348), and Memory  = 307.91, p < 0.001, \u03b72 = 0.844) were significant. According to post hoc pairwise comparisons with Bonferroni correction, the mean MFRT scores of the left and right ATL groups were significantly worse than those of the HC group . There was no significant difference in the mean MFRT scores between the left and right ATL groups (p = 1.000). In addition, the mean scores for oblique view and noise-masked view trials were significantly worse than those for the frontal view trial . There was no significant difference in the mean MFRT score between oblique view and noise-masked view trials (p = 0.852). Moreover, the mean MFRT score in the three-image tasks was significantly lower than that in the one-image tasks (p < 0.001).The MFRT results are summarized in 2 = 0.066), whereas the simple main effect of Group in the three-image tasks  = 8.62, p < 0.001, \u03b72 = 0.235) was significant. In contrast, the simple main effect of Memory was significant in each group  = 77.52, p < 0.001, \u03b72 = 0.581; right ATL group: F  = 142.97, p < 0.001, \u03b72 = 0.720; HC group: F  = 95.27, p < 0.001, \u03b72 = 0.624). Post hoc Bonferroni contrast analysis tests revealed that the mean MFRT scores in the left and right ATL groups were significantly worse than those in the HC group on the three-image tasks . There was no significant difference in the mean MFRT scores between the left and right ATL groups on the three-image tasks (p = 0.927). In addition, the mean MFRT score on the three-image tasks was significantly worse than that on the one-image tasks in each group .In the interaction between Group and Memory, there was no significant simple main effect of Group in the one-image tasks  = 1.97, p = 0.149, \u03b72 = 0.291; three-image tasks: F  = 11.12, p < 0.001, \u03b72 = 0.288). Furthermore, simple main effects of Memory were also significant for each view  = 99.39, p < 0.001, \u03b72 = 0.640; oblique view: F  = 126.23, p < 0.001, \u03b72 = 0.693; noise-masked view: F  = 223.72, p < 0.001, \u03b72 = 0.800). Post hoc Bonferroni contrast analysis tests revealed that the mean MFRT score for the oblique view was significantly worse than those for the frontal view and noise-masked view on the one-image tasks . In addition, the mean MFRT scores for the oblique view and noise-masked view were significantly worse than those for the frontal view on the three-image tasks . There was no significant difference in mean MFRT scores between the oblique view and noise-masked view on the three-image tasks (p = 0.868). Moreover, the mean MFRT score on the three-image tasks was significantly worse than that on the one-image tasks for each view .In the interaction between View and Memory, simple main effects of View in both the one-image and three-image tasks were significant  = 11.31, p < 0.001, \u03b7The PI20 results are shown in We investigated facial memory ability and self-awareness of face identification difficulties in patients with TLE after ATL using MFRT and PI20. The major findings were as follows: (1) the MFRT performance in patients with TLE after ATL was significantly worse than that in HCs regardless of the resected side; and (2) the PI20 score was higher in patients with TLE after left ATL but was not higher in patients with TLE after right ATL than in HCs.As previous studies have demonstrated, RMF performance was normal in TLE patients after left ATL in the current study , 22. On The discrepancy between the RMF and MFRT results in the TLE patients after left ATL is likely to have arisen from differences between the two tests. The RMF stimuli contained abundant noninternal facial feature information, such as hairline and clothes, whereas the MFRT stimuli were hair-removed. Given that the face images used in the MFRT contain less nonfacial feature information, the clues for learning and recognizing face images were more limited than in the RMF. In addition, the MFRT requires participants to recognize face images that are different from the learned face images. We suggest that this requires the ability to complement or compensate for incomplete face images. However, the TLE patients after left ATL exhibited good facial memory performance, despite the less informative face images on the one-image tasks. The poor performance on the MFRT in the TLE patients after left ATL only became obvious on the three-image tasks. These findings indicate that difficulties in learning or remembering less informative face images in TLE patients after left ATL were accompanied by an increased working memory load, which is coincident with one of our hypotheses.Previous studies of patients with focal hippocampal damage due to anoxia or encephalitis, amnestic mild cognitive impairment, or Alzheimer\u2019s disease and functional MRI studies have indicated that the hippocampus plays a crucial role in higher-order visuospatial working memory , 42, 43.In addition, a previous study reported that patients with medial temporal lobe lesions that were involved in not only the hippocampus but also the perirhinal cortex, which was involved in almost all patients after ATL in the present study, showed impaired discrimination of unfamiliar faces presented from differing viewpoints . MoreoveThe PI20 scores in patients with TLE after left ATL were higher than those in the HC group. This finding could not be predicted from the results of the RMF, since the RMF scores in patients with TLE after left ATL were not different from those of HCs in both the current and previous studies , 22. We In the present study, the performance of the patients with TLE after right ATL on both the RMF and MFRT was significantly lower than that of the HC participants. This finding is coincident with previous studies and our hypothesis \u201325. It iAlthough there was no significant difference in the mean visual memory index of the WMS-R between the TLE patients after right ATL and after left ATL, the mean intelligence quotient index of the WAIS-III in the TLE patients after right ATL tended to be lower than that after left ATL. The mean level of performance intelligence in the TLE patients after right ATL was not abnormal. However, it was possible that the performance on the MFRT in these patients was influenced by their intelligence.Surprisingly, the PI20 scores in the patients with TLE after right ATL were not higher than those in the HC participants. The TLE patients after right ATL did not seem to be aware of face identification impairment in their daily life, despite their significantly reduced objective unfamiliar face identification task performance. One possible explanation for this finding is that patients with TLE after right TLE might have anosognosia for visual memory impairment. Previous studies have demonstrated that very few TLE patients are aware of their significant memory decline after right ATL , 48. SawOur study has several limitations. First, we did not validate the developed neuropsychological tests, including the MFRT, famous face identification task, and Japanese version of RMF. We did not evaluate test-retest reliability for these neuropsychological tests. Therefore, we could not conclude whether the differences in performance on these neuropsychological tests among the TLE patients and HC participants were evident. In addition, as mentioned above, the recognition scores of the famous face identification task we created demonstrated a ceiling effect in the TLE patients and HC participants. Unfortunately, it is highly likely that we could not reliably detect deficits in famous face recognition in patients after right ATL.Second, we did not implement neuropsychological tests, including the MFRT and PI20, before surgery. Thus, we could not investigate whether the MFRT performance in the patients with TLE was reduced due to ATL or whether stronger awareness of difficulties in face recognition in daily life was derived from a left ATL.Third, we only evaluated facial memory ability. However, in patients with hippocampal damage, impaired higher-order visuospatial perception or recognition has been observed when working memory demand is increased, even though face stimuli were not used \u201340. TherFinally, some TLE patients did not undergo PI20 before either or both MFRT and RMF were implemented. Therefore, the results of MFRT and/or RMF could affect the PI20 scores in these patients. Moreover, the PI20 score is influenced by various factors, including memory function and face perception. The daily demand for face recognition in social life may be altered by occupation and/or household . EnvironIn conclusion, our results suggest that the ability to remember faces with less internal and/or external information is impaired when visuospatial working memory load increases in patients with TLE who underwent ATL, regardless of the resected side. However, validation of the MFRT is necessary to confirm our findings. In the future, we aim to investigate memory of not only unfamiliar but also familiar faces and related multiaspect information about patients with TLE before and after ATL to elucidate whether ATL affects facial memory ability, which could help improve the quality of life and surgical outcome of ATL in patients with TLE.S1 Table(DOCX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file."}
{"text": "The mitogen-activated protein kinase (MAPK) family is an important bridge in the transduction of extracellular and intracellular signals in different responses at the cellular level. Within this MAPK family, the p38 kinases can be found altered in various diseases, including cancer, where these kinases play a fundamental role, sometimes with antagonistic mechanisms of action, depending on several factors. In fact, this family has an immense number of functionalities, many of them yet to be discovered in terms of regulation and action in different types of cancer, being directly involved in the response to cancer therapies. To date, three main groups of MAPKs have been identified in mammals: the extracellular signal-regulated kinases (ERK), Jun N-terminal kinase (JNK), and the different isoforms of p38 . In this review, we highlight the mechanism of action of these kinases, taking into account their extensive regulation at the cellular level through various modifications and modulations, including a wide variety of microRNAs. We also analyze the importance of the different isoforms expressed in the different tissues and their possible role as biomarkers and molecular targets. In addition, we include the latest preclinical and clinical trials with different p38-related drugs that are ongoing with hopeful expectations in the present/future of developing precision medicine in cancer.  Currently, a large part of the resources allocated to global health are being directed towards the study and prevention of cancer, as it is one of the main causes of death worldwide . As a reThanks to these efforts, it has been possible to relate the appearance of various types of cancer with the mutational state of some important genes. By identifying certain molecular patterns that do not function properly, it would be possible to successfully intervene with different therapies aimed at correcting or blocking these alterations involved in the development and survival of tumor cells . SpecifiIn 1991, the sequences of the first MAPKs studied in mammals  were revealed, identifying them as member enzymes of this newly discovered specific family of protein kinases . MAPKs aMAPK signal transduction pathways are ubiquitous and highly conserved regulatory mechanisms in eukaryotic cells which promote coordinated and integrated responses to numerous stimuli through protein kinase-type receptors, leading to important cellular physiological effects. MAPKs phosphorylate target substrates at Ser or Thr residues, followed by a Pro residue, suggesting limited specificity . This spIn recent years, the cross-interaction of the JNK and p38 families has been studied in greater depth, elucidating their dual role in different types of cancer, depending in many cases on the cellular context and the specific individual . Both thIn this review, we highlight the broad functionalities of the different components of the p38 MAPK molecular signaling pathway, considering the role of even the less known p38 isoforms, in the development of numerous cancer-related processes, highlighting their dual role in multiple tumor types and their great viability as cancer biomarkers or potential therapeutic agents.MAPK14, MAPK11, MAPK12, and MAPK13, which code for p38\u03b1, p38\u03b2, p38\u03b3, and p38\u03b4, respectively [MAPK14 is expressed in almost all cellular types, whereas the other isoforms are expressed in specific tissues. For example, p38\u03b2 is mainly found in the brain, p38\u03b3 is expressed in skeletal muscles, and p38\u03b4 is observed in kidneys, the pancreas, the small intestine, and testis [Four genes with high sequence homology have been identified that code for MAPK/p38: ectively , with p3ectively . p38\u03b1/MAd testis . In gened testis . It has d testis ; howeverd testis . It shoud testis . TherefoThe great complexity of the p38 MAPK pathway lies in addition to its mechanism of action, in its regulation at various levels by activators and inhibitors of different natures.In mammals, p38 isoforms are activated in response to different stresses produced in the cellular environment as well as the liberation of inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-\u03b1) in processes of oxidative stress during UV radiation exposition, hypoxic conditions, or during ischemia . As with the other MAPK families, the p38 pathway involves the canonical cascade activation of certain small proteins of the Rho GTPase family, such as Rac or Cdc42, which are upstream of the MAPK module discussed above . Some ofOn the other hand, there is a noncanonical p38 activation pathway. This occurs, for example, in T cells, where p38 can be activated by T-cell receptor (TCR)-mediated phosphorylation at a Tyr323 residue by the 70-kDa protein kinase \u03b6-chain-associated protein kinase (ZAP70) .As mentioned before, the p38 module is also bound by scaffolding proteins that play a crucial role in the specificity of its signaling. These proteins do not possess catalytic activity themselves, but regulate the activity of the p38 cascade by promoting the conformational changes necessary for its activation and controlling its cellular localization . p38 activation occurs by dual phosphorylation at Thr180 and Tyr182 residues located in the activation loop, therefore one way to de-phosphorylate these sites is by the actuation of phosphatases at several levels, acting also upstream of dephosphorylate MAP2Ks. Within this group are members of the dual specificity phosphatases (DUSP) family of phosphatases, which includes MAPK phosphatases (MKP). Notably, these phosphatases are activated even by stimuli that activate p38 . DUSP4 iMicroRNAs (miRNAs) are small, conserved noncoding RNAs that act at the post-transcriptional level, inducing the silencing of complementary mRNA targets once the Argonaute proteins assemble in miRNA-induced silencing complexes (miRISCs) . Most ofOn the other hand, the p38 MAPK pathway has been also involved in cervical cancer through the miR-409-3p/CDK8 axis . In gliaThese findings suggest that miRNAs may become the main regulators of the long-term specificity and sensitivity of p38 and other signaling pathways. However, since there are numerous identified miRNAs that regulate the p38 MAPK pathway in different tumor models with different consequences, its use as biomarkers or therapeutic targets is still far from being readily applied in clinical practice.MAPK p38 can be found in both the cytoplasm and the nucleus, and its location determines its signaling specificity . p38 is In response to inducers of DNA damage, such as radiotherapy or chemotherapy used in cancer treatment, the nuclear localization of p38 depends on its phosphorylation, but does not depend on its own kinase activity . Since pThe p38 substrates include other protein kinases and transcription factors, 55% of which are found in the nucleus . Most ofIn conclusion, most studies have suggested that p38 regulates the inhibitors or activators of transcription, in addition to chromatin modulation, favoring or not the transcription of many genes implicated in different cellular processes .On the other hand, the cytosolic substrates of p38 include proteins with antiproliferative functions such as p57Kip2 and cyclin D1/3, and apoptotic functions such as Bax and BimEL. Moreover, p38 can regulate cell survival by phosphorylation of caspase-3 and caspase-8  Figure .There is strong evidence for a role of p38\u03b1 as a tumor suppressor, mainly mediated by the negative regulation of the cell cycle and the activation of apoptosis. Moreover, the induction of terminal differentiation is involved in its role as a tumor suppressor . HoweverAs we know, uncontrolled proliferation is a characteristic process in cancer, and the p38 MAPK pathway plays a key role in regulating cell cycle progression at different points by mechanisms that can depend on transcription machinery. In addition, it can modulate antagonic cellular programs for cell survival, differentiation, or cell death induction, resulting in significant effects on the progression of several types of cancers .HRAS, functioning as an anti-tumor defense mechanism by inducing p53 phosphorylation [As discussed before, p38\u03b1 can negatively regulate cell cycle progression at the G1/S and G2/M transitions by different processes, such as the downregulation of cyclins and the upregulation of cyclin-dependent kinase (CDK) inhibitors, as well as the modulation of the tumor suppressor p53 . p38 MAPrylation . This furylation . This efrylation . Howeverrylation . These orylation . It has A quiescent state known as cancer dormancy, induced by p38\u03b1 through inflammatory and antiapoptotic signals, such as the liberation of IL-6 cytokine, has been also suggested as a mechanism involved in drug resistance. This different activity of p38\u03b1 could be relevant for tumor suppression, leading to more differentiated and less transformed cells in cancer cell lines, such as in renal carcinoma or colon cancer . Ultimately, it is likely that the different effects of p38\u03b1 pathway activation depends on cell type differences, along with the duration, quality, and intensity of the stimulus and its communication and interaction with other signaling pathways.MAPK11 in keratinocytes could promote a decrease in c-Myc expression [On the other hand, it has been shown that p38\u03b2 could be involved in cell proliferation, thanks to its relationship with integrin-\u03b1v, as it maintains cell proliferation in keratinocytes by controlling the translation of c-Myc FAK, p38\u03b2, and p90RSK1. Therefore, it has been shown that either chemical inhibition or genetic interference of p38\u03b2 pression .MAPK13 gene [The analysis of the expression of p38\u03b3 and p38\u03b4 in both cell lines and patient samples indicate that they have pro- and antioncogenic roles in tumor development and outcome . For exaK13 gene ,70.Chronic inflammation plays a key role in cancer development by promoting cancer survival, along with angiogenesis and cell invasion and metastasis. Studies have shown that these processes are controlled by the p38 MAPK pathway, as that these enzymes regulate the activity and expression of some inflammatory mediators such as different proteases and cytokines that contribute to cancer development and progression .The tumor microenvironment (TME) is the collection of cells , molecules (such as cytokines and chemokines), and blood vessels that surround and feed a tumor. In addition, the stroma, which dynamically supports the TME, is formed by the extracellular matrix, a different type of cells, including fibroblasts, infiltrated immune cells, and histological structures such as the blood vessels or connective tissue  Figure .It has been shown that p38\u03b1 regulates the activation of cyclooxygenase 2 (COX2), which has a proinflammatory activity that has significant effects on cancer progression, such as nonmelanoma skin cancer, breast cancer, or glioma ,74,75. IIn addition, p38\u03b1, by other independent mechanisms, can induce the expression of metalloproteinases, which are involved in matrix formation, remodeling, or degradation by metastatic cancer cells, with the participation of the vascular endothelial growth factor A (VEGF-A), which facilitates the tumor survival through angiogenesis induction. For tumor growth, the formation of new vessels close to the cells is indispensable, a process known as angiogenesis . AngiogeImportantly, the downstream activator of p38: MKK6 may or may not have metastatic effects, depending on the experimental model ,88. FinaOn the other hand, there is evidence that p38\u03b1 may have tumor suppressor functions through genetic studies that have identified that MKK4 (a MAP2K shared by both the JNK pathway and p38) may have a relevant role in this type of process. Thus, loss-of-function alleles for MKK4 were found in some types of cancer such as lung, breast, colon, prostate, and even pancreatic cancer; however, there are also studies indicating that this loss could also promote tumorigenesis ,91.The tumor suppressor activity of p38\u03b1 has been studied in relation to the occurrence of the overexpression of several negative modulators of p38 MAPK signaling in various human tumor types, such as the phosphatases PPM1D and DUSP26 ,92,93 anBy studying the expression of p38\u03b3 and p38\u03b4 in knock-out mouse models and mouse cancer models, the tumor suppressor role of these isoforms in mouse embryonic fibroblasts has been demonstrated, such that their deficiency could increase cell migration and metalloproteinase-2 secretion, thereby modifying cell contact inhibition. Furthermore, decreased expression of p38\u03b3 in K-Ras-transfected fibroblasts resulted in increased cell proliferation and tumorigenesis in trials conducted both in vitro and in vivo . The proCross signaling between pathways plays a crucial role in cell regulation, depending on the cellular context. As noted above, the JNK and p38 MAPK pathways share several upstream regulators, therefore there are many stimuli that can activate both pathways at the same time . In someTherefore, most studies support the idea that therapeutic inhibition of the p38 MAPK pathway must depend on the cellular context, cancer type, and tumor stage, as well as the intensity and duration of the signal, together with cross signaling between MAPK pathways.Early detection and diagnosis of cancer is crucial for the survival of cancer patients. Tumor biomarkers are crucial in reflecting molecular differences between certain types of cancers, sometimes enabling detection at an early stage\u2014a consequent advantage in improving a patient\u2019s prognosis. Therefore, many studies have focused on the discovery of new biomarkers targeting the MAPK pathway and, in this sense, the p38\u03b1 isoform is one of the most studied; however, in recent years, isoforms p38\u03b2, p38\u03b3, and p38\u03b4 have been recognized with a potential role in cancer diagnosis.The p38\u03b3 isoform is associated with invasion, clinical stage, metastasis, and chemosensitivity. Some studies have shown that overexpression of this isoform is associated with a poor prognosis in patients with cellular hepatocarcinoma  and may MAPK13 gene [It has been shown that p38\u03b4 is overexpressed in cholangiocarcinoma, invasive human cutaneous squamous cell carcinoma (SCC), head and neck SCC, and prostate cancer ,109,110.K13 gene ,111,112.K13 gene . In addiK13 gene . Thus, tK13 gene . MAPK11 gene expression in specific female tissue cancers has been determined with in-silico studies [The p38\u03b2 is an isoform that has been recently incorporated in cancer studies and showed an interesting role in cancer development. For example, this isoform has been proposed as a key target of the Pok\u00e9mon proto-oncogene, a transcription factor involved in tumorigenesis and metastasis in liver cells . Further studies . Finally studies .The p38 pathway is a pleiotropic cascade with different actions in the proper functioning or otherwise of cells in the body, such as in response to growth factors or stress, during carcinogenesis or metastasis. For this reason, p38 plays a fundamental role in the approach to conventional cancer therapies, such as radiotherapy or chemotherapy and targeted treatments, using certain small molecules that act as inhibitors of key steps in the signaling pathway, although they can induce resistance from the first dose due to the positive selection of those tumor cells that are able to compensate the specific target pathway.A large percentage of patients are treated with ionizing radiation for cancer. The effect of exposure to this radiation on both normal and tumor tissues has been extensively studied in recent years. The early effects of chemotherapy result in the induction of apoptotic cell activation, with excessive secretion of proinflammatory cytokines because of this process, as well as the increased recruitment of blood cells and platelets, the activation of coagulation systems, and increased vascular permeability . The molFor example, it has been shown that, in human dermal microvascular endothelial cells, radiation-induced apoptosis is largely mediated by p38  and thisApoptosis is a mechanism of cell death in response to anticancer agents and, as mentioned before, p38 plays a key role in this process, modulating the chemotherapeutic response to both conventional and targeted therapies. P38 has been implicated in cell apoptosis that is mediated by cis-platinum and 5-fluorouracil (5-FU) in breast and colon cancer cell lines  and has P38 contributes to chemotherapeutic resistance in several tumor types, e.g., in response to doxorubicin, cis-platinum, or other agents that target DNA damage, as p38 will promote cell survival of p53-deficient cancer cells through MK2-mediated cell cycle arrest ,129. In Antiestrogen tamoxifen is a key treatment for hormone-sensitive breast cancers, and the activation of the p38 pathway is related to resistance and poor prognosis . HoweverIn terms of p38\u03b3-related drugs, it has been shown that this isoform can regulate the signal transduction of drugs targeting DNA topoisomerase II \u03b1, which represent an important class of antitumor drugs in the clinic, thus enhancing their therapeutic activity . However, it has been shown that the role of the p38 pathway in the response to chemotherapy depends on the type of tumor, since each tumor microenvironment also participates in the neoplasia. The involvement of this pathway in tumor development involves several cell types within the tumor microenvironment, as discussed before. Thus, several p38 inhibitors have been shown to inhibit cancer progression by halting processes such as angiogenesis, for example, in an in vivo experimental model of prostate cancer , as wellIn addition, genotoxic, ROS-generating drugs such as doxorubicin induce the liberation of IL-6 from endothelial cells in a mechanism that depend on p38 activation, contributing to the induction of senescence in tumor-associated stromal cells ,138. TheAll these studies demonstrate the viability of therapy directed against the p38 pathway as a target for cancer, and are summarized in the following section. There are many clinical trials evaluating the role of p38 as a biomarker and target for different diseases, with a considerable percentage dedicated to cancer treatment, where p38 is considered a promising target, either using p38-specific inhibitors alone or in combination with other chemotherapeutic agents  Table 1Table 1.However, one cannot overlook the fact that p38 is involved in many biological activities, and its inhibition could lead to unwanted and unknown side effects, therefore efforts are currently focused on discovering agents that specifically target p38 or a component of the pathway that is deregulated and is the trigger for a particular disease. For example, small peptides are being developed that target VEGFR-2, thereby inhibiting the activation of p38 by VEGF, and thus inhibiting p38-dependent angiogenic effects . In this regard, it has also been shown that the combination of p38 inhibitors with conventional chemotherapy drugs could be a promising approach in antitumor therapy. One of the most prominent is the p38\u03b1 and p38\u03b2 inhibitor LY2228820/Ralimetinib, which has antiangiogenic effects in vitro and in vivo  and has Because of the involvement of p38\u03b1 in proinflammatory cytokine production and cytokine receptor signaling, the development of a large number of small p38\u03b1 MAPK inhibitor molecules is ongoing . These hMoreover, treatment resistance should be considered in cancer therapy, and attempts should be made to limit it. Thus, the inhibition of the p38 pathway has been shown to limit resistance to irinotecan in colon adenocarcinoma . HoweverAlmost all clinical trials to date have failed and therefore no compound has yet been approved, which has been due to systemic side effects on the heart, liver, or nervous system , which cTherefore, efforts should focus on gaining a better understanding of the contribution of p38 to the development of cancer and other diseases, both at the cellular and organismal level, to develop better compounds that specifically target the different components of this MAPK signaling pathway to improve responses to treatment and reduce the drawbacks of conventional inhibitors.MAP kinases are involved in the response of cancer cells to a wide range of stimuli, both external and internal. In the last 25 years, many important advances have been made in our understanding of the regulation and functionality of the p38 MAPK pathway. Due to genomic and proteomic strategies, the different components of the p38 MAPK pathway have been identified that could play a key role in oncogenic processes when they are altered, making them potential targets for the development of antitumor drugs. However, these strategies have been hampered by the activation of compensatory pathways in cancer cells and the high degree of crossover and dual functionality that exists between the other different MAPK families (ERK and JNK), thus giving rise to a strong resistance to the most common treatments. These kinases are highly regulated at various levels, where numerous miRNAs have been described as interfering in different steps of signaling. However, numerous reports have shown that the use of miRNA as biomarkers or drug targets has been unsuccessful due to differences in the methodology of the studies, the lack of standard methods for normalization, the different miRNA processing, and the inability to discriminate among closely related miRNAs, among others.Given the latest advances in gene therapy, it is not impossible to think that cancer, which can be defined as a disease caused by different genetic predispositions, could be fought by modifying certain aberrations in the genome or by inducing certain tumor suppressor genes, a revolutionary process also known as epigenome editing. This would be possible thanks to the CRISPR-Cas9 method that could inhibit oncogenes such as the p38\u03b3 gene in some tumors, which could be a promising cancer therapeutic strategy . SeveralIt should be noted that immunotherapy has become an important cancer treatment in recent years. These include immunosuppressants that target PD-1 on T cells and PD-L1 on tumor cells. The aim of this antitumor therapy is to activate CD8+ T cells so that they can kill tumor cells specifically. However, when this occurs, there are immune checkpoints aimed at preventing these processes, resulting in immunosuppression and consequent immune escape from tumors . It has However, a better understanding of the contribution of p38 to disease at both the cellular and organismal levels is needed and can be achieved by better defining the specific functions of this signaling pathway in specific tumors. In addition, future therapies may also involve collateral cancer processes such as directly related as angiogenesis or the tumor microenvironment modulation. Moreover, the existence of several isoforms that have been located at different levels of expression depending on the tissues and the degree of tumorigenicity opens the door to explore new diagnostic strategies based on the different expression and/or activity of these peculiar kinases. Recent studies have shown that the differentiation and quantification of individual alternative isoforms could improve insights into cancer diagnosis and outcome. However, there are limitations in the traditional methods used for determining isoforms, such as the level of transcription, low level of expression, and cross-linking of activity.Nevertheless, there is no doubt that, in the coming years, the development of new drugs targeting p38 will be able to improve therapeutic efficacy in diseases such as cancer in a successful way, in view of favoring a medicine of greater precision."}
{"text": "RB1 gene was the first tumor suppressor gene cloned from humans by studying genetic lesions in families with retinoblastoma. Children who inherit one defective copy of the RB1 gene have an increased susceptibility to retinoblastoma. Several years after the identification of the human RB1 gene, a targeted deletion of Rb was generated in mice. Mice with one defective copy of the Rb gene do not develop retinoblastoma. In this manuscript, we explore the different roles of the Rb family in human and mouse retinal development in order to better understand the species-specific difference in retinoblastoma susceptibility.The RB1 was acutely inactivated in the developing human fetal retina, p107 was not upregulated in a compensatory manner.We found that the Rb family of proteins  are expressed in a dynamic manner during mouse retinal development. The primary Rb family member expressed in proliferating embryonic retinal progenitor cells in mice is p107, which is required for appropriate cell cycle exit during retinogenesis. The primary Rb family member expressed in proliferating postnatal retinal progenitor cells is Rb. p130 protein is expressed redundantly with Rb in postmitotic cells of the inner nuclear layer and the ganglion cell layer of the mouse retina. When Rb is inactivated in an acute or chronic manner during mouse retinal development, p107 is upregulated in a compensatory manner. Similarly, when p107 is inactivated in the mouse retina, Rb is upregulated. No changes in p130 expression were seen when p107, Rb or both were inactivated in the developing mouse retina. In the human retina, RB1 was the primary family member expressed throughout development. There was very little if any p107 expressed in the developing human retina. In contrast to the developing mouse retina, when RB1 loss, but mice require both Rb and p107 gene inactivation.We propose that intrinsic genetic compensation between Rb and p107 prevents retinoblastoma in Rb- or p107-deficient mice, but this compensation does not occur in humans. Together, these data suggest a model that explains why humans are susceptible to retinoblastoma following  The timing of cell cycle exit is coordinated with cell-fate specification and differentiation in the developing central nervous system (CNS) to ensure that the appropriate number of neurons and glia are generated in the correct proportion. If too many neural progenitor cells exit the cell cycle during the early stages of development, then the overall number of cells is reduced, and the ratio of cell types shifts ,2.Rb1 thymidine ([3H]thy) with p107  was significantly higher than that in stage-matched wild-type retinas . The mean proportion of p107+ cells in the P2 Rb-/- retinas (7.3% \u00b1 1.1%) was also significantly higher than that in age-matched wild-type retina . The proportion of p107+ cells was lower than that of Rb+ cells, because there are fewer proliferating retinal progenitor cells at P2 than at E13.5 . To. ToRb allls Fig. . Those d to p107  were simRb inactivation [Rb at P3. Even though the majority (~ 95%) of cells in the P3 retina are postmitotic [Rb has been acutely inactivated are susceptible to deregulated proliferation. A similar experiment in which all proliferating retinal progenitor cells in the P3 retina were labeled with [3H]thy for 24 h prior to acute Rb inactivation showed the same result  after acute p107 inactivation , but one copy of p107 in Chx10-Cre;RbLox/-; p107+/- retinas is haploinsufficient to regulate proliferation. Specifically, in retinal progenitor cells, two copies of the p107 gene are required to compensate for loss of Rb, but only one copy of Rb is required to compensate for loss of p107.TUNEL assay results revealed no difference in the proportion of apoptotic cells in dk2 Fig.  and Sfrprp2 Fig. . TogetheRb;p107-deficient retinas thy-. If a retinal progenitor cell is more susceptible to deregulated proliferation after acute Rb inactivation, then we would predict that the majority of BrdU+ cells would be [3H]thy+. If both retinal progenitor cells and postmitotic cells divide after acute Rb inactivation, then the proportions of BrdU+, [3H]thy- cells and BrdU+, [3H]thy+ cells will reflect the proportions of postmitotic cells and mitotic cells in the starting population, respectively.The developmental studies and clonal analyses described above suggest that reciprocal compensation between Rb and p107 prevents deregulated retinal progenitor cell proliferation and retinoblastoma. However, we have shown that p107 compensation also occurs at P3 when the majority (93%) of cells are postmitotic Fig. . Therefothy Fig. , inactivtmitotic . If a po3H]thy+ cells  ectopically dividing cells were BrdU+, thy demonstrated that p107 is downregulated as cells exit the cell cycle. There were no defects in cell-fate specification or differentiation in p107-deficient retinas; thus, we propose that p107 primarily regulates retinal progenitor cell proliferation in the embryonic retina. Rb is expressed in postnatal retinal progenitor cells and differentiating neurons and glia. Consistent with a role in cell-fate specification or differentiation, Rb was expressed throughout the cell cycle and continued to be expressed as cells entered G0. In the absence of Rb, immature cells persist in the ONL and rod photoreceptors failed to form, a finding that indicates that Rb regulates the developmental processes in this specific sublineage.Immunostaining, in situ hybridization, real-time RT-PCR and immunoblot analyses revealed that p107 is expressed primarily in proliferating retinal progenitor cells of the embryonic mouse retina. Pulse-labeling with thy-labeling experiments, we predict that Rb and p130 are redundant in newly postmitotic cells of the INL and GCL. It is not known if p130 is required for terminal cell cycle exit in the developing retina, for maintaining cells in the postmitotic state or both.Our data suggest that Rb and p107 share the same role in regulating mouse retinal progenitor cell proliferation but the role of Rb is unique in rods. An extensive genetic analysis of p130 is beyond the scope of this manuscript, but based on our expression studies and [3H]thy-labeled dissociated cells and immunoblotting); these approaches provided the same overall picture of the dynamic changes in the expression of the Rb family during seven stages of mouse retinal development. Spencer and colleagues provided a more limited data set with internal inconsistencies. For example, immunostaining was done on samples from just three stages of mouse retinal development , and in situ hybridization was carried out for Rb but not for p107 or p130. Their real-time RT-PCR and immunoblotting data show high levels of p107 expression in the embryonic retinas, but no p107+ nuclei are detected in their immunostained sections. Clearly, all expression data are limited by the reagents being used. For example, it is possible that the Rb, p107 and p130 expression patterns in our study or that of Spencer and colleagues are broader than reported due to the limited sensitivity of the antibodies used. We have attempted to minimize such limitations by combining several independent approaches such as in situ hybridization, immunostaining, real time RT-PCR and dissociated cell staining. Nontheless, we anticipate that the retinal expression pattern of the Rb family will be further refined as new reagents become available.A recent study published by Spencer and colleagues characterized the expression of the Rb family in developing mouse retina and adult human retina . Our datThe Rb family proteins are expressed in a dynamic manner during mouse retinal development. We show here that p107 is expressed in proliferating retinal progenitor cells in the embryonic retina and is downregulated as cells exit the cell cycle and undergo terminal differentiation. Around birth, the expression of p107 tapers off, and that of Rb takes it place in proliferating retinal progenitor cells. Rb expression persists in postmitotic differentiating neurons and glia of the mouse retina. The p130 protein is expressed in the late postnatal period of mouse retinal development. As cells exit the cell cycle, they upregulate p130, and its expression persists in postmitotic differentiating cells of the INL and GCL. Based on the expression pattern alone, one might predict that proliferation would be deregulated in p107-deficient embryonic retinas and Rb-deficient postnatal retinas. However, we report here that when p107 is inactivated, Rb is upregulated in a compensatory manner in the embryonic retina. Similarly, when Rb is inactivated, p107 is upregulated in a compensatory manner in the postnatal retina. There is no evidence for compensation by p130 in either Rb- or p107-deficient retinas.p107 in this process. We show here that two copies of p107 are required for efficient compensation of Rb loss, but only one copy of Rb is required to compensate for p107 loss. This may indicate that there are different mechanisms for Rb and p107 gene compensation or that retinal progenitor cells exhibit different sensitivities to Rb and p107 protein levels. One intriguing possibility is that p107 haploinsufficiency for compensation may reflect the differential binding of Rb and p107 to different E2F family members.One of the interesting features of the compensation that we uncovered in this study is the haploinsufficiency of p107 is an E2F-responsive gene; therefore, the mechanisms for p107 compensation most likely involve a direct feedback loop thy labeling we used in our study compared to the BrdU labeling used by Chen et al. Alternatively, it could reflect the non-cell-autonomous effect of broad Rb inactivation in Pax6-Cre;RbLox/Lox; p107-/- retinas examined by Chen et al. compared with the more restricted mosaic Rb inactivation in Chx10-Cre;RbLox/-; p107-/- retinas that we investigated thy  or 10 \u03bcM BrdU for 1 h at 37\u00b0C. For these pulse-labeling experiments, retinas were dissected at four stages of development  and exposed to the label for various durations  to allow retinal progenitor cells to progress through the cell cycle. The p27Kip1 protein, which is upregulated at G1/G0, was used as an internal control  into the eye. Electroporation consisted of five 50-\u03bcsec pulses of 80 V each separated by 950-\u03bcsec recovery periods. As a control, retinas were electroporated with a plasmid lacking Cre. For explant cultures, the DNA was purified and resuspended in HBSS at 1 \u03bcg/\u03bcL.Electroporation consisted of five pulses of 25 V for 50 \u03bcsec each with 950-\u03bcsec recovery periods. For FACS purification of electroporated cells, retinas were dissociated as described previously ,43, resu\u00ae software (Applied Biosystems). TaqMan\u00ae probes were synthesized with 5'-FAM and 3'-BHQ. RNA was prepared using Trizol, and cDNA was synthesized using the Superscript system . Samples were analyzed in duplicate and normalized to Gapdh, Gpi1 and Mmt2 expression levels.Real-time RT-PCR experiments were performed using the ABI 7900 HT Sequence Detection System . Primers and probes were designed using Primer Express33P-labeled or DIG-labeled probes at the same specific activity and total mass). Hybridization was carried out at 60\u00b0C overnight, and the slides were washed in a series of SSC washes. The sections were then counterstained with a nuclear dye (Sytox green or PI) and dehydrated prior to immersion in autoradiographic emulsion. Exposure was carried out for 1 week before the blots were developed.For immunoblot analysis, retinas of C57Bl/6 mice were dissected at the following seven stages of development: E14.5, E17.5, P0, P3, P6, P12 and adult. Retinas were sonicated in RIPA with protease and phosphatase inhibitors  to clear the lysate. The debris was pelleted, and the concentration of each lysate was determined using a Bradford assay . Lysate (30 \u03bcg) was loaded onto each lane and transferred to nitrocellulose. Each antibody was used at a dilution of 1:1000, and HRP-conjugated secondary antibodies were used at 1:5000. The Amersham ECL system  was used to detect antibody binding per the manufacturer's instructions. For in situ hybridization, retinas from each stage of development were rapidly dissected, fixed for 1 h in 4% paraformaldehyde, cryoprotected in 30% sucrose/PBS and embedded in OCT before freezing on dry ice. Cryosections (14-\u03bcm thick) were cut, mounted on slides and dried. Tissue was rehydrated in and treated briefly with proteinase K. Slides were then acetylated in acetic anhydride/0.1 M TEA and dehydrated using an alcohol series. Prehybridization was carried out with formamide/SDS hybridization solution, and the riboprobes were added . Eyes were transported in RPMI medium on wet ice. Retinas were isolated and divided into 8 radial sections. Each section was placed on a polycarbonate filter and maintained in explant culture medium, as described previously for mouse retinas ,25,37. B4, embedded, sectioned and viewed by transmission EM.Bright-field and single-cell fluorescent images were obtained using a Zeiss Axioplan-2 fluorescent microscope with the Zeiss AxioCam digital camera. Fluorescent images of tissue sections were obtained using a Leica TCSNT confocal microscope. For EM, animals were anesthetized with avertin until a loss of deep tendon reflexes. Transcardial perfusion was performed with carboxygenated Ames Medium supplemented with 40 mM glucose to clear the vasculature, followed by perfusion with Sorenson's phosphate buffer (pH 7.2) containing 2% EM-grade paraformaldehyde and 1% EM-grade glutaraldehyde. Eyes were then harvested; a slit was made in the cornea to aid in diffusion; and the tissue was placed in 3% glutaraldehyde in Sorenson's phosphate buffer overnight. Tissue was washed with 0.2 M cacodylate buffer in 5% sucrose, post-fixed in 1% OsORetroviruses and retinal culture procedures have been extensively described elsewhere ,37,43.3H]-thymidine pulse-labeling experiment. All authors contributed to the preparation of the manuscript.SD carried out in situ hybridization for mouse and human Rb family genes and performed the square wave electroporation and FACS experiments on human and mouse retinae. BS analyzed the mouse retinae from the different genetic combinations of Rb and p107 deficient mice and performed the reciprocal compensation experiments. BS also assisted with single cell immunostaining and scoring and performed the real time RT-PCR these samples. RM developed and characterized the RB1 siRNA and performed the immunoblot experiments. DJ performed all EM analysis. MD performed the immunostaining of the Rb family in human and mouse retinae and performed the [Expression of the Rb, p107 and p130 during the cell cycle in retinal progenitor cells.Click here for fileHuman fetal retinal explant cultures.Click here for fileRb in the developing retina.Effects of acute inactivation of Click here for filep107 in the Rb-deficient retinae during development.Acute inactivation of Click here for fileExpression of Proliferation and Differentiation Markers in P0 Retinae Lacking Rb and/or p107.Click here for fileExpression of Proliferation and Differentiation Markers in P6 Retinae Lacking Rb and/or p107.Click here for fileExpression of Proliferation and Differentiation Markers in P12 Retinae Lacking Rb and/or p107.Click here for fileExpression of Proliferation and Differentiation Markers in P30 Retinae Lacking Rb and/or p107.Click here for fileClone composition and distribution from in vivo lineage analysis.Click here for fileAntibodies used for immunofluorescence on adult tissue.Click here for file"}
{"text": "The p53 gene is one of the best studied tumour suppressor genes. Recently we performed mutation analysis on the p53 gene in a large number of bone and soft tissue sarcomas, and found that approximately one-third of the sarcomas have some type of DNA alteration at the p53 locus . However, the expression of the p53 protein resulting from these alterations still remains to be clarified. In this study, p53 expression in the sarcoma tissues was analysed immunohistochemically using antibody PAb421 (Oncogene Science) and its relationship to DNA alterations was examined. Of 113 tumours, 29 (25.7%) showed positive staining for the p53 protein. These included 19 of 67 osteosarcomas, five of 20 chondrosarcomas, four of 11 malignant fibrous histiocytomas (MFHs) and one Ewing's sarcoma. In chondrosarcomas, most of the p53-positive tumours belonged to highly malignant and atypical tumour types , suggesting a role for p53 mutation in the progression of cartilaginous tumours. All the cases with a missense mutation showed strongly positive staining, while no immunoreactivity was observed in the remaining three-quarters with DNA alterations including gross rearrangement, frame-shift mutation, nonsense mutation or mutation at splicing site except in one case. These results demonstrated the dominance of the p53 mutations with null protein expression in bone and soft tissue sarcomas, showing a unique characteristic of these types of tumours compared with other malignancies such as colon carcinomas."}
{"text": "Recent data suggest that the function of p53 might also play a role in the regulation of organismal lifespan. Increased p53 activity leads to lifespan shortening in mice, while apparent reduction of p53 activity in flies leads to lifespan extension. Although the mechanism by which p53 regulates lifespan remains to be determined, these findings highlight the possibility that careful manipulation of p53 activity during adult life may result in beneficial effects on healthy lifespan.As guardian of the genome the tumor suppressor p53 controls a crucial point in protection from cellular damage and response to stressors. Activation of p53 can have beneficial (DNA repair) or detrimental (apoptosis) consequences for individual cells. In either case activation of  It has long been known that mice that are deficient for p53 are prone to develop tumors and die prematurely , surprisingly did not live longer than controls. Even more astonishing, instead of living longer, the mice with constitutively hyperactive p53 (through incorporation of a shortened form of p53 into the p53 tetramer), actually died earlier than their wild-type counterparts.Why does increased tumor protection not translate into longer lifespans for these animals? Several markers of aging, including muscle loss, osteoporosis, skin atrophy and defects in wound healing, were found to be greatly accelerated in mice with hyperactive p53. No markers of accelerated aging were found in the other two models, leading to the speculation that, rather than increased p53 activity per se, altered functionality of the p44(p24)/p53 complex might explain those premature aging phenotypes , but no Recently, a form of programmed cell death has been described that shares features of both apoptosis and necrosis and has subsequently been termed necroptosis (An alternative to losing important functionality in the brain by permanent cell loss would be to functionally disable or alter cells without actually killing them. In fibroblasts, replicative senescence constitutes such a pathway . ImportaFinally, p53 has been demonstrated to activate apoptosis in a transcription-independent manner through interaction with the pro-apoptotic Bcl-2 family member Bax , which cIn light of the fact that Dmp53 transcriptional profile changes during fly development, it might also be possible that Dmp53 function changes, exchanging function as a transcriptional regulator to that of a signaling pathway modulator through direct interaction. It is conceivable that p53 influences biological events other than apoptosis through these direct protein\u2013protein interactions, which might be a major mechanism in the adult fly. Interestingly, two-hybrid analysis has revealed protein interaction partners of Dmp53 that are involved in a variety of processes, including DNA repair and metabolism ("}
{"text": "Breast cancer subtypes identified in genomic studies have different underlying genetic defects. Mutations in the tumor suppressor p53 occur more frequently in estrogen receptor (ER) negative, basal-like and HER2-amplified tumors than in luminal, ER positive tumors. Thus, because p53 mutation status is tightly linked to other characteristics of prognostic importance, it is difficult to identify p53's independent prognostic effects. The relation between p53 status and subtype can be better studied by combining data from primary tumors with data from isogenic cell line pairs (with and without p53 function).The p53-dependent gene expression signatures of four cell lines  were identified by comparing p53-RNAi transduced cell lines to their parent cell lines. Cell lines were treated with vehicle only or doxorubicin to identify p53 responses in both non-induced and induced states. The cell line signatures were compared with p53-mutation associated genes in breast tumors.Each cell line displayed distinct patterns of p53-dependent gene expression, but cell type specific  commonalities were evident. Further, a common gene expression signature associated with p53 loss across all four cell lines was identified. This signature showed overlap with the signature of p53 loss/mutation status in primary breast tumors. Moreover, the common cell-line tumor signature excluded genes that were breast cancer subtype-associated, but not downstream of p53. To validate the biological relevance of the common signature, we demonstrated that this gene set predicted relapse-free, disease-specific, and overall survival in independent test data.In the presence of breast cancer heterogeneity, experimental and biologically-based methods for assessing gene expression in relation to p53 status provide prognostic and biologically-relevant gene lists. Our biologically-based refinements excluded genes that were associated with subtype but not downstream of p53 signaling, and identified a signature for p53 loss that is shared across breast cancer subtypes. The tumor suppressor p53 is mutated in 30% of breast cancers , but ratin vitro studies of gene expression have demonstrated that cell line models of luminal breast cancers show a strong stress response following chemotherapeutic treatment, with notable changes in p53-regulated genes such as p21 (Cip1). The same magnitude of p53-regulated responses was not observed for cell line models of basal-like breast cancer . Kaplan-Meier analysis showed that this signature significantly predicted OS  and RFS (p = 2.6 \u00d7 10-7) for the Chang et al. dataset, and disease-specific survival  for the Miller et al. dataset. We also performed multivariate analysis, using the Chang et al. dataset. Controlling for standard clinical predictors , Cox proportional hazards ratios were estimated for both OS  and RFS . Thus, independent of standard clinical predictors the p53 expression classifier significantly predicted both OS (p = 0.006) and RFS (p = 0.006).Kaplan-Meier survival analysis yielded highly significant survival differences between groups from Figure et al.), the gene expression classifier had 82% agreement with sequence-based mutation status. True mutation status data was not available for the Chang et al. data set, but our classifier had 82% agreement with sequence-based mutation status in the Miller et al. data set. We were able to examine the location and type of mutations and compare them to classifier results using the Miller et al. data. Of the 29 mutants incorrectly classified as wildtype, 25 (86%) were either missense mutations or in-frame insertions/deletions. This differs significantly (p = 0.02) from the percentage of mutations that were missense or in-frame among correctly classified mutants (58%). Among the missense tumors, mutations in DNA binding domains of the p53 protein were also significantly more frequent (p = 0.01) in tumors classified as mutant (87%) than wildtype (45%).In our training data set  and are likely to be regulated by multiple oncogenic pathways. Our aim was not to identify a new prognostic signature that improves on previously published signatures. Rather, we aimed to demonstrate that events that are downstream of functional p53 loss are clearly associated with prognostic outcome, and are therefore biologically relevant. The predictive accuracy of p53-dependent gene expression profiles  supportset al. and Miller et al. datasets. This high level of agreement across data sets attests to the fact that the signature is indicative of p53 across a wide range of cell backgrounds. If the signature were merely correlated with proliferation, ER status, or another tumor characteristic, then poor concordance with p53 mutational status would be expected in cross validation. The samples where gene expression and mutation status disagree may represent true differences in the functional p53 pathway. For example, the tumor BC606 was p53 wildtype by sequence but clustered with p53 mutants using the 52 gene classifier. This tumor overexpressed MDM2 mRNA (data not shown), a key negative regulator of p53. Among the false negatives (sequence mutant but wildtype expression signature), our analysis of the Miller et al. data showed that misclassified mutants had higher proportions of mutation types that are likely to be less deleterious (missense mutations and in-frame insertion/deletions).Our data analysis also showed that there was good agreement between mutation status and expression profiles. Using our 52 gene list, there was >80% agreement between p53 mutation status and p53 expression class in both the Sorlie In addition to identifying a stereotyped signature associated with p53 loss, these results demonstrate that the relative importance of p53-regulated functions such as cell cycle control, DNA repair, and apoptosis are subject to significant inter-individual variation. Each cell line displayed a unique p53 response signature. However, similarities according to cell type were also evident. Both of the HMEC-derived cell lines showed a greater response to p53 loss in the untreated state, while the MCF-7 and ZR-75-1 lines showed a stronger p53-regulated signature following DOX treatment. These results extend previous observations [Our data reconfirmed the complex relation between chemosensitivity and p53 status . Previouin vitro setting allowed for control of expression of p53 protein, breast cancer subtype, and p53-inducing events [in vitro approach is limited in that a small number of cell lines can be reasonably examined, representing only a handful of tumors. By combining the in vitro expression data with data from human tumors assayed before and after DOX treatment, we examined a much wider range of individual responses to p53 loss than cell line experiments could reasonably examine, and performed a controlled experiment that cannot be accomplished in humans. Previous studies have characterized p53-responses in breast cancer using gene expression data from tumors and statistical models to try to control the effects of breast cancer heterogeneity. For example, in Miller et al. [A strength of our study was the use of cell line experiments to control a range of variables that influence p53-response. The g events -29. Hower et al. , prolifer et al.  was deriMany of the genes associated with p53 loss in this analysis were of prior interest in breast cancer. For example, GATA3 is involved in growth control and maintenance of the differentiated state in breast epithelial cells and has been hypothesized to play a role in tumorigenesis of ER-positive breast tumors . p21  declare that they have no competing interests.MAT participated in study design, created the isogenic cell line pairs, performed toxicity assays, western blots, and microarrays, performed analysis and interpretation, drafted and revised the manuscript. JIH participated in study design, creation of cell line pairs, interpretation of results, and manuscript revisions. DSO performed survival and gene ontology analyses. XH performed microarrays. KAH participated in toxicity assays and manuscript revisions. CSB participated in interpretation and critical review of the manuscript. CP participated in study design and coordination, supervision of experimental conduct and analysis, interpretation of results, drafting and revision of the manuscript, and approved the final version. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Microsoft Excel spreadsheet (196 kB) containing lists of genes for which expression levels were significantly associated with p53 loss .Click here for fileCluster figure showing parents and RNAi-transductants for all four parent cell lines . Genes included are the 696 genes identified as significant by SAM analyses comparing parents to RNAi-transductants.Click here for file"}
{"text": "We show that the allelic composition of the glycolytic enzyme phosphoglucose isomerase(Pgi) has a significant effect on the growth of local populations, consistent with previously reported effects of allelic variation on flight metabolic performance and fecundity in the Glanville fritillary andColias butterflies. The strength and the sign of the molecular effect on population growth are sensitive to the ecological context , which affects genotype-specific gene flow and the influence of migration on the dynamics of local populations. The biological significance of the results forPgi is underscored by lack of any association between population growth and allelic variation at six other loci typed in the same material. In demonstrating, to our knowledge for the first time, that molecular variation in a candidate gene affects population growth, this study challenges the perception that differential performance of individual genotypes, leading to differential fitness, is irrelevant to population dynamics. These results also demonstrate that the spatial configuration of habitat and spatial dynamics of populations contribute to maintenance ofPgi polymorphism in this species.The dynamics of natural populations are thought to be dominated by demographic and environmental processes with little influence of intraspecific genetic variation and natural selection, apart from inbreeding depression possibly reducing population growth in small populations. Here we analyse hundreds of well-characterised local populations in a large metapopulation of the Glanville fritillary butterfly Pgi locus (an enzyme linked with flight metabolic performance) and local population growth within a metapopulation of butterflies.Researchers observe a link between the genetic composition of the The notion that a population's genetic composition influences its ecological dynamics is conceptually deeply rooted in population biology \u20133, but For gene, which encodes the cGMP-dependent protein kinase, influence foraging behaviour inDrosophila melanogaster larvae and adults. The analogous gene in honeybees influences the regulation of division of labour between forager and nurse bees  for populations with at least six copies ofPgi typed in the autumn 1995 sample and hence a minimum sample size of three larvae from three larval families (hence minimumtN was 3). The material included 139 such populations, but we lacked information for eight populations in 1996 and hence these populations had to be omitted. We added \u201c1\u201d to population size to allow the inclusion of populations that went extinct by 1996 (n = 34). Regional change in population sizes in the surroundings of the focal populationi is defined asi,trendN =i,tS1+/i,tS [i,*S is given byProportional change in population size  was calculated as/Si,t , whereSPgi-f allele in small and large habitat patches using the following results and assumptions. Among the 139 populations in 1995, the average sizes of \u201csmall\u201d and \u201clarge\u201d populations were 0.10 and 0.89 ha, respectively, using the limit ofA = 0.3 ha between the two size classes  gave the following daily probabilities of laying subsequent clutches, and the average clutch sizes: et al. ; hence wf and non-f females):f females before and after day 14, 0.05 and 0.3; non-f females before and after day 14, 0.05 and 0.15.In the experiment described by Hanski et al. , daily mL =f 0.2,Sf = 0.4, non-L =f 0.05, non-Sf = 0.4. The justifications are as follows. Hanski et al. [f females than old populations [f and non-f females, but it is likely that all butterflies have high emigration rate from very small patches (see [Proclossiana eunomia).Butterflies were assumed to stay in the natal patch until they became mated and had laid their first clutch, after which they emigrated with the following daily probabilities: et al.  found dalations . We do nes , which Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file."}
{"text": "Expression of the tumour suppressor gene p53 was examined in squamous cell carcinoma of the head and neck using two p53 antibodies, PAb 421 and PAb 1801. Elevated p53 expression was found in 67% of the 73 patients investigated. P53 expression was not found to correlate with whether the patient had been previously treated or not, nor any of the clinico-pathological parameters. However a correlation was found between the patients smoking history and positive p53 staining. Six out of seven non-smokers did not express p53 whereas 29 of 37 heavy smokers were found to have elevated p53 expression (P less than 0.005). Also, of a group of ten patients who had given up smoking more than 5 years ago, nine had elevated expression. Epidemiological studies have shown a correlation between heavy smoking and head and neck cancer. The present study indicate a genetic link for this correlation."}
{"text": "Rad51C mutant mice exhibit precocious separation of sister chromatids at metaphase II, a phenotype that reflects a defect in sister chromatid cohesion, not a lack of Holliday junction resolution. Here we discuss a model to explain how a Holliday junction resolution defect can lead to sister chromatid separation in mouse oocytes. We also describe other recent in vitro and in vivo evidence supporting a late role for RAD51C in homologous recombination in mammalian cells, which is likely to be resolution of the Holliday junction.DNA double strand breaks are efficiently repaired by homologous recombination. One of the last steps of this process is resolution of Holliday junctions that are formed at the sites of genetic exchange between homologous DNA. Although various resolvases with Holliday junctions processing activity have been identified in bacteriophages, bacteria and archaebacteria, eukaryotic resolvases have been elusive. Recent biochemical evidence has revealed that RAD51C and XRCC3, members of the RAD51-like protein family, are involved in Holliday junction resolution in mammalian cells. However, purified recombinant RAD51C and XRCC3 proteins have not shown any Holliday junction resolution activity. In addition, these proteins did not reveal the presence of a nuclease domain, which raises doubts about their ability to function as a resolvase. Furthermore, oocytes from infertile  The breaks generated as a result of this cleavage could be sealed by a DNA ligase, resulting in intact resolved products. A search for resolvases in other organisms resulted in the identification of one of the best-characterized resolvases, RuvC of E. coli [E. coli RuvC have been identified like CCE1 in yeast, RuvC in Lactoccocal bacteriophage and A22R in Poxvirus [Exchange of genetic material by homologous recombination during meiosis is essential for generating genetic diversity in living organisms. It also results in the formation of chiasmata, that are the cytological manifestation of crossovers and ensure proper chromosome segregation. Homologous recombination is important for error-free repair of damaged DNA as well . One of  E. coli ,6, which E. coli . RecentlPoxvirus -11. SomePoxvirus ,13. In aPoxvirus -16.Saccharomyces pombe raised hopes that the eukaryotic resolvase has been identified [S. pombe mus81 mutants are infertile due to failed meiotic recombination, which could be rescued by the expression of bacterial RusA resolvase [Saccharomyces cereviciae (Mus81-Mms4), Arabidopsis (AtMUS81/At4g30870) and humans (Mus81-Eme1 or Mms4) [S. pombe mutants, S. cereviciae mus81 mutants are partially fertile, and MUS81-deficient Arabidopsis plants and mice are fully fertile, with no defect in meiotic recombination [Although many resolvases have been identified in prokaryotes, few have been found in eukaryotes. The yeast CCE1 is encoded by a nuclear gene but functions as a resolvase in the mitochondria ,17. Idenentified . It has entified . Geneticesolvase . These sor Mms4) -26. Howebination -23,27,28bination .The idea that RAD51 paralogs may be involved in resolution of HJs came from the observation that the protein fraction with HJ resolution activity contained RAD51C and XRCC3, members of the RAD51-like protein family . This obArabidopsis [Studies in chicken B-lymphocyte DT40 cells have shown that only RAD51 is essential for cell viability, while loss of other RAD51 paralogs does not affect cell survival . Interesbidopsis -40. Deplbidopsis . These fbidopsis ,42. Hereirs3 defective in RAD51C was shown to lack the HJ processing function. Recently, mouse embryonic fibroblasts generated from Rad51c knockout embryos revealed marked reduction in HJ activity [XRCC3, irs1SF, also showed a defect in HJ resolution activity, while mutation in another RAD51 paralog, XRCC2, had no effect on HJ resolution [In a very significant study, Liu et al. demonstrated that RAD51C is required for the resolution of HJs in mammalian cells . After aactivity . Interessolution . AlthougMore recently, in a follow-up study, Liu et al. provide new biochemical evidence to link RAD51C and XRCC3 to the HJ processing function . Using aMlh1-deficient mice, which have a severely reduced number of crossover sites, also showed a marked reduction in RAD51C foci. This observation strengthens the notion that RAD51C foci formation during pachytene depends on the generation of crossovers, which is one of the last steps of homologous recombination. Surprisingly, co-localization of MLH1 and RAD51C foci on the bivalents was not observed. Also, XRCC3 foci were not detected on these bivalents. These observations raise concerns and are currently difficult to explain. The lack of co-localization of RAD51C and MLH1 may be due to temporal differences in their localization at crossover sites. It is interesting that, although no XRCC3 foci were observed on autosomal bivalents, XRCC3 foci, along with RAD51C and MLH1 foci, were present in the pseudoautosomal region of the sex chromosomes, a region on X and Y chromosomes that undergoes an obligatory crossover event. These foci in the pseudoautosomal region were not detected in Mlh1-deficient spermatocytes, supporting their dependence on a crossover event.In addition to the biochemical studies, Liu et al. have presented interesting immunofluorescence data showing the localization of RAD51C on meiotic chromosomes, which further supports its role in late stages of homologous recombination . Since vin vivo model system. In Drosophila, the loss of RAD51C-like protein encoded by spn-D gene results in a meiosis-specific defect and may play a role similar to DMC1 [Arabidopsis revealed that RAD51C is essential for repair of Spo11-induced DSBs during prophase I of meiosis [Rad51c-deficient plant meiocytes fail to synapse and become severely fragmented. These results support a role for RAD51C early in meiotic recombination but do not shed light on its role later in the process.It was surprising that Liu et al. did not detect RAD51C foci during the leptotene and zygotene stages of prophase I, where it is expected to play an important role based on its known function in the early stages of homologous recombination. However, failure to observe RAD51C foci does not rule out its functional importance at this stage. Different antibodies and more sensitive imaging methods may help resolve this discrepancy. In the meantime, it may be interesting to examine the consequence of the loss of these proteins in an  to DMC1 . Recent  meiosis ,46. Homoin vivo, as a defect in the former may result in the arrest of meiocytes. Because RAD51C-deficient mice die during embryogenesis and no suitable meiosis-specific Cre transgenic mouse line is available yet, it is a challenge to generate a suitable mouse model to study the meiotic functions of RAD51C. We recently reported the generation of a hypomorphic allele of Rad51c that results in a reduction of the protein level due to aberrant splicing [in vivo model system to study the role of RAD51C in meiotic recombination. In addition, the meiotic phenotype associated with the loss of RAD51C function is sexually dimorphic, showing an early meiotic defect in males and a late defect in females, which provides a unique opportunity to study the dual function of RAD51C in mouse meiosis.Because RAD51C is predicted to have a dual function, an early and a late role in homologous recombination, it may be difficult to demonstrate the latter function splicing . This abInfertile males revealed an early role for RAD51C in meiosis, marked by the spermatocyte arrest at leptotene and early zygotene stages, reduction of RAD51 foci at leptotene, and persistence of DNA breaks and unsynapsed chromosomes at pachytene. This finding is consistent with the known function of RAD51C in DSB repair but does not provide any evidence to implicate RAD51C in the late steps of the recombination.Brca2 in the gonads have been generated and shown to have a defect in the early stages of meiotic recombination [Brca2 mutant oocytes displayed a wide range of abnormalities, including a 2.5-fold reduction in the number of oocytes that progressed to metaphase II, compared to controls and major defects in chromosome segregation into polar bodies. It will be interesting to determine whether these oocytes display any PSSC defect at metaphase II.Histological examination of the ovaries of mutant females revealed a defect in ovulation, which could be partially rectified by hormonal treatment. The number of oocytes was markedly reduced, and the embryos that developed from these oocytes suffered from severe developmental defects. The reduced number of oocytes suggests that some oocytes may have been lost due to a defect in the early meiotic prophase I. While all oocytes obtained after superovulation progressed normally to metaphase I, they displayed a number of abnormal features at metaphase II. These abnormalities included aneuploidy and broken chromosomes, but most strikingly, precocious separation of sister chromatids  declare that they have no competing interests."}
{"text": "Patients with breast cancer treated with MPA often report an improvement in appetite. Similar appetite stimulation is seen in patients treated with some corticosteroids, but MPA has a potential advantage over these drugs in that it does not exert a catabolic effect. MPA  has therefore been compared with placebo in 60 patients with advanced malignant disease. Twenty-one patients in the MPA group and 20 in the placebo group were receiving chemotherapy. Patients were treated for 6 weeks and were assessed at weeks 0, 3 and 6 for appetite, energy, mood and pain using visual analogue scales. Nutritional status was assessed by the measurement of serum proteins and anthropometrics. Karnofsky score was recorded as a measure of performance status. There was a significant improvement in appetite in the MPA group between weeks 0 (pre-study) and 3 (P = 0.0002) and 0 and 6 (P = 0.015). There was no significant improvement in appetite in the placebo group. Supporting this finding was the significant increase in serum thyroid binding pre-albumin and retinol binding protein in the MPA group between weeks 0 and 3 and 0 and 6 (P = 0.023 and P = 0.039 respectively). These two parameters showed no significant change in the placebo group. There was no change in anthropometric measurements, weight, performance status, energy, mood or pain in either group. These data indicate that there was a significant increase in appetite in anorexic patients with advanced cancer treated with MPA which was reflected in increases in rapid turnover proteins reported to reflect nutritional status. However, this apparent increase in appetite did not result in improved weight, performance status, energy levels, mood or relief of pain. Further studies to investigate the effect of higher doses of MPA are indicated."}
{"text": "The availability of antibodies which recognise p53 protein in paraffin-embedded tissue has created the opportunity to use immunohistochemistry to study the expression of p53 in a wide variety of clinical material. In this paper we have investigated the relationship between the type of fixative and the pattern of p53 staining in mammary carcinoma. Optimal results were obtained from breast tissue fixed in phenol formol saline, methacarn or cold formol saline with positive staining for stabilised p53 protein occurring in 69/95 (73%) cases studied. Care must be taken in the interpretation of these results since positive staining for p53 protein is not always indicative of mutation of the p53 gene. Furthermore, a range of staining patterns is seen in mammary carcinomas, making interpretation difficult. Assessment of staining needs to be standardised in order that different studies can be compared. However, in breast carcinoma, p53 immunohistochemistry appears to give information relating to tumour grade and, independently, to prognosis."}
{"text": "As with miRNAs a decade ago, the scientific community recently understood that lncRNAs represent a new layer of complexity in the regulation of gene expression. Although only a subset of lncRNAs has been functionally characterized, it is clear that they are deeply involved in the most critical physiological and pathological biological processes. This review shows that in ovarian carcinoma, data already available testify to the importance of lncRNAs and that the demonstration of an ever-growing role of lncRNAs in the biology of this malignancy can be expected from future studies. We also underline the importance of their relationship with associated protein partners and miRNAs. Together, the available information suggests that the emerging field of lncRNAs will pave the way for a better understanding of ovarian cancer biology and might lead to the development of innovative therapeutic approaches. Moreover, lncRNAs expression signatures either alone or in combination with other types of markers  could prove useful to predict outcome or treatment follow-up in order to improve the therapeutic care of ovarian carcinoma patients. Ovarian cancer is the leading cause of death from gynecological malignancies worldwide. More than 230,000 new cases are diagnosed each year, at a late dissemination stage in most cases, leading to the death of about 140,000 women . Wh. Whcis  cell line was associated with a loss of cell polarity, as well as with increased capacities of adhesion to the peritoneum. This latter finding may be of some importance as spreading in the intraperitoneal cavity and peritoneal carcinomatosis invasion is a very common feature of ovarian cancer [n cancer .AB073614 is an lncRNA that was recently found to be overexpressed in ovarian cancer versus normal tissue in publicly available databases. The authors also checked for AB073614 expression in cancer tissue versus normal adjacent tissue in a cohort of 75 ovarian cancer patients and observed that AB073614 high expression was correlated with a shorter 5-year overall survival . In vivo, AB073614 downregulation in xenografted mice led to reduced tumor growth and reduced expression of proliferation- and invasion-related proteins (PCNA and MMP2 and MMP9 respectively). In vitro, AB073614 was greatly overexpressed in HO-8910 and OVCAR3 cell lines and its downregulation decreased proliferation and induced cell death in them. In addition, PCNA, MMP2 and MMP9 were also decreased in vitro in response to AB073614 downregulation, as well as members of key signaling pathways such as the phosphorylated forms of AKT and ERK. Although the mechanisms of action of AB073614 remain to be identified, it appears to be a key regulator of critical processes in ovarian carcinoma cells [ma cells .HOST2 is one of the five human ovarian cancer specific transcripts (HOSTs) identified by a SAGE study from a database of 137 SAGE libraries of normal and neoplastic ovarian tissue. Five tags were specifically expressed in ovarian cancer with one of them, HOST2, corresponding to a transcript lacking a coding capacity [myc, hmga2, dicer and imp3 [capacity . HOST2 wand imp3 .LSINCT5 has been shown to be overexpressed in many breast and ovarian cancer cell lines and tumor samples. LSICNT5 down-regulation decreases cell proliferation in breast cancer cell lines. A microarray analysis found that LSINCT5 down-regulation triggered the deregulation of 816 genes, of which 95 were deregulated at least 2-fold. Of those, the down-regulation of CXCR4 was postulated by the authors to be responsible for the effects on cell proliferation, but this remained to be demonstrated experimentally [mentally . In anotmentally . AlthougFAL1 was recently identified by Hu et al. [p21 (CDKN1A), leading to sustained proliferation and reduced senescence. Conversely, down-regulation of FAL1 by siRNA delivery in mice xenografted with ovarian carcinoma cells reduced tumor growth, decreasing cell proliferation and increasing apoptosis.u et al. . This lnPVT1 is an lncRNA located in the 8q24 chromosomal region. This region also harbors myc, which is amplified in 45% of ovarian carcinomas. Guan et al. [et al. [pvt1 and myc duplication in almost every tumor bearing myc amplification. Their analysis included more than 30 000 tumors from the Progenetix dataset and more than 10 000 from TCGA. When all cancers were considered, more than 18% of them presented the duplication of both pvt1 and myc, whereas fewer than 0.5% showed only myc or pvt1 duplicated. In more than 1 000 ovarian cancers from TCGA, more than 45% displayed both pvt1 and myc amplification, with only a few cases (<1%) having only myc or pvt1 duplicated. Interestingly, this study also suggested that the multiple miRNAs encoded in the pvt1 region did not play a significant role in MYC/PVT1-driven tumorigenesis [et al. [pvt1 inactivation impaired the development of tumor xenografts originating from the colon cancer cell line HCT116. This striking discrepancy might be due to the use of tumor cell lines from diverse origins (ovarian or colon cancer cells) and therefore to the existence of different cellular contexts. This underlines the need for further studies on the exact role of the myc/pvt1 couple in ovarian cancer.n et al.  reported [et al.  challengigenesis . In addiigenesis . Howeverigenesis , it woul [et al.  where pvXIST is a major player in the complex mechanism leading to heterochromatinization and subsequent transcriptional inactivation of one of the two X chromosomes in women, a phenomenon necessary for dosage compensation. A role for XIST has been described in several malignancies but its effects are dependent on the cancer type. For example, XIST expression is upregulated in glioblastoma tissue and stem cells and its knockdown exerts tumor suppressive functions. These tumor suppressive effects do not rely directly upon the effects of XIST but on miR-152, which is released from its interaction with XIST. Indeed, miR-152 binds to XIST and this miRNA is able to induce anti-tumor effects comparable to XIST downregulation, supporting a ceRNA relationship between miR-152 and XIST in this pathology [r = 0.653, P = 0.001). In the same study, a reduced expression of XIST in several ovarian cancer cell lines was associated with an increased resistance to paclitaxel. However, no modification in the response to cisplatin was observed in cell lines with reduced XIST expression. [athology . Conversathology . In ovarathology . A reducression. . OverallHOTAIR is an lncRNA expressed from the HOXC locus on chromosome 12. It has been implicated in the proximal and distal orientation during development by inducing the transcriptional silencing of genes in trans in the HOXD locus on chromosome 2 [vs. 61 months), similar to FIGO stage and histological grade [et.al [et.al [in vivo cell growth and/or capacity of adhesion to the peritoneum [mosome 2 . It has mosome 2 . In breamosome 2 . In humamosome 2 . In a coal grade . Howeveral grade . The fire [et.al  includedl [et.al  includedl [et.al . Similarl [et.al . Moreoveritoneum . Interesritoneum . This obritoneum .HOXA11-AS is an lncRNA located in the HOXA locus in chromosome 7 that harbors several coding and non-coding transcripts. HOXA coding genes regulate mullerian duct differentiation and are not expressed in ovarian surface epithelium under normal conditions. However, they are re-expressed in ovarian cancer, including some in a subtype-specific manner. For example, HOXA9 is re-expressed in serous, endometrial and mucinous subtypes, irrespective of grade, whereas HOXA10 is expressed in endometrial and mucinous but not serous subtypes [HOXA locus hosts 3 lncRNAs, namely HOXA10-AS, HOXA11-AS and HOTTIP. A recent study looked for variants of these 3 lncRNAs in a cohort of 1947 ovarian cancer patients (1201 of them of serous subtype) and 2009 control cases. No significant variant was identified, although the authors described an A>T variant in HOXA11-AS that was marginally associated with reduced ovarian cancer risk (p = 0.06). Expression of HOXA11-AS was reduced in ovarian tumor versus matched normal tissue in a cohort of 18 ovarian cancer patients, irrespective of variant status. Plasmid-based expression of HOXA11-AS, either with the \u201cA\u201d or \u201cT\u201d allele, in OVCA-433 and C19 ovarian cancer cell lines reduced proliferation, migration and invasion in vitro, as well as tumor burden in vivo in a xenograft model with C13 cells. Interestingly, expression of the \u201cT\u201d minor allele increased the inhibitory effects of HOXA11-AS in vitro and in vivo, in line with a putative reduced risk for ovarian cancer [trans regulation of distant genes. However, the mechanisms of the HOXA11-AS effects remain unknown, as well as the reason why the \u201cT\u201d allele displays an increased inhibitory effect. In this regard, the A>T variation does not change the secondary structure of HOXA11-AS. In view of this and given the lack of coding potential for HOXA11-AS, understanding why the \u201cA\u201d and \u201cT\u201d variants show significantly different activities could be of importance for further understanding the modes of action of HOXA11-AS.subtypes . The 5-pn cancer . The autANRIL is an lncRNA originating from the 9p21 chromosomal region in the same location as the p14, p15 and p16 genes, which play a central role in cell cycle regulation, apoptosis and senescence. The ANRIL transcript encompasses p14, p15 and p16 and is transcribed antisense to them. SNPs in the anril locus have been associated with a number of pathologies including cardiovascular diseases, diabetes and endometriosis [in vitro in ovarian cancer cell lines through the modulation of MET and MMP3, although the authors did not determine whether or not this regulation is dependent on p14, p15 and p16 [etriosis . ANRIL eetriosis  by epigeetriosis . The resetriosis  and prometriosis . A recen and p16 . It has  and p16 .MEG3 is an imprinted, maternally expressed lncRNA associated with PRC2, most likely via its EZH2 subunit [Meg3 promoter was reported to be methylated and MEG3 expression was absent or reduced in ovarian cancer tissues and cell lines. Its re-expression decreased OVCAR3 cell line growth and proliferation [brca1/2 [meg3 promoter methylation [ subunit . It is e subunit , which i subunit , 91. Its subunit . The Megferation . Interesferation , whereas[brca1/2 . It coulhylation . Howeverhylation . Anotherhylation . This obhylation . Howeverhylation .H19 is one of the most widely studied lncRNA and is at the center of a complex regulatory network involving the implication of miRNAs in diverse ways. H19 expression is restricted to fetal tissue and adult muscle under normal conditions [et al. [cis on the igf2 gene and in trans on the imprinted gene network controlling growth in mice [in vivo [nditions . Apart f [et al. ) through in mice , H19 has in mice  and to b in mice , 101 inc in mice . Alterna[in vivo .et al. in a muscle differentiation model [One role of H19 is through its ability to sponge the family of tumor suppressor let-7 miRNAs, as shown by Kallen on model . In ovaron model , suggestin vivo in mice where H19 overexpression increased the metastatic behavior of lung carcinoma cells [However, the involvement of H19 in cancer biology has been mainly described owing to its role as a precursor for miR-675. This miRNA displays an oncogenic role in cancer cell lines from various origins. The tumorigenic functions of miR-675 are mediated, at least in part, thanks to its ability to target directly the tumor suppressor RB in colorectal cancer  and hepama cells . TherefoBMI1 and EZH2, which are members of the PRC1 and PRC2 complexes respectively, and therefore partners in the action of lncRNAs in the control of gene expression, have been shown to be deregulated in ovarian cancer.BMI1 plays an oncogenic role in epithelial malignancies. It is frequently overexpressed in breast, cervical, endometrial and ovarian cancer [Several lines of evidence show that n cancer , 109. Inn cancer  and is an cancer . InteresEZH2, a methyltransferase subunit of the PRC2 complex, has been shown to be associated with advanced stages of ovarian cancer and to be independently associated with shorter overall survival in ovarian cancer patients [in vitro. In addition, miR-101 was found to target EZH2 directly in ovarian cancer cell lines. MiR-101 is under-expressed in ovarian cancer tissues and its low expression correlates with FIGO stage but not with tumor grade [In vivo, miR-101 restoration leads to the inhibition of growth of ovarian tumor xenografts. In another study using a cohort of patients with ovarian carcinoma, the authors confirmed several observations already made by Semaan et al. [in vivo. Finally, they demonstrated that miR-101 was less expressed in ovarian cancer cell lines resistant to cisplatin than in their sensitive counterparts and that forced miR-101 ectopic expression re-sensitized resistant cells to cisplatin, which is in agreement with the EZH2-induced resistance to cisplatin described in ovarian cancer cells [p21 and to an increase in p21 transcription [Furthermore, an elevated expression of patients . EZH2 knor grade . In vivon et al. . They foer cells . These fcription , which icription .Another interesting miRNA in the context of EZH2 is let-7. In ovarian cancer, most let-7 family members appear to be down-regulated , lncRNAs expression signatures could be used to predict outcome or response to treatment in order to improve the therapeutic care of ovarian carcinoma patients. The recent discovery that miRNAs are present in blood and other body fluids and the potential use of circulating miRNAs as diagnostic or prognostic tools in ovarian carcinoma have opened up new perspectives for the use of easily accessible biomarkers. Regarding lncRNAs, no study has yet reported their presence in serum or plasma from ovarian cancer patients. However, proof of concepts for the meaningful use of lncRNAs present in body fluids has been obtained with the characterization of urinary PCA3 levels as a diagnostic tool in prostate cancer , and cirSince several lncRNAs are overexpressed (See Table Another strategy to modulate lncRNAs activity, when it relies on chromatin modification mediated by PRC1 and PRC2 complexes, is to use the pharmacological inhibitors of these proteins. Specific inhibitors of EZH2 methyltransferase activity such as GSK126, EPZ005687 and EI1 have been recently developed . Therefoet. al. presented an original strategy in which they used a construct of diphtheria toxin under the control of the H19 promoter. The transfection of this plasmid, BC-819, led to the selective killing of malignant H19-overexpressing cells. Local IP administration of the plasmid vectorized with PEI has reached phase I in ovarian carcinoma patients, with no reported toxicity and encouraging preliminary clinical data [Furthermore, the characterization of the genes and pathways modulated by lncRNAs could lead to the identification of potential therapeutic targets for which inhibitors are under development or already available in clinical practice. In addition, the identification of the regulators of lncRNAs expression might provide interesting tools. Using this concept, Mizrahi cal data , 101. ThIn summary, the emerging field of lncRNAs has created new opportunities of investigation for a better understanding of ovarian cancer biology. Although limited to a few examples so far, the huge amount of lncRNAs whose roles and functions have yet to be unraveled holds great promise for major improvements in the understanding and future management of this disease."}
{"text": "Fear is an important emotional reaction that guides decision making in situations of ambiguity or uncertainty. Both recognition of facial expressions of fear and decision making ability can be impaired after traumatic brain injury (TBI), in particular when the frontal lobe is damaged. So far, it has not been investigated how recognition of fear influences risk behavior in healthy subjects and TBI patients. The ability to recognize fear is thought to be related to the ability to experience fear and to use it as a warning signal to guide decision making. We hypothesized that a better ability to recognize fear would be related to a better regulation of risk behavior, with healthy controls outperforming TBI patients. To investigate this, 59 healthy subjects and 49 TBI patients were assessed with a test for emotion recognition  and a gambling task (Iowa Gambling Task (IGT)). The results showed that, regardless of post traumatic amnesia duration or the presence of frontal lesions, patients were more impaired than healthy controls on both fear recognition and decision making. In both groups, a significant relationship was found between better fear recognition, the development of an advantageous strategy across the IGT and less risk behavior in the last blocks of the IGT. Educational level moderated this relationship in the final block of the IGT. This study has important clinical implications, indicating that impaired decision making and risk behavior after TBI can be preceded by deficits in the processing of fear. In social situations human information processing capacity is to a large extent taken up by the processing of emotional information that is crucial for making adequate decisions \u20133. In siStudies from different fields suggest that the experience of emotions is an important process linked to emotion perception. Theories of \u2018embodied cognition\u2019 state that facial emotion perception involves perceptual, somatovisceral, and motoric re-experiencing of the relevant emotion in one's self . In a stA frequently used task to measure risky decision making is the Iowa Gambling Task (IGT) . The IGTThe ethical committee of the University of Groningen approved of the collection of data of healthy volunteers. Assessment of TBI patients was part of the inclusion procedure for a study on behavioral sequelae after TBI, which was approved by the medical ethical committee of the University Medical Center Groningen, the Netherlands. All participants gave informed written consent prior to study inclusion for their information to be used for research, and were treated in accordance with the declaration of Helsinki.The patient group consisted of 49 moderate to severe TBI patients. At the time of assessment, all patients were outpatients who were seen in the sub-acute or chronic stage for clinical follow-up by the trauma neurologist or the rehabilitation physician of the University Medical Center Groningen. Clinicians referred patients for assessment of behavioural and social cognitive deficits.TBI patients were included if they were free from neurological conditions other than TBI, psychiatric conditions and substance abuse pre- or post-injury. Mean age of the 49 patients was 44.6 years . The group consisted of 38 male and 11 female patients. Mean educational level was 5.1  primary school only to 7) university level). There was no significant relationship between age and educational level of TBI patients . For 43 patients data were available concerning the duration of Post Traumatic Amnesia (PTA); the mean PTA duration was 25.9 days (SD 25.1), with a range from 1 to 112 days. For 25 patients Glasgow Coma Scale scores were available, ranging from 3 to 15, with a mean of 8.6 (SD 4.0). Of 42 patients imaging data (MRI or CT) with regard to frontal damage was available: 27 patients had visible lesions to the frontal lobe, while 15 patients did not have frontal lesions. Mean time since injury (TSI) was 104.8 months (SD 103.3), with a broad range of 4 to 402 months.2 = 2.8, p =.10).Normal healthy volunteers participated in the study, matched to patients based on age, gender and educational level. They were recruited through newspaper advertisement and from social networks of the investigators. They were free from alcohol or drug abuse, and had no psychiatric or neurological history. Data were collected from 59 participants, 37 men and 22 women. Mean age of the group was 43.5 years . Mean educational level was 5.4 . Age of control subjects appeared to be negatively related to their educational level . Patients and healthy controls did not differ significantly in age , distribution of educational level  or distribution of gender (XDecision making was assessed using the Iowa Gambling Task (IGT). The current Dutch computerized version of the IGT is an implementation based on the original description . ParticiDeck A and B are \u2018risky\u2019 and will eventually lead to a loss of money and deck C and D are \u2018safe\u2019 and will lead to profit. The frequency and magnitude of gains and losses differ for each deck and must be learned by the participant during the task. See Cards are drawn 100 times. For each 20 draws, the number of cards drawn from advantageous decks (C+D) are subtracted from the number of cards chosen from the disadvantageous decks (A+B) resulting in 5 net block scores.Recognition of basic emotions  was measured using the Ekman 60 Faces Test from the Facial Expression of Emotion- Stimuli and Tests (FEEST) ,51. The The course of performance in the IGT was analyzed using General Linear Model, Repeated Measures Analyses, with Difference contrast and Group as a Between-subjects variable. Box\u2019 test was used to test the homogeneity of variance-covariance matrices assumption which was always met. When the assumption of sphericity was not met, a Greenhouse-Geisser correction was applied. When variances were equal, Bonferroni post-hoc procedure was used, with unequal variances Games-Howel post-hoc test was applied. When patients and healthy controls were compared, T-tests were used for parametric data and Mann-Whitney U tests for ordinal data. Pearson\u2019s correlation was used for parametric data and Spearman\u2019s rho for ordinal data. The influence of emotion recognition on IGT performance was analyzed with GLM Repeated Measures, with net block scores (CD-AB) and choices B as outcome measures. Fear, Anger, Sadness, Disgust, Happiness, Surprise, Age and Educational level were entered as covariates; Group and Gender were used as Between-subjects-factors. These analyses were subsequently performed with Fear as emotion recognition covariate together with the covariates age and educational level. Group and Gender were entered as Between-subjects-Factors. In analyzing the course of net block scores and the course of choices for deck B in TBI patients, duration of PTA and TSI were entered as covariates, while the presence/absence of frontal lesions was entered as Between-subjects-factor. Alpha levels were two-tailed with p <. 05. All statistical analyses were performed with IBM SPSS Statistics 20.Repeated measure analysis on the 5 net block scores (C+D\u2014A+B) of the IGT showed an overall increase of positive net block scores from block 1 to block 5  = 3.2, p =.02) for both patients and healthy controls  = 0.5, p =.74). Healthy controls and TBI patients differed significantly in their performance on the IGT  = 5.4, p =.02). Healthy controls made more advantageous choices than TBI patients in Block 3  = 3.9, p =.05), while this difference just did not reach significance in block 5  = 3.5, p =.07).Age appeared to influence the course of net block scores in both patients and controls  = 2.6, p =.05; Block*age*group, F =.71, p =.57). Educational level or gender did not significantly interact with course of net block scores  = 1.6, p =.17; Block*gender, F = 1.9, p =.12) in both healthy controls and TBI patients  = 1.2, p =.29), Block*education*group, F =.32, p =.84).In TBI patients, PTA duration  =.34, p =.79), TSI  =.46, p =.71) and the presence of frontal lesions  =.10, p =.96) did not affect the course of net scores from block 1 to block 5. See In healthy controls, women had significantly higher scores on fear recognition than men . Healthy females were significantly better than healthy males in recognizing anger and disgust . In addition, total FEEST scores were significantly higher in healthy females than in healthy males .In healthy controls, higher educational level was significantly related to higher fear recognition , higher anger recognition , higher recognition of disgust  and higher total FEEST score . Age was significantly related to total FEEST score, with higher age related to poorer emotion recognition .In TBI patients, there were no significant differences between men and women in the recognition of the six basic emotions. Age of patients was not significantly related to emotion recognition. Higher educational level was significantly related to higher fear recognition ability  and to poorer recognition of surprise . PTA duration was not significantly related to emotion recognition ability. TSI was not significantly related to emotion recognition, except for a significant positive correlation between TSI and recognition of anger . Mean emotion recognition scores of patients with (n = 27) and without (n = 15) frontal lesions did not differ significantly.Repeated measure analysis on the influence of emotion recognition ability on the course of net block performance (A+B\u2014C+D) in the IGT showed that fear recognition was significantly related to the course of decision making in the IGT  = 3.2, p =.02). Recognition of anger  =.69, p =.58), disgust  =.78, p =.52), happiness  = 1.2, p =.33), sadness  =.44, p =.75), and surprise  =.72, p =.56) were not significantly related to the course of decision making on the IGT. Three way interactions with block, emotions and group were not significant, indicating no differences in the influence of emotion recognition on the course of decision making for healthy controls and TBI patients. Three way interactions with demographical variables , block and emotions showed no significant effects,Both fear recognition and decision making were significantly worse in TBI patients compared to healthy controls. In healthy controls, but not in TBI patients, fear recognition correlated significantly with total profit on the IGT .Fear recognition scores were related to the course of net block performance (A+B\u2014C+D) in the IGT for both healthy controls and TBI patients. GLM repeated measure analyses showed a main effect of block  = 3.3, p =.01), an interaction between fear recognition and the course of net block score  = 5.9, p =.00) but no interaction between course, fear and group  = 1.1, p =.38). Thus, fear recognition ability significantly interacted with the course of choice behavior in the IGT in healthy controls and patients. Post-hoc contrast showed the interaction between fear and choice behavior in the IGT to be significant in block 4  = 4.3, p =.04) and block 5  = 15.2, p =.000). This effect was similar for both groups  =.05, p =.83; Block5 vs previous, Block*Fear*Group, F = 2.1, p =.15). Higher fear recognition ability was related to more advantageous choices in the last blocks of the IGT in both healthy controls and TBI patients.Age was significantly related to the course of net block scores in the IGT for patients and controls. Age, however, did not interact significantly between fear recognition and net block scores in the IGT  = 2.0, p =.10). Educational level was positively related to fear recognition ability in patients. Educational level also interacted with fear recognition and net block scores in the IGT  = 2.8,p =.03), which appeared to be similar for patients and controls. Contrasts showed that this interaction effect was present in the last block of IGT  = 4.6, p =.04). When age was also entered into this model, it did not appear to significantly moderate the relationship between educational level and fear recognition  = 2.0, p =.10).Fear recognition ability was significantly better in female compared to male controls. Gender, however, did not interact significantly between fear recognition and course of IGT net block scores  = 2.1, p =.09). This was found in patients and healthy controls  = 1,5, p =.13).With regard to risky choices, healthy controls and TBI patients differed significantly in the amount of total choices for deck B. See Repeated measure analysis on the influence of emotion recognition ability on the course of choices for deck B in the IGT showed that only fear recognition was significantly related to the course of choices of deck B  = 4.5, p =.002). Recognition of anger  = 1.6, p =.19), disgust  =.45, p =.76), happiness  =.78, p =.53), sadness  =.74, p =.55), and surprise  =.28, p =.88) were not significantly related to the course of choices for deck B on the IGT. Three way interactions with block, separate emotions and group were not significant, indicating no differences in the influence of emotion recognition on the course of choices for deck B for healthy controls and TBI patients.Analyzed as a separate emotion, fear recognition ability significantly interacted with the course of choice behavior of deck B in both patients and controls  = 7.3, p =.000; Block*Fear*Group, F =.54, p =.69). Post-hoc contrast showed that fear recognition interacted with choosing deck B in block 3  = 12.8, p =.001) and block 5 of the IGT  = 13.9, p =.000). This interaction was similar in patients and healthy controls. Further analyses showed a negative correlation in both groups, with higher fear recognition abilities related to choosing deck B less often .Age did not interact significantly with the course of choices for deck B  = 1.3, p =.10), nor with fear recognition and the course of choices for deck B  = 1.1, p =.33). Educational level interacted with the course of choices for deck B  = 2.5, p =.05), but there was no significant three-way-interaction between educational level, fear recognition and the course of choices for deck B  = 2.1, p =.09). Gender was neither related to the course of choices for deck B  = 1.4, p =.25), nor did it show an interaction between fear recognition and course of choices for deck B  = 1.2, p =.32).In TBI patients, the presence of frontal lesions was neither related to the course of choices for deck B  = 0.1, p =.95) nor did it show an interaction between fear recognition and choices for deck B  =.11, p =.95). PTA-duration was not related to the course of choices for deck B  =.29, p =.83). Also, PTA-duration did not interact with fear recognition ability in influencing the course of choices for deck B  = 1.4, p =.25). TSI was neither related to the course of choices for deck B  =.62, p =.60) nor did it interact with fear recognition ability in influencing the course of choices for deck B  =.81, p =.49).This is the first study that investigated and indeed found a relation between facial fear recognition ability and gambling behavior in both healthy subjects and patients with moderate to severe TBI. In both groups, a higher ability to recognize fear, but not a higher ability to recognize other basic emotions, was related to the development of a more advantageous strategy and to the course of risky choices across blocks in the IGT. As expected, patients with TBI were significantly more impaired in recognizing fear from faces than healthy subjects \u201315. MoreIn both TBI patients and healthy controls decision making on the IGT was related to the ability to perceive fear. This relationship between perception and action is thought to be mediated by the ability to subjectively experience fear ,28. A reIn the present study, we also investigated in TBI patients and healthy controls whether fear recognition influences decision making under ambiguity or whether it is related to more deliberate behavior. Maia and McClelland (2004) identified three levels of awareness in the IGT . At the In the current study, fear recognition was not only related to overall decision making in both groups, but in particular to risky choice behavior at the end of the IGT. In both TBI patients and control subjects we found that fear was explicitly used as a warning signal not to choose disadvantageously. Heilman and colleagues (2010) also found in healthy subjects that explicit use of fear was related to risk behavior in the IGT . In the As expected, we found that TBI patients performed more poorly on the IGT than healthy controls. Originally, the IGT was developed to assess deficits in affective decision making after ventromedial prefrontal cortex (VMPFC) damage . HoweverIn both healthy controls and TBI patients, a negative effect of age on net block performance on the IGT was found. Performance on the IGT is based on localized but also on distributed mental information processes such as memory functioning, which are sensitive to age ,50,72. WAccording to Yechiam and colleagues (2005) different psychological components can underlie poor performance on the IGT, including a motivational tendency to attend to gains, a learning process to attend to recent outcomes and a response tendency with poor consistency of choices . With reThe present study design has some limitations. Fear recognition and risk behavior were measured, with the assumption that a better identification, experience and regulation of fear underlies behavior. It would have been valuable to additionally measure the ability of subjects to experience and appraise fear, either by questionnaires or by physiological measures. This might also give more insight into the specific deficits in the experience of fear in TBI patients. The advantage of the inclusion of an emotion recognition task is, however, that it allows objective measurement. Different from questionnaires, its responses do not depend on patients\u2019 insight, which can also be impaired after TBI. The patient group used in the current study is a selection of patients with moderate to severe injury and behavioral difficulties. It is important to also analyze this relationship in a larger and more heterogeneous sample of TBI patients. The majority of the TBI patients in this study was male, and the inclusion of more female patients is preferred. In a greater sample of male and female patients, gender differences with regard to the influence of fear recognition on decision making can be analyzed in greater detail and with more power. However, male dominance is a characteristic of the TBI population .In conclusion, the most important finding of the present study is that fear recognition is related to deliberate choice and risk behavior in both healthy controls and TBI patients suggesting strongly that deficits in behavior are preceded by deficits in emotion processing. This is a clinically relevant finding because deficits in emotion processing might be the target of research and treatment for TBI patients with risky and inappropriate behavior. So far, only few studies have investigated the effect of training of underlying deficits in emotion processing in TBI patients ,77,78. A"}
{"text": "Gastroenteropancreatic neuroendocrine carcinomas (GEP-NECs) are aggressive, rapidly proliferating tumors. Therapeutic response to current chemotherapy regimens is usually short lasting. The aim of this study was to examine the expression and potential clinical importance of immunoreactive p53 protein in GEP-NEC.Tumor tissues from 124 GEP-NEC patients with locally advanced or metastatic disease treated with platinum-based chemotherapy were collected from Nordic centers and clinical data were obtained from the Nordic NEC register. Tumor proliferation rate and differentiation were re-evaluated. All specimens were immunostained for p53 protein using a commercially available monoclonal antibody. Kaplan-Meier curves and cox regression analyses were used to assess progression-free survival (PFS) and overall survival (OS).All tumor tissues were immunoreactive for either one or both neuroendocrine biomarkers (chromogranin A and synaptophysin) and Ki67 index was >20% in all cases. p53 immunoreactivity was only shown in 39% of the cases and was not found to be a prognostic marker for the whole cohort. However, p53 immunoreactivity was correlated with shorter PFS in patients with colorectal tumors  in a univariate analysis as well as to poorer PFS  and OS  in patients with colorectal tumors with distant metastases, a correlation which remained significant in the multivariate analyses.In this cohort of GEP-NEC patients, p53 expression could not be correlated with clinical outcome. However, in patients with colorectal NECs, p53 expression was correlated with shorter PFS and OS. Further studies are needed to establish the role of immunoreactive p53 as a prognostic marker for GEP-NEC patients. Their proliferative capacity is high, with Ki67 proliferation index >20% and/or mitoses >20 per 2 mm2 .GEP-NECs account for approximately 35\u201355% of all extra-pulmonary NECs. They are mainly located in the esophagus, stomach, pancreas, colon and rectum, but in 30% of cases, they present as tumors of unknown primary location .The present WHO 2010 classification for NENs G3 has been debated for not being optimal as it assumes that all G3 tumors are poorly differentiated. Furthermore, the WHO G3 group includes all tumors with Ki67 index above 20% as one disease entity. In recent publications, the presence of tumors that are well-differentiated, but with Ki67 index >20% and with a better prognosis than poorly differentiated GEP-NECs was demonstrated . HoweverThere is a reported increase in incidence of GEP-NECs over the years, but there is still a lack of effective treatment resulting in persistent poor survival for these patients , 6. In tPlatinum-based chemotherapy has been used as a first-line treatment for GEP-NECs since the nineties and the Nordic, European and North American Societies of neuroendocrine tumors (NETs) recommend combination chemotherapy with cisplatin/carboplatin and etoposide \u201310. In tTP53 is a known tumor suppressor gene normally present in all human cells and the p53 pathway is usually activated by different types of stress signals due to e.g. DNA damage [TP53 are common and occur in many cancer types, including NEC: 70\u2013100% of tumor cells have been shown to be mutated in high grade pulmonary NECs [TP53 mutations are associated with poorer clinical outcome, treatment resistance and higher degree of metastases in different types of cancer [TP53 have been shown to result in an immunohistochemically detectable expression of the p53 protein; since the mutated protein is not degraded, it accumulates into tumor cell nuclei [A damage . The tumA damage . Mutatioary NECs . Furtherf cancer \u201315. Mutal nuclei .A few studies have investigated the immunohistochemical expression of p53 protein in carcinomas and most of them have shown heterogeneity in the outcome , 17, 18.We hypothesized that immunohistochemical expression of p53 protein is associated with shorter progression-free survival (PFS) and OS and might be of prognostic relevance in GEP-NEC patients.This cohort included patients diagnosed with poorly differentiated GEP-NEC with a primary tumor located in the gastrointestinal tract or a cancer of unknown primary (CUP). CUP was defined as NEC with predominant abdominal metastases but where no primary tumor could be identified.Tumor specimens were collected retrospectively based on availability from the Nordic NEC Study, resulting in 124 GEP-NEC patients treated with a platinum-based chemotherapy at the Nordic Centers, and diagnosed 1999\u20132011. Clinical data was obtained from the Nordic NEC register [Formalin-fixed paraffin-embedded (FFPE) material included: 40 needle biopsies, 20 surgical biopsies and 64 surgical specimens. All tumors were immunoreactive (IR) for CgA and/or synaptophysin and all tumors had Ki67 index >20%. An endocrine pathologist (LG) recalculated the frequency of Ki67 IR tumor cells in all tumor samples and the morphological differentiation (well vs. poorly) was re-assessed by a panel of experienced neuroendocrine pathologists.Fifty-seven tumor specimens were retrieved from primary tumors and 67 from metastases. Exclusion criteria were Ki67 index <20% and the diagnose of a mixed adenocarcinoma-neuroendocrine carcinomas (MANEC) based on the WHO definition describing these tumors as having an exocrine and endocrine component where the neuroendocrine component is present in at least 30% of the tumor .Patients were divided into groups depending on location of the primary tumor: esophagus, stomach, pancreas, colon and rectum, or CUP. Four tumors were located in the esophagus, 11 in the stomach, 28 in the pancreas, 31 in colon and 17 in rectum. In 33 cases, the primary tumor was unknown.The clinical variables chosen to be investigated in the statistical analyses included age (median age 60 years), Ki67 index, LDH, therapeutic response  and performance status (according to the Eastern Cooperative Oncology Group consensus (ECOG)).Specimen size was included to evaluate if larger specimens yielded more tumor cells IR for p53. A sub-analysis for PFS and OS was done for patients with distant metastases for each group of primary tumor location.FFPE tissue specimens were cut into approximately 4-\u00b5m thick sections and attached to positively charged glass slides .Before immunostaining, the sections were treated in a pressure cooker reaching maximum temperature of 121\u00b0C using Tris-HCL buffered saline, pH 9.0 as retrieval solution. The sections were incubated with a primary monoclonal antibody  at room temperature for 30 minutes (dilution 1:100). A polymer-detection system was used  according to manufacturer\u2019s instructions. Diaminobenzidine was used as chromogen.The Ki67 index was calculated in a light-microscope at a magnification of x40, with a square grid in one of the ocular to facilitate the cell counting. At least 2000 tumor cells were counted in the areas with the highest tumor cell proliferation. In small biopsies, containing less than 2000 tumor cells, all tumor cells were counted. The Ki67 index was expressed as the percentage of IR tumor cells.The presence of p53 immunoreactivity was semi-quantitatively estimated (in percent) by assessing the area of IR tumor cells vs the total tumor area by light-microscopy at a magnification of x40. Ten percent or more p53 nuclear IR tumor cells in the tumor area was considered as positive outcome based on the results from a previous study performed with the same antibody .Photographs were taken using a Zeiss Observer Z1 microscope and the Axiovision software .TP53 mutation, was used as a positive control, and omission of the primary antibody was used as a negative control.Colon adenocarcinoma tissue, with a known The defined event was death from any cause. PFS was defined as the time between date of first treatment and date of tumor progression and OS was defined as time from diagnosis of locally advanced or metastatic disease until date of death; or if event was not found, censored at date of last observation.Kaplan-Meier plots were used for PFS and OS analysis, and the log-rank test was used to compare curves separated according to expression of p53. Cox proportional regression was performed for the estimation of hazard ratios (HRs) and confidence intervals (CIs).Spearman correlation was used to assess the correlation of p53 protein expression to clinicopathological variables. For the statistical analyses all variables were dichotomized: p53 IR vs. non-IR, age \u226460 years vs. >60 years, Ki67 \u226555% vs. \u226455% LDH normal vs. high and performance status ECOG 0+1 vs. 2+3.All statistical analyses were performed using IBM SPSS statistics software .Local ethics committees in the Nordic countries from which tissue samples were collected approved the research protocol.The study was approved and the need for consent was waived by the local ethics committee, Regionala etikpr\u00f6vningsn\u00e4mnden , in Uppsala, Sweden.Of the 124 tumors stained, 39% (n = 48) were p53 IR . The freColon primary was the tumor group with most frequently p53 IR cells followed by pancreas, CUP, rectum, stomach and esophagus. In both the p53 IR and p53 non-IR groups, approximately 80% had distant metastases and Ki67 was >55% in a majority of patients in both groups .Based on the p53 staining, two different patterns could be distinguished: one with scattered cells and the other with densely packed cells. Five of the 48 p53 IR tumors (10%) showed a scattered pattern, where 20\u201340% of the tumor cells were p53 IR. The remaining 43 (90%) had a homogenous pattern where 60\u2013100% of the tumor cells were IR .Statistical analyses of the whole cohort with all clinical variables dichotomized showed that p53 immunoreactivity was positively correlated with Ki67 with a higher frequency of p53 IR cells for patients with Ki67 index above 55%. The tumor specimen size correlated positively with p53 immunoreactivity in the whole cohort i.e. large specimens were more often IR. There was no correlation between p53 expression and small cell or large cell morphology.For patients with colorectal tumors, a positive correlation was found between p53 immunoreactivity and performance status showing that those with p53 IR tumors had a poorer performance status compared to those with non-IR tumors. For patients with colorectal tumors and distant metastases, p53 immunoreactivity correlated negatively with treatment response. No significant result was obtained for correlation with response from sub-analysis of patients with metastatic primaries in esophagus, the gastric mucosa or pancreatic or in the CUP subgroup.Spearman\u2019s correlations are presented in Kaplan-Meier analysis, dichotomized for p53 immunoreactivity, including all 124 patients did not show any differences in PFS and OS  . In the In univariate analysis, p53 immunoreactivity showed a significant correlation with shorter PFS in colorectal patients  and patients with colorectal tumors and distant metastases showing p53 immunoreactivity had a significantly shorter PFS and OS  compared to patients with non IR- tumors .In the group of colorectal patients with distant metastases, these associations remained significant in multivariate analysis adjusted for age, Ki67 index, LDH and the ECOG for performance status . The ranDCR after platinum-based chemotherapy in this cohort was 67% .In the group of patients with colorectal tumors with distant metastases, a positive correlation was found between p53 immunoreactivity and disease control by chemotherapy . For patThe p53 positive control showed IR tumor cells. When omitting the primary antibody, the immunoreactivity was completely abolished.To our knowledge this is the largest study investigating p53 protein expression as a possible prognostic marker for GEP-NEC patients. We found that patients with colorectal NECs with distant metastases, expressing p53, had a significantly shorter PFS and OS, than those lacking p53 expression. This is in line with the correlation between performance status and response within the same group. Furthermore, the positive correlation between p53 expression and Ki67 indicates that p53 may be a marker for poorer prognosis .In accordance with these findings, others have also suggested that p53 expression might be useful to distinguish between GEP-NECs and tumors belonging to the recently recognized category of well differentiated G3 NENs [Two immunohistochemical staining patterns were observed for p53 expression. One subgroup of tumors exhibited scattered p53 IR tumors cells, where 20\u201340% of the tumor cells were IR, while another subgroup displayed densely packed tumor cells with 60\u2013100% of tumor cells showing immunoreactivity. This is in accordance with a study of p53 in gastric cancer where similar staining patterns were observed. However, as in this study, no correlation was seen between these patterns and clinicopathological parameters , i.e. thWe found that tumor specimen size correlated positively with p53 protein expression in the way that surgical specimens were more frequently IR for p53 than needle biopsies. This finding is rather unexpected since patients undergoing surgery are more likely to have a better performance status and expected longer OS, and presence of p53 IR tumors is hypothesized to be associated with poorer outcome. One possible explanation might be that biopsies can be less representative than larger specimens.TP53 gene have been observed in a significant number of cases, suggesting it may have a role in NEC development [TP53 gene was confirmed in a recent report, in which next-generation sequencing of 50 cancer-related genes in 23 NEC tumors showed several different mutations, but with the presence of TP53 mutation in the majority of tumors. A high number of tumors with TP53 mutation has also been demonstrated in pancreatic NEC-patients [Genetically, GEP-NECs show chromosomal instability. Alterations in the elopment , 23. FreTP53 mutation in tumors, and consequently, provides a quicker screening method to choose appropriate treatment [Results from a study of ovarian carcinoma demonstrate that p53 expression can be used as an alternate marker for reatment . Howeverreatment .There was a significant correlation between response to chemotherapy and p53 expression in patients with colorectal tumors with distant metastases; patients with a p53 expression had a poorer response to platinum-based chemotherapy. These patients also had a worse prognosis in general, and patients with p53 IR tumors scored lower on the ECOG scale for performance status.In conclusion, p53 expression was correlated with poorer survival and poorer response to chemotherapy in patients with colorectal NECs, especially for those with distant metastases. To confirm the possible prognostic role of p53 in GEP-NECs, additional genetic studies are necessary. These should preferably be prospective and include genotyping to compare genetic information with p53 immunoreactivity and other possible biomarkers such as RB1, ATRX and DAXX to further investigate the relationship to clinical outcomes."}
{"text": "S. cerevisiae has been particularly useful. This seemingly naive model allows the expression of a functional human p53 and thus the assessment of p53 intrinsic transcriptional activity. The aim of this article is to review the various contributions that the budding yeast has made to the understanding of p53, p63 and p73 biology and to envision new possible directions for yeast-based assays in the field of cancer as well as other p53-family-related diseases.Since its discovery in 1979, p53 has been on the forefront of cancer research. It is considered a master gene of cancer suppression and is found mutated in around 50% of all human tumors. In addition, the progressive identification of p53-related transcription factors p63 and p73 as well as their multiple isoforms have added further layers of complexity to an already dense network. Among the numerous models used to unravel the p53 family mysteries,  Saccharomyces cerevisiae has proven to be a precious tool to unravel its mysteries. 37 years after p53 original discovery, this article reviews the various uses of S. cerevisiae and envisions new possible applications for already existing yeast-based assays, as well as the creation of original yeast models dedicated to the study of the p53 family.Mutation of p53 is the most common genetic alteration in human cancers , 2, makiS. cerevisiae took part in the early identification of p53 function. In order to verify the suspected transcription activation role of p53, R.W. O\u2019Rourke et al. tested its ability to activate the transcription of the CAT (chloramphenicol acetyltransferase) reporter gene. The amino-terminal fragment of p53 was indeed able to induce transcription and p53 was thus proposed to be a transcription factor of the tumor suppressive response . Al. Al32]. Although p63 is scarcely found mutated in cancers, mutations in this gene are strongly linked to developmental syndromes such as ADULT  and EEC  . The majBaker's yeast allows the expression of a functional human p53 protein, which is isolated from its natural mammalian partners making it a seemingly ideal model to study p53 intrinsic transcription factor activity and the impact of its mutations.Homo sapiens, Mus musculus, Xenopus laevis, Danio rerio, Drosophila melanogaster and Caenorhabditis elegans) using a firefly reporter system under the control of various p53-REs sequences . Significant variations in sequence specificity, temperature sensitivity or transcriptional potential of p53 were highlighted between these different species [Yeast has also been used to assess the divergence of p53 transcriptional activity throughout evolution. M. Lion et al. compared the transcriptional response of p53 from several species , apoptosis-inducing factor (YNR074C or AIF1), endonuclease G (NUC1) or Bcl2-like proteins (Ybh3p) [NUC1 [S. cerevisiae, p53-WT, but not p53-R248W, induces signs of apoptosis which are associated with the repression of thioredoxin genes (TRX1/2) and the production of high levels of reactive oxygen species [ACT1 which over-expression is known to be toxic in yeast [in levuro may appear limited, it provides interesting elements to the understanding of p53 family transcriptional role. And even if the quest for a p53 orthologue in yeast remains unsuccessful to date, answers may come from digging deeper in the function of the older evolutive member p63, which has been implicated in female germline protection during meiosis in vertebrates [In yeast, the apoptotic machinery seems conserved, since several key elements homologues have been identified that include caspases ( (Ybh3p) . Confirmp) [NUC1 . Moreove species . In addiin yeast , 61. Of in yeast . p53 hasin yeast . The exitebrates . This futebrates , 66 and M. trossulus [Yeast two-hybrid (Y2H) allows the identification of protein-protein interactions and has been used to explore the vast interaction network of p53. Y2H allowed the identification and characterization of some of p53 most prominent partners like Mdm2. Mdm2 is an E3-ligase which is a core regulator of p53 in charge of maintaining cellular p53 levels low in the absence of stress by promoting its ubiquitination and consequently its degradation by the 26S proteasome. Mdm2 was also found to inhibit p53 transcriptional activity by binding to its transactivation domain . Y2H wasrossulus . Two othrossulus . Severalrossulus , BAF60 [rossulus , Ki-1/57rossulus  or influrossulus . In addirossulus , 53BP1 [rossulus  or MBP1 rossulus . More rerossulus . Among trossulus .Due to their late identification, p63 and p73 are not renowned for their large social networks but can be expected to close the gap in a near future. At least two p63 partners have been discovered using Y2H: Stxbp4  and SetdInteractions within the p53 family have also been subjected to Y2H experiments and showed strong homotypic interactions  but much lower heterotypic interactions due to the diversification of their respective tetramerization domains . Howevervia its C-terminal domain in yeast, although no RNA sequence or structure specificity was identified. Specific p53-RNA interaction might indeed be dependent on post-translational modifications of its C-terminal domain that do not occur in yeast [Yeast three-hybrid, a variant of the yeast two-hybrid, allows the detection of interactions between proteins and RNA. Due to the highly basic composition of the p53 C-terminal domain, K.J.-L. Riley et al. looked for possible RNA sequences able to associate with p53. They showed that p53 is indeed able to bind RNA sequences in yeast .in vitro, many have fallen short in vivo [The discovery of drugs targeting p53 remains highly dynamic but in spite of multiple promising candidates identified  in vivo . Two appMdm2 interacts with p53 in yeast and induces a diminution of its transcriptional activity. However Mdm2 does not induce a significant degradation of p53 in yeast, but can bind to its transactivation domain. Mdm2 would thus interfere with transcription cofactors such as 53BP1, through a competitive mechanism. These results indicate that, although most of the p53 pathway is absent from yeast , the intADE2 reporter gene by Firefly or Renilla luciferase reporters allowing a miniaturization of the assay and hence a high throughput. Compounds targeting the p53-Mdm2 interaction (Nutlin and RITA) are also effective in yeast and restore p53 transcriptional activity, thereby validating the yeast model for further screening. In contrast, PRIMA-1, which restores mutant p53 transcriptional activity in vitro, happens to be inactive in yeast [in silico for their ability to bind Mdm2 and then in yeast for their ability to restore p53-dependent growth inhibition. Several candidate drugs were isolated that include pyranoxanthone that turned out to be also active in mammalian cells [In order to use yeast as a model for pharmacological screening, FASAY has once again been adapted by replacing the in yeast . Recentlan cells , \u03b1-Mangoan cells . The saman cells  and led an cells . Nutlin-an cells . Unlike an cells .p53 has travelled across numerous models, most of the time with its mammalian companions, and seems to fit in everywhere but it definitely found a second home in baker's yeast. From the early years of p53 characterization to recent high-throughput pharmacological screenings, yeast has proven to be a versatile tool, which helped unraveling p53 mysteries. Now 21 years old, FASAY has undergone various transformations and evolutions to accompany the new questions about p53, p63, p73, their mutants and isoforms and it will probably not stop there as the p53 field is far from drying out. The p53 family remains an \u201cunresolved puzzle\u201d  of whichIn a sense, p53 really recalls the disease it contributes to cause. In the early developments of chemotherapy, the question asked was: is it possible to destroy cancerous cells while preserving the sane ones? With p53 one can wonder: how can we disable its mutants or isoforms while preserving its functional elements? The answer will not come easily, but baker's yeast is likely to help solving this critical issue."}
{"text": "Acetylcholine and dopamine are neurotransmitters that play multiple important roles in perception and cognition. Pharmacological cholinergic enhancement reduces excitatory receptive field size of neurons in marmoset primary visual cortex and sharpens the spatial tuning of visual perception and visual cortical fMRI responses in humans. Moreover, previous studies show that manipulation of cholinergic or dopaminergic signaling alters the spatial tuning of macaque prefrontal cortical neurons during the delay period of a spatial working memory (SWM) task and can improve SWM performance in macaque monkeys and human subjects. Here, we investigated the effects of systemic cholinergic and dopaminergic enhancement on the precision of SWM, as measured behaviorally in human subjects. Cholinergic transmission was increased by oral administration of 5 mg of the cholinesterase inhibitor donepezil, and dopaminergic signaling was enhanced with 100 mg levodopa/10 mg carbidopa. Each neurotransmitter system was separately investigated in double-blind placebo-controlled studies. On each trial of the SWM task, a square was presented for 150 ms at a random location along an invisible circle with a radius of 12 degrees of visual angle, followed by a 900 ms delay period with no stimulus shown on the screen. Then, the square was presented at new location, displaced in either a clockwise (CW) or counterclockwise (CCW) direction along the circle. Subjects used their memory of the location of the original square to report the direction of displacement. SWM precision was defined as the amount of displacement corresponding to 75% correct performance. We observed no significant effect on SWM precision for either donepezil or levodopa/carbidopa. There was also no significant effect on performance on the SWM task  for either donepezil or levodopa/carbidopa. Thus, despite evidence that acetylcholine and dopamine regulate spatial tuning of individual neurons and can improve performance of SWM tasks, pharmacological enhancement of signaling of these neurotransmitters does not substantially affect a behavioral measure of the precision of SWM in humans. Spatial working memory (SWM) refers to the short-term storage of locations of items not currently present in the environment for immediate use. The limits on working memory can be quantified by measuring capacity (the amount of information that can be remembered) as well as precision . In the domain of visual SWM, precision is often quantified as the average distance in the visual field between the encoded location and the location reported during retrieval.Neural correlates of SWM precision have been described in macaque dorsolateral prefrontal cortex (dlPFC). Here, neurons exhibit sustained spiking activity during a delay period between encoding and retrieval, and the magnitude of this activity varies as a function of the remembered location  completed the donepezil study, and 20  completed the levodopa/carbidopa study. One female subject from the donepezil study and two female subjects from the levodopa/carbidopa study were excluded from the analyses because their calculated SWM thresholds were greater than the maximum displacement we tested (described in \u201cStimuli and Task\u201d section below).Subjects were not enrolled in the study if they reported that they smoked tobacco, were taking any drugs that could affect cholinergic (for the donepezil study) or dopaminergic (for the levodopa/carbidopa study) function, or had a history of substance abuse, heart arrhythmia or heart problems, neurological or psychiatric illness, or liver disease. Because levodopa/carbidopa can cause hypotension, blood pressure was measured just before administration of levodopa/carbidopa (or placebo). Participants were required to have a resting blood pressure reading between 100/60 mmHg and 140/90 mmHg and a pulse rate above 60 bpm to continue in the experiment. Participants\u2019 ages ranged from 18 to 27 (donepezil study) and from 19 to 31 (levodopa/carbidopa study).We employed a double blind within-subject experimental design in which each subject ingested either placebo or an active drug  on different days. Carbidopa was co-administered in order to inhibit peripheral metabolism of levodopa, thereby allowing more levodopa to cross the blood-brain barrier  they did not take during the second session. Participants waited 3 h after ingesting donepezil and 45 min after ingesting levodopa/carbidopa to begin the SWM task, intervals that correspond to the time to reach peak plasma concentration after oral ingestion for each drug . Across all eccentricities, the mean hit rate was 61%, and the mean correct reject rate was 75%. It should be noted that we used a 500 ms stimulus presentation time in this control experiment instead of the 150 ms stimulus duration used in the SWM experiments, as 150 ms is not enough time for the subjects to make an eye movement to the target while it was still being displayed. This 150 ms stimulus duration was selected to discourage eye movements to the stimulus to be remembered during the SWM task.During the SWM experiment, participants were encouraged to take breaks whenever they wanted to, and they communicated this by either withholding their response or informing the experimenter, who would then pause the experiment after the subject\u2019s response. Additionally, the experimenter explicitly asked participants if they wanted to take a break every time they completed 20% of the trials .For the baseline session, the set of displacements was 0.3, 1, 2, 3, 4, 6, 8 and 10 degrees (defined as the polar angle between the encoded stimulus and the probe). Performance was plotted as a function of this displacement angle Figure  to comput(37) = \u22121.49, p = 0.14). We therefore included all collected data in our analyses.For the pharmacology sessions, displacements ranged from 0.3 to 12 degrees of polar angle, with the intervening displacements at 10%, 30%, and 60% above and below the subject\u2019s threshold (computed from the baseline session). The baseline session had 960 trials, and the pharmacology sessions had 1080 trials each. Due to experimenter error, for a subset of the participants (10 in the donepezil study and seven in the levodopa/carbidopa study), data were not collected at a displacement of 60% above threshold. In order to estimate the effect of this missing data, we removed the 60% above threshold data point from those subjects with a complete data set and then recomputed the thresholds. We found that there was no significant difference between thresholds calculated from the complete data set and those from the data that were missing the 60% above threshold value  = 0.10, p = 0.73) or levodopa/carbidopa (t(17) = 0.49, p = 0.12; Figure To assess stability of SWM precision across multiple testing sessions, we compared threshold displacement (measured in units of degrees of polar angle) for the two pharmacology sessions in each study (acetylcholine and dopamine) using paired t(17) = \u22120.25, p = 0.81) or between levodopa/carbidopa and placebo (t(17) = 0.80, p = 0.44; Figure We observed no significant difference in SWM precision thresholds between donepezil and placebo (t(17) = 0.46, p = 0.65; levodopa/carbidopa: t(17) = 0.50, p = 0.62; Figure We also examined the effects of cholinergic and dopaminergic enhancement on overall task performance (percent correct) and again observed no significant drug effects (donepezil: t(17) = 1.21, p = 0.24) or levodopa/carbidopa (t(17) = 0.50, p = 0.62) on lapse rate  or levodopa/carbidopa /(SWM placebo threshold + SWM drug threshold)) for each study. This contrast index will have a value of zero when the drug has no effect on displacement threshold, positive values when the drug enhances precision (decreases threshold), and negative values when the drug reduces precision (increases threshold). This correlation was not significant for either donepezil (t(35) = \u22120.70, p = 0.48) or between the left and right hemifields (t(35) = \u22121.25, p = 0.21). Lower visual field advantages in perception have often been measured for stimuli on or near the vertical meridian  = 0.71, p = 0.47).Finally, we explored whether SWM precision varies across different locations in the visual field. There is a well-established lower visual field advantage in performance for a variety of visual perception tasks  = 2.24, p = 0.03; Figure t(17) = \u22121.21, p = 0.23; Figure t(17) = 1.52, p = 0.15; Figure The oblique effect is another well-studied anisotropy in visual perception across visual field locations Appelle, , charactThe purpose of this study was to investigate the effects of cholinergic and dopaminergic enhancement on SWM precision, using the cholinesterase inhibitor donepezil and the dopamine metabolic precursor levodopa, respectively. We found no detectable effects of enhanced acetylcholine and dopamine signaling on either SWM precision or task performance.At the single neuron level, local administration of acetylcholine reduces excitatory receptive field size in marmoset V1  could reveal cholinergic and/or dopaminergic effects on a behavioral measure of SWM precision in humans.The acute dose of donepezil that we used was 5 mg, corresponding to the lowest dose prescribed clinically for daily administration. While it is possible that cholinergic effects on SWM precision would be observed at higher doses of donepezil, previous studies in our lab have documented statistically significant effects of a single dose of 5 mg donepezil on spatial extent of fMRI responses in visual cortex  in the donepezil study and 0.60 degrees (19.1% change from placebo) in the levodopa/carbidopa study in order to produce a significant drug effect at Another consideration is that we used a delay period of 900 ms without a visual mask. In principle, persistence of the sensory response to the stimulus to be remembered could have aided subjects\u2019 performance on the SWM task. However, this type of retinal persistence, often studied in the psychological literature as iconic memory, fades after 300 ms (Sperling, Much of the evidence for cholinergic and dopaminergic effects on spatial tuning of working memory representations comes from studies of macaque dlPFC neurons. Although the SWM task we employed is very similar to that used in the macaque studies, recent evidence from human patients with lesions to dlPFC has raised questions about the homologies between humans and macaques in this region (Mackey et al., These lesion results are supported by a recent transcranial magnetic stimulation study in which disruption of human dlPFC did not affect accuracy of memory-guided saccades (Mackey and Curtis, Our results support a distinction between the limits of spatial resolution in visual cortical neurons and visual perception and the corresponding limits in SWM representations. Although there are clear cholinergic effects on spatial resolution at the level of single neurons (Roberts et al.,  (Harewood et al., We also found no evidence for visual field asymmetries in the precision of SWM, a result that also indicates fundamental differences between the spatial resolution of perception and memory. Previous studies have documented a clear lower visual field advantage in visual crowding tasks (He et al., Thus, even though SWM representations must be derived from perceptual representations to some extent, we have found fundamental dissociations between the spatial resolution of SWM and perception, both in their associated neurochemical mechanisms as well as visual field anisotropies.ANHS, JAC and MAS designed the experiment and gave final approval of this version of the manuscript to be published. ANHS and JAC collected data and created figures for the article. ANHS and MAS created data analysis procedures, interpreted data, and drafted and edited 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. The handling editor is currently co-organizing a Research Topic with one of the reviewers AD, and confirms the absence of any other collaboration."}
{"text": "It is therefore of great interest to identify targets of p53 (wild type and mutant) that are responsible for this phenotype during reprogramming, as these could be exploited for therapeutic use, that is, formation of induced pluripotent stem cells with high reprogramming efficiency, but no oncogenic potential. Here we studied the transcriptional changes of microRNA in a series of mouse embryonic fibroblasts that have undergone transition to induced pluripotent stem cells with wild type, knock out or mutant p53 status in order to identify microRNAs whose expression during reprogramming is dependent on p53. We identified a number of microRNAs, with known functions in differentiation and carcinogenesis, the expression of which was dependent on the p53 status of the cells. Furthermore, we detected several uncharacterised microRNAs that were regulated differentially in the different p53 backgrounds, suggesting a novel role of these microRNAs in reprogramming and pluripotency.The tumour suppressor p53 plays an important role in somatic cell reprogramming. While wild-type p53 reduces reprogramming efficiency, mutant p53 exerts a gain of function activity that leads to increased reprogramming efficiency. Furthermore, induced pluripotent stem cells expressing mutant p53 lose their pluripotency  Thus, p53 plays an important role in maintaining a pool of stem cells with an intact genome and moreover prevents of reprogramming cells with faulty genome.27Recently, p53 is emerging as a key regulator in the process of reprogramming from somatic to induced pluripotent stem (iPS) cells as well as being involved in stem cell maintenance.27 p53R172H (R175H in human) is a conformational mutant that results in a misfolded p53 protein. This study showed that p53 depletion or the expression mutant p53 increases reprogramming efficiency.27 However, cells expressing p53R172H in addition to their augmented pluripotency in vitro exhibited carcinogenic potential in vivo. When injected into nude mice, p53R172H expressing iPS cells lost their differentiation capacity and gave rise to aggressive sarcomas, while p53 KO iPS cells maintained pluripotency and led to the formation of benign teratomas, thus displaying a novel GOF for mutant p53.27We have previously studied the reprogramming efficiency of a series of MEFs with different p53 status, that is, p53 wt, p53 knock out (KO) and mutant p53R172H cells.43 but also influence reprogramming of stem cells and the maintenance of an undifferentiated cellular stage.45It is of great interest to generate iPS cells with a high reprogramming efficiency, but low tumorigenic potential for therapeutic use. As p53 was shown to be important in both reprogramming and maintaining genomic integrity of iPS cell, it provides an interesting target for manipulation of the reprogramming pathway. It is thus of interest to dissect the mechanisms and players regulated by p53 in these pathways. In addition to controlling the expression of protein coding genes, p53 was shown to control the transcription of a number of microRNAs (miRNAs). Expression of miRNAs is altered in many pathological conditions including cancer, where different miRNAs exhibit oncogenic and tumour suppressive properties. Moreover, miRNAs are key regulators of development; for example, miR-34a is fundamental for neuronal and muscle differentiation,In this study, we set out to examine miRNAs that are differentially regulated in cells during reprogramming depending on their p53 status, aiming to identify miRNAs that play a role in this process and that could be directly targeted to help optimise iPS cells. This would allow the generation of cells that have intact p53, which protects their genomic integrity, but at the same time exhibit high reprogramming efficiency. To this end, we performed a microarray screening of miRNA expression before and after three factors driven reprogramming of wt, KO and mutant p53 cells and identified several miRNAs whose expression is dependent on the p53 status of the cell.et al.27To identify miRNAs that are targeted by either wt or mutant p53 during reprogramming, we performed microarray analysis on a series of iPS cells and their parental MEFs with different p53 . The MEFs were reprogrammed by introduction of Oct4, Sox2, Klf4 as described in our previous study by Sarig All Cq values above 32 were considered noise background and excluded from the analysis. The results for a given sample were normalised by the geometric mean of the relative quantities of all targets expressed in the same sample . Targets that were up- or downregulated more than twofold were considered to be changed in the analysis.In a first step we looked at the miRNAs that were up- or downregulated in all three conditions during reprogramming . A high 47 Furthermore, several of these miRNAs have been implicated in both stemness and cancer , while in this study the iPS cells were generated using only three reprogramming factors  since c-Myc has been correlated with carcinogenesis of iPS cells in vivo.89Of note, our microarray screening did not highlight miR-34a as upregulated during reprogramming, as previously reported.In conclusion, our study highlighted a group of miRNAs driven by wt and mutant p53 that might be important for reprogramming, tumorigenesis and loss of genomic integrity. While a high number of miRNAs identified in our screen have already been associated with cancer development or differentiation and stemness , others 27 Briefly, MEFs were prepared from 13.5 days postcoitum embryos and maintained in DME, supplemented with 10% FCS, 1\u2009mM sodium pyruvate, 2\u2009mM\u2009l-glutamine, 0.1\u2009mM nonessential amino acids, 0.1\u2009mM \u03b2-mercaptoethanol and antibiotics. Reprogramming was induced using the three factor protocol  as described previously.27Primary MEFs were prepared from wt, p53 mutant or p53 KO E13.5 embryos as reported previously.RNA was extracted using Trizol , following manufacturer's instruction.n=3) were collected and pooled together for technical reasons; these were analysed with three technical replicates, obtaining always a mean error lower than 5% of the measure. Because this variation derives from a technical measure, and not from a biological replicate, it has not been reported on the histograms. Therefore, we did not show any statistics on these qualitative results.Microarray analysis was performed by Biogazelle . All Cq values above 32 were excluded as noise background. The results for a given sample were normalised by the geometric mean of the relative quantities of all targets that are expressed in that sample . Three biological samples .MicroRNAs targets were predicted by using the miRanda Software (microrna.org). Only target sites of conserved miRNAs and with good mirSVR score were taken into consideration."}
{"text": "Human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT)\u2014a critical enzyme of the viral life cycle\u2014undergoes a complex maturation process, required so that a pair of p66 precursor proteins can develop conformationally along different pathways, one evolving to form active polymerase and ribonuclease H (RH) domains, while the second forms a non-functional polymerase and a proteolyzed RH domain. These parallel maturation pathways rely on the structural ambiguity of a metamorphic polymerase domain, for which the sequence\u2013structure relationship is not unique. Recent nuclear magnetic resonance (NMR) studies utilizing selective labeling techniques, and structural characterization of the p66 monomer precursor have provided important insights into the details of this maturation pathway, revealing many aspects of the three major steps involved: (1) domain rearrangement; (2) dimerization; and (3) subunit-selective RH domain proteolysis. This review summarizes the major structural changes that occur during the maturation process. We also highlight how mutations, often viewed within the context of the mature RT heterodimer, can exert a major influence on maturation and dimerization. It is further suggested that several steps in the RT maturation pathway may provide attractive targets for drug development. The accessory proteins negative regulatory factor (Nef) p66 have been interpreted to indicate the formation of a stable, symmetric p66/p66 homodimer containing two structurally and magnetically equivalent, stable RH domains -labeled p66 in our lab obtained under conditions similar to those of Sharaf et al. [Importantly, all of the NMR spectra that have been obtained for the Ile-labeled p66/p66\u2019 homodimer reveal many subunit-specific chemical shift differences, consistent with the conclusion that the p66/p66\u2019 homodimer exists as a structural heterodimer, and consistent with the above conformational selection process in which two conformationally distinct p66 monomers form an initial homodimer. Fortuitously, despite their close structural similarity, the RH and RH\u2019 domains initially present in the p66/p66\u2019 homodimer yield many separate resonances, allowing their separate conformational evolution to be individually tracked . In cont domains . Howeverf et al.  are domiM , many of these factors can exert their effects by influencing the earlier domain rearrangement process, rather than by directly influencing the monomer association step. Even perturbations that interfere with the subsequent subunit-specific RH domain unfolding step can influence the reversibility of the entire maturation pathway, and hence the fraction of p66 that is dimeric. Therefore, dimer stability provides a readout for multiple steps in the RT maturation pathway.extended conformation, and the second corresponding to dimerization of polymerase molecules in the E and M conformations p51MM and p51C conformations , this additional enhancement of the E/C ratio will enhance dimer formation  perturbations of the structure of the dimer interface that are transmitted from the NNRTI binding pocket ,83; and extended structure of the polymerase domain [In addition to the NNRTIs, other active site ligands, including dsDNA, hybrid RNA\u2013DNA, and metal\u2013nucleotide complexes are each expected to stabilize the dimer as a result of selective binding to the e domain ,88,89. Te domain ,90). In As in the case of symmetric multimers such as HIV protease, the metamorphic RT polymerase domain must accommodate a pair of mutations for each mutational variation in the p66 precursor. This constraint can be mitigated in protein regions with differential sensitivity to mutations. For example, the \u03b27\u2019\u2013\u03b28\u2019 loop on p66\u2019 (or p51) plays an important role at the dimer interface , while tNumerous studies have shown that RT mutations can influence dimer stability ,95,96,97E/M conformer ratio [M, Leu289 is solvent exposed, while in the structure of apo RT which serves as a model for p66E, Leu289 is located in a small fingers:thumb interface and makes hydrophobic contacts with fingers residues Phe61, Ile63, and Leu74 in the predominant closed conformation . Structural analysis indicates that the buried Asp110 and Asp186 residues in p51 make multiple H-bond and salt bridge contacts with other residues, while the buried Trp402 and Trp406 stabilize the compact conformation due to hydrophobic interactions. Trp406 N\u03b5-H is also involved in stabilizing H-bond interactions in the compact p51 subunit. The role of the aspartyl residues of the YMDD motif in stabilizing the compact conformation of p51 has also been demonstrated by studies of Mulky et al. [extended conformation as neutral or slightly destabilizing with the additional possibility that W402R and W406R may be capable of dimer stabilization due to the formation of inter-subunit H-bond interactions. Consequently, while the L234A mutation is expected to lower the E/C conformational ratio of p66, the rescue mutations are expected to increase this ratio, primarily due to destabilization of the compact conformation. In summary, the effects of the dimerization inhibiting and rescue mutations studied by Tachedjian et al. [Tachedjian et al.  further am VADAR  for resiy et al. . Alternan et al.  appear tn et al.  and illuM and p66C, introduction of the larger side chains in the compact conformer will lead to local structural disruption that may be transmitted further into the structure, perturbing the dimer interface. The corresponding region in p66 in the apo structure is somewhat less crowded, but more importantly, exhibits substantial intrinsic flexibility that allows the accommodation of large NNRTIs. Movement of side chains in the NNRTI site of the E conformation can thus allow the accommodation of mutations requiring substantially greater side chain volume. It was determined that the most extreme side chain expansion (corresponding to a G190W mutation) was able to enhance dimer stability nearly as effectively as the NNRTI efavirenz (EFV) [E/C conformer ratio, similar to the mechanism discussed above for NNRTIs  . Consistnz (EFV)  shows thr NNRTIs . This exAs outlined above, subunit-specific RH domain unfolding is the final step in the maturation of the p66/p66\u2019 homodimer. Mutations that significantly destabilize the RH domain can result in premature unfolding and proteolysis that effectively uncouples this step from dimer formation. Precursors containing mutations at the Phe440\u2013Tyr441 cleavage site or in the segment between the domain N-terminal Tyr427 residue and the cleavage site have been reported to result in excessive formation of p51 and low or even absent levels of the RT heterodimer ,53,112. M illustrated in Abram et al. identified T477A as a rescue mutation for F440V and several other mutations that destabilize the RH domain and lead to premature processing ,113. BasThe RT maturation processes outlined above are mostly accomplished within the virion after it has separated from the host cell. Various lines of evidence indicate that the viral polyproteins exist primarily as a linear assembly of folded proteins and protein domains ,115, adoThe structures of the RT heterodimer and its p66/p66\u2019 homodimer precursor A are arrThe polyprotein structure of pro-Pol results in cooperative dimerization, in which formation of an initial homodimer facilitates more extensive dimer formation . Since i20 lowers the equilibrium dissociation constant for p66 by about 30-fold. Formation of a complex with double-stranded RNA (dsRNA) formed from vRNA annealed with tRNA(Lys3) requires an extended conformation of the polymerase domain, which will stabilize the dimer and presumably accelerate the maturation of the p66\u2019 subunit shown in In addition to the higher polyprotein concentration in the virion, other interactions are likely to facilitate RT dimerization. There is evidence that a pre-initiation reverse transcription complex (RTC) that includes viral RNA (vRNA), tRNA(Lys3), and RT is formed within the virion ,126. In In addition to supporting dimer formation, the high concentration of p66 within the virion can compensate for many p66 mutations that reduce but do not prevent dimer formation. Thus, Wapling et al.  observedThe virion pH is not well regulated, making the intra-virion pH subject to variations as a result of changes in the viral medium . Since Reverse transcriptases derived from HIV-1 and HIV-2 show only ~60% sequence identity ; howeverThe coding economy that results from reliance on a metamorphic RT comes at the cost of constraints on mutational variability and reliance on a complex maturation pathway. Recent NMR, crystallographic, and modeling studies have provided initial insights into this pathway, and suggest that maturation may be an attractive target for therapeutic intervention. In contrast, nucleoside drugs that target mature RT can also interfere with activities of host polymerases, and in some cases have been shown to inhibit the mitochondrial polymerase, leading to associated HIV lipodystrophy . In the The feasibility of targeting the final, subunit-specific RH domain unfolding step using an RH-active site-directed ligand has recently been demonstrated . As discThe RH:thumb\u2019 interface\u2014which is proposed to be absent in the initial homodimer structure A\u2014represe"}
{"text": "There is increasing importance placed upon noninvasive assessment of gut inflammation. These tools are likely to be the key in differentiating intestinal inflammatory disease from functional disorders and in monitoring the response to intervention in individuals with known inflammatory conditions. Although various noninvasive markers are currently available, they have limitations and do not provide ideal utility. This review focuses on emerging markers of gut inflammation, highlighting the potential of specific markers. Historically, noninvasive assessments of gut inflammation have utilized tests such as the detection of fecal white blood cells  and wholPyruvate kinase, a key enzyme in the glycolytic pathway , can be High levels of M2-PK were documented in 81 adults diagnosed with IBD  Table 1Table 1. Fecal M2-PK was assessed in a group of Polish children with IBD . Seventyp = 0.0007) [In a recent Australian study, mean fecal M2-PK levels were also higher in 17 children with active CD than in 21 healthy controls ( 0.0007) . HoweverHigh fecal M2-PK levels also were demonstrated in children with active UC . M2-PK aIn 2014, Czub et al.  who statOsteoprotegerin, also known as osteoprotegrin, (OPG) is a basic glycoprotein that is found either as a 60\u2009kDa monomer or as a 120\u2009kDa dimer. OPG is a cytokine receptor and belongs to the TNF superfamily , 17. OPG\u03baB (RANK) and its ligand (RANKL). OPG acts as a decoy receptor for RANK [\u03b1) [OPG has a well-established role in bone turnover. The equilibrium of osteoclast and osteoblast activity is coordinated primarily by the receptor activator of nuclear factor-for RANK , 20\u201322. for RANK  promotinRANK [\u03b1) . This isRANK [\u03b1) . HoweverRANK [\u03b1) .OPG also may have a role in IBD pathogenesis, quite separate to the role in bone metabolism. The OPG/RANKL/RANK triad may contribute to mucosal and systemic inflammation , 20, 22.Recently, a small number of studies have demonstrated that OPG can be a useful marker of inflammation in the context of IBD. Nahidi et al.  evaluate\u03b1 inhibitor infliximab in adult patients with IBD. In this study, OPG levels also decreased with treatment, in correlation with falling CRP levels. It is important to note that OPG levels did not fall acutely and were only significantly lower 22 weeks after the commencement of therapy. The authors suggest that in this setting, OPG is representative of the inflammatory response and not bone turnover.Galliera et al.  measuredIn addition, Sylvester et al.  recentlyThere are, however, conflicting reports on fecal OPG expression. In a preliminary investigation, Skinner et al.  report tMyeloperoxidase (MPO) is one of a number of proteins stored in and released from neutrophil secretory granules: it is stable for at least 3 days in stool . One potp < 0.001) [MPO levels were greatly increased in a cohort of 55 Indian patients with UC compared to 74 healthy controls (0.42 units versuss 0.06 units: < 0.001)  Table 1p < 0.001< 0.001)   1, is released from immune cells in the setting of inflammation. It has been described as an alarmin, with key inflammatory properties.r = 0.77). Furthermore, fecal concentrations reflected endoscopic severity in a subset of 16 children with clinically inactive disease but active mucosal findings. These results were reproduced by the same group in adult patients with IBD [r = 0.763, p < 0.001 for the Simple endoscopic score for Crohn's disease (SES-CD) and r = 0.44, p < 0.05 for endoscopic Mayo subscore). Palone et al. [One study has assessed HMGB1 in a cohort of children with IBD . Fecal lwith IBD . Howevere et al.  thereforHMGB1 has previously been shown to reflect intestinal inflammation in the context of enteric infection . In addiChitinase 3-like 1 (CHI3L1) binds chitin, an abundant polysaccharide. The expression of CHI3L1 is upregulated in various cell types such as colonocytes and lamina propria macrophages . This prp = 0.01). In addition to its presumed role in IBD, CHI3L1 was suggested by Chen et al. [Aomatsu et al.  demonstrn et al.  to play Although there is some rationale to consider that this protein may be a useful marker, further assessment and validation is required.\u03b2 defensin group are produced in the colon by epithelial cells and plasma cells. An initial study completed in French children demonstrated that human \u03b2 defensin (HBD) 2 could be measured in stool samples [p = 0.0002), especially those with UC   . HoweverThe matrix metalloproteinases (MMP) are a family of key biological mediators involved in tissue degradation and restitution. A number of studies have examined the roles of MMP proteins in IBD , 52: theMMPs are expressed in areas of inflammation and ulceration in the gut, and several MMPs are overexpressed in IBD , 53\u201357. MMP was also monitored in patients with CD managed with infliximab. Interestingly, MMP-2 serum levels increased, both in responders and nonresponders to treatment, and this was hypothesised to be due to increased intestinal cell turnover . Garg etp < 0.001 for both relationships) and were also associated with CRP levels (p = 0.002). Furthermore, MMP-9 levels correlated with fecal calprotectin (p = 0.014).Most interest in MMPs as disease markers of IBD has focused on MMP-9. Kofla-Dlubacz et al.  showed tDespite MMP-9 being elevated in both CD and UC, the performance of MMP-9 as a disease marker appears to be better in UC and pouchitis . Kolho eSeveral other MMP's are also elevated in IBD. MMP-7 and MMP-13 mRNA levels were elevated in biopsy specimens from CD and UC patients . Serum MOne study has assessed human deoxyribonucleic acid levels in stool of 36 individuals with UC . ExcretiIn addition to DNA, RNA or more specifically microRNA (miRNA) has the potential to be used as a disease marker in IBD. miRNAs are small noncoding RNA molecules that are capable of regulating gene expression at the posttranscriptional level. Expression patterns have been described in intestinal biopsies collected from IBD patients with a number of specific miRNA reported to be upregulated in both CD and UC \u201375. In CHowever, there are several difficulties that need to be overcome to compare these studies. For example, concurrent medications, the inflammatory status, and the location of the biopsy need to be considered when interpreting the role of miRNAs , 80. FurA biomarker can be described as a product that provides a measurable indication of the presence and/or severity of disease or physiological state of an organism. The holy grail of a biomarker is that it is disease specific, correlates highly with disease severity, and can provide both diagnostic and prognostic indications. To date, several promising fecal biomarkers for IBD have been identified. However, none of the currently described markers are disease specific so the search for better biomarkers of IBD continues. A number of promising novel biomarkers have been identified, and this article has described a portion of the most promising novel markers. However, there are many more potential biomarkers of IBD and further work is required to determine if any of the newly identified markers will be equal to, or surpass, the utility of currently available markers. The aims of the tables are to describe and compare the current knowledge in the use of novel markers: as markers of diagnostic tests for IBD , as markThe initial identification of calprotectin as a biomarker of IBD was greeted with great enthusiasm and promise that it would impact clinical care. However, after nearly 30 years of calprotectin research, the incorporation of this biomarker into a routine clinical care has been slow. Calprotectin has not replaced current nonspecific inflammatory markers, but when used, it is generally used as an adjunct disease marker. Therefore, it is reasonable to suspect a long wait for new IBD biomarkers to appear in the clinic. Nevertheless, biomarkers of IBD have a bright future, in research studies at least, as prognostic indicators and in the search for personalised and improved IBD clinical care.There also are several promising markers described here that likely reflect different cellular sources and different aspects of the IBD response. Currently, no single marker appears to be sought after highly sensitive, diagnostic, and prognostic IBD specific indicator. However, a number of novel biomarkers have been evaluated in just one or two cohorts: these all require more extensive assessments in various settings to establish their roles in identifying, monitoring, and predicting disease behaviour.More clinical studies are required to ascertain the full potential of fecal biomarkers in IBD. Although such evaluations should focus on specific biomarkers, comparative assessments are also required, yet there remains plenty of potential for novel biomarkers to impact clinical care."}
{"text": "TMEM230 has been associated with autosomal dominant Parkinson\u2019s disease (PD). Subsequent studies have remained negative, and none of previous described mutation has been reported anymore. We investigated the implication of this gene in the PD in a population of 703 PD patients and 695 unrelated healthy controls from southern Spain. Thirteen variants were found, twelve of them observed only in controls or in patients and controls, and one (c.190A>G) observed only in one patient. Subsequent analysis of this variant indicates that probably it is not pathogenic. In addition, we found a variation in the 3\u2019-UTR (rs183551373) and related with the miRNA hsa-miR-4299 but it was observed only in healthy controls. Our results suggest that variants in TMEM230 gene are not associated with the development of PD. PARK2, LRRK2 or SNCA, among others, are well documented as causative genes or risk factors for PD [Parkinson\u2019s disease (PD) is a complex pathology in which both environmental and genetic factors are involved. More than 18 genes, like TMEM230 gene has been described to be a cause of autosomal dominant PD in a large family from North American [TMEM230 (p.*184ProGlyext5*) was identified as being related with PD in 7 familial cases. Several additional studies in different populations have failed to find neither causative mutation nor risk factors in TMEM230 associated with PD [Recently, a missense mutation (p.Arg141Leu) in  with PD   of the nature of the study and signed a written consent before blood collection.TMEM230 (NM_001009923 and NM_001009925) and their intron boundary regions were analyzed using high-resolution melting analysis in a LightCycler 480-II (Roche). Samples showing abnormal melting profiles were sequenced in an ABI3500 Genetic Analyzer (Applied Biosystem). The identified variants were annotated according to the longest isoform (NM_001009923). A variant call of sequenced samples was made using Variant Reporter software v1.1. (Applied Biosystem).We included a total of 703 PD patients from the Movement Disorders Unit of the Hospital Universitario Virgen del Roc\u00edo , and 695 healthy controls (HC). One hundred forty-eight patients had familial PD . PD was TMEM230. However, another variant found in the present study, c.191T>C, carrying the same effect was present in two healthy controls and in one PD patient. Another novel variant was identified in 3\u2019UTR region, c.*670T>C but it was present in patients and control subjects. None of these variations were identified in the familial cases. Neither of the known variants previously related with PD have been found in this study.We detected a total of 13 genetic variants (two not reported before) in our population: two at 3\u2019-untranslated region, one in 5\u2019-untranslated region, one intronic variant, four synonymous variants, and five nonsynonymous variants . Ten werThree frequent variants  were present in PD case cohort and control cohort with similar frequencies.TMEM230 but neither of them seems to be related with the development of PD, since they are present only in HC or in both patients and HC. The exception has been c.190A>G, which is present in a single patient. This variation causes the loss of the ATG start codon in the isoform 2 of TMEM230 , therefore, it could be disease causing. However, the variant c.191T>C, with similar consequences in the protein , is present in healthy controls in this study and in previous ones [TMEM230 protein is also methionine, which would act as an alternative start codon to generate a novel protein that lacks only the first amino acid. So, it is expected that this variation does not cause a significant alteration of the protein. In addition, a variation in the 3\u2019-UTR (rs183551373) has been related with the miRNA hsa-miR-4299 but it was found only in healthy controls in our population and, thereupon, the pathogenicity of this variant seems not be probable.In this study we have reported 13 variants in TMEM230 is very low and, therefore, TMEM230 do not play a major role in familial and sporadic PD patients in southern Spanish population which can have important implication in clinical investigation.Our findings suggest that the incidence of pathogenic variations in S1 Table(PDF)Click here for additional data file."}
{"text": "Economic bubbles are an empirical puzzle because they do not readily fit the notion of an efficient market. We argue that bubbles are associated with a conflict and a gap in the allocation of effort during negotiation by sellers and buyers. We examined 21 experimental asset markets where in one condition players could buy and sell and in the other they could either buy or sell. The results indicated that when making concurrent buying and selling decisions the mean number of asks for sellers was 71% higher than the number of bids for buyers. Similar findings emerge in a re-analysis of data from Lei et al. (2001). Importantly, bubbles only emerged in markets where the number of asks was larger than that of bids. These findings indicate that bubbles are associated with increased negotiation effort when acting as a seller and diminished effort when acting as a buyer. Economic bubbles are an empirical puzzle because they do not readily fit the notion of an efficient market . BubbIn light of the surprising ubiquity of economic bubbles, understanding the formation and mitigation of bubbles has been the subject of extensive experimental research. Still, most studies of experimental asset markets have focused on processes shared by buyers and sellers \u20137, 10\u201315Buyers and sellers\u2019 effort is for all likelihood associated with their pricing performance. There is an extensive literature in psychology on the relationship between effort and performance \u201323, whicSeveral empirical findings support the notion that selling is associated with greater effort. First, the literature suggests that sellers view the task of trading more from the point of view of preventing a loss (of the object) than from that of obtaining a gain, while buyers view the task more from a gain than a loss perspective , 25. ForConsistent with the notion that selling is associated with more cognitive effort, studies have shown that in pricing of lotteries participants acting as sellers tend to be more sensitive to the lottery\u2019s expected value than those acting as buyers e.g., \u201338) and  and 38])These theoretical accounts also suggest some moderators for the postulated cognitive differences between buyers and sellers. Increased attention and effort on the part of sellers should be more impactful when attentional resources are limited. This is implied by the diminishing marginal benefit of attentional investment ,23. AddiIn the current study we evaluated these predictions in an experimental asset market. In order to study the effect of concurrent buying and selling we compared a market where traders either buy or sell versus one where they can perform both tasks. As a proxy for negotiation efforts, we examined the volume of price proposals  which is considered to reflect the extent of one\u2019s efforts at negotiating different prices ,40. ConsTo pilot these research questions, we re-analyzed data from a standard experimental asset market  collectet(27) = 2.75, p = .01). There was no significant difference between the number of asks and bids in the Buy OR Sell condition (t(21) = 0.65, p = .52).Using Lei et al.\u2019s  data, wet(36) = 1.94, p = .07). High buying proposals significantly predicted excessive transaction prices above the fundamental value  = 0.38, p = .02); however, there was no direct relation between the market\u2019s asks minus bids index and excessive transaction prices (r(48) = 0.15, p = .30). The latter analyses are sensitive to confounds since they are pooled across the two experimental conditions (Buy AND Sell vs. Buy OR Sell). However, the sample size was too small to conduct an analysis controlling for condition (n\u2019s \u2264 10 in some of the cells). In our main study we conducted a more thorough analysis that controls for experimental condition.We then compared markets where the mean number of asks was larger than that of bids, to those where the number of bids was larger (there were five markets in the former and two in the latter category). Buying price proposals in markets dominated by asks were somewhat higher compared to their counterparts, though the effect was only marginally significant  and none had prior experience in asset market experiments. Their average age was 24.7; ages ranged between 18 and 40. Experimental sessions lasted approximately 75 minutes, and payment ranged between NIS 25 and 100 depending on performance. There were eight participants in each session; three sessions included only seven participants each due to insufficient show ups.Following Lei et al. (2001 ) particit equaled the expected value of the dividend (30 tokens) multiplied by the remaining rounds (30 \u00d7 (13-t)). The number of assets in one\u2019s possession and the cash balance were accumulated during rounds. The asset had no value for the participants after the experiment had ended, while experimental currency was converted into real currency at a rate of NIS 1 per 200 tokens.The experimental asset market was based on the parameters of Lei et al.\u2019s (2001 ) experimIn the Buy OR Sell condition participants were assigned to the role of either buyers or sellers. Buyers were only permitted to purchase, while sellers were only permitted to sell. At the beginning of the experiment, each seller was endowed with 20 units of the asset (referred to as \u201cstocks\u201d) but no experimental tokens. Each buyer was endowed with 7,200 tokens  and no units of the asset. Because each unit of the asset paid on average 360 tokens during the 12 rounds, the expected value of the initial endowment was identical for buyers and sellers in this condition. In the Buy AND Sell condition all participants could buy and sell, and each participant was endowed with 10 units of the asset and 3,600 tokens at the beginning of the experiment. The expected value of the initial endowment in this condition was thus the same as in the Buy OR Sell condition.The sequence of events in all sessions was as follows. Upon arrival, participants completed an informed consent statement and received written instructions consisting of a description of the task and an explanation of the dividend structure. The instructions were based on those described in Lei et al. (2001 ), translThe experimenter read these instructions out loud. Participants were then encouraged to ask questions. This was followed by a short quiz regarding the value of a single asset one round ahead and 12 rounds ahead. If participants did not complete the test properly they were given further explanation until they were able to answer correctly. Next, there was a 4 minute training session in which participants practiced using their designated perspective in the experimental task . The initial endowment in the training session was identical to that given in the main experiment but monetary decisions were not incentivized. Participants then received a piece of paper stating their initial endowments and the possible actions available to them . This was followed by the 12 rounds asset market experiment with continuous double-auction rules.A accepted a pending bid from participant B then all asks for A and all bids for B were removed. Thus, each price proposal pertained to a single unit of the asset . At theAs in our re-analysis of Lei et al. (2001 ), we calt) refers to the mean transaction price in round t, and FV(t) refers to the fundamental value in round t.For descriptive purposes, we also calculated two common market-level indices of bubbles , 40: Bubt(21) = 2.79, p = .01). The results are thus consistent with those of Smith et al. (1998 [The mean prices in each trading round appear in l. (1998 ) in thatt(19) = 1.82, p = .084; Amplitude: t(19) = 1.84, p = .091).We also examined whether differences in the market indices of bubbles emerged between conditions. For both indices of bubble duration and bubble amplitude there was a marginally significant difference between conditions, with longer and more varied bubbles in the Buy OR Sell condition than in the Buy AND Sell condition (Duration: t(21) = 8.14, p < .001).Another difference between market conditions was the number of transactions in each condition, which was considerably higher in the Buy AND Sell condition (3.78\u00b10.36) than in the Buy OR Sell condition (0.72\u00b10.07). This difference was significant even at the market level (t(69) = 4.80, p < .001). There was also a significant difference in responsiveness in the predicted direction, with buyers being 32% more likely to accept an ask than sellers were to accept a bid (paired-sample t(69) = 2.81, p = .006). As in the data of Lei et al. (2001 [t(93) = 0.74, p = .46) and their responsiveness (t(93) = .07, p = 0.94).As shown in l. (2001 ), these W = 4.92, p < .001). Similarly, the median buyer responsiveness was 1.65 compared to 0.92 for sellers, a significant difference . An analysis of changes in the mean number of bids and asks over time (in different rounds) appears in the S1 Supplementary section.The effects in the Buy AND Sell condition did not seem to be due to extreme values. The median number of asks in the Buy AND Sell condition was 6.71 compared to 3.58 for bids, and the main effect was replicated in a non-parametric test  characterized by more asks than bids, versus those with more bids than asks. This enabled a direct contrast of an environment where selling or buying proposals are more dominant. In all, there were 13 markets where asks outnumbered bids and 8 markets where bids outnumbered asks . The mean pricing characteristics of the two groups are shown in F = 6.07, p = 0.01) and also by higher selling proposals  = 4.04, p = 0.047). Buyers in these markets also accepted higher price proposals  = 6.34, p = .01), as did sellers  = 6.49, p = .01).As in the data of Lei et al. (2001 ), marketF = 11.26, p = .001), as well as by sellers  = 16.65, p < .001). Indeed, for markets dominated by higher number of asks transaction prices were significantly above the FV (transactions accepted by buyers: t(81) = 2.72, p = 0.008; by sellers: t(81) = 3.53, p = 0.010). In contrast, in markets dominated by higher numbers of bids transaction prices accepted by either buyers or sellers were not significantly different from the FV (t(33) = -1.50, p = 0.14; t(33) = -1.36, p = 0.18, respectively). Thus, transaction prices significantly exceeded the FV only in those sessions that had greater numbers of asks than bids. As shown in Markets with higher number of asks were further characterized by higher positive deviations from the FV (price\u2014FV), both when transactions were accepted by buyers  = 6.51, p = .01). No such effect emerged for transactions accepted by buyers  = 2.68, p = .11). We also looked at the mean price of the transactions at the market level in markets with more asks than bids and vice versa. FV. When regressing market price on condition and market type, there was a positive association between asks-dominated markets and mean market price . The effect was not significant, however, likely due to poor statistical power (df = 19).Finally, in line with the differences observed at the market level for bubble duration and amplitude, there was also an effect of condition on excessive prices for transactions accepted by sellers: The positive disparity between transaction prices and the FV was higher in the Buy Or Sell condition than in the Buy AND Sell condition . However, the Buy OR Sell condition was associated with higher accepted prices both for buyers and for sellers  = 15.39, p < .001; F = 4.46, p = .04). With respect to the magnitude of buying proposals, there was also an interaction between condition and selling to buying volume  = 9.57, p = .002) with the effect of market type being stronger in the Buy AND Sell condition. A similar interaction emerged for the price accepted as a seller  = 7.00, p = .01) and for excessive prices above the FV in transactions accepted by buyers  = 5.26, p = .02).There was no effect of condition on the magnitude of buying and selling proposals (r(67) = 0.40, p < .001) and showed a marginally significant correlation with the average price accepted as a buyer (r(63) = 0.21, p = 0.08). The difference between the number of asks and bids was also marginally correlated with excessive prices above the FV (price\u2014FV) in transactions accepted by buyers (r(67) = 0.21, p = .08), but not sellers (r(68) = 0.05, p = 0.68).The Buy AND Sell condition allows assessment of the relation between individuals\u2019 pricing decisions and the volume of their bids and asks. We therefore examined whether individuals\u2019 pricing decisions were predicted by the disparity between their own number of asks compared to bids (#Asks\u2013#Bids). The results indicated that the difference between the number of asks and bids was strongly correlated with the magnitude of buying proposals for a given individual (r(163) = 0.25, p = 0.001).Given these individual differences it was interesting to evaluate to what extent the tendency of transaction prices to exceed the FV in the Buy AND Sell condition was predicted by the individual\u2019s index of #Asks\u2013#Bids, or by the markets\u2019 (session\u2019s). To examine this, we conducted a regression analysis for predicting excessive transaction prices (price\u2014FV) using two variables: 1) whether a session had higher number of asks or bids , 2) the individual\u2019s #Asks\u2013#Bids. The results are presented in The current findings indicate a basic difference between the volume of buying and selling proposals: When individuals can act as both sellers and buyers, the number of asks far exceeds the number of bids. Secondly, in our new study the disparity between the number of asks and bids appeared to predict the magnitude of bubbles. In markets dominated by asks, buyers as well as sellers had higher price proposals and accepted higher prices, and extant bubbles developed, as evidenced in transaction prices being above the fundamental value of the asset. In markets dominated by bids, there were no significant differences between transaction prices and the fundamental value of the asset. These findings echo those observed in our re-analysis of Lei et al.\u2019s (2001 ) experimDifferences in the numbers of bids and asks did not emerge when participants could only buy or only sell. This is consistent with notion that a selling perspective captures more attention when there is a competition in priorities owing to the limited cognitive resources available . Still, Interestingly, despite the fact that larger differences between numbers of bids and asks emerged in the Buy AND Sell condition than in the Buy OR Sell condition, excessive transaction prices were higher in the Buy OR Sell condition (for transactions accepted by buyers). This indicates that other factors besides the volume of proposals modulate the difference between conditions with respect to excessive pricing. One relevant factor may be trading experience. Because assets in Buy AND Sell condition could be rebought and resold, participants gained much more trading experience in this condition (as reflected in the larger number of transactions). It is well known that experience moderates the emergence of bubbles ,13,46. FThe present findings constitute a departure from the standard assumptions about buying and selling proposals in economic theory. The standard way to interpret price proposals in economics is that a surplus of asks represents high supply, leading to reduced prices; whereas a surplus of bids represents high demand, leading to increased prices ,48. We fOne clear implication based on the current findings therefore concerns the relation between price proposals and bubbles. The findings suggest that reducing the gap between the numbers of bids and asks might be helpful in reducing bubbles. Obviously, in actual asset markets involving non-algorithmic trading, it is not possible to affect differences in the number of bids and asks without regulating the market. However, one could potentially reduce asymmetries by modifying the salience of bids and asks, for instance by using distributed averaging algorithms or a closed order book wherein only the current bid/ask spread is presented. Examining the effect of such presentation changes on asymmetries in pricing is an interesting endeavor for future studies.An obvious limitation of our research is that it focuses on experimental asset markets. First, in this setting participants have relatively little experience: With extended experience, differences between buyers and sellers, such as the endowment effect, tend to diminish \u201353. SecoIn sum, in the studied experimental asset markets, the outcome of one\u2019s effort in negotiating prices through price proposals depended on one\u2019s trading perspective. Greater numbers of asks than bids were predictive of the emergence of bubbles, whereas greater numbers of bids than asks were not. More markets conformed to the former type, however, giving way to bubbles on the aggregate level. The findings highlight the relevance of theories of attention and effort to studies of economic markets.S1 Text(DOCX)Click here for additional data file."}
{"text": "The ubiquitin\u2013proteasome system (UPS) and autophagy are two distinct and interacting proteolytic systems. They play critical roles in cell survival under normal conditions and during stress. An increasing body of evidence indicates that ubiquitinated cargoes are important markers of degradation. p62, a classical receptor of autophagy, is a multifunctional protein located throughout the cell and involved in many signal transduction pathways, including the Keap1\u2013Nrf2 pathway. It is involved in the proteasomal degradation of ubiquitinated proteins. When the cellular p62 level is manipulated, the quantity and location pattern of ubiquitinated proteins change with a considerable impact on cell survival. Altered p62 levels can even lead to some diseases. The proteotoxic stress imposed by proteasome inhibition can activate autophagy through p62 phosphorylation. A deficiency in autophagy may compromise the ubiquitin\u2013proteasome system, since overabundant p62 delays delivery of the proteasomal substrate to the proteasome despite proteasomal catalytic activity being unchanged. In addition, p62 and the proteasome can modulate the activity of HDAC6 deacetylase, thus influencing the autophagic degradation. Nearly 30% of newly synthesized proteins in the cell are misfolded under normal conditions . Two sysp62 is an autophagy substrate that is used as a reporter of autophagy activity. Recently, p62 was also shown to deliver ubiquitinated proteins, such as tau, to the proteasome for degradation. In addition, it can shuttle between the nucleus and cytoplasm to bind with ubiquitinated cargoes and facilitate nuclear and cytosolic protein quality control. Other functions of p62 are gradually being revealed, emphasizing its importance in the proteolytic system. This review focuses on the role of p62 in linking the ubiquitin\u2013proteasome system and autophagy pathway upon ubiquitinated protein degradation Fig.\u00a0.Fig. 1ThThe ubiquitin\u2013proteasome system (UPS) plays a critical role in the degradation of short-lived, misfolded and damaged proteins. This is necessary to maintain protein homeostasis, cell cycle control , inflammThe progress of proteolysis also requires ubiquitin to covalently attach to substrates. This 76-amino acid protein can form an isopeptide bond between its C-terminal glycine (G76) and a lysine residue within the target molecules or ubiquitin itself . UbiquitAutophagy is a highly evolutionarily conserved degradation system in eukaryotes . It was The autophagic degradation model of eukaryotes is emerging through more recent research . AutophaAutophagy-related gene (Atg) proteins play essential roles in autophagy. They are known as the \u2018core machinery\u2019 . More thp62 was the first selected autophagy adaptor discovered in mammals , 21, 22.p62 is a multifunctional protein consisting of an N-terminal Phox-BEM1 domain (PB1), a ZZ-type zinc finger domain, a nuclear localization signal (NLS), an export motif (NES), an LC3-interacting region (LIR), a Keap1-interacting region (KIR), and a C-terminal ubiquitin-associated domain (UBA) , 25 , the Ras/MAPK pathway, the JNK/c-Jun pathway and some chemical compounds  . ProteasUbiquitination is integral to the proteolytic system. Ubiquitin possesses 76 amino acid residues and is highly conserved from yeast to humans. It also possesses some non-proteolytic functions, including vesicle trafficking via ubiquitination of membrane proteins, protein kinase activation, DNA repair and chromatin dynamics through monoubiquitinated histone. A common mechanism involves ubiquitin or polyubiquitin chains recruiting ubiquitin receptors to perform biological functions .Ubiquitin is involved in three degradation pathways . The triage of ubiquitinated proteins is probably based on their location, the ubiquitin chain length and the linkage types. The three degradation pathways may be interdependent on the ubiquitin pool in the cell . Based oThe K48 chain is a classical sign of proteasomal degradation . K63 is It should be noted that K48 is still the common linkage targeting proteins to the proteasome. During proteasomal degradation, the length of the substrate proteins determines whether the process is mediated by mono- or polyubiquitination . The exiThe UBA domain of p62 can bind K48 and K63 (with a higher affinity for K63) , 44, 45.Drosophila to mice suggest that the inactivation of autophagy by pharmacological or genetic inhibition causes the accumulation of ubiquitinated proteins in the brain [Determining the overall contribution of UPS and autophagy to ubiquitinated protein degradation is a topic of considerable interest. Studies with subjects ranging from he brain , 49, skehe brain . In theihe brain , 50. DifDrosophila [As mentioned previously, p62 delivers ubiquitinated cargoes for autophagic degradation via the C-terminal UBA domain or the LIR domain, and then the PB1 domain promotes the process . Activatosophila ).In HeLa cells, p62 overexpression has no influence on the autophagic vacuole number and p62 knockdown does not have an effect on autophagic flux , 56, 57.Drosophila [p62 is localized to ubiquitin-positive inclusions, which is a common phenomenon that can be observed in some diseases, such as neurodegeneration . Collectosophila . The totosophila . Therefoosophila , 21, 55.p62 is widely used as a predictor of autophagic flux , since iAnother issue is the degradation process. It has been reported that Pan-cathepsin inhibitors and bafilomycin A1 can stop the degradation of p62 . We shouDuring starvation, the expression level of p62 does not always inversely correlate with autophagy activity. Not only can autophagy be induced, but p62 transcriptional synthesis is also active upon starvation , 66. p62The significance of p62 restoration might be the integration of different proteins to fulfill specific functions, since the other domains of p62 get involved in many signal pathways. Besides, p62 knockdown likely activates autophagy through mTORC1 inhibition in response to starvation, since p62 is a positive regulator of mTORC1 . This crNatura et al. used the proximity ligation assay (PLA) to reveal that p62 and the proteasome are co-localized in situ under basal conditions. They also found that p62 aggregates contain inactive proteasome, ubiquitinated proteins and autophagosome upon proteasome inhibition . It has The N-terminal PB1 domain of p62 might interact with Rpt1 and S5a/Rpn10 of the 26S proteasome and collaborate with the C-terminal UBA domain of p62 by binding ubiquitinated proteins to facilitate UPS degradation , 68. p62These studies indicate that p62 is also involved in the proteasomal degradation of ubiquitinated proteins in the nucleus via its NLS and NES domains and in the cytosol via its PB1 domain. Therefore, it naturally contributes to both nuclear and cytosolic protein quality control. Besides, the PEST domain serves as a proteolytic signal for rapid degradation, leading to short intracellular half-lives, which may relate to proteasome function . For insPharmacological inhibition of UPS enhances p62 transcription , 71 and Overexpressed p62 can enhance protein aggregation and has a protective effect on cell survival as described above. p62 deletion barely decreases the amount of ubiquitinated puncta in autophagy normal cells. Although p62 is not necessary for all the formation of ubiquitinated aggregation, it still plays a crucial role in aggregate degradation.Proteasome inhibition can activate autophagy, in which p62 is the bridge . First, The S351 of KIR is phosphorylated, leading to a rising affinity of p62 for Keap1 and followed by sequestration of Keap1 on the cargoes . SubsequNF-E2-related factor 2 (Nrf2), a transcription factor that controls the expression of an abundance of anti-oxidant genes, is degraded by the proteasome via the Cul3\u2013Keap1\u2013E3 ligase complex under basal conditions. However, during oxidative stress, p62 expression is upregulated by the nuclear import of Nrf2 resulting from the blocked interaction between Keap1 (a negative regulator of Nrf2) and Nrf2. The increased p62 can compete with Nrf2 for Keap1 at the Nrf2-binding site, forming a positive feedback loop . All of While proteasome deficiency enhances autophagy, autophagy inactivation compromises the ubiquitin\u2013proteasome system due to surplus p62, which delays proteasomal substrate delivery to the proteasome with no changes shown in proteasomal catalytic activity . It is pThe terms \u2018aggresome\u2019, \u2018aggregate\u2019, and \u2018inclusion bodies\u2019 are used to describe misfolded protein granules in cells. In 1998, the aggresome was defined as a \u2018pericentriolar membrane-free, cytoplasmic inclusion containing misfolded ubiquitinated proteins encased in a cage of intermediate filament proteins that co-localize with the microtubule organizing center (MTOC)\u2019, in which the autophagosome and lysosome fuse . The \u2018agAlthough the aggresome was initially used to describe the disease-associated inclusion bodies formed in neurodegenerative diseases, e.g., Lewy bodies in Parkinson\u2019s disease and hyaline inclusion bodies in amyotrophic lateral sclerosis (ALS), the relevance of the aggresome to inclusions in disease is still disputable . Almost There is a common consensus that misfolded proteins aggregate and are concentrated in the aggresome, which is removed via the autophagy\u2013lysosome pathway . DegradaBecause the UPS, autophagy and p62 are the interdependent elements of the protein quality control system, they must act in a networked manner to maintain proteostasis. p62 may serve as an integration center for multiple functions, including the formation of the autophagosome, the delivery of ubiquitinated proteins to the proteasome, and aggregate formation for autophagic clearance. It is also involved in several signaling pathways , 89. In"}
{"text": "In normally proliferating cells, p53 protein levels and function are tightly controlled by main regulators, i.e., MDM2 (mouse double minute 2) and MDM4 proteins. p53 plays an important role due to its ability to mediate tumor suppression. In addition to its importance as a tumor suppressor, p53 coordinates diverse cellular responses to stress and damage and plays an emerging role in various physiological processes, including fertility, cell metabolism, mitochondrial respiration, autophagy, cell adhesion, stem cell maintenance and development. Interestingly, it has been recently implicated in the suppression of autoimmune and inflammatory diseases in both mice and humans. In this review based on current knowledge on the functional properties of p53 and its regulatory pathways, we discuss the potential utility of p53 reactivation from a therapeutic perspective in oncology and chronic inflammatory disorders leading to autoimmunity. The promdm4 and mdm2 KO mice is accomplished by an apoptotic outcome in the mdm2 KO and mostly by a growth arrest response in the mdm4 KO [In normally proliferating cells, p53 is tightly controlled by the function of its main regulators, MDM2 (mouse double minute 2) and MDM4 proteins, which perform non-redundant activities . Indeed, mdm4 KO ,24,25. T mdm4 KO .The role of p53 in human cancer has been the object of intensive investigations. Leading to evidence on the different mechanisms by which this protein mediates tumor suppression . Recent A variety of tumor suppressive mechanisms underlie the onset and progression of cancers. To inhibit tumor proliferation effectively, fine sensors are needed to single out the normal versus neoplastic growth . p53 serAs evidence of its protective role against tumor development, loss or mutation of p53 predisposes to a variety of spontaneous and induced tumors in animal models ,45. MostIn those tumors that retain the wild-type p53 protein, its pathway is, however, abrogated, leading to aberrant p53 inactivation and avoidance of the tumor suppressor response . These iRegarding the different approaches to reactivate p53, whenever this protein is found as awild type in tumors and the p53-regulatory pathways are defective, the approach to its reactivation is the use of small molecules or peptides to inhibit the N-terminal interaction between p53 and MDM2 or MDM4. Most of these molecules induce p53 activation by disruption of its binding to MDM2, either acting in the p53 binding cleft of MDM2  or by dWhenever p53 is mutated, such mutations lead to structural defects that prevent its appropriate folding, thus abolishing its binding to DNA. The ability of a molecule that acts as a chaperone to restore and stabilize the correct protein conformation was exploited . This aIt must be emphasized that other strategies in the field of cancer therapeutics might be linked to the p53 pathway. One example is evident in the reactivation of the tumor suppressor retinoblastoma susceptibility gene (RB) through RB-inducing agents. RB protein plays an important role in faithful chromosome segregation, checkpoint control, apoptosis, senescence and terminal differentiation. This protein suppresses tumor formation by means of its multiple biological functions. Moreover, RB has been shown to bind to MDM2, the negative regulator of p53. By doing so, RB positively regulates p53 apoptotic activity . AnotherThe role of p53 in inflammation has been extensively in experimental animal models especially in reference to non-organ specific autoimmune diseases ,67,68,69\u2212/\u2212p53 macrophages exhibited a higher innate immune response to lipopolysaccharide (LPS) and interferon \u03b3 (IFN \u03b3) compared to +/+p53 mice. They provide evidence that p53 deficiency increases the incidence of T1D caused by p53-mediated inhibition of proinflammatory cytokines and of total and phosphorylated signal transducer and activator of transcription STAT-1.As regards to organ-specific autoimmune diseases, the putative role of the p53 protein has been investigated in insulin-dependent diabetes  . In moreCD4-Cre p53 fl/fl mice or p53 conditional KO mice. They found that aged p53/cKO mice spontaneously developed several inflammatory diseases such as thyroiditis, sialoadenitis, interstitial pneumonitis, hepatis, gastritis and glomerulonephritis. Moreover, they observed that in p53/KO mice the development of inflammatory lesions was paralleled by the reduction of FoxP3 CD4+CD25+ Treg. These data strongly support the putative effect of p53 in inducing the expression of FoxP3, i.e., a master regulator of Treg, by binding to the promoter and the conserved noncoding DNA sequence-2 of the Foxp3 gene. Interestingly, p53\u2019s effects appeared to be on the development and maintenance of Treg rather than on their suppressive function [In the light of the foregoing, the mechanism by which p53 is involved in the suppression of autoimmunity development remains to be further elucidated. While unravelling this issue, we have to bear in mind that, as for many transcription factors, the net effect of the protein comes from the balance of its expression in cells of the immune system as well as in non-immune cells such as the pancreatic islets in T1D. As regards several functionally distinct peripheral T cell subpopulations, they play a pivotal role in the control of the immunological response, i.e., effector, regulatory and memory T cells. In normal conditions, T regulatory cells (Treg) are present in the peripheral blood where they represent approximately 2%\u20135% of the total pool. FoxP3 (forkhead box P3) Treg originate in the thymus as a naturally arising subset and in the peripheral compartment as an inducible population . Treg plfunction .\u2212/\u2212p53 mice decreased the activity of signal transducer and activator of transcription 5 (STAT-5), lowered the level of STAT-5 and compromised Treg cell differentiation. CD4+ T cells from \u2212/\u2212p53 mice were less prone to differentiate toward Treg expressing lower FoxP3 levels. They also demonstrated that p53 regulates Treg cell differentiation by interacting directly with STAT-5. In inflammatory conditions p53 suppressed Th17 cell differentiation skewing T cells toward Treg through STAT-5 signaling. In mice affected by collagen-induced arthritis, administration of a p53 overexpression vector or an antagonist of MDM2 controlled arthritis development [Of note, Park et al.  investigelopment . Consistelopment .Interestingly, activation-induced cell death (AIDC) of T lymphocytes is a recognized mechanism of immune regulation ensuring the control of the size of the activated pool of T cells subsequent to its expansion . CaspaseSeveral studies also document the effect of p53 in human experimental models of inflammation. NK-\u03baB and p53, known to exert opposite effects in cancer cells, coregulate proinflammatory cytokine secretion in primary human monocytes and macrophages, thus modulating their control on tissue microenvironment . A novelGene expression microarrays analysis of patients with benign multiple sclerosis (BMS) revealed a signature with 30 p53 target genes. Among these, 19 had expression consistent with activation of p53 protein . FurtherIn recent years, longitudinal observational studies have highlighted a bidirectional and dynamic link between cancer and autoimmunity, especially for non-organ-specific rheumatic diseases . The assThe underlying reason for this increased risk remains to be elucidated at the molecular level although in some patients a common inciting trigger may act for both cancer and \u201cparaneoplastic\u201d autoimmune disease .Skp2 (S-phase kinase associated protein 2) and ErbB2  involved in cancer progression, was detected in p53-dependent DNA damage responses in human breast and colon cancer cells [Recent evidences further support the double role of p53 in cancer and autoimmunity. As regards the expression of FoxP3, as master regulator suppressing breast cancer oncogenes er cells . In the Remarkably, to further unravel this issue, it is interesting to observe the secondary autoimmune responses detected in cancer patients. In relation to this phenomenon, p53 Abs were detected in the sera of patients affected by pancreatic adenocarcinoma, serous ovarian and breast cancer . These hDiscovery of new molecular targets for treatment of pathological conditions at increased incidence worldwide is an area in which considerable efforts are being employed to develop new and safe therapeutic agents . In the Recently, in addition to reactivating p53 by targeting the interface between the protein and MDM2 or MDM4, novel strategies have been proposed that interfere with MDM2/MDM4 heterodimerization  Figure . These cNevertheless, as for every new area of drug discovery, safety and efficacy concerns must be addressed both in preclinical and clinical settings, especially when a certain cell type has to be targeted and/or a biological effect is desired. Side effects can be produced by inappropriate dosing, such as suppressive effects on wound healing or tissue repair following toxic or ischemic damage or impaired host defense responses . Thus, aIn the light of the foregoing, activation or restoring of p53 pathway has already reached the step of clinical trial exploitation . As rega"}
{"text": "S. cerevisiae or TER94 in Drosophila) is one of the most abundant cytosolic ATPases. It is highly conserved from archaebacteria to eukaryotes. In conjunction with a large number of cofactors and adaptors, it couples ATP hydrolysis to segregation of polypeptides from immobile cellular structures such as protein assemblies, membranes, ribosome, and chromatin. This often results in proteasomal degradation of extracted polypeptides. Given the diversity of p97 substrates, this \u201csegregase\u201d activity has profound influence on cellular physiology ranging from protein homeostasis to DNA lesion sensing, and mutations in p97 have been linked to several human diseases. Here we summarize our current understanding of the structure and function of this important cellular machinery and discuss the relevant clinical implications.p97/VCP (known as Cdc48 in  ATPases associated with various cellular activities) ATPase family, which functions generally as essential chaperones to promote protein folding or unfolding. Cdc48 was initially identified in S. cerevisiae as a cell cycle regulator, which upon inactivation, leads to a cell cycle arrest at the G2-M transition stage  has been used given the partial localization of this enzyme to the endoplasmic reticulum (ER) surface , Golgi, mitochondria, and endosomes  for these domains   to the overall ATPase activity  that threads polypeptides through a central tunnel  present in gp78  domain of the peptide-N-glycanase (PNGase) bound by a 10-residue peptide from the p97 C-terminus dubbed as PUB-interacting motif (PIM) Figure . PNGase Several p97-adaptor assemblies have also been examined by Cryo-EM  or that bound by p47 (PDB:1S3S) or other adaptors showed no obvious change in the structure of p97 upon adaptor binding  can act in conjunction with Ufd1 and Npl4 to recruit substrates to Cdc48 in MAD  by extracting polypeptides from mitochondrial outer membrane   for lysosomal degradation via autophagasomes. However, the precise function of p97 in this process is controversial, mainly because the substrate(s) regulated by p97 is unclear. Several studies suggest p97 as a positive autophagy regulator because its inhibition causes a phenotype reminiscent of what appears to be an autophagasome maturation defect  [also named IBMPFD ], FALS , CMT2Y  , the bones , and/or the brain . To date, more than 40 mutations covering 29 different positions in p97 have been reported in MSP1/IBMPFD patients   is a progressive neurodegenerative disease. It mainly affects the motor neurons in the brain and spinal cord, resulting in death from respiratory failure. While most ALS cases were caused by sporadic mutations, about 10% are considered \u201cfamilial\u201d because often more than one individual in a family develops the disease. Mutations in at least 18 genes have been identified in familial ALS. Among them, p97 mutations account for less than 2%  is an autosomal dominant axonal peripheral neuropathy characterized by distal muscle weakness and atrophy associated with length-dependent sensory loss. Like ALS, CMT is a clinically and genetically heterogeneous disorder and is divided into subtypes based on genetics, pathology, and electrophysiology of the disease (Dyck and Lambert, in vitro as it preferentially kills cancer cells isolated from patients; and it can synergize with the proteasome inhibitor Bortezomib to induce apoptosis in cancer cells (Wang et al., Given the important roles played by p97 in diverse cellular processes, specific inhibitors of p97 can be useful tools for dissecting the mechanism of p97 action. Early chemical screens focusing on compounds that inhibit ERAD identified two structurally related chemicals (Fiebiger et al., More recently, chemical screens in search of compounds directly targeting p97 have been conducted. Chou and colleagues reported the first reversible p97 D2 inhibitor, DBeQ (Chou et al., In addition to the above-mentioned inhibitors, efforts from several groups have resulted in a large collection of p97 inhibitors Figure  Yi et a. Among tin vitro GFP unfolding assay represent a major step toward fully elucidating the mechanism of this important enzyme. Another key question is to understand the hierarchical organization of cofactor binding in the context of the ATPase cycle and substrate binding cycle. Moreover, animal models bearing disease-associated mutations are needed in order to better appreciate the connections between p97 dysfunction and human diseases. The recent advance in CRISPR technology should dramatically ease the development of these animal models. Finally, given the promising anti-cancer effect of p97 inhibitors, it is anticipated that more p97 inhibitors will be sought, and studies in this direction may one day produce a new class of anti-cancer agent.Through years of studies, we have accumulated a large body of knowledge on the structure and function of p97/Cdc48. Specifically, the identification of new p97 cofactors and substrates has revealed a whole new set of biological functions for this essential chaperone system, and it is anticipated that future studies will further expand the p97 functional repertoire. By contrast, mechanistic dissection of the molecular nature of the \u201csegregase\u201d activity has lagged behind, and many fundamental questions remain unresolved. Among them, the most intriguing one is how conformational changes in p97 generate the proposed \u201csegregase\u201d activity. The recently developed TZ and WT prepared the figures and tables, prepare part of the manuscript. DX and YY 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."}
{"text": "The present study investigated differences in the pickup of information about the size and location of an obstacle in the path of locomotion. The main hypothesis was that information about obstacle location is most useful when it is sampled at a specific time during the approach phase, whereas information about obstacle size can be sampled at any point during the last few steps. Subjects approached and stepped over obstacles in a virtual environment viewed through a head-mounted display. In Experiment 1, a horizontal line on the ground indicating obstacle location was visible throughout the trial while information about obstacle height and depth was available only while the subject was passing through a viewing window located at one of four locations along the subject\u2019s path. Subjects exhibited more cautious behavior when the obstacle did not become visible until they were within one step length, but walking behavior was at most weakly affected in the other viewing window conditions. In Experiment 2, the horizontal line indicating obstacle location was removed, such that no information about the obstacle (size or location) was available outside of the viewing window. Subjects adopted a more cautious strategy compared to Experiment 1 and differences between the viewing window conditions and the full vision control condition were observed across several measures. The differences in walking behavior and performance across the two experiments support the hypothesis that walkers have greater flexibility in when they can sample information about obstacle size compared to location. Such flexibility may impact gaze and locomotor control strategies, especially in more complex environments with multiple objects and obstacles. To successfully navigate through complex environments in the natural world, humans and other animals must adapt their movements to the conditions that are encountered not only at the present moment but also in the near future. For example, a safety conscious hiker faced with a choice of which of two routes to follow may prefer the one that is initially more difficult if the alternative option eventually leads to a steep slope that lacks secure footing. A hiker who lacks the foresight to account for the dangerous terrain that lies farther ahead will eventually be faced with an unfortunate dilemma: to turn back, thus making an energetically costly corrective adjustment, or to attempt to scale the unsafe slope without injury and at great energetic cost. Similarly, anticipation plays an important role over shorter time-scales as the hiker chooses where to place his or her feet. Sudden and unexpected changes to foot placement are energetically costly, and introduce instability that could lead to falling. Avoiding these sudden adjustments requires the advanced sampling of the visual information necessary for guiding gait in the presence upcoming obstacles in a feedforward manner.In the present study, we investigated the visual information that walkers use to guide the placement and trajectory of the feet when approaching and stepping over an obstacle. The specific aim was to determine whether there are differences in when visual information about obstacle location and visual information about obstacle size  are needed. We present the results of two experiments designed to test the hypothesis that there is a greater flexibility in the time at which walkers must sample information about obstacle size compared to obstacle location.Perhaps the most clear-cut example of visual information sampled at one point in time being used at a later time to negotiate obstacles is when guiding the trail foot over the obstacle . Once thInterestingly, walkers use visual information in a feedforward manner well before the obstacle passes out of view. Despite the demands for precision in placement of the feet in front of and behind the obstacle \u20134 and inAlthough the act of moving the legs over the obstacle does not appear to require continuous visual sampling, it remains possible that walkers rely on on-line control to guide placement of the feet in front of the obstacle, especially given that variability in foot placement before the obstacle can lead to an increase in the risk of tripping , 3, 16. Although visual occlusion leads to differences in the kinematics of the approach to and step over an obstacle, we contend that it would be premature draw the conclusion that walkers rely on on-line rather than feedforward control to guide placement of the feet on the last step before the obstacle for the following reasons. First, subjects were still capable of successfully crossing the obstacle under such conditions. They simply adopted a more cautious strategy. Second, Patla  Of course, there is a limit to how far in advance such information can be sampled. When vision was occluded three steps before reaching the obstacle, subjects cleared the obstacle but with a higher safety margin . OccludiFor the purposes of the present study, there are two important points to take away from the prior research. First, although it remains possible that visual information is used in an on-line manner during some phases of obstacle crossing , the evidence also indicates that walkers are capable of guiding the lead and trail legs over the obstacle without concurrent visual information. Second, walkers perform best when information about the obstacle is available up until (or shortly before) placement of the trail foot before the obstacle.The previous research summarized above suggests that walkers are most successful in obstacle crossing when they are able to sample information about the obstacle at a particular point during the approach\u2014viz. while initializing and executing the last step before the obstacle. The information that walkers sample at this time appears to be that which specifies the location of the obstacle and is needed to guide placement of the trail foot before the obstacle. When crossing obstacles that have non-negligible height and depth, however, the walker must adapt his or her behavior to the dimensions of the obstacle as well as its location. Hence, in addition to information about obstacle location, walkers must also pick up information about the extent of the obstacle. That is, obstacle crossing requires the detection of both exproprioceptive information  and exteroceptive information  , 15. WheThe primary aim of this study was to test the hypothesis that there is flexibility in the time that information about obstacle size can be sampled. Studies of both humans  and non-Walkers may also be able to pick up information about obstacle size later in approach. Humans do not place their trailing foot closer or farther from the obstacle across variations in obstacle height . If otheOur approach was to instruct subjects to step over obstacles in a virtual environment viewed through a head-mounted display (HMD). To test the flexibility in when information may be sampled, we manipulated the availability of visual information about the obstacle\u2019s size. In Experiment 1, the obstacle\u2019s location was always visible by displaying a narrow line at the base of the front edge of the obstacle. In this regard, our approach is similar to that in previous studies in which information about obstacle dimensions was removed while information about obstacle location was made available throughout the approach  and the full vision control condition.Fifteen undergraduate students from Rensselaer Polytechnic Institute volunteered to participate in the study. Subjects reported that they had normal or corrected-to-normal vision and did not have any visual or motor impairments. The protocol was approved by the Institutional Review Board at Rensselaer Polytechnic Institute and all subjects gave informed consent prior to participation.The experiment was conducted in a 6.5 m \u00d7 9 m laboratory. The HMD was an nVis SX111 stereoscopic head mounted display , with a resolution of 1280 pixels \u00d7 1024 pixels  per eye and a diagonal field of view of 111 degrees. Head position and orientation were tracked using an Intersense IS-900 motion tracking system . Data from the tracking system were used to update the position and orientation of the simulated viewpoint. Movements of the rest of the body were tracked using a 14 camera Vicon motion-capture system running at 120 FPS. Subjects wore tight fitting, stretchable clothing to which 34 retro-reflective markers were affixed in accordance Vicon full-body marker template. Subjects completed the experiment barefoot in order to avoid any irregularities caused by differences in footwear. The four Vicon markers normally used to record head position in the full-body template were instead attached to the HMD. The cables from the HMD and tracking system were bundled together and held by the experimenter, who walked alongside the participant as he or she moved to ensure that the cables did not interfere with the subject\u2019s movement.The virtual environment was created using Vizard Virtual Reality Toolkit  running on an Alienware Area-51 PC , and consisted of a flat, grass-textured ground plane underneath a black sky . Bamboo Subjects\u2019 feet were also rendered in the virtual environment and visible through the HMD see . The dimPrior to the beginning of each session, the subject\u2019s leg length was measured as the distance from the greater trochanter of the femur to the floor, and orientation of the virtual foot was aligned to match that of the real-world foot. Subjects were instructed to begin each trial by pressing a button on a hand-held wireless mouse while (1) standing within the visually indicated start box and (2) facing the end post. If these two conditions were met at the time of the button press, a virtual obstacle or line appeared along the subject\u2019s walking path. At the same time, an auditory go-signal indicated that the subject was to begin walking towards the post at a comfortable speed, without stopping, and while stepping over the virtual obstacle along the way.On some trials, the obstacle was visible only when the subject\u2019s head was within a spatially defined viewing window. When the subject\u2019s head was outside of the viewing window, the obstacle was invisible and was replaced by a thin line on the ground at the location of the obstacle\u2019s front edge. Collisions between the virtual feet and the obstacle were continuously monitored. If any part of either foot collided with any part of the obstacle, an auditory \u201cthud\u201d was played through the speakers.The height, depth, and location of the obstacle were manipulated as independent variables. The values of all three variables were specified in units of leg length (LL) to ensure that the kinematic demands of obstacle crossing would be similar across subjects whose body dimensions varied.Each experimental session included 126 trials, 46 of which were full vision trials and 80 of which were viewing window trials. Full vision trials varied in obstacle height , depth , and distance of the obstacle from the start box . Subjects also performed an additional full vision trial to ensure an even number of trials, which was necessary for us to run even and odd numbered trials in opposite directions in the laboratory. On this additional trial, the obstacle was placed at 3.6 LL and had a height and depth of 0.1 LL.On viewing window trials, information about the obstacle\u2019s dimensions was only available when the subject\u2019s head was inside one of four spatially defined windows. These windows spanned increments of 0.7 leg lengths from the obstacle . Obstacle height , depth , and location  also varied across trials. The selected values of height and depth correspond to the extremes of the ranges used on full vision trials. All independent variables were manipulated within-subjects. All trial types were pooled and presented in a different fully randomized order to each subject. The entire experimental session lasted approximately 45 minutes.Subjects completed two short practice blocks prior to beginning the experiment. The first practice block comprised 20 full vision trials: 2 obstacle heights  \u00d7 2 obstacle depths  \u00d7 5 obstacle locations . In the second practice block, subject completed eight trials, two in each viewing window condition, with both obstacle height and depth set to 0.1 LL. Practice trials were intended to provide familiarity with the virtual environment, and were not included in the analysis.In interpreting the findings below, it is often helpful to know where the subject was in the gait cycle while the obstacle was visible in each viewing window condition. The size of the viewing windows (0.7 LL) was chosen to roughly match average step length during steady state walking  so that Subsequent to data collection, Vicon Nexus was used to convert marker positions into joint positions. Data were filtered using a seventh-order low-pass Butterworth filter with a cutoff at 4 Hz. Steps were identified using a velocity based kinematic analysis presented in  and usedWe calculated two measures of obstacle crossing behavior: maximum foot elevation and obstacle clearance. Maximum foot elevation was estimated based on the height of the toe marker at the peak of its trajectory during the crossing step. Obstacle clearance was based on the distance from the toe marker to the top of the obstacle at the moment that the toe marker crossed the front face of the obstacle.Our approach to analyzing the data relied on mixed-effects regression modeling using the \u201cnlme\u201d package in R. For each dependent measure, we built a set of linear models with various combinations of the three independent variables  and in some cases their interactions as predictors. To keep the number of levels of height and depth consistent across all five viewing window conditions, we excluded data from the middle height and middle depth conditions in the full vision condition. Decisions about which models to build and compare were motivated by the theoretical predictions. For most dependent measures, we first compared a model with height as a predictor against the baseline (intercept-only) model. If the height model is superior to the baseline model, this is equivalent to finding that the main effect of height is statistically significant. To test the main hypothesis of the study, we then compared the model with height against a model with both height and viewing window  as predictors. If behavior (as measured by the dependent variable) is influenced by viewing window or the height \u00d7 viewing window interaction, the more complex model will be superior to the simpler model. Thus, by comparing the two models, we can determine whether viewing window influenced behavior or if the effect of height varied across viewing window.Model fit alone is not an adequate metric for determining which of two models is better, since more complex models always better fit the data. Instead, we compared models using the log-likelihood ratio comparison (in the form of a chi-squared significant test) and the change in Akaike Information Criterion (AIC). AIC is a widely used metric of the quality of a model because it captures both the goodness of fit and the complexity of the model. When reporting the difference in AIC (dAIC) between two models, negative values indicate that the model of interest is superior to the comparison  model.After analyzing the effects of height and viewing window, we repeated the process for depth and viewing window. Thus, these were treated as separate analyses. Technically, it is possible to combine height, depth, and viewing window into a single analysis for each dependent measure. However, such an approach would have required consideration of models with higher-order interaction terms . This was not necessary because the height \u00d7 depth interaction was not statistically significant for any of the dependent measures.Number of steps On average, subjects took 5.59 (SE = 0.13) steps to reach the obstacle in the full vision condition. The mean number of steps was slightly greater in each of the viewing window conditions  compared to full vision. Indeed, the model with viewing window as a predictor had a significantly lower AIC than the baseline model (\u03c72(4) = 13.83, p<.01, dAIC = -5.83). Adding height (\u03c72(1) = 1.19, p = .28, dAIC = -.81) or both height and height \u00d7 viewing window (\u03c72(5) = 1.53, p = .91, dAIC = 8.47) did not significantly improve the model. However, the model with depth and viewing window was superior to the model with viewing window (\u03c72(1) = 6.02, p<.05, dAIC = -4.02), reflecting a tendency to occasionally take an additional step during approach to deep obstacles (M = 5.73) compared to shallow obstacles (M = 5.64). We attribute this difference to the tendency  to place the feet slightly closer to the obstacle when it is deeper.Walking speed In general, subjects gradually accelerated between steps N-4 and N-1, reaching peak walking speed at step N-1 before decelerating on the step over the obstacle (\u03c72(4) = 128.23, p<.01, dAIC = -120.28). Adding height (\u03c72(1) = 1.01, p = .31, dAIC = 0.97) or depth (\u03c72(1) = 0.22, p = .64, dAIC = 1.78) did not improve the model relative to the step-number model, suggesting that subjects did not walk at different speeds in different obstacle conditions. However, AIC decreased when viewing window and viewing window \u00d7 step number were included (\u03c72(20) = 98.01, p<.01, dAIC = 58.01). Inspection of obstacle . The chaObstacle height For lead foot placement, the model with height as a predictor was not significantly different than the baseline model (\u03c72(1) = 0.21, p = .65, dAIC = 1.791) (see \u03c72(5) = 5.56, p = .35, dAIC = 4.44), nor did it improve when the model included height, viewing window, and the height \u00d7 viewing window interaction (\u03c72(9) = 15.46, p = .07, dAIC = 2.54). Although the difference between the full model and the baseline model is close to statistically significant, the change in AIC is positive, consistent with a poorer model. Taken together, the analysis reveals no evidence that lead foot placement was influenced by obstacle height, viewing window, or their interaction.791) see . This is\u03c72(1) = 1.95, p = .16, dAIC = 0.05), nor the model with height and viewing window (\u03c72(5) = 3.40, p = .64, dAIC = 6.60), nor the model with height, viewing window, height \u00d7 viewing window (\u03c72(9) = 10.95, p = .28, dAIC = 7.05) was significantly better.Similarly, there was no evidence that trail foot placement was affected by either variable or their interaction. The model with the lowest AIC was the baseline model. Neither the model with height  = 13.13, p<.01, dAIC = -11.13). Adding viewing window did not significantly improve the model (\u03c72(4) = 7.03, p = .13, dAIC = 0.97), nor did adding both viewing window and the depth \u00d7 viewing window interaction (\u03c72(8) = 8.96, p = .35, dAIC = 7.04). Thus, subjects placed their lead foot closer to the obstacle when it was deeper, but lead foot placement was not significantly affected by viewing window or the depth \u00d7 viewing window interaction.ctor see . The dep\u03c72(1) = 13.79, p<.01, dAIC = -11.79), but not significantly different from the model with viewing window (\u03c72(4) = 1.48, p = .83, dAIC = 6.52) or the model with viewing window and the depth \u00d7 viewing window interaction (\u03c72(8) = 13.79, p = .09, dAIC = 2.22).A similar pattern of results was observed for trail foot placement. The model with the lowest AIC was the one with depth as a predictor. This model was significantly better than the baseline model , but subjects tended to place their feet slightly closer to the obstacle when it was deeper. However, neither lead nor trail foot placement were affected by the viewing window manipulation or its interactions with height or depth. The fact that subjects adapted foot placement to variations in obstacle depth in the VW-4 and VW-3 conditions provides compelling evidence that walkers are able to adapt to variations in obstacle dimensions even when the relevant information is sampled several steps in advance.The next set of analyses focuses on obstacle crossing and is organized by foot .Lead foot elevation and clearance Let us first consider the effects of obstacle height and viewing window on lead foot obstacle crossing (\u03c72(1) = 15.47, p<.01, dAIC = -13.47). This confirms that subjects were able to discriminate obstacles of different heights in the virtual environment and adapt their behavior as they do in the real world. Had subjects not been able to perceive variations in obstacle height , they could have successfully performed the task by elevating their feet to the same height . The increase in lead foot elevation with obstacle height suggests otherwise.crossing . The mod\u03c72(8) = 20.05, p<.05, dAIC = -4.05), which suggests that the manipulation of obstacle height affected lead foot elevation differently depending on when the obstacle was visible. To unpack the interaction, we conducted four 2 (VW-# vs. Full) \u00d7 2  interaction contrasts using Dunnett\u2019s correction. The interaction was significant (p < .05) in the VW-1 condition but not in any of the other viewing window conditions. This likely reflects adoption of a cautious strategy in the absence of information about obstacle height. If so, this would suggest that walkers are most successful in obstacle crossing when they can sample information about obstacle height prior to placement of the trail foot before the obstacle, which is consistent with the results of the analysis of walking speed.However, the model with the lowest AIC was the full model that included height, viewing window, and their interaction. This model was significantly better than the height-only model (\u03c72(1) = 49.82, p<.01, dAIC = -47.82)  = 26.18, p<.01, dAIC = -24.18), which is consistent with the findings of Patla and Rietdyk [\u03c72(4) = 8.77, p = .06, dAIC = -0.77) nor the full model (\u03c72(8) = 15.26, p = .06, dAIC = 0.74) were significantly better than the depth-only model. Thus, unlike the effect of obstacle height, the effect of obstacle depth did not significantly vary across viewing window conditions. The pattern of results was similar  = 6.42, p<.05, dAIC = -4.42). As with the lead foot, the increase in trail foot elevation was less than the change in obstacle height, resulting in a significant decrease in trail foot clearance (\u03c72(1) = 33.81, p<.01, dAIC = -31.81). Mean trail foot maximum elevation was greater for taller obstacles in each of the viewing conditions except for VW-1, mirroring the pattern of results observed with the lead foot. However, the model of maximum foot elevation with viewing window and height \u00d7 viewing window was not significantly better than the height model (\u03c72(8) = 9.86, p = .27, dAIC = 6.14). In other words, the effect of obstacle height did not vary across viewing window conditions as it did for the lead foot. This is likely due to the greater variability in trail foot elevation.bstacles . This wa\u03c72(1) = 12.02, p<.01, dAIC = -10.02) (see \u03c72(8) = 18.22, p<.05, dAIC = -2.22), suggesting that obstacle depth may have affected trail foot elevation differently across viewing window conditions. Specifically, inspection of Trail foot elevation also increased with obstacle depth (.02) see . The mod\u03c72(1) = 3.60, p = .06, dAIC = -1.60) or viewing window \u03c72(4) = 9.23, p = .06, dAIC = -1.24) (see \u03c72(1) = 4.09, p<.05, dAIC = -2.09) (see Collision rates for the lead foot were below 2% on average and were not significantly affected by obstacle height (.24) see . Subject.09) see .\u03c72(1) = 7.72, p<.01, dAIC = -5.72) but was not significantly affected by viewing window (\u03c72(4) = 0.71, p = .95, dAIC = 7.29) or depth (\u03c72(1) = 0.04, p = .84, dAIC = 1.96) (see Trail foot collision rate increased with obstacle height (.96) see . The ratSeveral conclusions can be drawn from these analyses. First, when the obstacle was visible throughout the trial, subjects were able to adapt foot placement and trajectory to variations in the position, height, and depth of the obstacle, much like they do in the real world. The only measures on which performance noticeably differed were foot elevation during obstacle crossing and the frequency of collisions involving the trailing foot.Second, for most dependent variables, adding viewing window, height \u00d7 viewing window, and/or depth \u00d7 viewing window did not improve the model, suggesting that walking behavior was at most weakly affected by the absence of obstacle dimension information during most of the approach. The largest effects were observed when such information was not made available until subjects were within one step length of the obstacle. In this condition , the obstacle first appeared on average shortly before placement of the trailing foot before the obstacle see . The facThird, there was no evidence that walking behavior or obstacle crossing performance were affected when obstacle dimension information was available early in the approach and then removed . As in full vision, subjects adapted foot placement to obstacle depth but not height, elevated their feet to a greater height for taller and deeper obstacles, and maintained a comparable rate of collisions even in the VW-4 condition. This provides compelling evidence that walkers are able to sample information about obstacle size in advance and use that information several steps later to guide obstacle crossing.Taken together, the findings of Experiment 1 support the hypothesis that walkers have considerable flexibility in the timing of the sampling of information about obstacle size. Such information can be sampled early when the walker is several steps from the obstacle, which implies an ability to use information that was detected several steps earlier. Alternatively, information about obstacle size can be sampled later, shortly before placement of the trail foot before the obstacle, which reflects the fact that accommodating variations in obstacle size does not require adjustments until shortly before the step over the obstacle is initiated.In Experiment 1, our focus was on information about obstacle size and how such information can be sampled in a brief glance at any point between two and four steps in advance. Our hypothesis, which was motivated by previous research on obstacle crossing, is that such flexibility in the timing of visual sampling is specific to information about obstacle size. That is, information about obstacle location, unlike information about obstacle size, is most useful when it is sampled at a particular point in time during approach\u2014shortly before placement of the trail foot before the obstacle. However, the difference is likely to be a matter of degree; that is, walkers may have some flexibility in terms of when they can sample information about obstacle location, but less flexibility than is afforded for information about obstacle size.The design of Experiment 1 does not allow us to compare such differences in flexibility because information about obstacle location was continuously available in all five conditions. The results of the previous studies summarized in the introduction provide some insight into the range of distances within which location information is most useful. However, any conclusions would be stronger if they were based on a more direct comparison using the same experimental paradigm. To this end, we conducted a second experiment that was similar to Experiment 1 but with the visibility manipulation applied to both size and location information. In other words, the obstacle was either visible or not. There was no persistent line indicating the position of the front edge of the obstacle. If information about obstacle location is most useful when it is sampled while the walker is within a narrow range of distances from the obstacle, then unlike in Experiment 1, differences between the viewing window conditions and the full vision condition should be widely observed.Nine undergraduate students from Rensselaer Polytechnic Institute volunteered to participate in the study. Subjects reported that they had normal or corrected-to-normal vision and did not have any visual or motor impairments. The protocol was approved by the Institutional Review Board at Rensselaer Polytechnic Institute and all subjects gave informed consent prior to participation.The methods were identical to those of Experiment 1, with the exception that the line at the front edge of the obstacle was removed in Experiment 2. As such, information about obstacle location, height, and depth was only available when the subject\u2019s head was within the viewing window.As in Experiment 1, timing of the viewing windows was determined by the position of the subject\u2019s head from the obstacle, defined in units of leg length. The actual time at which the appearance and disappearance of the obstacle were triggered with respect to the events in the gait cycle is presented in Whereas the viewing window manipulation had little effect on walking behavior during the approach phase in Experiment 1, differences between the VW conditions and the full vision condition were apparent in Experiment 2.Number of steps The number of steps that subjects took before crossing the obstacle was significantly affected by viewing window (\u03c72(4) = 95.77, p<.01, dAIC = -87.77). As depicted in \u03c72(1) = 0.05, p = .82, dAIC = 1.95 compared to the model with viewing window), but adding depth did (\u03c72(1) = 5.50, p<.05, dAIC = -3.50). As in Experiment 1, the mean number of steps was slightly greater during approaches to deeper obstacles.Walking speed The manipulation of viewing window also affected walking speed (see \u03c72(24) = 386.539, p<.001, dAIC = -338.539 compared to the baseline model). Adding either height (\u03c72(1) = 0.15, p = .70, dAIC = 1.85) or depth (\u03c72(1) = 1.30, p = .25, dAIC = 0.70) did not further improve the model. These analysis suggest that walking speed was consistent across changes in obstacle dimension but that the walking speed profile varied depending on when the obstacle was visible. As depicted in peed see . The modOn their own, these findings could be interpreted as evidence that subjects proceeded cautiously due to the absence of information about obstacle size, obstacle location, or both. However, in Experiment 1, which differed from Experiment 2 only in that information about obstacle location was visible throughout each trial, walking speed in the VW conditions was nearly identical to full vision on each step with the exception of step N-1. This suggests that it was the absence of obstacle location information (not size information) that led subjects in Experiment 2 to walk initially at a slower speed.Interestingly, walking speed was slower overall compared to Experiment 1 even in the full vision condition. We attribute this difference to the adoption of a more cautious strategy in the VW conditions of Experiment 2, and the fact that full vision trials were randomly interleaved with and less frequent than trials in which obstacle visibility was manipulated. Thus, the tendency to walk slower apparently carried over to full vision trials.Obstacle height As in Experiment 1, placement of the lead and trail feet before the obstacle on the last step before crossing was consistent across obstacle height (\u03c72(1) = 0.26, p = .61 dAIC = 1.74 for the lead foot; \u03c72(1) = 0.01, p = .96, dAIC = 2.00 for the trail foot; see \u03c72(4) = 116.51, p<.001, dAIC = -108.51) and trail foot (\u03c72(4) = 72.70, p<.001, dAIC = -64.70) placement.Contrasts with Dunnett\u2019s correction between each VW condition and the full vision condition revealed significant differences (p < .05) in the VW-2 and VW-1 conditions for the lead foot and in the VW-1 condition for the trail foot see . Thus, iIn the VW-2 condition, subjects placed their lead foot significantly closer to the obstacle compared to full vision, but trail foot placement was not significantly different than full vision. These effects were observed for each combination of obstacle height and depth. This makes sense because in the VW-2 condition, the obstacle did not appear on average until shortly before the end of the lead foot\u2019s last step before the obstacle see . Thus, sIn the VW-1 condition, both the lead and trail feet were placed significantly closer to the obstacle than in full vision. This also makes sense because in the VW-1 condition, the obstacle did not appear until after placement of the lead foot and shortly before placement of the trail foot.Obstacle depth Lead foot placement was not significantly affected by obstacle depth (\u03c72(1) = 2.41, p = .12, dAIC = -0.41) but trail foot placement was significantly closer when the obstacle was deeper (\u03c72(1) = 9.64, p<.01, dAIC = -7.64) (see \u03c72(4) = 110.00, p<.001, dAIC = -102.00) and trail foot (\u03c72(4) = 69.13, p<.001, dAIC = -61.13) placement..64) see . Adding Contrasts with Dunnett\u2019s correction between each VW condition and the full vision condition revealed significant differences (p<.05) in the VW-2 and VW-1 conditions for the lead foot and in the VW-1 condition for the trail foot. It is noteworthy that subjects placed their trail foot closer to the obstacle when it was deeper in the VW-4 condition, as they did in the full vision condition see . This prThe results of the analysis of foot placement before the obstacle are not particularly surprising. Nevertheless, the contrast with the results of the same analysis for Experiment 1  highlighLead foot elevation and clearance As shown in \u03c72(1) = 4.60, p<.05, dAIC = -2.60) and deeper (\u03c72(1) = 6.34, p<.05, dAIC = -4.34). The tendency for subjects to increase the maximum foot elevation for taller and deeper obstacles was also found in Experiment 1. However, foot elevation in Experiment 2 was consistently greater than it was in Experiment 1 even in the full vision condition, despite the fact that trials in the full vision condition were identical in both experiments. This clearly reflects the adoption of a more cautious strategy due to the increased difficulty of the task in Experiment 2.\u03c72(4) = 33.46, p<.001, dAIC = -25.46). Further adding height \u00d7 viewing window resulted in an increase in AIC, suggesting that the effect of obstacle height did not significantly vary across VW conditions. Contrasts with Dunnett\u2019s correction between each VW condition and the full vision condition were significant (p<.05) only in the VW-1 condition. Thus, subjects\u2019 ability to elevate their lead foot over short and tall obstacles was affected only when the obstacle did not become visible until the last step. This was also the case in Experiment 1. However, whereas lead foot elevation in VW-1 was greater than or comparable to that in full vision in Experiment 1 (see Adding viewing window as a predictor significantly improved the model of lead foot elevation relative to the height-only model (Trail foot elevation and clearance Trail foot elevation was significantly affected by both obstacle height and viewing window (\u03c72(5) = 25.93, p<.001, dAIC = -15.93 compared to the baseline model) but the interaction between these two variables was not statistically significant (\u03c72(4) = 8.09, p = .08, dAIC = -0.09) (see .09) see . Contras\u03c72(5) = 28.38, p<.001, dAIC = -18.38 compared to the baseline model) but the interaction between depth and viewing window was not statistically significant (\u03c72(4) = 5.26, p = .26, dAIC = 2.74).The analysis of the effects of depth and viewing window on trail foot elevation revealed a similar pattern of results see ; that isTaken together, this analysis suggests that walkers are able to properly elevate their trail foot over obstacles of varying heights and depths as long as visual information about the obstacle was available between roughly two and three step lengths in advance. If such information is available before or after this portion of the approach phase, the walker\u2019s ability to properly elevate the trail foot will be degraded. Furthermore, because these effects were observed in Experiment 2 but not in Experiment 1, we can conclude that the degradation is due to the absence of information about obstacle location specifically.\u03c72(1) = 3.51, p = .06, dAIC = -1.51) or depth (\u03c72(1) = 2.39, p = .12, dAIC = -0.39), but the addition of viewing window as a predictor did improve the model (\u03c72(4) = 11.93, p<.05, dAIC = -3.93). This effect is likely to be a consequence of the reduced foot placement distance before the obstacle in the VW-1 condition.As in the full vision condition of Experiment 1, collisions were rare for the lead foot but more frequent for the trail foot see . Lead fo\u03c72(1) = 5.49, p<.05, dAIC = -3.49) but was not affected by the manipulation of obstacle depth (\u03c72(1) = 1.55, p = .21, dAIC = 0.45). The manipulation of viewing window also affected trail foot collision rate (\u03c72(4) = 15.64, p<.01, dAIC = -7.64), in particular in the VW-1 and (to a lesser degree) the VW-4 conditions.Trail foot collision rate was significantly greater for taller obstacles  in Experiment 2 compared to Experiment 1. Taken together, the results indicate that obstacle crossing performance degrades when information about obstacle location is not available when it is needed to guide placement of the feet in front of the obstacle. This contrasts with the information about obstacle size, which can be sampled at any point during the last few steps before placement of the lead foot before the obstacle, as demonstrated in Experiment 1. Thus, walkers have less flexibility in when they can sample information about obstacle location compared to information about obstacle size.To successfully cross an obstacle in the path of locomotion, humans must adapt their gait to both the location of the obstacle and its dimensions . Walkers are most successful in obstacle crossing when they can sample information about location shortly before placement of the trail foot in front of the obstacle, which makes sense given that precise foot placement plays a key role in minimizing the risk of tripping , 3, 16. The ability to use information about obstacle size that was detected several steps in advance was demonstrated in Experiment 1, in which behavior in the VW-4 condition was similar to that observed in the full vision condition on measures of number of steps, walking speed, lead and trail foot placement, maximum foot elevation, foot clearance, and number of collisions. On measures where behavior was affected by manipulations of obstacle height and depth in the full vision condition, behavior was similarly affected in the VW-4 condition. In both conditions, subjects placed their feet slightly closer to the obstacle when it was deeper but not when it was taller, and elevated their feet to a greater height when the obstacle was taller and when it was deeper. Although behavior in the VW-4 condition was also similar to the full vision condition in Experiment 2, when the line marking obstacle location was absent, performance in both conditions was degraded compared to the corresponding conditions in Experiment 1. Thus, the ability to use information that was sampled in advance is specific to information about obstacle size.Why are walkers able to use information about obstacle size that is sampled in advance but need information about obstacle location later during the approach? One possibility is that size and position  differ in their stability. Information about properties that do not vary from moment to moment, such as obstacle size, may be sampled well in advance because their stability makes it possible for that information to be useful at a later time. In contrast, information about properties that vary from moment to moment, such as the walker\u2019s position relative to the obstacle, must be sampled around the time that it is needed for making gait adjustments. A similar account has been proposed to explain why the transport and manipulation components of reaching and grasping are affected differently when there is a delay between viewing the object to be picked up and initiating movement of the hand (see ). Of couThe ability to use information about obstacle size later in the approach was apparent in the similarity of behavior in the VW-2 and full vision conditions of Experiment 1. Although the obstacle did not appear until shortly before placement of the lead foot, subjects generally elevated their feet to a greater height for taller and deeper obstacles as they did in the full vision condition. Nevertheless, some of these effects were weaker than they were in the full vision condition, suggesting that subjects\u2019 ability to adapt to changes in obstacle dimensions was slightly diminished in this condition. When the obstacle did not appear until the last step before crossing (VW-1 condition), subjects were even less able to tailor foot elevation to variations in obstacle height and depth. Thus, the last point at which subjects are able to adapt gait to obstacle size appears to be shortly before placement of the trail foot in front of the obstacle.Why are walkers able to adapt to changes in obstacle size even when the relevant information is not made available until shortly before obstacle crossing? Even when information about obstacle size is available throughout the approach, walkers apparently do not make any adaptations to gait that are specific to obstacle size prior to the step over the obstacle.  As such, not knowing the dimensions of the obstacle prior to the initiation of obstacle crossing does not impair performance in the way that not knowing the location of the obstacle does.The findings of the present study demonstrate that walkers are capable of adapting gait to variations in obstacle height and depth even if information about these characteristics is sampled several steps in advance. One might wonder about the significance of this ability during real-world obstacle crossing given that walkers must sample information about obstacle location later during the approach anyway (to guide foot placement in front of the obstacle), and could sample information about obstacle dimensions at the same time. In other words, what is the functional significance of estimates of obstacle dimension based on information that is detected well in advance if those estimates are updated later during the approach, when information about obstacle location must be sampled?Although walkers need information about obstacle location when they are closer to the obstacle, they do not necessarily need to look at the obstacle to pick up that information. In many complex environments, there are other objects that are visible in the upper visual field that could be used to keep track of one\u2019s position relative to the obstacle without actually looking at it. For example, if the obstacle is a fallen branch that lies on a hiking path near the base of a tree, the walker could perceive his or her changing position relative to the branch without looking down or relying on peripheral vision by detecting information about the relative position of the tree, much like subjects in the study by Rietdyk and Rhea  relied oThis could be especially useful when there are multiple obstacles in the path of locomotion. In such situations, walkers must shift their gaze to future obstacles before stepping over the obstacle that is most immediately in front of them . The abiThe present study is one of a small number on obstacle crossing to be conducted in a virtual environment (VE) viewed through a head-mounted display (HMD). VEs offer a potentially powerful tool for investigations of visually guided locomotion over complex terrain, as they enable researchers to manipulate visual information in a manner that is not possible in the real world. Of course, there are also some drawbacks. Misperceptions of size and egocentric distance , latDespite these differences, subjects were consistent in the placement of their lead and trail feet in front of the obstacle, elevated their feet to a greater height for taller and deeper obstacles, and avoided making obstacle contact with the lead foot on the vast majority of trials. Thus, in many respects, obstacle crossing behavior in the present study was similar to that observed in the real world. Given the potential for using VEs in both basic research on locomotion as well as in the diagnosis and treatment of disorders that affect mobility , further research on obstacle crossing behavior in virtual environments, as well as comparisons of behavior in real and virtual environments, would make a valuable contribution.Although walkers need information about both the location and size of an upcoming obstacle to guide successful crossing, only information about obstacle location is needed at a specific point in time during approach. The success with which walkers are able to negotiate obstacles is less dependent upon when information about obstacle height and depth is sampled. They can sample information about obstacle size several steps in advance and use that information to adapt the placement and trajectory of the feet during obstacle crossing. Alternatively, they can sample such information as late as the last step before obstacle crossing and still elevate their feet to different heights depending on the dimensions of the obstacle."}
{"text": "The role of p53 as \u201ca guardian of the genome\u201d has been well established in somatic cells. However, its role in pluripotent stem cells remains much more elusive. Here, we discuss research progress in understanding the role of p53 in pluripotent stem cells and in pluripotent stem cell-like cancer stem cells. The p53 protein, which plays a key role in embryonic stem cells, was first discovered in 2005. Landmark studies of p53-related reprogramming elucidated this protein\u2019s importance in induced pluripotent stem cells in 2009. The p53-related safety concerns in pluripotent stem cells have been raised in stem cell-based therapy although the use of iPSCs in therapeutic application is promising. Because cancer stem cells have profiles similar to those of pluripotent stem cells, we also describe potential strategies for studies in cancer stem cells and cancer treatments. The new discoveries of p53 family proteins in pluripotent stem cells have made possible stable progress in stem cell transplantation efficiency and safety, as well as treatment strategies targeting cancer stem cells based on pluripotent stem cell technology. Although just a fairy tale, the theme of the story can be interpreted to mean \u201cregeneration medicine\u201d, one of the hottest research topics today. The existence of endogenous pluripotent stem cells and embryonic stem cells (ESCs) holds great promise for regenerative medicine. Induced pluripotent stem cells (iPSCs) were first generated from mouse somatic cells in 2006 through viral insertion of four transcription factors: Oct4, Sox2, Klf4, and cMyc .A year later, it was observed that human adult cells were also capable of being converted into pluripotent stem cells under similar conditions , 3. Due However, despite the great potential of pluripotent stem cell-based cell therapy, some important issues regarding safety concerns have been raised, especially their ability to induce tumorigenesis and teratoma formation . To addrThe protein p53 has been described as \u201ca guardian of the genome\u201d that protects somatic cells from tumorigenesis. However, whether p53 plays the same role in pluripotent stem cells is much more elusive. While efficiency in iPSC generation has been achieved, tumor-related safety studies involving iPSCs are limited. In fact, some iPSC generation methods may even undermine iPSC safety with respect to tumorigenesis. The current use of iPSCs in clinical trials is therefore limited due to tumorigenic risks. We thus discuss the role of p53 in pluripotent stem cells for clinical application and in pluripotent stem cell-like cancer cells, and how pluripotent stem cell technology can be used for cancer treatment.While the tumor-suppressor p53 activation in somatic cells during stress results in cell-cycle arrest and apoptosis, its activities in pluripotent stem cells remain unexplored. Different roles of p53 in ESCs, however, continue to be discovered. At the end of 2004, p53 was found to play a key role in the differentiation of mouse embryonic stem cells , and thiWhen DNA damage occurs in ESCs, phosphorylation of p53 at serine 315 occurs leading to high expression of the p53 protein. In particular, two sequences within the Nanog promoter region are regulated by p53 . Our extUnder normal conditions, with an intact p53 signaling pathway, ESCs can be differentiated by shifting culture conditions from mouse ESC (mESC) medium with mouse leukemia inhibitory factor (Lif) to mouse embryonic fibroblast (mEF) medium supplemented with retinoic acid (RA) Fig.\u00a0 24]. Un. Un24]. In contrast, the differentiated cells could be de-differentiated or reprogrammed back to ESC status when the p53 switch is turned off in humanized p53S315A knock-in mouse ESCs Fig.\u00a0. This meIn contrast to the findings noted above, recent studies have revealed that p53 might be involved in negative feedback pathways in the absence of DNA damage in mouse ESCs. For example, Abdelalim and Tooyama found that p53 was required for mouse ESC self-renewal . In addiThe detailed mechanism of the pluripotency switch by p53 protein isoforms was elegantly discovered by Ungewitter and Scrable in 2010 . This stThese two p53 homologues have specific functional differences from p53 and from one another. Mice lacking p63 are born with developmental defects of the limbs and skin caused by impaired ectodermal differentiation during embryogenesis , while pTypically, iPSCs are generated through insertion of viral genome into the sequences of a set of four pluripotency-associated genes: Oct4, Sox2, cMyc, and Klf4 \u20133. Such However, iPSC generation was initially a very slow and inefficient process, lasting approximately 2\u00a0weeks for mouse cells and 4\u00a0weeks for human cells. Furthermore, the original conversion of somatic cells into iPSCs occurred at an extremely low rate: approximately 0.01% in human  and 0.1%p53 expression not only reduces iPSC generation efficiency, but its deletion also replaces the reprogramming factors. It has been reported that deletion of c-Myc from the four-factor gene cocktail could generate iPSCs but that the yield is dramatically low-efficient iPSCs. However, a greatly improved reprogramming efficiency (by 10%) of parental cells generated into iPSCs was seen if p53 was switched off . More reThe p53 pathway switches on when DNA damage occurs; hence, only cells having no DNA damage will be able to undergo reprogramming , 43. p53All iPSC generation proteins  are expressed either individually or collectively in many cancer cell types , 45. HowAccordingly, a recent study found that the production of pluripotent stem cells, which is similar to the creation of the so-called \u201ccarcinogenic foci,\u201d can also be used as an experimental model for the study of cancer development and generation of possible treatment strategies . SimilarSome studies have linked iPSCs and pluripotent stem-like cancer cells. Kitajima et al. observed that deleting retinoblastoma protein (Rb) and N-ras genes in combination with oncogenic Trp53 mutations resulted in cancer stem-like cells, showing elevated expression of embryonic genes, carcinogenic identities, and sensitivity to cancer stem cell target compounds . In manyIf these data are representative of tumor or cancer stem cell initiation, then strategies based on pluripotent stem cell technology investigating cancer stem cells might be developed for cancer study and treatment Fig.\u00a0. In Fig.Shown as a red sphere, the tumor stem cell can freely transfer from a point in the developmental hill to some other point, including pluripotent stem-like and metastasis-like status. Once the normal activity of p53 is reactivated, the pluripotent stem-like status can be restricted and the cancer stem cell loses most of its stem cell properties. Previous findings have shown that activation of p53 by nutlin, a small-molecule antagonist of MDM2, leads to the rapid differentiation of human ESCs . BecauseA well-designed paper published a discovery that the undifferentiated state was induced by Rb-p53 double inactivation in mouse somatic cells in 2015 . Using iThe protein p53 has long been established as a key factor for the safety of adult cells. However, recent studies of pluripotent stem cells, including ESCs and iPSCs, have found that p53 is also a key player in pluripotent stem cell differentiation and in reprogramming to generate induced pluripotency. Given what is currently known regarding the generation of stem cells, p53-related activities must be carefully assessed in pluripotent stem cell-based research and applications.Pluripotent stem cells, including ESCs and iPSCs, are capable of transforming into malignant teratomas, carcinomas, or CSCs once p53 is not controlled. In contrast, the activation of p53 alone or in association with other factors is capable of inhibiting cancer via inhibition of stem cell-related mechanisms. As such, the use of p53 is a potential therapeutic strategy in the management of cancer by maintaining normal and genetically safe cells.Reactivation of p53 might control and rescue cells from switching into cancer stem cells with malignant pluripotency. However, due to other p53 protein isoforms, such as the deleted p53 isoform mentioned above , we shouAs discussed above, undifferentiated reprogramming resulting from Rb-p53 double inactivation might be a major target of the cancer stem cell treatment strategy. The triple abnormality RN6 cells showed not only pluripotency but also cancer formation potential. RN6 cells with pluripotency and tumorigenesis have already been found to be sensitive to agents that target against cancer stem cells. Targeting the pluripotency as a whole to deal with cancer stem cells, similar to the cell RN6 in which the p53 proteins might be at the center of the regulation target, would be a good strategy. Since p53 switches off pluripotency during differentiation plays major roles in pluripotent stem cells and in pluripotent cancer cells, the activated p53 switch in cancer stem cell management should be applied to cancer treatment."}
{"text": "The transcription factor p63 is a master regulator of epidermal development. Mutations in p63 give rise to human developmental diseases that often manifest epidermal defects. In this review, we summarize major p63 isoforms identified so far and p63 mutation-associated human diseases that show epidermal defects. We discuss key roles of p63 in epidermal keratinocyte proliferation and differentiation, emphasizing its master regulatory control of the gene expression pattern and epigenetic landscape that define epidermal fate. We subsequently review the essential function of p63 during epidermal commitment and transdifferentiation towards epithelial lineages, highlighting the notion that p63 is the guardian of the epithelial lineage. Finally, we discuss current therapeutic development strategies for p63 mutation-associated diseases. Our review proposes future directions for dissecting p63-controlled mechanisms in normal and diseased epidermal development and for developing therapeutic options. TP63 gene, belongs to the p53 gene family. Distinct from the leading member of the gene family, p53, that plays an important role in tumor suppression, the role of p63 in cancer is not fully understood. However, p63 has been shown to be a key regulator of epidermal development. This has been demonstrated in various animal models and by p63 mutation-associated human diseases. For example, complete deletion of p63 in mice results in the absence of the epidermis and epidermal related appendages, as well as defects in other epithelial-related tissues [TP63 cause several developmental disorders, and many of these diseases manifest skin abnormalities [The transcription factor p63, encoded by the  tissues \u20133. In humalities \u20137. Classmalities \u201310. Sevemalities \u201316. AmonDifferent p63 isoforms have been shown to play roles in various cells and tissues, such as the epidermis, oocytes, muscles and cochlea. As such, this review discusses p63 isoforms identified so far, with a focus on isoforms that are expressed in epidermal cells. Subsequently, we dedicate most of this review to the most studied topic in p63 biology: the role of p63 in epidermal development and related diseases. Specifically, we provide an overview of p63 mutation-associated developmental diseases with epidermal phenotypes. We discuss in-depth key molecular and cellular mechanisms by which p63 controls gene regulation to define epidermal identity, and speculate that the affected cell fate contributes to various disease states. Finally, we review current efforts and future perspectives in developing therapeutic strategies for treating these diseases.\u0394N  in 1997, as it is homologous to p53 in rat epithelial tissues . One yeaTP63 transcript was detected, rather than being degraded completely via the nonsense-mediated decay mechanism that is common for stop mutations. This incidental observation led to the identification of an alternative translational start site downstream of all stop mutations. This alternative translational start site gives rise to the \u0394\u0394N protein variant in AEC/RHS patient keratinocytes, and it is also present in keratinocytes of healthy controls, although at a lower level than the \u0394N isoform. At the C-terminal end, two other isoforms, \u03b4 and \u03b5, were identified by bioinformatic analyses using the Alternative Splicing Prediction Data Base, ASPicDB (http://www.caspur.it/ASPicDB/index.php) and validated by reverse transcription quantitative PCR (RT-qPCR) in several cell lines including keratinocytes, HEK293 cells and in some tissues, such as the muscle and the brain [In recent years, several novel isoforms have been reported. At the N-terminal end of the p63 protein, an alternative translational start site located in the 4th exon gives rise to a \u0394\u0394N isoform that lacks the first 26 amino acids of the \u0394N isoform in epidermal keratinocytes Fig.\u00a0. This ishe brain . The \u03b4 ihe brain . In the he brain . IsoformExpression patterns and functions of TA and \u0394N isoforms during epidermal commitment and subsequent stratification have been under debate, although it is generally agreed that the \u0394N isoform is highly expressed in basal epithelial cells. One report showed that the TA isoform was expressed first and subsequently required to initiate epidermal stratification, but it was counter-balanced by the \u0394N isoform during stratification . In contAs recently developed genomic approaches allow detecting active promoters and expressed exons, these techniques greatly facilitate the identification of isoform expression at the transcript level. Several independent analyses using RNA-seq, ChIP-seq of RNA polymerase II, DNA hypersensitivity sites (DHS)-seq and cap analysis gene expression (CAGE) , 22, 29 As for p63 C-terminal isoforms, the \u03b1 isoform is the predominant one in most p63-expressing cells , althougTP63 have until now only been associated with ectodermal-related disorders manifested with three hallmark defects: ectodermal dysplasia, limb malformation and orofacial clefting [\u0394N domain or C-terminal TID or SAM domain of the \u0394Np63\u03b1 isoform [Although p63 isoforms are expressed in a range of tissues of different germ layer origins, germline mutations of clefting . These d isoform  Fig.\u00a01)TP63 have\u0394N and C-terminal SAM domains of p63. For ADULT syndrome, the amino acid R298 in the DNA-binding domain is the most significant hot spot of mutations [Among these disorders, EEC syndrome is the prototype of p63 mutation-associated disorders, as it exhibits defects of all three hallmarks. About 30% of EEC patients have skin defects, and the skin is often thin and dry and sometimes resembles dermatitis. Other affected tissues with ectodermal origin include hair, teeth, nails, and lacrimal ducts Table\u00a0. These putations . Rather utations . Anotherutations .Table\u00a01PAmong all p63 mutation-associated syndromes, the most severe skin phenotypes are observed in AEC/RHS syndromes Table\u00a0. About 7TP63 mutations. This notion is supported by several lines of evidence. First, TP63 mutations associated with human syndromes are heterozygous, and mostly missense mutations [TP63 gene was lost in a larger genomic deletion region, the patient did not present any ectoderm dysplasia phenotype [TP63 mutations in human disease, two knock-in mouse models carrying Trp63 (encoding p63 in mice) mutations recapitulating EEC (R279H in human) [R279HN. Heterozygous Trp63R279HN mice exhibited phenotypes that are similar to those observed in EEC syndrome patients. This includes cleft palate, anomalies of the distal limbs, defective tooth morphogenesis, and dystrophic nails. Although no major skin abnormalities were detected in Trp63R279HN mice, primary keratinocytes from these mice exhibit reduced proliferation and increased senescence. Furthermore, an increased level of p63 mRNA and protein was detected in Trp63R279HN mice, which is consistent with observations reported in the epidermis of p63 EEC patients [It is generally believed that dominant negative or gain-of-function is the disease mechanism of utations . In a dihenotype . Second,henotype , 38, 39.n human)  and AEC n human)  were reppatients . These ppatients .Due to the striking epidermal phenotype of p63 knockout mouse models and of p63 mutation-associated human diseases, the molecular and cellular role of p63 in epidermal keratinocytes has been extensively studied. It has been shown that p63 is important in both keratinocyte proliferation and differentiation , 44. HigThe identification of p63 target genes has been a major effort in the field, either through dedicated candidate gene studies or through genome-wide explorative approaches. These studies identify the genomic binding regions of p63 and of p63-regulated genes most often by either knock-down or overexpression of p63 , 55, as As a master regulator of epidermal development, p63 regulates transcriptional programs not only through its target genes but also via higher order regulation. In recent years, a number of studies have shown that p63 plays a role in epigenetic regulation in epidermal keratinocytes via several independent mechanisms. p63 can recruit HDAC1/2 to control the repression of cell cycle arrest genes, such as p21 and 14-3-3\u03c3 . p63 wasIn addition to regulating chromatin factors and nuclear envelop-associated components involved in higher order gene regulation in keratinocytes, it has become evident that p63 plays a master role in regulating enhancers. It should be noted that many studies discussed below took unbiased genomic approaches rather than p63-centered approaches, yet they came to the conclusion that p63 is the most significant transcription factor that controls epidermal enhancers. Bao et al. showed that genomic regions of p63-bound enhancers detected in keratinocytes are nucleosome-enriched and inaccessible in cells where p63 is not expressed Fig.\u00a0a, and thIn summary, p63 is likely to regulate gene expression in epidermal keratinocytes at several levels: it regulates direct target genes involved in keratinocyte proliferation and differentiation; it directly regulates chromatin and epigenetic factors; and it might act as a pioneer factor to shape the chromatin and enhancer landscape, and thereby regulate global gene expression.Consistent with the recent proposed pioneer role of p63 in epidermal development, classical studies in mice nearly two decades ago showed that p63 is essential for proper epidermal commitment , 3. Two Recently, spatio-temporal expression of p63 during normal mouse embryonic development was analyzed . p63 expAlthough the essential role of p63 in epidermal commitment has been established, the gene networks and regulatory mechanisms of epidermal commitment that are upstream and downstream of p63 are not yet fully understood. A closer look into molecular pathways during epidermal development reveals the interaction of p63 and bone morphogenetic protein (BMP) signaling . The \u0394NpTaken together, in vivo and in vitro studies have demonstrated that p63 is essential for epidermal commitment. The molecular pathways and gene regulatory mechanisms that act during epidermal commitment remain to be elucidated. It will be particularly interesting to investigate whether p63 functions as a pioneer factor during this process, either alone or most probably together with other co-regulators, and how p63 mutations affect this process.Somatic cell transdifferentiation is a process in which transient ectopic expression of lineage master regulators can induce conversion into a different somatic cell fate without passing through a pluripotent configuration \u201381. GiveTwo studies have also suggested that p63 plays a role in cell reprogramming to a pluripotent cell state. In one study, the authors reported that p63 enables reprogramming to pluripotency in mouse embryonic fibroblasts . In thisSo far, gene regulatory programs during these cell fate conversions have not yet been identified. The precise role of p63 in gene regulation during cellular reprogramming, especially towards an epithelial fate or during a switch between different epithelial fates, is important for understanding its master regulatory function controlling epithelial cell identity. Beyond exploring the fundamental biological role of p63, the examples discussed in this review also hold great potential for developing therapeutic options, e.g. converting human epidermal keratinocytes to LSCs for cornea regeneration or to other rare epithelial types.Other than cosmetic surgery and correction , there iMET was shown to interact with the mutant p53 core domain and promote its folding to the wild-type conformation [Initially designed to treat cancers, the small compound APR-246/PRIMA-1ormation , 90. Thiormation . Given tormation , the potormation , 93. Sheormation . As p63 In addition to small compounds, small interfering RNAs (siRNA) have also been explored for their therapeutic potential in treating p63 mutation-associated diseases. Novelli et al. reported the identification of a panel of siRNAs specifically targeting the p63 EEC R304W mutant transcripts. These R304W-targeting siRNAs can rescue defects during corneal epithelial differentiation of hiPSCs carrying the R304W mutation . SimilarIn addition to approaches discussed in this review, rapidly emerging technological tools, such as genome editing, have provided new possibilities for developing therapeutic options for p63 mutation-associated diseases. Further in vivo studies and clinical trials are necessary to validate these therapeutic options and eventually to treat patients.Since the discovery of the transcription factor p63 nearly two decades ago, its master regulatory role in the development of the epidermis has been extensively studied in cellular and animal models and at normal and diseased states. Although many isoforms have been identified for the p63 protein, the \u0394Np63\u03b1 is the most abundant isoform in epidermal cells and is probably responsible for the bulk of the functional p63 activity. p63 plays its key role in epidermal commitment and terminal differentiation, not only by regulating its direct target genes, but also by shaping the chromatin state, in particular, the enhancer landscape. Recent in-depth molecular analyses suggest that p63 functions as one of the pioneer factors for epidermal cell identity. This model is supported by classical cellular and in vivo studies as well as recent transdifferentiation studies where p63 is required for epithelial cell lineages. Testing this pioneer factor model during epidermal commitment at the molecular level and identifying key regulators that cooperate with p63 are critical topics for future research. It is plausible that p63 mutations involved in human disorders may deregulate gene expression by disrupting the pioneer factor function and thereby the epithelial epigenetic landscape, subsequently giving rise to epidermal phenotypes. Current and ongoing efforts to understand the role of p63 in normal and diseased conditions are not only important for the fundamental insights into gene regulation and epidermal development, but also for the development of novel therapeutic strategies to treat patients with p63 mutation-associated and other related diseases. Although therapeutic development for these diseases is still at its infancy, encouraging results of small molecule compounds and siRNA approaches have opened new possibilities, and should be extended further to eventually benefit patients."}
{"text": "MiR122 and miR33a/b* expression was analyzed by qRT-PCR. Additionally, miR122 and miR33b* circulating levels were analyzed in 122 women. Hepatic miR33b* expression was increased in MO compared to ModO and controls, whereas miR122 expression was decreased in the MO group compared to ModO. In obese cohorts, miR33b* expression was increased in nonalcoholic steatohepatitis (NASH). Regarding circulating levels, MO patients with NASH showed higher miR122 levels than MO with simple steatosis (SS). These circulating levels are good predictors of histological features associated with disease severity. MO is associated with altered hepatic miRNA expression. In obese women, higher miR33b* liver expression is associated with NASH. Moreover, multiple correlations between miRNAs and the expression of genes related to lipid metabolism were found, that would suggest a miRNA-host gene circuit. Finally, miR122 circulating levels could be included in a panel of different biomarkers to improve accuracy in the non-invasive diagnosis of NASH.Specific miRNA expression profiles have been shown to be associated with nonalcoholic fatty liver disease (NAFLD). We examined the correlation between the circulating levels and hepatic expression of miR122 and miR33a/b*, the key lipid metabolism-related gene expression and the clinicopathological factors of obese women with NAFLD. We measured  Nonalcoholic fatty liver disease (NAFLD), the hepatic consequence or precursor of type 2 diabetes and metabolic syndrome, is the most common chronic liver disease in developed countries ,2,3. TheIn recent years, microRNAs (miRNAs) have emerged as important post-transcriptional regulators of lipid metabolism and other pathological conditions . These sThus, the aim of the present study was to analyze the liver expression of miR33a/b* and miR122 in women in relation to the degree of obesity and also to the absence/presence of NAFLD. We assessed the relationship between the liver expression of these miRNAs and the expression of the main genes involved in lipid metabolism in these cohorts, and compared the potential of the circulating levels of these miRNAs with the gold-standard method, liver biopsy, as the diagnostic and prognostic biomarkers of differentiation between simple steatosis and steatohepatitis in obese patients.Here, we present the main results obtained in response to our four objectives.First, we describe the main characteristics of our cohort, including anthropometric, biochemical  parameters and, finally, parameters of liver histology.2), moderately obese (ModO) (BMI 32\u201338 kg/m2), and morbidly obese (MO) subjects (BMI > 40 kg/m2). Biochemical analyses indicate that ModO and MO women had significantly higher levels of homeostatic model assessment 2-insulin resistance (HOMA2-IR), glucose, insulin, HbA1c, and triglycerides than the control group. High-density lipoprotein cholesterol (HDL-C) was significantly lower in the ModO and MO patients than in the control group. However, ModO women had significantly higher levels of fasting glucose than the MO group. Secondly, we classified the ModO and MO cohorts according to the liver pathology into normal liver (NL), simple steatosis (SS) and nonalcoholic steatohepatitis (NASH). Their characteristics are also described in Patients\u2019 baseline characteristics, given in The first objective of the present study was to analyze the liver expression of miR33a/b* and miR122 in women in relation to the degree of obesity. First of all, we want to clarify that, in the present work, we included the study of miR33b* liver expression instead of miR33b because the first accumulates to higher steady-state levels in the liver than miR33b and, moreover, it has been little studied.p = 0.012 and p = 0.041; p = 0.003; p = 0.425 and p = 0.674).Our results indicate that the gene expression of miR33b* was significantly greater in MO women than in ModO and control women , in order to facilitate the understanding.p < 0.001). Later, we classified the NAFLD cohort into SS and NASH. miR33b* expression was significantly higher in NASH and SS than in NL . Furthermore, the gene expression of miR33b* was significantly higher in NASH than in NL .In addition, we explored the association between miRNAs expression in liver and histopathological features. We found that miR33b* correlated positively with hepatocyte ballooning and lobular inflammation : p < 0.001; ATP binding cassette transporters A1 (ABCA1): p = 0.018; ATP binding cassette transporters G1 (ABCG1): p < 0.001, while the expression of the genes involved in lipid oxidation was diminished in both ModO and MO women compared to the normal-weight controls : p = 0.033, carnitine O-octanoyltransferase (CROT): p = 0.003; The results of the liver expression of genes related to lipid metabolism depending on the degree of obesity are shown in SREBP2 and ABCG1 gene expression was significantly higher in NAFLD than in NL . Furthermore, gene expression of SREBP2 was significantly higher in NASH vs. NL  were significantly up-regulated in NASH vs. SS  was higher in NASH vs. SS (p = 0.042 and p = 0.029). ACC1 gene expression was also increased in NASH vs. NL  and a gene related to lipid oxidation (CROT). We also found a positive correlation between the liver expression of miR33a and PPAR\u03b1. However, we found negative correlations between the expression of this miRNA, and ABCG1 and CROT. Finally, we found no correlation between miR122 expression and the expression of genes involved in lipid metabolism.In The last objective of our work was to study the potential of the circulating levels of these miRNAs as diagnostic and prognostic biomarkers of differentiation between simple steatosis and nonalcoholic steatohepatitis. We also evaluated them according to BMI and to liver histology.miR33a according to degrees of obesity and liver histology so we no longer determine their circulating levels.No significant differences were found in the hepatic expression of p < 0.001; p < 0.001). Moreover, circulating levels of miR122 were significantly higher in patients with ModO than in those with MO . Moreover, the most important result was that MO patients with NASH showed significantly higher circulating levels of miR122 than MO with SS . We found no correlation between serum levels and hepatic expression of the other miRNAs studied.When we studied the correlation between miRNAs and their expression in liver, a significant correlation was detected between serum and hepatic r = 0.430, p < 0.001) and negative correlation with HDL-C . A significant correlation was also observed between miR122 and liver enzymes . The correlation of miR33b* circulating levels with glucose, triglycerides and AST was also positive .miRNA 122 circulating levels revealed a strong positive correlation with glucose , receiver operating characteristic (ROC) curves were obtained. The accuracy with which miR122 discriminates NAFLD from non-NAFLD subjects and an advanced disease from a mild clinical form showed an average area under the curve of receiver operating characteristics (AUROC) of about 0.82 and 0.76, respectively .The binary logistic regression model that contained miR122 serum levels adjusted for age, BMI, HDL-C, triglycerides, AST, and ALT as covariates showed that miR122, along with ALT, made the highest contribution to the detection of the hepatocellular ballooning presence in population studied and was expressed by the exponent values of the beta coefficients (exp (B) or odd ratio: 2.19 for miR122; 1.03 for ALT; miR122 and miR33a/b*, the key lipid metabolism-related gene expression, and the clinicopathological factors of ModO and MO patients with NAFLD. We also investigated whether circulating miRNA levels are good predictors of NAFLD severity.In a previous study, we demonstrated a clear relationship between NAFLD and genes related to the de novo synthesis of fatty acids in a cohort of women with MO . ConsidemiR33b* and the down-regulation of miR122. In this sense, recent studies report the functional involvement of miRNAs in metabolic and endocrine pathways and adipogenesis [Nowadays, the relationship between human liver miRNA expression and degrees of obesity has not been studied yet. The present work provides evidence that morbid obesity is associated with altered hepatic miRNA expression in humans, specifically the up-regulation of ogenesis ,29. The ogenesis ,35,36. Aogenesis .miR33b* expression has been found in several human tissues including the liver [miR33b* liver gene expression in both ModO and MO women.Although there are numerous studies on the role of miRNAs in viral hepatitis and hepatocellular carcinoma in humans , relativhe liver , its relmiR33a and miR122 expression between groups classified by liver histology. However, some authors have reported that liver miR122 expression is decreased in NASH [Another finding is that there were no significant differences in  in NASH ,26 and m in NASH . These dmiR33a and miR33b work in concert with their host genes, SREBP2 and SREBP1, to ensure that a cell\u2019s metabolic state is balanced in different in vitro studies [ABCG1 expression was increased in NASH vs. SS. In ModO patients, SREBP1c and ACC1 expression, genes related to liver lipogenesis, SREBP2 and ABCG1, genes related to lipoprotein secretion, and CPT1\u03b1, related to fatty acid oxidation, were increased in NASH vs. SS. In addition, we found a positive correlation between miR33a and PPRA\u03b1 expression and a negative correlation with ABCG1 and CROT. In this respect, miR33a and miR33b, intronic miRNAs located in the SREBP2 and 1 genes, respectively, have recently been shown to regulate lipid homeostasis in concert with their host genes [miR33a and miR33b and their passenger strands, miR33a*/b*, and demonstrated that they share a similar lipid metabolism target gene network. They also showed that miR33 and miR33* represses key enzymes involved in cholesterol efflux (ABCA1 and NPC1) and fatty acid metabolism (CROT and CPT1\u03b1) in human hepatocyte cell lines [miR33a correlated inversely with ABCA1 protein levels. Moreover, miR33 target gene, CROT, correlated inversely with miR33a. However, regarding miR33b*, in our study, we found positive correlations between this miRNA and genes related to lipoprotein secretion and to lipid oxidation. These discrepancies could be explained because our study is conducted in a cohort of obese women, whereas Goedeke et al. studied this relationship in human hepatic cell line [Some authors have previously established that  studies ,40. In ost genes ,40,41,42ll lines , in agrell lines , in a MOell line . It is kell line . Therefoell line . AltogetmiR122, and suggested that the lower expression of miR122 in liver is a consequence of a high rate of release into the circulation [miRNAs can also circulate freely in blood, so we evaluated the microRNA circulating levels of miR33b* and miR122 in our cohort. We detected a correlation between serum and hepatic miR122 expression, in agreement with other authors , which sculation .Of particular interest among our findings is that circulating miR122 and miR33b* levels were higher in ModO and MO women than in normal-weight subjects. Moreover, circulating levels of miR122 were higher in patients with ModO than in those with MO. In this respect, previous studies have shown that several miRNAs seem to be involved in the regulation of adiposity and insulin sensitivity. Wang et al. showed that elevated circulating miR122 was positively associated with obesity and insulin resistance in young adults . Other agold standard for the histopathological diagnosis of NAFLD, its invasive nature means that it is difficult to use and unsatisfactory. Therefore, it is essential to search for NASH biomarkers. To do so, we examined circulating miRNAs and their association with the stratification of histological disease severity, and we found that miR122 levels were increased in MO patients with NAFLD compared with MO with NL. It should also be noted that MO women with NASH showed significantly higher circulating levels of miR122 than MO with SS. Similarly, Pirola et al. [Although liver biopsy is still the a et al.  analyzeda et al. ,48. Morea et al. . As the a et al. ,51.We also explored the association between miRNA circulating levels and the severity of NAFLD histology and found a positive correlation between miR122 and lobular inflammation and hepatocellular ballooning. ROC analysis revealed that miR122 has the potential to distinguish NASH from SS, because it predicts hepatocellular ballooning or lobular inflammation with an AUROC of 0.76. Although this predictive value is not good enough for an ideal biomarker, the ability of miR122 to predict NASH is quite similar to that of other studies ,53,54. HOur cohort has made it possible to establish clear relationships between obese women with NAFLD and altered hepatic miRNA expression, without the interference of such confounding factors as gender or age. However, these results cannot be extrapolated to men or overweight subjects.2) (MO), 30 moderately obese women (BMI 32\u201338 kg/m2) (ModO), and 30 normal-weight control women (BMI < 25 kg/m2). Liver biopsies were obtained from the morbidly obese women during planned bariatric surgery, from the moderately obese women during laparoscopic cholecystectomy for benign gall bladder disease or laparoscopic hiatus hernia repair and from the normal-weight women by means of percutaneous biopsy. Of 30 normal-weight control women, we only obtained liver biopsies from 8 women. All biopsies were performed for diagnostic indications. The pathology service of the hospital provided the control liver samples, taken with informed consent, and once they had been shown to be normal, the minimal excess sample was used to perform gene expression analysis.The study was approved by the institutional review board . All participants gave written informed consent for participation in medical research. In this study we included 62 morbidly obese women ; (2) MO with NL histology (n = 22); (3) MO with simple steatosis (SS) ; (4) MO with nonalcoholic steatohepatitis (NASH) ; (5) ModO with NL histology (n = 9); (6) ModO with SS ; and (7) ModO with NASH .According to their liver pathology and BMI, patients were sub-classified into the following groups: (1) Liver controls: normal-weight subjects with normal liver (NL) histology . cDNA was synthesized using a High Capacity RNA-to-cDNA Kit (Applied Biosystems).The liver samples obtained were conserved in RNAlater  for 24 h at 4 \u00b0C and then stored at \u221280 \u00b0C. TaqMan Assays predesigned by Applied Biosystems  were used for the detection of To quantify miRNA from liver samples, we used the miRNeasy Mini Kit  for the isolation of total RNA. The specific primers used\u2014hsa-miR33a, hsa-miR33b*, hsa-miR122\u2014were all from Applied Biosystems, and values were normalized with the housekeeping gene hsa-miR-16.elegans miR39 miRNA mimic was added to the serum samples before RNA was extracted (miRNeasy Serum/Plasma Spike-In Control).To quantify miRNA from serum samples, we used the miRNeasy Serum/Plasma Kit (Qiagen). The housekeeping gene C. All reactions were carried out in duplicate in 96-well plates using the 7900HT Fast Real-Time PCR systems (Applied Biosystems).t-test or one-way ANOVA. The strength of association between variables was calculated using Pearson\u2019s method or Spearman\u2019s \u03c1-correlation test. Area under the receiver operating characteristic curve (AUROC) was used as an accuracy index for evaluating the diagnostic performance of the selected miRNA. To analyze the potential of circulating miRNAs to designate hepatocellular ballooning in liver, we performed a binary logistic regression model. miR122, with a non-normal distribution, were log transformed for this analysis. p values <0.05 were considered to be statistically significant.All the values reported are expressed as mean \u00b1 SEM and were analyzed using SPSS version 23.0 . Differences between groups were calculated using Student\u2019s In conclusion, there is also a clear relationship between the presence of NASH and higher miR33b* liver expression in both ModO and MO women. Moreover, miR33a/b* seem to act as \u201cmicro-controllers\u201d of hepatic lipid metabolism in NAFLD. Finally, the circulating levels of miR122 could be included in a panel of different biomarkers to improve the accuracy of the non-invasive diagnosis of NASH."}
{"text": "These roles are achieved by interaction with other proteins, but particularly by interaction with DNA. As a transcription factor, p53 is well known to bind consensus target sequences in linear B-DNA. Recent findings indicate that p53 binds with higher affinity to target sequences that form cruciform DNA structure. Moreover, p53 binds very tightly to non-B DNA structures and local DNA structures are increasingly recognized to influence the activity of wild-type and mutant p53. Apart from cruciform structures, p53 binds to quadruplex DNA, triplex DNA, DNA loops, bulged DNA and hemicatenane DNA. In this review, we describe local DNA structures and summarize information about interactions of p53 with these structural DNA motifs. These recent data provide important insights into the complexity of the p53 pathway and the functional consequences of wild-type and mutant p53 activation in normal and tumor cells. Tp53 mutations and inactivation of this gene plays a critical role in malignant transformation [p53 is one of the most intensively studied tumor suppressor proteins and its regulation and relation to cancer has been reviewed extensively ,2,3. Theormation ,3. As a ormation ,5,6. It The discovery of B-DNA structure by Watson and Crick in 1953 showed the basic structure of DNA . FurtherHairpin and cruciform formation is dependent on DNA sequence and requires inverted repeats in the nucleic acid sequence. The length of this repeat should be six or more nucleotides ,15. High2+ ions, which relieves the electrostatic repulsion [Taq DNA polymerase [S. cerevisiae and triplex formation reveals single stranded DNA  and less by dimers (15%) or monomers (6%), but are also bound by higher oligomeric p53 forms (33%) . T4 endosolvases . They inficiency . Althougficiency ,121. Intficiency ,122 and ficiency ,123. By ficiency . The strficiency . Interesnd ~4500  shows a nd ~4500 .Hemicatenanes are thought to be important intermediates for DNA replication, repair and recombination. This structure is created by a denaturated DNA duplex that renatures in the presence of HMGB1 or HMGB2  and is rMammalian telomeres are organized into large duplex loops in vivo (T-loops) . In vitrTriplex-forming sequences occur in many gene promoters, for example IL2R, DNA-POL1 and MYC , which sMyc promoter and by luciferase transactivation assay in cell lines that this p53 binding leads to represssion of transcription [Recent research has highlighted the importance of quadruplexes in multiple cellular processes, including DNA replication, telomere maintenance and the binding and activity of transcription factors ,134. Comcription . It was cription .DNA binding is fundamental for the ability of p53 to act as a tumor suppressor. p53 has diverse activities in transcription, repair, recombination, replication and chromatin accessibility. These functions are realized through versatile modes of p53 interaction with DNA . p53\u2013DNAAn important part of the complex role of p53 in cancer development is DNA binding of mutant p53. Despite the partial or complete loss of sequence specific DNA binding, mutant p53 proteins can induce or repress transcription of mutant p53-specific target genes and various mutant p53 proteins can bind to oligonucleotides mimicking non-B DNA structures . Mutant It was demonstrated that the stability of p53 is enhanced by its binding to local DNA structures. These structures can therefore remarkably change p53 dependent transactivation as was demonstrated for triplex structures in promotor regions . Besides"}
{"text": "The gamma-aminobutyric acid (GABA)-glutamate hypothesis of schizophrenia suggests a neurotransmitter imbalance \u2013 reduced GABA and increased glutamate - which causes disruption of the modulation between inhibitory GABAergic interneurons and excitatory glutamatergic neurons. In the left superior temporal gyrus (STG) both hyperactivation and increased glutamate levels have previously been associated with auditory hallucinations in schizophrenia patients. However, the STG GABA-glutamate imbalance by simultaneously measuring GABA and glutamate in the same subjects has not previously been tested, and was therefore the aim of the present study. We hypothesized reduced GABA and increased glutamate in the patients relative to controls. Furthermore, reduced GABA and elevated glutamate in STG should be related to severity of auditory hallucinations in these patients.The current study tested 23 schizophrenia patients  and 53 healthy controls. The sample included both female and male participants above the age of 18. All patients were on medication, and they were tested at different times relative to the treatment onset. Magnetic resonance spectroscopy (MRS) was used to acquire data from voxels in the right and left superior temporal gyrus (Heschl\u2019s gyri) with a 3T GE 750 Discovery MR scanner. PRESS and MEGA-PRESS sequences were applied to measure glutamate and GABA, respectively. Voxel tissue water was used as reference for glutamate and GABA. Scores on the Positive and Negative Syndrome Scale (PANSS) were also collected, and used to differentiate hallucinating from non-hallucinating patients.Separate 2(Group) x 2(Hemisphere) ANOVAs were estimated for GABA and glutamate. No main or interaction effects came out significant for GABA <2.7, p>0.1, \u03b72<0.03). The analysis for glutamate resulted in a main effect of Hemisphere =24, p<0.001, \u03b72=0.25) in which the right STG showed overall higher concentrations than the left STG. In addition, an interaction effect between Group and Hemisphere was found =5.22, p=0.03, \u03b72=0.07). Bonferroni Post-hoc analysis showed significantly elevated glutamate levels in patients relative to controls in the right STG only (p=0.005). Furthermore, a multiple regression analysis was estimated between severity of hallucinations (PANSS P3 item) at the time of testing, and GABA and glutamate values in left and right STG. Although the overall model fit was non-significant, an approximate significant correlation was found between hallucination severity and left STG glutamate levels .The present study found higher glutamate levels in schizophrenia patients relative to healthy controls in the right STG. In spite of no overall differences in glutamate in the left STG, as initially hypothesized, glutamate levels in this region was found to predict severity of auditory hallucinations. One could speculate that the additional neuronal activity associated with auditory hallucinations elevate glutamate to \u2018normal levels\u2019 corresponding to that of healthy controls. The increased glutamate levels in the right STG seems (linearly) unrelated to auditory hallucinations and future analysis should test whether other symptoms are related to this finding. Overall, the study found only limited support for the GABA-glutamate hypothesis; In spite of increased glutamate in one of the regions, GABA was not found to be reduced in patients and was unrelated to auditory hallucinations."}
{"text": "Cellular stress-induced temporal alterations\u2014i.e., dynamics\u2014are typically exemplified by the dynamics of p53 that serve as a master to determine cell fate. p53 dynamics were initially identified as the variations of p53 protein levels. However, a growing number of studies have shown that p53 dynamics are also manifested in variations in the activity, spatial location, and posttranslational modifications of p53 proteins, as well as the interplay among all p53 dynamical features. These are essential in determining a specific outcome of cell fate. In this review, we discuss the importance of the multifaceted features of p53 dynamics and their roles in the cell fate decision process, as well as their potential applications in p53-based cancer therapy. The review provides new insights into p53 signaling pathways and their potentials in the development of new strategies in p53-based cancer therapy. Cells are constantly bombarded by a variety of endogenous and environmental stress that results in cellular damage. This usually leads to cell cycle arrest allowing the damage to be repaired by cellular repair mechanisms. However, in an occasion of severe damage, cells have to directly activate a \u201csuicide\u201d apoptotic death program to induce cell death, preventing initiation of oncogenic processes. Thus, cells have two possible fates\u2014to survive or die. Failure in making the right decision in determining cell fate can ultimately lead to the development of diseases such as cancer. The cell fate decision machinery is composed of multiple complex signaling pathways, in which p53, a key tumor suppressor, plays a central role in coordinating the multiple cellular signaling pathways as well as determining cell fate .P21 gene and pro-apoptotic genes such as BAX, BAK, PUMA, and P53AIP1 [As a transcription factor, p53 is activated by various types of stimuli. This selectively regulates the transcription of p53 target genes that subsequently mediate cellular responses to stress. In the past two decades, hundreds of p53 target genes have been identified. These include the cyclin-dependent kinase inhibitor  P53AIP1 . The pro P53AIP1 . While tIn this review, we discuss the progress in an emerging new field in cell biology that explores a p53-mediated cellular signaling network via the dynamics of key signaling molecules in the network ,8, whereIn the 1990s, it was observed that treatment of rat neuronal precursors with epidermal growth factor (EGF) and neural growth factor (NGF) can lead to cell proliferation and differentiation, respectively. Further investigation revealed that both stimuli activated the same protein kinase, the extracellular signal-regulated kinase (ERK), with a distinct temporal feature\u2014i.e., EGF induces a transient ERK activation\u2014whereas NGF triggers a sustained ERK activation ,11,12. TFurthermore, several studies support the notion that cells can translate different p53 dynamical patterns into a specific type of downstream cellular responses. It has been found that IR-induced transient p53 pulses are associated with activation of pro-cell cycle arrest genes that result in cell-cycle arrest, whereas prolonged p53 pulses induced by UV are involved in upregulation of pro-apoptotic genes that can lead to apoptosis . This isAs a transcription factor, p53 can activate pro-cell cycle arrest and pro-apoptotic genes as well as induce the expression of genes that regulate its own gene expression and the stability of its protein. This results in the formation of a feedback loop leading to the pulsatile dynamics of p53 protein level.The first identified feedback loop in the p53 network is the p53 mouse double minute 2 (Mdm2) negative feedback loop. Discovered in 1992, Mdm2 was initially proposed as an oncoprotein because it functions as a p53-specific E3 ubiquitin ligase that promotes ubiquitination and subsequent proteasomal degradation of p53 . Later iWIP1 gene can result in the dynamics induced by UV-like stimuli [p53 can be activated by a variety of stimuli including IR, UV radiation, activated oncogenes, hypoxia, and chemotherapeutic drugs. However, our current understanding about the p53-mediated stress response mainly results from the response to double-strand DNA breaks (DSBs) induced by IR and single-strand DNA breaks (SSBs) induced by UV radiation ,24,28. T stimuli . It has  stimuli . Thus, tBecause IR-induced p53 pulses are fixed in amplitude and duration, it was initially proposed that the decision of cell fate is governed by the frequency of p53 pulses . It is cWith the discovery of multiple functional feedback loops that result in different p53 dynamics (transient or sustained pulse), an emerging question is how the p53 dynamics determine cell fate in a coordinated manner? To address this, several computational models that integrate transient and sustained p53 pulses have been developed to understand the cell fate decision process. The models have shown that in response to UV radiation, p53 exhibits a single prolonged pulse that is associated with the irreversible cell fate\u2014apoptosis . In contIt is noteworthy that the majority of studies on p53 dynamics focus on how p53 responds to stimuli, i.e., exogenous DNA damaging agents such as IR and UV radiation. Few studies explore how p53 reacts to intrinsic or spontaneous DNA damage caused by normal physiological processes. With quantitative time-lapse microscopy of individual human cells (MCF7 cell line), a recent study showed that p53 exhibits excitable spontaneous transient pulses in non-stressed cells to respond to spontaneous DNA damage with a dynamic pattern similar to that in response to IR . This fiIn general, p53 dynamics are designated as the variation in p53 protein level in response to stimuli. However, this notion has been challenged by the fact that the maintenance of a steady level of p53 protein induced by IR with a p53-stabilizing agent only can switch cell fate from survival to irreversible senescence . This isAs a transcription factor, p53 is continuously transported among the nucleus, cytosol, and mitochondria. Export of p53 from the nucleus to mitochondria is thought to be a negative regulation of p53 transcription activity by excluding the protein from its target genes. Yet, recent studies have shown that non-transcriptional mitochondrial p53 proteins also play an important role in p53 dynamics at the spatial level of the protein ,37,38.Because of its regulatory roles in gene transcription, p53 proteins are mainly located in the nucleus, where they bind to the promoter and/or enhancer of the target genes to regulate gene expression. Yet, a small portion of p53 protein is located in mitochondria to perform its non-transcriptional function. It appears that such a small amount of p53 in mitochondria is sufficient to mediate mitochondrial apoptosis effectively ,39. UponIt appears that nuclear and mitochondrial p53 are orchestrated to determine the fate of cells that are damaged by IR . This haThe posttranslational modifications of p53\u2014including ubiquitinylation, phosphorylation, methylation, and acetylation \u2014play impDiscovery of the dynamics of p53 ubiquitinylation also led to the discovery of the dynamics of other types of post-translational modifications of p53 . Thus faAcetylation of p53 occurs on several lysine residues at the C-terminal domain of p53 including K370, K372, K373, K381, and K382, and this is carried out by a p53 co-factor, p300/CBP. The modification mainly results in increased DNA binding of p53, promoting the activation of its target gene. In contrast, methylation of p53 by methyltransferases at histidines, arginines, and lysines, suppresses transcriptional activity of p53 . InteresRecent studies have shown that besides its crucial role in regulating gene expression, p53 can also activate its target miRNAs, which include miR-192, miR-194, miR-215, miR-107, and miR-605 ,52. InteOur current understanding of p53 dynamics is limited to the pulsatile switches of its protein level. However, the interplay among different types of p53 dynamics is critical in governing a specific cellular damage response. Based on this, p53 dynamics can be designated as a multi-faceted dynamic network comprised of all variables that can be measured over time. These include p53 protein level, its activity, subcellular localization and posttranslational modifications. We therefore propose a model that illustrates the roles of multiple dynamic features of p53 in governing cell fate . In non-The central role of p53 in governing the cell fate decision and preventing tumorigenesis makes it a promising target for cancer therapy. Over the past decades, p53-based cancer therapeutic strategies have mainly focused on p53 gene-transfer therapy with an adenovirus vector, or on restoring p53 activity using small molecule drugs that interact with mutant p53 protein and alter the conformation of the protein . These tHowever, it should be noted that such cancer therapeutic strategies may be complicated by the multiple roles of Mdm2. This is because Mdm2 can mediate translocation of p53 from the nucleus to mitochondria to initiate rapid apoptosis ,37. ThisIn a scenario where the mitochondrial p53-mediated apoptotic pathway fails to be initiated, a nuclear p53-mediated apoptosis can be initiated as an alternative pathway to promote cell death. A critical step of this process is to promote p53 transactivation of pro-apoptosis genes. Based on the fact that p53 transcriptional activity is determined by both its protein level and posttranslational modifications, two strategies can be employed to alter p53 transcriptional activity. One is to switch p53 pulses from a transient mode to a sustained mode. Defects in this switch have been identified in human cancers in a tissue-specific manner. For example, Wip1, a phosphatase, is essential for the initiation of transient p53 pulses. Thus, it acts as an oncogene in many cancers such as breast cancer, glioma, and colon carcinoma . In contThe dynamics of p53 have emerged recently as a new field of cell signaling. However, its biological significance remains to be elucidated. With the development of fluorescent labeling and time-lapse technology, studies on p53 dynamical profiles in protein level, spatial location, and posttranslational modifications will provide new insights into the biological function and implications of p53 dynamics in cancer treatment. Because p53 dynamics have been mainly studied in IR-induced DNA damage models, many of which are based on computational and mathematic approaches, additional experimental results that support these models are in urgent need. These include the results of p53 dynamics induced by other stimuli such as ROS, heavy metals, and chemotherapeutic drugs. In addition, because the measurements of p53 dynamics are currently restricted in cultured cells, the dynamic profiles of this protein need to be further verified in a biological system such as fruit flies, worms, zebra fish, mice, and humans, if it is feasible. Nevertheless, our knowledge about dynamic interactions among various cell signaling networks and the dynamics of the components of the network will provide valuable guidance to manipulate and control cell signaling pathways for achieving therapeutic purposes in both cancer and other human diseases such as neurodegenerative diseases."}
{"text": "N = 201) were assessed on the BCET and a number of other neuropsychological tests that have been demonstrated to rely on aspects of executive processing  and the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). Internal consistency of the total BCET was good; however, interpretable solutions for existing subscales were not discerned. The BCET total score demonstrated positive associations with tests of executive functioning; however, it was also significantly associated with more general aspects of neuropsychological functioning suggesting that it does not solely assess executive processes in ABI patients undergoing rehabilitation. Hierarchical multiple regression suggested that the BCET accounted for significant additional variance in community integration after severity of disability, executive functioning, and more general aspects of neuropsychological status were statistically controlled. While the subscale structure of the BCET may be somewhat inconsistent, the total scale score accounts for some unique variance in pragmatic rehabilitation outcome and may be a useful tool in postacute rehabilitation assessment protocols.The purpose of the present study was to examine the convergent and divergent validity of the Biber Cognitive Estimation Test (BCET) in individuals with ABI undergoing postacute rehabilitation and to assess the measure's ability to account for unique variance in community integration following rehabilitation. Participants with ABI referred for postacute rehabilitation ( The ability to provide reasonable numerical estimates from available but incomplete information is an ability not normally acquired directly through formal education, yet it is an important aspect of problem solving that is regularly called upon in everyday life. Previously acquired information may not always have direct relevance to a particular estimation problem and, as such, problems in providing reasonable or plausible cognitive estimations are related not simply to an abstract problem in processing numbers, but also to probabilistic judgements (mapping components) arising from domain-specific reasoning and general heuristics , 2. FailThe first formal examination and detailed assessment of estimation abilities were performed on individuals with lesions to the frontal lobes . This laCommunity integration of people with ABI rests on the premise that such individuals should have the same rights and be involved in community life like any other member of their community. It is generally considered to represent a person's ability to carry out everyday activities in their home and community, enjoy interaction with its members, and participate in some aspect of productivity in the community. While community integration is also the ultimate goal of rehabilitation following ABI, formal examination of the construct and its possible relationship to variation in community integration outcomes in the context of rehabilitation have not been undertaken to date. This mandates a comprehensive examination of a widely used measure of cognitive estimation and its influence on more distal community integration outcomes.There is no clear consensus on the use of a particular measure of cognitive estimation that has been rigorously developed and standardised. In terms of measurement of the construct, it has been observed  that theThe aims of the study were as follows: firstly, to examine the latent variable structure of the BCET in relation to the subscale assessment domains of quantity, time/age, length, and weight in a sample of people referred for postacute rehabilitation following acquired brain injury; secondly, given the question posed by previous research as to whether estimation is associated more with performance on executive or more general cognitive function assessments, to investigate the convergent and divergent validity of cognitive estimation using established neuropsychological tests that are considered to represent executive and more general measures of cognitive function; thirdly, to assess what aspects of cognitive function measured by the battery of neuropsychological tests may be responded to differently in an impaired estimation group relative to an unimpaired estimation group; and finally, to investigate whether the BCET adds any unique variance to prediction of community integration, given that community integration is the ultimate goal of rehabilitation, and impairments in estimation ability may have real-world consequences in terms of such community integration aims .A total of 201 participants who had sustained an acquired brain injury and who were referred for postacute neurorehabilitation services in the South and Mid-West of Ireland participated in the study. All participants engaged in a programme of accredited and individualized Home and Community Based Rehabilitation (HCBR). HCBR is defined by the Commission of Accreditation of Rehabilitation Facilities (CARF), as a programme of postacute rehabilitation that provides integrated, case managed, and outcome focused rehabilitation. In the HCBR model, services are developed from a comprehensive needs assessment and focus on the expectations and outcomes identified in treatment planning with the person served by the programme. The positive effects of such individualized community based programmes are well established .Inclusion criteria were participants accessing ABI-specific neurorehabilitation services (home and community rehabilitation), confirmed medical diagnosis of ABI, age over 18 years and not older than 65 years at study end, and English as a first language. Exclusion criteria were the presence of aphasia/communication, comprehension, or significant visual difficulty that necessitated an alternative assessment protocol.The principal purpose of recruiting ABI rather than a single cause of brain injury  was that it would reflect the possible ecological or real-world rehabilitation usage of the measure of cognitive estimation and permit an examination of its ability to predict the ultimate goal of rehabilitation (community integration) over and above the standard assessments undertaken in this group.n = 150; 74.6%; women n = 51; 25.4%) and were aged between 19 and 63 years . Age at onset of brain injury ranged from 1 to 60 years , and duration of injury ranged from 2 years to 20 years . Twenty percent of participants (n = 40) completed more than 14 years of education, 33% (n = 67) 12 to 14 years, and 38% (n = 76) 8 to 12 years, and 9% of participants (n = 18) completed less than 8 years of formal education. Participants' functional disability was assessed on the Mayo Portland Adaptability Index [All participants gave informed consent. Participants were predominantly male , 35% had stroke (n = 71), and 15.5% (n = 31) had other diagnoses including neoplasm , anoxia , encephalitis , and aboulia . Due to the small number of participants in some subcategories of brain injury, 3 categories were created for subsequent analysis: traumatic brain injury, stroke, and a third category comprising neoplasm, anoxia, aboulia, and encephalitis.49.5% of participants sustained a TBI . T-scores were used as an assessment of the severity of functional limitations such that scores less than 50 indicated mild to moderate limitations, while scores above 60 were suggestive of more severe limitations arising from ABI. One hundred and four participants (51.7%) had disability in the moderate to severe range, while 97 participants (48.3%) had disability scores in the mild to moderate category.Mean MPAI The study was approved by the local research ethics committee  and all participants provided informed consent commensurate with the declaration of Helsinki. The study adhered to standardised reporting guidelines for cohort studies (STROBE). All participants completed the following assessment measures.The BCET  is a 20-The RBANS  is a briParticipants were required to generate as many words as possible that either begin with a particular letter, for example, F, A, and S (phonemic fluency), or that belong to a specific semantic category, fruits, vegetables, and animals (semantic fluency) within a specified time interval. These measures are commonly used to assess the organisation and integrity of lexical and semantic memory representations and executive functions. Phonemic and semantic verbal fluency relies on a partially different network of brain regions , and theThe Modified Six Elements Test (MSET)  consistsThe TMT  was usedCommunity Integration of participants was assessed by use of the Community Integration Questionnaire (CIQ) . The CIQp's > 0.11). Fourteen cases had some missing data from their assessments. No participant had missing data in excess of 7% on the assessment measures, and no participant had missing data on the neuropsychological assessments. To assess whether the data was Missing Completely at Random (MCAR) or whether there was an underlying pattern to this missing data, Little's MCAR test was utilised. MCAR values ranged from \u03c72 = 6.399, p = 0.93, to \u03c72 = 78.016, p = 0.97, which suggested that the small amount of missing data was randomly missing. Therefore, these cases were retained and the standard expectation-maximisation procedure was utilised to maximise power. Means and standard deviations were calculated for the main measures and subscales as appropriate. Analysis of Variance with Scheffe post hoc test was used to examine the effects of educational attainment and cause of ABI on BCET scores. Independent samples t-test was used to examine mean differences between scores on the neuropsychological measures and disability total score as a function of BCET cut-off scores, and Chi Square analysis examined the association between the BCET cut-off and levels of disability severity. Relationships between cognitive estimation and demographic variables, neuropsychological measures, disability, and community integration were undertaken by Pearson correlation coefficient. Latent variable structure of the BCET was undertaken via the Categorical Principal Components Analysis (CatPCA) program. Logistic regression analysis was used to examine neuropsychological factors associated with cognitive estimation scores that were below threshold, and hierarchical multiple regression was used to explore whether the BCET accounted for unique variance in community integration. All data were analysed using SPSS 24.Q-Q plots and Shapiro-Wilk's test were used to examine the distribution of data. Because of the nonnormal distribution of trail-making scores in both raw  and derived measures  and in the Modified Six Elements Test (MSET), transformation was applied before further analysis to render this data suitable for parametric analysis. Observed data showed no significant deviation from normality , with subscale scores being acceptable for estimates of distance/length (\u03b1 = 0.73) and weight (\u03b1 = 0.69) and somewhat lower for quantity (\u03b1 = 0.57) and time/age (\u03b1 = 0.62). Correlations between each of the four domains and the total scale score were as follows: quantity (r = 0.62), weight (r = 0.60), time/age (r = 0.55), and distance/length (r = 0.71).Descriptive statistics (means and standard deviations) for the BCET, TMT, verbal fluency, MSET, and the six indices of the RBANS are presented in In order to appropriately examine the latent variable structure of the BCET, categorical PCA (CatPCA) was utilised. CatPCA is a relatively new algorithmic model and can be considered the nonlinear equivalent of PCA. It is especially suitable for the dimension reduction problem with categorical variables, as it accounts for the nature of items, the different role of items in determining the measure, and the possible multidimensionality of the underlying concept , 55. TheA scree plot was firstly examined to determine the most parsimonious number of components to retain in the analysis. Nonlinear PCA solutions are not nested, so the position of the scree plot \u201celbow\u201d can move with the number of components. In the present analysis, different dimensionalities consistently placed the elbow at the fourth component.Component loadings are presented in Figures 1Examination of the biplots suggest that, graphically, the variables do not seem to form distinct or particularly meaningful groups/clusters that might conform to the original 4 estimation domains or that represent some new groupings with clinical meaning.Following examination of the biplots, item analysis was used to examine the distribution of responses to each item of the BCET. Examination of the percentage of correct and incorrect responses  shows thRotation options are not available in the CatPCA program and given the well-documented difficulty in interpretation of unrotated solutions, the transformed variables were additionally saved and utilised as input variables to permit a rotated solution . The comAcross the subscale assessment dimensions of quantity, weight, time, and distance, individual compatible items did not consistently fall into these established dimensions. Moreover, the factors produced were also not interpretable in any obvious manner . Given the difficulty in obtaining a fully interpretable solution in relation to the proposed subscales  as suggested in Figures 1F, 6.84, p = 0.001) and Scheffe post hoc tests suggested that the effect was largely monotonic, with individuals who had attained a postgraduate education scoring significantly higher on the BCET (BCET mean (SD) for primary school leaver 13.29 (3.17), secondary school leaver 14.36 (2.79), and postgraduate 16.33 (2.49)).Mean total BCET scores rose with educational attainment. This represented a significant effect  and with an older age at onset , but not with duration with ABI .Higher (better) BCET scores were associated with increasing age of the participant , 4.73, p = 0.01).One-way ANOVA suggested that people who had sustained a TBI scored more poorly on the BCET, mean (SD) TBI = 14.04 (2.44), than people who had sustained a stroke, BCET mean SD = 15.44 (3.19), or other causes, mean SD = 15.9, 3.10 (T score (mean (SD) for mild/moderate disability = 15.09 (2.97); mean (SD) for severe disability = 14.25 (2.81); t = 1.58, p = 0.11).BCET total score did not significantly differ as a function of the severity of participant's disability as assessed by MPAI-4 r = .42, p = 0.001), phonemic fluency , TMT B , and the TMT difference score  but not with the TMT ratio score (r = \u2212.07). Additionally, the BCET was also highly correlated with more general indices of neuropsychological functioning including the TMT A  and the 6 scales of the RBANS . Higher BCET scores were also associated with increased community integration .t = \u22124.62, p = 0.001), on verbal fluency (mean (SD) = 21.45 (11.34) versus 26.07 (11.85); t = \u22122.68, p = 0.006) and semantic fluency (mean (SD) = 13.40 (4.25) versus 15.63 (4.71); t = \u22123.28, p = 0.001) and on RBANS indices of immediate memory (mean SD = 67.04 (18.34) versus 79.14 (21.27); t = \u22124.05, p = 0.001), visuospatial functioning (mean SD = 79.60 (19.03) versus 88.96 (23.20); t = \u22122.91, p = 0.004), language (mean SD = 77.91 (13.57) versus 88.96 (23.20); t = \u22122.95, p = 0.004), attention (mean SD = 72.24 (17.56) versus 84.56 (17.41); t = \u22124.57, p = 0.001), and delayed memory (mean SD = 66.34 (21.67) versus 79.15 (18.48); t = \u22124.09, p = 0.001). The BCET cut-off was not significantly associated with scores on the TMT A mean SD = 69.34 (49.88) versus 57.06 (43.21) and TMT B (mean SD = 204.44 (157.36) versus 184.39 (143.60); t = \u22121.76, p = 0.07) or its difference (mean SD = 149.01 (102.11) versus 110.87 (107.04); t = \u22121.19, p = 0.23) or ratio score (mean SD = 3.32 (2.33) versus 2.56 (2.20); t = \u22121.29, p = 0.19). The cut-off score was not significantly affected by severity of disability . Participants with a younger age at onset were more likely to score above the BCET cut-off (mean SD = 29.43 (13.85) versus 37.83 (13.91); t = \u22124.08, p = 0.001). A forward conditional logistic regression analysis including the statistically significant univariate results above suggested that BCET cut-off scores were predicted by a younger age at onset of ABI (odds ratio (OR) = 1.04, 95% CI = 1.01\u20131.06, Wald = 10.49, p = 0.001) poorer scores on the MSET  and poorer scores on RBANS attention index . The model discriminated the unimpaired cognitive estimation group from the unimpaired estimation group with an accuracy of 76.8%.Using the established cut-off for the BCET (scores below 15.6 indicating impairment in estimation abilities), 55.7% of participants had scores in the unimpaired range, with 44.3% scoring in the impaired range. The estimation-impaired group scored significantly lower on the MSET (mean (SD) = 1.93 (1.1) versus 2.76 (1.2); Given the BCET's apparent lack of specificity in assessment in terms of performance based cognitive functioning, the BCET total score was regressed onto participants' community integration scores to examine if the functions assessed by the BCET may explain unique variance in more pragmatic social integration outcomes when entered on the final step in the model. Therefore, a hierarchical multiple regression analysis was undertaken with variables that correlated with community integration as IVs. Thus, severity of disability as assessed by MPAI was entered on step (1), RBANS total score on step (2), MSET on step (3), semantic fluency on step (4), TMT B (as it was the most highly correlated of the TMT measures) on step (5), and BCET on step (6).Results  suggesteThe current study comprehensively examined the convergent and divergent validity of estimation abilities operationalised through the Biber Cognitive Estimation Test (BCET) in a large sample of individuals with acquired brain injury undergoing postacute rehabilitation. The study examined the latent variable structure of the BCET, reported an item analysis of responses, and addressed whether performance on executive or more general assessments of neuropsychological functioning were more strongly associated with the BCET total score and with its impairment cut-off score. This is also the first study to date to examine the effects of estimation abilities on what is usually considered to be the end point of rehabilitation, namely, the person's level of integration into their community.The latent variable structure of the BCET was examined by Categorical Principal Components Analysis, a relatively new algorithmic model which was considered to be more appropriate than more traditional models given that CatPCA can reveal nonlinear relationships by quantifying categorical or nonlinearly related variables to reveal relational structures among the observed variables. Despite examining the BCET with a number of procedures including CatPCA variance accounted for, examining the coordinates for each item on each dimension in relation to the centroid, the use of biplots, and utilising rotation procedures, none of these represented the information in the data sufficiently closely such that it might be reduced in a meaningful way to the smaller number of summary variables . Neither were there any obvious theoretically informed justifications for interpretation of the factors, such as components representing a process of estimation that may have relied upon direct procedural experience  versus a process that may have relied on more remote declarative/semantic procedures to effect an estimate . While it was judged prudent from a psychometric perspective to restrain further analysis of the BCET in the current study to its total score, the prospect that the areas of estimation targeted by the subscales would be isomorphic in clinical practice with rehabilitation patients may not represent a sensible assumption in any case . For exaThe current study found a relationship between BCET estimation performance, education, and age, in that more years of formal education, being older, and sustaining their injury at an older age tended to be associated with better estimation performance. While the original development and validation study for the BCET  did not r = .28). Poorer MSET performance suggests a difficulty in alerting the individual to changes that should be taken into account during the task and to faulty reevaluation of goals and monitoring tasks in progress [r = 0.42 and r = 0.39, resp.). Of the RBANS subscales, immediate memory and attention could be considered more legitimately to require aspects of executive function in their performance, particularly updating semantic working memory. Similarly, in terms of discriminating an impaired estimation group from an unimpaired group, patients whose BCET scores placed them in the impaired category were significantly more likely to have a younger age at onset of their brain injury, lower MSET scores, and poorer RBANS attention index scores.In terms of the relationship between the BCET and the more established tests of executive function, the BCET showed a stronger relationship with tests involving planning and set shifting  than with the verbal fluency component of executive functions. Both the MSET and the TMT could be considered to map onto the response inhibition and set shifting components of Miyake et al.'s  model ofprogress , which wThe distal goal of postacute rehabilitation is to integrate the person into their community. While the assessed model accounted for a relatively modest proportion of the variance in community integration (21%), some important findings were uncovered. Namely, that the RBANS total score accounted for the majority of the variance in community integration, with severity of ABI-induced disability, MSET, TMT B, and semantic fluency not making any significant contribution to the model. The BCET however did account for unique variance in community integration after it was entered on the last step of the model accounting for an additional 4% of the variance. While modest, it nonetheless suggests that the BCET may be assessing something distinctive and of potential value in this area. Further work needs to be undertaken to develop the assessment protocol for cognitive estimation including various forms of presentation that may involve visual cueing as an attention orientation strategy. Similarly, goal management interventions  may provThe study has some limitations. Firstly neuroimaging data was not available on participants and therefore no detailed formal information can be provided from a structural-functional perspective on anterior versus posterior injuries or in terms of issues of laterality and their possible effects on BCET performance. However, since brain injury patients with moderate to severe injuries typically have large lesions affecting fairly large tracts of frontal and temporoparietal cortex, it is also possible that multiple functional systems may be affected which would limit the ability of the BCET to localise function in such samples in any case. Secondly, variability in the ages at which participants sustained their brain injury, while reflecting the nature of routine referrals to ABI postacute rehabilitation, may have influenced the results given issues of brain plasticity in more established versus more recent injury. Thirdly the sample comprised people who had sustained brain injuries of varying causes; thus, standard severity measures which might be utilised in TBI cases were not sensible to be used  and that criteria for severity in CVA such as Rankin criteria are not routinely utilised in the Republic of Ireland. Therefore, a valid and reliable measure of disability arising from acquired brain injury (MPAI-4) was used in order to provide a sympathetic measure that would have relevance to all types of ABI. The presence of clinical severity information from the time the person sustained their injury in addition to specific and contemporary lesion location information would have been a helpful adjunct to the study.This is the first study to examine the effects of estimation abilities on community integration in a rehabilitation sample of people with acquired brain injury. The study has also provided detailed information on estimation assessment in this patient group. While the latent structure of the BCET in the current study did not readily map onto the four subscale indices of quantity, weight, distance/length, and time/age and to describe the BCET solely as a test of executive functioning in the current rehabilitation sample would not be sensible, the measure has some merit in terms of its association with pragmatic outcomes and may have a continued role in assessment protocols in postacute rehabilitation."}
{"text": "Diffuse large B cell lymphoma (DLBCL), currently the most common type of non-Hodgkin lymphoma (NHL), is an aggressive B cell neoplasm that typically presents in older adults as a rapidly enlarging mass. The enlarging mass typically represents a lymph node, although extranodal disease can occur in a significant percentage (40%) of cases. The most common extranodal sites of involvement include the gastrointestinal tract and skin; primary bladder lymphoma represents only 0.2% of extranodal non-Hodgkin lymphomas. We report a case of diffuse large B cell lymphoma occurring in the bladder of an 83-year-old gentleman with an initial presentation of hematuria. This neoplasm displayed large, atypical cells with vesicular chromatin and prominent nucleoli that involved the bladder mucosa with invasion into muscularis propria, prostate, and urethra. Positive staining for p63 initially raised suspicion for poorly differentiated urothelial carcinoma; however, lack of staining for pancytokeratin and positive staining for LCA, CD20, CD79a, and PAX-5 confirmed the diagnosis of diffuse large B cell lymphoma. Though it does not occur in all cases, p63 can be positive in a significant percentage of cases of DLBCL; therefore, a diagnosis of lymphoma remains an important entity on the differential diagnosis of aggressive and particularly poorly differentiated neoplasms. Diffuse large B cell lymphoma (DLBCL) represents the most common type of non-Hodgkin lymphoma (30\u201340% of NHL cases) . This agWe present a case of an 83-year-old gentleman with a past medical history of prostate cancer status after radiation treatment, hypertension, BPH, hyperlipidemia, and renal impairment who presented with gross hematuria. A transurethral resection revealed an infiltrating, poorly differentiated tumor with abundant apoptosis and mitotic activity showing invasion into the muscularis propria. Due to the patient's history of prostate carcinoma, immunohistochemical stains were performed. The malignant cells were positive for p63 and negative for PSA, cytokeratin 7 (CK7), and cytokeratin 20 (CK20); therefore, a diagnosis of poorly differentiated urothelial carcinoma was favored. A CT scan of the abdomen and pelvis was remarkable for a remarkably thickened bladder wall with ill-defined boundaries and dilation of the distal right ureter. Small pelvic lymph nodes were also visualized.Subsequent sampling revealed an aggressive neoplasm with invasion to bladder muscularis propria, prostate, and urethra. The tumor displayed large malignant cells with vesicular chromatin and prominent nucleoli arranged in a mildly cohesive pattern . Based oDiffuse large B cell lymphoma may present in extranodal sites. This case illustrates the importance of considering a diagnosis of lymphoma when confronted with a poorly differentiated neoplasm. Expression of p63 by lymphoma cells presents a potential diagnostic pitfall. Review of the literature reveals several reports of p63 expression in high-risk diffuse large B cell lymphoma. P63 is positive in a significant amount of DLBCL cases, with percentages ranging from 15 to 70% depending upon the study examined [ p63 gene, homolog of the p53 tumor suppressor gene, is located on chromosomes 3q27-3q28 and encodes proteins responsible for p53 activation and apoptosis [The p53 is widely acknowledged as a tumor suppressor gene and its mutations are responsible for tumor progression, the role of p63 as a cancer oncogene continues to be studied. Two particular groups of p63 isoforms, TAp63 and \u0394Np63, have received further study in the development of neoplasia.P63 comprises a group of six different proteins produced through alternative splicing of a single mRNA transcript . These i p53 target genes and inducing apoptosis. The \u0394Np63 isoform lacks this N-terminal domain and likely has a dominant-negative effect on p53 [The TAp63 isoform contains an N-terminal transactivation domain with sequence homology to the p53 transactivation domain. This isoform is responsible for transactivatingt on p53 . RT-PCR t on p53 . This imRecent studies , 10 have p63 gene responsible for creating these various protein isoforms is located on chromosomes 3q27-3q28. Interestingly, the Bcl-6 gene is also located on chromosome 3q27, adjacent to the p63 locus. Given that rearrangements in the Bcl-6 gene are seen in approximately 30% of diffuse large B cell lymphomas [ p63 to Bcl-6 may contribute to its potential involvement in DLBCL tumor progression [As previously noted, theymphomas , some auSeveral studies have adopted the task of analyzing p63 expression in diffuse large B cell lymphoma and its prognostic impact, fueled by an earlier study by Di Como et al. that showed high levels of p63 in a subset of DLBCL 25%) and follicular lymphoma (22%) . These s% and fol P63, homolog of the tumor suppressor p53, has an important role in the development and differentiation of stratified epithelia; as such, it is usually interpreted as a marker of epithelial differentiation/carcinoma. However, as illustrated by this case, p63 can be positive in a significant subset of cases of diffuse large B cell lymphoma. Of particular note, the isoform TAp63 appears to be involved in the development of lymphoma, while the \u0394Np63 isoform is typically seen in cases of squamous cell carcinoma. Numerous studies support this phenomenon and correlate p63 expression with DLBCL tumor progression; its impact on prognosis and survival remains to be seen.This case illustrates the importance of considering hematologic malignancies in the differential diagnosis of poorly differentiated neoplasms and serves as a reminder that no single immunohistochemical marker is entirely specific for a certain tumor type. Incorporation of morphology and immunophenotype is vital to final diagnosis."}
{"text": "The p53 tumor suppressor continues to be distinguished as the most frequently mutated gene in human cancer. It is widely believed that the ability of p53 to induce senescence and programmed cell death underlies the tumor suppressor functions of p53. However, p53 has a number of other functions that recent data strongly implicate in tumor suppression, particularly with regard to the control of metabolism and ferroptosis (iron- and lipid-peroxide-mediated cell death) by p53. As reviewed here, the roles of p53 in the control of metabolism and ferroptosis are complex. Wild-type (WT) p53 negatively regulates lipid synthesis and glycolysis in normal and tumor cells, and positively regulates oxidative phosphorylation and lipid catabolism. Mutant p53 in tumor cells does the converse, positively regulating lipid synthesis and glycolysis. The role of p53 in ferroptosis is even more complex: in normal tissues, WT p53 appears to positively regulate ferroptosis, and this pathway appears to play a role in the ability of basal, unstressed p53 to suppress tumor initiation and development. In tumors, other regulators of ferroptosis supersede p53\u2019s role, and WT p53 appears to play a limited role; instead, mutant p53 sensitizes tumor cells to ferroptosis. By clearly elucidating the roles of WT and mutant p53 in metabolism and ferroptosis, and establishing these roles in tumor suppression, emerging research promises to yield new therapeutic avenues for cancer and metabolic diseases. TP53 has been the most heavily studied human gene since its discovery nearly 40\u2009years ago (The tumor suppressor gene ears ago . The maiears ago . Howeverears ago . A summavia the transactivation of SCO2 (cytochrome c oxidase assembly), which plays a direct role in oxidative phosphorylation (GLS2 (Glutaminase 2); this enzyme allows glutamine usage as an energy source for the mitochondria  or arginine (R72). This amino acid variation can impact p53 function with regard to cell fate after stress. In response to DNA damage, the P72 variant of p53 predominantly triggers cell cycle arrest, while the R72 variant predominantly induces cell death, or apoptosis , 19. DesR72 mice . InteresR72 mice . These fR72 mice .Cpt1c-deficient mice display delayed tumor development and higher survival rates (LPIN1) is another p53 target gene; LPIN1 is necessary for proper adipocyte development and is induced under low nutrient conditions , a critical p53 transcriptional target. The mechanism through which p21 delays ferroptosis has yet to be elucidated, but it is believed that the conservation of intracellular glutathione may be a contributing factor for reduced ferroptosis sensitivity. The authors conclude that the p53\u2013p21 axis enables cancer cells to survive under conditions of metabolic stress, such as cystine deprivation, by suppressing the onset of ferroptosis (A study recently published by Tarangelo and colleagues shows that p53 negatively regulates ferroptosis in cancer cells . This grroptosis . A recenroptosis . The bidMDM2 and MDM4). The Pro47Ser variant (hereafter S47) is the second most common SNP found in the p53 coding region (after Pro72Arg) that alters the amino acid sequence of the protein. To better elucidate the impact of this variant on p53 function and cancer risk, the Murphy group generated a humanized p53 knock-in mouse model, in which exons 4\u20139 of murine p53 were replaced by human p53 exons containing either the wild type or the S47 variant (Gls2 (glutaminase 2) and Sco2  in the TP53 gene and other proteins known to regulate this pathway (such as  variant \u201355. The  variant . In mousand Sco2 . Consistand RSL3 . This deSLC7A11, which inhibits sensitivity to ferroptosis (SLC7A11 appear to predominate in the regulation of this gene. For example, the master antioxidant transcription factor NRF2 can also regulate the expression of SLC7A11 at the transcriptional level, and NRF2 has been implicated as a key player in protecting cancer cells against ferroptosis. For example, inhibition of NRF2 in hepatocellular cancer cells increases the anti-cancer activity of Erastin and Sorafenib in vivo (SLC7A11, and thus show increased sensitivity to ferroptosis. Notably, overexpression of SLC7A11 in mutant p53 models led to drug resistance, suggesting that levels of SLC7A11 expression must be considered when targeting mutant p53 driven cancers with ferroptosis-inducing compounds (Wild-type p53 negatively regulates the expression of the cystine importer  in vivo . Mutant ompounds . In suppompounds . These dThe role of p53 in metabolism is quite clear and possibly even intuitively obvious: WT p53 limits glucose metabolism and lipid synthesis, while mutant p53 appears to do the opposite. The contribution of its metabolic role to tumor suppression by p53, and to the ability of mutant p53 to drive tumor progression, remains to be unequivocally proven. The role of p53 in the regulation of ferroptosis, and the contribution of this function, to tumor suppression is even less clear. While compelling data from mouse models supports the premise that p53 regulates the sensitivity of cells to ferroptosis, this may be restricted to the ability of basal p53 to suppress spontaneous tumor development, and in oncogene-stressed mouse models, it is clear that senescence and apoptosis play the predominant role. Similarly, p53 may regulate ferroptosis sensitivity in a cell type-specific manner. More studies in animal models, with attention to ferroptosis in different tissues, need to be done to more fully understand the role of p53 in ferroptosis and ferroptosis in tumor suppression. Additionally, a clearer idea of what p53-target genes play a role in sensitivity to ferroptosis needs to be attained. Resolution of these questions should provide for much needed novel avenues to combat tumors with mutant p53.KG, SB, TB, AB-K, C-PK, and MM each wrote one to two paragraphs of this article. KG and SB did the figure. KG and MM outlined the chapter.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. The reviewer OAF and handling Editor declared their shared affiliation."}
{"text": "Cell Death & Differentiation leading scientists in the p53 field elucidate some of these questions taking us a step ahead towards our understanding of this versatile protein.1TP53 is the most frequently mutated gene in cancer and its study helped to define fundamental principles underlying tumorigenesis. However, despite over three decades of accumulating knowledge , we stilCDKN1A) or association with MDM2 \u2013 the main p53 regulator \u2013 which acts as a repressor by its own.2 In particular, as shown by Engeland in this Issue, p21 connects the p53 pathway with the DREAM transcriptional repressor complex which includes members of the retinoblastoma family of tumor suppressors \u2013 whose inclusion within the DREAM is regulated by the cyclin-dependent kinases that are inhibited by p21. The DREAM controls hundreds of cell cycle-associated targets, further expanding the number of genes affected by p53 modulation, and connecting p53 to the regulation of all cell cycle checkpoints.3 Espinosa also discusses findings further detailing the functions of p53 trans-activation and carboxy terminal domains required for its transcriptional activator role.2 Interestingly, p53 emerged as a pioneer transcription factor able to bind a core set of enhancers even when the chromatin is closed, overriding epigenetic regulatory landscapes. Not all of the p53 binding, however, seems productive: rather, it becomes functional upon changes of the chromatin environment, which are time and context dependent. This explains why, although p53 binding to chromatin is largely invariable, the p53-regulated programme is extremely variegated.2P53 controls the expression of wide gene sets owing to its transcriptional activity leading to both mRNA up- and downregulation. However, its ability to act as a direct repressor of transcription has been long disputed. Here Espinosa and colleagues provide evidence that such repressive effects are mainly attributable to downstream effectors such as microRNAs or p21 . So, they suggest that these two types of mechanisms are the main contributors to the loss of function phenotype characterized by impaired DNA binding and transcriptional ability, which blunt p53 tumor suppressor activities.4 Some of the most common missense mutations are likely due to the effect of environmental mutagens, such as UV rays or cigarette smoke, which act in a tissue-specific manner, whereas methylation of CpG residues within the TP53 sequence seems a more general mechanisms increasing p53 mutational rate in most tissues, which also explains while arginine residues (encoded by four CG-containing codons) are among the top most frequently mutated. The reason why only specific mutations, over all the methylated CpG residues within the TP53 sequence, are selected could depend on the structural and biological relevance of the affected residues and their contribution in fostering tumorigenesis.4Differently from other tumor suppressors that are mostly inactivated by deletions or truncating mutations, 5 Also, p53 mutants that acquire oncogenic activity through a gain-of-function (GOF) mechanism have an increasingly recognized role. Kim and Lozano in this issue explore a wide set of p53 mutants and the mechanisms whereby they gain new oncogenic functions. Such mutants mostly act through protein-protein interactions with other transcription or chromatin remodelling factors affecting vast downstream gene networks. These interactions, which so far have been mostly described in cell lines, are highly context dependent determining various degrees of GOF activity in different tumor types with mutated p53. Interestingly, it seems that some p53 mutants acquire similar GOF activities binding common transcription factors and transactivating the same gene targets. This could have translational relevance because it would ideally limit the set of targets which should be tackled to suppress the new oncogenic GOFs.5 Overall, determining the mechanisms whereby p53 loss of function, DN and GOF activity drive tumor development and progression is crucial to definitively establish the tumor suppressor function of p53.Beyond loss of function, p53 mutants can drive tumorigenesis through other mechanisms: in heterozygosity, when both the wt and mutant alleles coexist, the mutant proteins can act as dominant negative (DN) over the wt, forming heterotetramers with impaired transcriptional ability.6 In particular, as discussed in this Issue by Strasser and colleagues, the ability of p53 to induce apoptosis in transforming cells through the direct activation of pro-apoptotic proteins (such as PUMA and NOXA) was initially regarded as the main cancer preventing function of p53. However, in contrast with Trp53 null mice that develop tumors with complete penetrance within the first year of life, mice depleted of all the (known) main downstream effectors of p53-mediated apoptosis, do not develop spontaneous tumors, even when coupled to the loss of p53 G1/S cell cycle arrest and senescence effectors, suggesting that full p53 antitumoral potential relies on further capabilities.7Induction of apoptosis, cell cycle arrest and senescence are the \u2018classical\u2019 outputs of p53 activation that seem to guarantee tumor suppression.6So, how does p53 really protect from cancer development? Kaiser and Attardi, provide some clues derived from the analysis of genetically engineered mice modelling p53 alterations, which help to reconcile older and new studies. The type of stress that challenges p53 action seems to be the key: whereas the above-mentioned classical outputs of p53 activation are needed to respond to acute DNA damage cues, incipient cancer cells are more likely exposed to other stresses such as nutrient and oxygen deprivation, oncogenic signalling, replication or oxidative stress, and also low levels of genotoxic, chronic DNA damage. To face these, p53 might induce common outcomes  through alternative routes \u2013 triggering metabolic changes, autophagy, promoting DNA repair \u2013 in a context-dependent manner. Here the authors analyze both cell autonomous and non-autonomous emerging functions of p53 and its downstream targets, which also play a part in the p53 antitumoral response.8But p53 does not act alone as commander in chief when it comes to cell fate decisions. The fine tuning of the cellular responses to a wide range of stimuli is achieved through the cross-talk between complex gene networks and signalling pathways. As an example, Oren and colleagues here discuss the cross talk between the p53 and the Hippo tumor suppressor pathway, which \u2013 similarly to p53 \u2013 regulates a variety of cellular processes. The authors highlight how the direct interaction between core components of both p53 family and Hippo pathways, and their regulation of common targets, is coordinated to modulate the response to different stresses and maintain homeostasis between stemness and differentiation, cell death and proliferation, anabolism and catabolism. Alterations of either pathway, as occurring in cancer cells, disrupt this balance with profound effects on cell fate.9Regardless of the mechanism, p53 tumor suppressor functions protect organisms against cancer development by ensuring genome stability through DNA repair or eliminating damaged cells that might undergo neoplastic transformation. However, factors that limit indefinite proliferation protect from cancer but foster aging, the gradual process of cell and tissue deterioration. Depletion of functional cells, stem cell exhaustion and the induction of a chronic inflammatory microenvironment by senescent cells, all feature aging. Although intimately related, the link between p53 and aging is still unclear with p53 activity being found associated to both premature aging and increased longevity. To clarify this conundrum Wu and Prives analyzed the mechanisms whereby p53, and its main regulator MDM2, could affect aging by regulation of genomic stability, senescence, cross talk with metabolic pathways (such as the insulin/IGF1 and mTOR pathways), mitochondrial dysfunction and regulation of reactive oxygen species, at the cellular level. Also, based on the analysis of mouse models with modulated p53-MDM2 axis, they propose a model to infer the relation between lifespan and cancer resistance as a function of p53 stability achieved through the loss of MDM2 regulation or chronic stress. Hyperactivation of p53 could provide greater cancer resistance but at the cost of accelerated ageing. Therefore, the underlying mechanisms of this delicate balance will have to be carefully determined when considering anti-aging approaches as well p53-reactivating strategies against cancer.Another layer of complexity, hampering our understanding of p53 physiological and pathological functions is added by the roles, overlapping or disjointed, of the other family members p63 and p73 and their respective isoforms. The presence of an highly conserved DNA binding domain shared by the three members of the p53 family explains some functional redundancy in their regulation of gene expression. However, p63 and p73 can also forms heterotetramers that are more stable than individual tetramers. Indeed both proteins are found to co-occupy DNA target sites throughout the genome, as discussed here by Melino and colleagues, who argue that studying these genes singularly could provide only a restricted view of their global functions. Melino and colleagues also report on latest findings implicating p63 and p73 in the regulation of cell metabolism which also contributes to the p53-family-mediated flexible response to stress. Besides tumor-related functions the authors present recent evidence establishing p73 as the master regulator of ciliogenesis whose defects underlie various human diseases. This overlooked function finally explains disparate multi-organ defects previously observed in p73 knock out models providing a unifying view that will help to better characterize other non-oncogenic functions of p73 such as infertility neurodevelopment and inflammation."}
{"text": "However, we provide evidence for a preservation of the corresponding genes in two animals unable to synthesize cholesterol (auxotrophs): Drosophila melanogaster and Caenorhabditis elegans.It is known that primary sequences of enzymes involved in sterol biosynthesis are well conserved in organisms that produce sterols bona fide orthologs of several ERG genes in both organisms using a series of complementary approaches. We have detected strong sequence divergence between the orthologs of the nematode and of the fruitfly; they are also very divergent with respect to the orthologs in organisms able to synthesize sterols de novo (prototrophs). Interestingly, the orthologs in both the nematode and the fruitfly are still under selective pressure. It is possible that these genes, which are not involved in cholesterol synthesis anymore, have been recruited to perform different new functions. We propose a more parsimonious way to explain their accelerated evolution and subsequent stabilization. The products of ERG genes in prototrophs might be involved in several biological roles, in addition to sterol synthesis. In the case of the nematode and the fruitfly, the relevant genes would have lost their ancestral function in cholesterogenesis but would have retained the other function(s), which keep them under pressure.We have been able to detect ERG24 and ERG25 in D. melanogaster and genes encoding factors involved in intracellular protein trafficking and folding and with Start1 involved in ecdysteroid synthesis. These potential functional connections are worth being explored not only in Drosophila, but also in Caenorhabditis as well as in sterol prototrophs.By exploiting microarray data we have noticed a strong expressional correlation between the orthologs of  DrosophilaCaenorhabditis elegans possesses several genes encoding proteins with regions similar to Hh and potentially undergoing cholesterylation Cholesterol and other sterols such as ergosterol and phytosterols are universal components of eukaryotic plasma membranes and are absent from the membranes of prokaryotes. These sterols  modulate membrane fluidity Saccharomyces cerevisiae, which involve ERG (i.e. from ERGosterol) genes and the respective ERG proteins.Yeast, plants and mammals synthesize sterols through a series of complex reactions that occur in the endoplasmic reticulum (ER) and, therefore, most of the involved enzymes have transmembrane domains . We outlERG25, ERG26, and ERG27, in cooperation with ERG28p. Finally, ERG6p (C-24 methylase) converts zymosterol to fecosterol, which is further transformed into ergosterol by ERG2p, ERG3p, ERG5p and ERG4p The first three steps of sterol biosynthesis are catalized by ERG9p , ERG1p  and ERG7p (lanosterol synthase), respectively. These proteins are essential for aerobic viability and their absence results in an inability to synthesize ergosterol. The third enzyme, ERG7p, converts squalene epoxide into lanosterol, the first cyclic component of the cholesterol biosynthesis cascade (i.e. the first sterol). The remaining enzymes of the pathway metabolize lanosterol into ergosterol. The sequential action of ERG11p (lanosterol demethylase) and ERG24p (C-14 reductase) leads to 4,4-dimethylzymosterol. They are also essential in aerobic conditions Recent results show that, in yeast, ergosterol biosynthetic enzymes display specific protein-protein interactions and form a functional complex called ergosome. Proteins ERG11p, ERG25p, ERG27p and ERG28p, appear to form a core center of the complex and would interact with other enzymes of the pathway D. melanogaster and C. elegans cannot synthesize sterols de novoC. elegans takes sterols from animal feces or yeast and plant remnants Drosophila obtains sterols from the diet: ergosterol from yeast and phytosterols from plants Most animals synthesize cholesterol. However, some animals such as dauer larva formation and molting depend on sterols. Cholesterol, or more likely its derivatives, seem to act as hormones. Indeed, recent papers report the identification of natural steroid ligands for the DAF-12 nuclear receptor As already noted, sterols in these animals are required as hormone precursors and/or developmental effectors C. elegans, the distribution and transport of cholesterol in vivo has been studied by using dehydroergosterol (DHE), a fluorescent analog which mimics many cholesterol properties C. elegans might be involved in the structural and functional organization of the plasma membrane cell types that are richer in this lipid In D. melanogaster with Anopheles gambiae and prototrophs has suggested that these insects have lost most of the genes involved in sterol synthesis Drosophila is unable to synthesize cholesterol. However, in a previous work we have shown that Drosophila contains an ERG28 ortholog that has undergone a process of acceleration in its evolution, and is undetectable using the current techniques for ortholog detection by sequence homology C. elegans and D. melanogaster, not only for ERG28, but most of the genes/enzymes involved in the sterol synthesis pathway, in the light of new genomic and functional data.A previous comparative genome analysis of ERG gene orthologs in C. elegans and D. melanogaster following their order in the sterol synthesis cascade . This protein, which also exists in C. elegans, contains two functional domains: monooxygenase and UbiH (2-polyprenyl-6-methoxyphenol hydroxylase and FAD-dependent oxidase). This is also the case in yeast ERG1p. However, in a reverse BLAST, CBG19254 recognized with very high score yeast Coq6 and only marginally ERG1p . A similar behavior was observed for GA20231-RA from D. pseudoobscura, so we did not proceed to a further analysis of these sequences.We failed to detect the squalene synthase (ERG9p) homolog, which catalyzes the first committed step in cholesterogenesis. In the search for squalene epoxidase (ERG1p) in a BLAST using ERG1p from yeast, we detected the gene CBG19254 in For lanosterol synthase (ERG7p), which follows in the classical pathway, we could not gather any convincing evidence for the existence of orthologs in the fruitfly and the nematode.Drosophila . However, the Drosophila genes are likely to belong to other Cyp subfamilies (not Cyp51). Cyp51 is probably missing, which is in agreement with the results of Tijet, Helvig & Feyereisen P450 gene superfamily. Cyp51 is also absent in C. elegansThe following enzyme in the pathway is a sterol 14\u03b1-demethylase, ERG11p/Cyp51, involved in the biosynthesis of cholesterol, phytosterols and ergosterol. Thus, it is the only cytochrome P450 having an ortholog common to animals, plant and fungi ERG24 orthologs in D. melanogaster (CG17952) and in C. elegans (B0250.9) were easily found. The corresponding proteins contain the ERG4-24 domain. D. melanogaster produces three isoforms that are longer than the yeast ortholog, a peculiarity that they share with the human ortholog. The ortholog of ERG24 in mammals encodes the Lamin B receptor (LBR), a nuclear envelope protein first described in vertebrates. LBR bears extensive structural similarities with the members of the sterol reductase family (ERG24p and ERG4p). Human LBR (hLBR) cannot restore ergosterol biosynthesis in an ERG4 yeast mutant, whereas it is able to restore ergosterol prototrophy in an ERG24 mutant. This strongly suggests that hLBR is a sterol C14-reductase hLBR gene causes an autosomal recessive disease called hydrops-ectopic calcification-\u2018moth-eaten\u2019 (HEM). This mutation leads to high levels of cholesta-8,14-dien-3-beta-ol in cultured skin fibroblasts, which is compatible with a deficiency of the cholesterol biosynthetic enzyme 3-beta-hydroxysterol delta(14)-reductase Potential LBR gene arose from an ancestral gene coding for a soluble nuclear protein  and that the rest of the protein evolved from another gene, similar to yeast ERG24. Indeed, the C-terminal hydrophobic domain of LBR can be retained in the endoplasmic reticulum when expressed in transfected cells, as expected for the ortholog of ERG24 in mammals. In turn, the N-terminal domain is transported to the nucleus The hLBR contains two major domains: a \u223c220-amino-acid N-terminal segment highly charged, and a hydrophobic C-terminal half with eight putative transmembrane segments Drosophila CG17952 gene is the ortholog of vertebrate LBRDrosophila LBR (dLBR) possesses a highly charged N-terminal domain of 307 amino acids followed by eight transmembrane segments. Transmembrane segments 1\u20136 are similar in length and position to the transmembrane domains 1\u20136 of hLBR. However, the putative membrane domains 7 and 8 of dLBR are shorter than those of hLBR. Thus, dLBR is expected to have a topological organization similar to that of its vertebrate orthologs Drosophila lamin B, a function residing in the N-terminal domain. Not unexpectedly, dLBR does not display sterol C14 reductase activity when expressed in the yeast ERG24 mutant. This shows that, during insect evolution, although the enzymatic activity of this protein has been lost, its capacity to bind lamin B has not. However, depletion of dLBR by RNA interference does not lead to any obvious effect on nuclear architecture, or viability, of treated cells and embryos. Thus, although dLBR might be important, it is not a limiting component of the nuclear architecture in Drosophila cells, at least during the first days of development ERG24 ortholog in C. elegans. It would be interesting to experimentally assess if it has kept LBR activity.Recent functional studies show that the D. melanogaster (CG1998/dERG25A and CG11162/dERG25B) and C. elegans (F49E12.9/ERG25A and F49E12.10/ERG25B) using the sequence of yeast ERG25p as a starting point. In both organisms the duplicated copies of ERG25 are located in the same chromosome (chromosome II for C. elegans and chromosome X for D. melanogaster). The two paralogs in D. melanogaster are separated by 0.25 Mb and contain a different number of predicted exons. Namely, CG1998 contain 6 exons, while CG11162 contains only 2. However, the last intron of both genes interrupts the coding sequence at very similar positions  were also easily found in both C. elegans (ZC8.1) and D. melanogaster (CG7724) was obtained in BLAST searches taking the sequence from yeast as the starting point  orthologs. However, a BLAST with C32D5.12 detected as the first hits NSDHL in man and ERG26 in yeast (ERG26p (C-3 dehydrogenase) belongs to the 3\u03b2-hydroxysteroid dehydrogenase family and convincing evidence for the presence of orthologs in in yeast . Thus, iet al.Drosophila and C. elegans, although several oxidoreductases were detected.In agreement with Breitling C. elegans (C14C10.6) was hardly detectable by BLAST starting with yeast sequences. This precludes the use of standard phylogenetics methods to show orthology. However, further evidence of sequence relatedness was gathered using Psi-BLAST with the yeast sequence against the Metazoa division of Genbank D. melanogaster, which is the ortholog of ERG28p according to our previous results \u221227). Reverse Psi-BLAST also suggested orthology (i.e. significant scores). The sequence of C14C10.6 is so divergent that it had no match in the conserved domain database (CDD). Moreover, we also computed the hydrophobic profiles for some of the orthologs As outlined above, ERG28p might tether many other ERG proteins to the ER. The ERG28p ortholog of C. elegans (H14E04.1) was easily detected by BLAST with protein sequences from either yeast or A. thaliana (SMT1). We used the plant sequence since no clear ERG6 homolog could be detected in human. The issue with Drosophila turned out to be more complex because, when starting the search with the yeast sequence, we detected CG8067 marginally (E>1). This was even worse when starting with the sequence of A. thaliana. However, when using the C. elegans sequence as a starting point, the first significant hit in Drosophila (E<10\u22126) was CG2453, which proved to be the ortholog of yeast Coq5, but not of ERG6p. Finally, considering i) the similar lengths of the previously marginally detected CG8067, of H14E04.1 and of SMT1 proteins and ii) their similar pI, we have preferred CG8067 as the most likely ortholog. Indeed, the hydrophobic profiles of SMT1 and the protein encoded by CG8067 displayed a strong correlation. Namely, we obtained an R\u200a=\u200a0.43 with a p-value 10\u221216.The ortholog of ERG6p in C. elegans (W08F4.3) was marginally detected by BLAST with the yeast and the human protein . However, W08F4.3 was found to contain a sigma1-receptor domain when compared with the CDD. This strengthens the idea that this gene is the ortholog of OPRS1 and ERG2. The situation in Drosophila was more complex. When starting our BLAST with either yeast or human sequences, we detected the sequence HDC14735 (DAA04220) very marginally (E\u226b1) and no conserved domain was found. However, when starting with the C. elegans sequence, it came as the best hit with E\u200a=\u200a0.002 . Although not significant, this result was taken as suggestive of similarity. The results were improved using Psi-BLAST. Again, we computed the hydrophobic profiles for the various potential orthologs and we found strong correlations . Therefore, we propose that the orthologs of ERG3 are potentially missing in both organisms.In BLAST searches with yeast ERG3p, we detected again CG1998 and CG11162 in For ERG5p, which belongs to the big Cyp protein family, no clear orthologs could be established. However, three potential candidates were found: CG4321-PA (Cyp4d8), CG3540-PA (Cyp4d14) and CG8859-PA (Cyp6g2). In the reverse BLAST, they all matched ERG5p as the best scoring hit in yeast.Finally, the search for ERG4p orthologs led to the same ERG24 orthologs in the nematode and the fruitfly. Moreover, with ERG4p the BLAST scores were worse than with ERG24p. Thus, either ERG4 orthologs are missing or they have been replaced by ERG24.ERG orthologs mentioned above undergo a selective pressure, we have examined the ratio of the number of non-synonymous substitutions per non-synonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks). The ratio Ka/Ks is indicative of the mode of evolution operating on the sequences. If selection is dominantly purifying, then we expect few non-synonymous substitutions per background synonymous changes and hence, a low ratio. If selection is absent, then a ratio of unity is expected  ratios show that these genes are under selective pressure . This property is important for enzymes because protein-protein and enzyme-substrate interactions are often electrostatic in nature. Thus, we should expect the pI of an enzyme to be similar in different organisms if protein-protein and/or enzyme-substrate interactions are conserved. In order to test this idea we gathered the protein sequences of the orthologs of ERG2, 6, 24, 25, 26 and 28. Next, we asked whether the corresponding vergence . SimilarERG orthologs that we have described above for both D. melanogaster and C. elegans. Co-expression can be assessed by determining the correlation coefficient. The correlation coefficient can be artificially inflated by flat profiles (no changes in the expression of the relevant genes). To avoid this, we focused on experiments where the genes of interest display strong variation  and it was not possible to proceed further with the analysis.Co-expression is indicative of i) physical interaction between proteins and ii) of membership to the same complex or molecular process tion see . Thus, wD. melanogaster ERG orthologs were correlated (dLBR (ERG24) and CG1998 (ERG25) with an associated p-value of 10\u221216 (after a Bonferroni correction). Such a p-value means that only one correlation coefficient out of 1016 is expected to be as high as 0.89 just by chance . Considering the maximum number of possible correlations for the 14000 transcripts in the microarrays (representing the Drosophila genome), such a high R cannot be found by chance. The behavior of dLBR and CG1998 might be reminiscent of the situation in yeast because ERG24p and ERG25p are supposed to interact, according to Epistatic MiniArray Profiling experiments First, we asked whether the expression profiles of the rrelated . The strERG orthologs in Drosophila. However, it seems that only dLBR and CG1998 still \u201cremember\u201d their ancestral belonging to the sterol biosynthesis pathway. Poor expressional correlation among the rest of the ERG orthologs also suggests that the corresponding proteins either have lost their ability to physically interact in order to form stable complexes, or they do so in conditions/moments not covered by the microarray experiments explored here. Then, we focused our attention on dLBR and CG1998 by determining which other genes were expressionally correlated with them. For this, we gathered 84 genes displaying R\u22650.875 with respect to both genes. For a correlation involving 51 data points, this R cut-off is associated with a safe p-value of 10\u221213 after correction , chaperone cofactors or unfolded protein-binding factors. A similar analysis conducted using g\u2236profiler \u22125). The existence of expressional correlation does not imply any causality. In fact, from this exploration it is not possible to determine whether dLBR and CG1998 somehow interact with other partners to participate in intracellular protein trafficking or folding, or on the contrary, they undergo the action of the latter. Since dLBR has been shown to be a nuclear protein ERG orthologs. However, no unifying theme emerged from this analysis . All in all, the strong expressional correlation between dLBR and CG1998 with proteins involved in intracellular protein trafficking or folding, and the absence of such correlation with other Erg orthologs  suggest that the involvement of dLBR and CG1998 in both processes is worth exploring.In the analysis using DAVID, the most overrepresented class included genes encoding membrane proteins, often targeted to the ER, where sterol biosynthesis takes place in prototrophs  was found for Start1. Interestingly, Start1, which is involved in intra-mitochondrial sterol transport, is expressed ubiquitously. However, in situ hybridization demonstrates a stronger expression in the prothoracic gland, where ecdysteroids are synthesized from cholesterol. These and other observations are consistent with the idea that Start1 plays a key role in the regulation of ecdysteroid synthesis In a previous paper, given the structural similarity between cholesterol and ecdysteroids, we had proposed that divergent ERGp orthologs might somehow participate in the synthesis of the latter ERG genes in Drosophila melanogaster and Caenorhabditis elegans. In spite of their sequence divergence with respect to the corresponding orthologs in sterol prototrophs, they still are under selective pressure. Since insects are unable to synthesize cholesterol de novo, an appealing way to explain this evolutionary acceleration is that ERGp orthologs have other biological functions in addition to sterol synthesis. This is clearly the case of the LBR, which is also a reductase in sterol prototrophs. Shut-down of cholesterogenesis in insects and nematodes would have allowed these proteins to evolve as much as their other functions were not compromised ERG24 and ERG25 in D. melanogaster and genes encoding factors involved in intracellular protein trafficking and folding. This is compatible with our idea that ERGp might be involved in other biological roles in addition to sterol synthesis. The potential link between ERG proteins and intracellular protein trafficking and folding deserves experimental exploration not only in Drosophila and Caenorhabditis but also in sterol prototrophs. Moreover, the potential link between dLBR, CG1998 and Start1 is to be explored in D. melanogaster. This is compatible with our previous idea of a potential implication of these proteins in the synthesis of ecdysteroids. We hope that this genomic exploration and the hypotheses prompted here might open new avenues of experimental research.In conclusion, we detected a preservation of We used BLASTp For some distant homologues we computed the hydrophobic profiles and calculated the Pearson correlation coefficient \u201cR\u201d for various pairs of profiles. The values of R range between 1 (perfect match between the profiles) and \u22121 (profiles are mirror images). Strong positive correlation is not a proof of orthology but strengthens the idea of structural relationship at the protein level.The protein-coding nucleotide sequences were first translated into amino acid sequences and aligned. Then, this alignment was used to define the alignment of the corresponding nucleotide sequences to avoid frame-shifting indels as alignment artifacts http://www.expasy.ch/tools/pi_tool.html). Then, we asked whether the corresponding Drosophila and Caenorhabditis sequences displayed outlier pI values. In short, we performed an extreme studentized deviate test to determine whether one of the values in the pI list was a significant outlier from the rest.We have investigated whether the pI of presumably orthologous proteins were similar. In order to test this, we gathered the protein sequences of the orthologs of ERG2, 6, 24, 25, 26 and 28 listed in the HomoloGene division of the NCBI database and estimated their pI ("}
{"text": "Due to the extent of functional overlap within the Rb family, it has been difficult to assess which functions are exclusive to individual members and which are shared. Like its family members, p107 can bind a variety of cellular proteins to affect the expression of many target genes during cell cycle progression. Unlike Rb and p130, p107 is most highly expressed during the G1 to S phase transition of the cell cycle in actively dividing cells and accumulating evidence suggests a role for p107 during DNA replication. The specific roles for p107 during differentiation and development are less clear, although emerging studies suggest that it can cooperate with other Rb family members to control differentiation in multiple cell lineages. As a tumor suppressor, p107 is not as potent as Rb, yet studies in knockout mice have revealed some tumor suppressor functions in mice, depending on the context. In this review, we identify the unique and overlapping functions of p107 during the cell cycle, differentiation, and tumorigenesis. Rb tumor suppressor was first identified as the gene whose loss causes hereditary retinoblastoma in children . HoweveRb-related genes can be found across multiple species, including humans, mice, chickens, reptiles, flies, and even some plants. Most unicellular and lower organisms have only one Rb-related gene, while higher organisms tend to have two or three family members, perhaps reflecting an increasing complexity of cell cycle control in these species. For example, the unicellular alga Chlamydomonas reinhardtii only contains one Rb-like gene (mat3), whose loss leads to a deregulation of proliferation and a reduced cell size [Whi5 appears to play a functionally similar role to Rb, despite a lack of sequence homology [Rb-related gene, although recently a second Rb-related gene was identified in maize and rice [Caenorhabditis elegans also contains one Rb-like gene, lin-35, which in sequence homology is more similar to p107 than to Rb [Rb-related genes in Drosophila, RBF1 and RBF2 [lin35 in C. elegans, these two genes are more similar to mammalian p107 and p130 than they are to Rb itself. Interestingly, the gene duplication events in plants and flies are only two examples of many duplication events within the Rb-family over the course of evolution as additional duplication events have occurred in Gallus gallus (chicken), Danio rerio (zebrafish), and Anolis carolinensis (lizard)  as it was originally named, was identified through its interaction with SV40 Large T antigen and adenovirus E1A . StructuUnlike Rb and p130, p107 levels are generally, but not always, low in quiescent and differentiated cells and higher when cells proliferate -37. p107Of the three Rb family members, p107 is thought to be the most heavily regulated at the transcriptional level ,36. p107p107, like p130 and Rb, is a substrate for Cyclin/Cdk kinase activity during cell cycle progression, which is thought to cause p107 to release E2F transcription factors and relieve repression of target gene promoters -54. In qin vivo, p107 preferentially binds to E2F4 and E2F5 at the promoters of E2F target genes in cycling cells [in vivo. Future experiments should aim to identify more extensive sets of target genes bound by individual Rb family members in various cellular contexts. Experiments such as genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-Seq) with antibodies specific to p107, p130, and Rb in different cell types or at different phases of the cell cycle may help to shed light on this question.Once bound to E2Fs, Rb family members repress transcription through a variety of methods. The Rb family can directly recruit chromatin modifying enzymes such as histone deacetylase HDAC1  derived from these animals display no significant cell cycle defects [p107-deficient mice in a Balb/c background show a severe postnatal growth deficiency, as well as myeloid hyperplasia in the spleen and liver. MEFs and myoblasts derived from these animals exhibit increased proliferation that was associated with constitutive expression of Cyclin E [-/- p107pre-adipocytes could not upregulate Rb, which is required to initiate differentiation in vivo through interaction with Pgc1\u03b1 [ defects . InteresCyclin E . These mth Pgc1\u03b1 .-/- p107mouse may be due to compensation from p130 or Rb. -/-;p130-/- p107embryos die at birth with multiple defects in tissue development. Interfollicular keratinocytes from -/-;p130-/- p107newborns show impaired terminal differentiation in the epidermis, decreased numbers of hair follicles, and a developmental delay in hair, whisker, and tooth formation [p107 and p130 seem to play overlapping roles during embryonic development in the mouse, and one reason for the lack of severe phenotypes in the ormation . These d-/-;p130-/- p107embryos as well as -/-;p27-/- p107embryos display defects in ossification of the long bones and chondrocyte proliferation [p107 has also been implicated in the control of bone and cartilage development. Double knockout feration ,103. Durferation . Overexpferation . Biochemin vitro and in vivo [In the adult mouse brain, p107 expression is unique from the other Rb family members in that it is restricted to cycling progenitor cells in the ventricular zone, and its expression decreases as these cells begin to differentiate into cortical neurons ,105. Rb  in vivo . This apHes1, a key downstream target of the Notch signaling pathway, since p107-deficient animals display increased Hes1 signaling  is increased in neural progenitors of p107-deficient brains. Recently, p107 was shown to repress E2F3 activity at the promoter of Fgf2, an essential growth factor that modulates the population of neural precursors in the developing brain [-/- p107brains, as increased FGF2 would be expected to promote survival. Therefore, the mechanisms driving apoptosis in -/- p107progenitors may be independent of FGF2 signaling, and have yet to be identified.p107 may also be acting through the FGF growth factor signaling pathway to control the numbers of neural precursors in embryonic and adult brains Figure . FGF is ng brain . HoweverThe two examples in chondrocytes and neurons suggest a scenario in which FGF signaling can indirectly activate p107, which could then repress FGF signaling through direct promoter binding. Although this feedback loop between p107 and FGF has yet to be identified within one specific cell type, it suggests the potential for a complex regulatory system for p107 and growth signaling during development.p107 shortened the lifespan of -/- Rbembryos from birth to E14.5, and further increased the abnormal levels of proliferation and apoptosis that are present in the central nervous system and the ocular lens of Rb-deficient embryos. Mutation of p107 in an Rb-deficient background also caused heart development defects that were not seen in the Rb-deficient or p107-deficient embryos alone. These heart defects are likely the result of blood vessel endothelium and endocardial cell proliferation in the absence of both Rb and p107. Additionally, the cerebellar architecture is severely disrupted in adult mice lacking both Rb and p107 in the dorsal mid-hind brain junction, and these mice exhibit impaired terminal differentiation and migration defects in granule cell precursors, in addition to increased granule cell apoptosis upon maturation [Recent studies have uncovered functions of p107 that overlap with Rb during development. Additional loss of turation . While tturation , a basicturation . Thus, pRb is mutated in a variety of sporadic and familiar human cancers, most notably in pediatric retinoblastoma and osteosarcoma. Mutations in p107 itself have not been observed in human tumors [p107 in human tumors, it still may play a role in tumorigenesis, as mutations in upstream regulators of the Rb family are common [n tumors . So far,n tumors -118. Dese common ,120. Inae common . This fip107 do not develop spontaneous tumors [+/-;p130-/- p107and -/-;p130+/- p107mice revealed no spontaneous loss of either p107 or p130 allele and no obvious tumor phenotypes [-/-;p130-/- p107mice has not been described, these studies suggest that p107 and p130 do not by themselves have tumor suppressor functions in the mouse. However, these observations do not exclude the possibility that p107 and/or p130 may act as tumor suppressors in other contexts.It is clear that p107 is not a strong tumor suppressor by itself, as mice with mutations in s tumors . Since penotypes . While cRb loss, p107 can contribute some tumor suppressor functions within a cell  of the retina showed that Rb expression was redundant with p130 . In the  retinas . Thus, iigenesis . The mecRb-heterozygosity results in retinoblastoma with 100% penetrance in humans but is not sufficient to cause retinoblastoma in mice. This difference in mice may be due to the ability for other family members, namely p107, to compensate for the loss of Rb in this context [p107 in response to Rb loss, whereas mouse retinal cells do [p107 promoters contain two tandem E2F binding sites. The mouse promoter has a single point mutation in the proximal 3' E2F site that may affect the binding of E2Fs or other transcriptional machinery that is recruited there [p107 in response to Rb loss in specific contexts, while the other cannot. These differences in p107 transcriptional regulation may also be seen within the different tissues of the same organism. For example, if deregulation of the Rb pathway can be found in almost all human tumors, why, then, do Rb-heterozygous patients primarily only develop a narrow spectrum of tumors, mainly retinoblastoma and osteosarcomas? This discrepancy may be explained by the fact that some tissues can upregulate p107 in response to Rb loss, whereas others cannot. Indeed upregulation of p107 is seen in several cell types upon Rb loss, including mouse retinal progenitors [p130 is generally not seen in response to Rb or p107 or both [ context . Interescells do . This died there . Alternagenitors , keratingenitors , hepatocgenitors , and lungenitors ,126. Int or both .To fully understand the tumor suppressor functions of Rb in human tumors, it is important to understand the functions of each of the family members, both individually and as a group. In particular, p107 functions in cell cycle control and tumor suppression have remained elusive.During cell cycle progression, p107 function may be divided into two categories; those that require E2F and those that do not. ChIP-Seq for Rb family members and E2F family members in normal cells would shed light on the normal binding patterns of these proteins and may identify promoters that are regulated by individual Rb family members or by several at once. Expanding upon this, one could then compare the binding profiles of the Rb family members in normal cells to that of tumor cells, cells in different phases of the cell cycle, or cells from different tissues. p107 can also interact with several other key transcription factors such as Sp1, B-myb, c-myc, and Smad3. The significance of these interactions is not well understood, and several questions remain. For example, is p107's ability to regulate the cell cycle mainly exerted through E2F repression or can the interaction with other transcription factors also arrest the cell cycle independently of E2F\u03b1 One way to answer these questions would be to take advantage of the fact that E2Fs bind different regions of p107 than do the other transcription factors.Evidence for p107 function during S phase suggests that it may play a critical role outside of the control of G1. Rb and p130 have both been implicated in the control of G0 and G2 -136, so p107 mutations to mice carrying mutations in other known cancer causing genes outside of the Rb pathway. Furthermore, no studies have examined whether p107 mutation and DNA damaging agents can contribute to faster or more aggressive tumors. Future studies should clarify the specific contexts in which p107 can act as a tumor suppressor, with or without compound mutation of Rb.It has yet to be determined whether p107 can serve as a tumor suppressor in the context of other mutations outside of the Rb pathway. To date, there have been no studies crossing mice with Although much has been uncovered since the discovery of the Rb family, the complexities in functional overlap, regulation, and tumor suppressor abilities of each of the Rb family members is only just beginning to be explored. The use of transgenic, knock-in, and knock-out mouse studies, as well as in vitro cell culture systems will be critical to increase our understanding of the role of these genes during multiple cellular functions, and these techniques will continue to reveal the subtle and distinct ways in which these proteins can interact with each other as well as the hundreds of other proteins known to associate with them. More analysis of how the Rb family normally functions is needed to understand their functions within a single cell, in addition to their tumor suppressor capabilities.Rb: Retinoblastoma; Cdk: Cyclin-dependent kinase; HDAC: histone deacetylase; DHFR: dihydrofolate reductase; BMP: bone morphogenetic protein; FGF: fibroblast growth factor; VZ: ventricular zone; ChIP: chromatin immunoprecipitation.The authors declare that they have no competing interests.SW and JS designed and wrote the manuscript together. Both authors have read and approved the final manuscript."}
{"text": "Several studies have focused on the role of p53 inactivation in cervical cancer, either by inactivating mutations in the TP53 gene or by degradation of the p53 protein by human papillomavirus (HPV). In this study, primary cervical carcinomas from 365 patients were analysed for presence of HPV using both consensus primer-sets and type-specific primer-sets. Nineteen samples were determined to have no or low virus load, and were selected for further analyses: mutation screening of the TP53 gene using constant denaturant gel electrophoresis (CDGE) followed by sequencing, and protein expression of p53, MDM2 and p21 using immunohistochemistry (IHC). Mutations in the TP53 gene were found in eight samples (42%). Elevated p53 protein expression was significantly associated with presence of a mutation (P < 0.007). P21 protein expression was detected in 16 of the 19 carcinomas. No p21 expression was seen in normal cervical tissue. Two samples, both with wild-type p53, had elevated MDM2 expression. Compared with a previous study from our group, of mainly HPV-positive cervical carcinomas, in which only one sample was found to contain a TP53 mutation, a significantly higher mutation frequency (P < 0.001) was found among the carcinomas with no or low virus load. Although p53 inactivation pathways are not detected in every tumour, our study supports the hypothesis that p53 inactivation, either by binding to cellular or viral proteins or by mutation, is essential in the development of cervical carcinomas."}
{"text": "Prior research has identified the lateral occipital complex (LOC) as a critical cortical region for the representation of object shape in humans. However, little is known about the nature of the representations contained in the LOC and their relationship to the perceptual experience of shape. We used human functional MRI to measure the physical, behavioral, and neural similarity between pairs of novel shapes to ask whether the representations of shape contained in subregions of the LOC more closely reflect the physical stimuli themselves, or the perceptual experience of those stimuli. Perceptual similarity measures for each pair of shapes were obtained from a psychophysical same-different task; physical similarity measures were based on stimulus parameters; and neural similarity measures were obtained from multivoxel pattern analysis methods applied to anterior LOC (pFs) and posterior LOC (LO). We found that the pattern of pairwise shape similarities in LO most closely matched physical shape similarities, whereas shape similarities in pFs most closely matched perceptual shape similarities. Further, shape representations were similar across participants in LO but highly variable across participants in pFs. Together, these findings indicate that activation patterns in subregions of object-selective cortex encode objects according to a hierarchy, with stimulus-based representations in posterior regions and subjective and observer-specific representations in anterior regions. a.d., the Arab scholar Ibn al-Haytham suggested that visual experience was not veridical, but inherently subjective. During the last few decades, this observation has given rise to one of the core questions in visual neuroscience: how does the subjective experience of visual stimuli relate to their neural representations in the brain? It is well-known that visual shape is represented in a brain region called lateral occipital complex (LOC). However, do these representations reflect physical or perceptual stimulus characteristics? We presented observers with a set of complex visual stimuli and obtained three measures of similarity for these stimuli: a physical similarity measure based on stimulus parameters; a behavioral similarity measure based on discrimination performance; and finally a neural similarity measure based on multivariate pattern analyses in LOC. We found that in anterior LOC, neural stimulus similarities correlated with subjective perceptual similarities, but not with physical stimulus similarities; the reverse was true in posterior LOC. In addition, neural similarities were consistent across participants in posterior LOC, but highly variable across participants in anterior LOC. Together these findings suggest a two-part answer to the question of how cortical object representations relate to subjective experience: anterior regions appear to contain subjective, individually variable shape representations, whereas posterior regions contain stimulus-based shape representations.As early as 1031  How does the subjective experience of visual shapes relate to the neural representations of these shapes in the brain? Using psychophysics, functional MRI, and multivariate pattern analysis methods, this study shows that activation patterns in anterior, shape-selective brain regions reflect perceptual shape similarities, whereas patterns in posterior regions reflect physical similarities. What is the neural code for object shape? This question has been at the core of systems neuroscience for decades. In monkeys, inferotemporal (IT) cortex has been shown to contain cells selective for complex shapes ; in humaA number of previous studies suggest that the coding of objects in high-level visual cortex may reflect subjective perceptual experience of shapes. For instance, LOC adapts across changes in low-level physical stimulus properties that leave perceived shape unaltered, but not across changes that affect perceived shape ,7. FurthFMRI studies of visual processing have traditionally focused on mean activation levels, looking for brain regions showing a difference in activation between different stimulus conditions. More recent studies, in contrast, have illustrated the importance of the distributed pattern of activation in representing information about stimulus conditions \u201314. HaxbWe used a combination of human fMRI and psychophysics to test the hypothesis that distributed activation patterns in LOC reflect perceived shape. We created a novel artificial shape space, in which physical similarity was controlled by gradual, parametric changes in aspect ratio and skew. Perceptual similarity was measured by psychophysical discrimination performance between the shapes, and neural similarity was measured by the correlations between the fMRI activation patterns of these shapes in LOC.  We founWe created a novel artificial stimulus space consisting of four complex objects A. Each sTo study the relationship between perceptual, physical, and neural similarities in pFs and LO, we obtained three similarity measures for each pair of stimuli as follows. First, for each of the six possible pairs of nonidentical stimuli, physical similarity was measured by the inverse pairwise distances of the four shapes in the aspect ratio/skew space , followed by a 1,500 ms response period. The proportion of trials on which a particular participant responded \u201cidentical\u201d to a pair of stimuli that were in fact different was used as a measure of the perceptual similarity of that pair of stimuli. An example of a perceptual similarity matrix is shown in p < 10\u22124). LOC can be subdivided into a posterior portion, LO, on the lateral surface of occipitotemporal cortex; and an anterior portion, pFs, on the fusiform gyrus of the temporal lobe [Finally, to obtain a measure of neural similarity, we scanned the brains of eight participants using fMRI. Since we had a specific hypothesis about neural coding in object-selective cortex, we first identified the human object-selective region LOC in an independent localizer scan, using the standard comparison of intact versus scrambled everyday objects  . The purpose of this task was to keep participants' attention focused on the stimuli. We then extracted the spatially distributed activation patterns of each individual stimulus from the two LOC ROIs, on a voxel-by-voxel basis. Thus, in each participant and for each ROI, we obtained four vectors, each representing the voxelwise activation pattern of one particular shape in that ROI. We then computed the correlations between each of the six pairs of activation patterns for nonidentical stimuli, separately for pFs and LO. This resulted in six correlation coefficients for each ROI, one for each possible pair of the four shapes. An example of a neural similarity matrix is shown in Thus, we obtained physical, perceptual, and neural similarity measures for each possible pair of stimuli. We next compared these six-element similarity matrices to one another, by computing their correlation coefficients within participants and ROIs B. A highThe results confirmed this hypothesis, with an interesting twist. Neural and perceptual similarities were positively correlated in pFs, with an average correlation of 0.35 across participants, whereas in LO the average neural-perceptual correlation was only 0.001 . Converst(5) = 5.66, p < 0.001), but not greater than zero in LO (t(5) = 0.10, p = 0.38). Conversely, the correlations between neural and physical similarities were significantly greater than zero in LO (t(5) = 2.66, p < 0.05), but not in pFs (t(5) = 0.70, p = 0.29). A two-way analysis of variance (ANOVA) with region of interest (pFs versus LO) and correlation type  as factors revealed a significant interaction of ROI and correlation type  = 13.79, p < 0.005), confirming the dissociation between these ROIs: neural pattern similarities in pFs correspond to subjective shape similarities, while neural pattern similarities in LO correspond to physical shape similarities.To quantify these results, we initially applied the Fisher z transformation to all correlation coefficients. This method transforms the non-normally distributed correlation coefficients into normally distributed variables, which allows the use of standard analysis of variance methods  for det. StatistF = 1.86, p = 0.17; LO: F = 0.17, p = 0.92). Moreover, these results cannot be due to task performance, since critically the perceptual similarity measure was obtained in a separate testing session, while in the scanner participants performed an easy one-back task. These results were not due to differential mean signal levels for any of our stimuli, for two reasons: first, the correlation analysis does not take into account mean levels of activation; second, there were no differences in mean signal between the four stimuli in either region of interest  = 0.92, p = 0.26; t(25) = 7.83, p < 0.00000005; t(25) = \u22122.80, p < 0.05).As predicted, inter-participant reliability was low in pFs  does not depend on whether they correlate with the behavioral similarities within participants (tested above).p < 10\u22124); and the parahippocampal place area, PPA [p < 10\u22124). In none of these regions did the neural similarities exhibit significant correlations with either perceptual or physical similarity =2.77, p < 0.05), showing that pairs of stimuli that a particular participant confused with high probability were also perceptually similar for the other participants. However, at the same time the fact that this correlation was not extremely high leaves room for subjective, observer-specific patterns of perceptual similarities.The psychophysical same-different task used outside the scanner to obtain a measure of perceptual similarity was made as difficult as possible by presenting stimuli for extremely short durations (17 ms each) and using both forward and backward high-energy noise masks (50 ms each). Nevertheless, the performance level was high, with an average of 93% \u00b1 1% correct performance on the same-different task. However, this level of performance corresponds to an average of 44 \u00b1 9 errors over the course of the psychophysical experiment; this number of errors, distributed over the four stimuli used, proved sufficient to obtain a reliable measure of perceptual similarity. In support of this claim, the inter-participant reliability of perceptual similarity was on average r = 0.38; this value was significantly different from zero (t(5) = 2.75, p < 0.05). Thus, perceptual similarity correlated with physical similarity, but not perfectly, again leaving room for the neural similarity measures to correlate with either the perceptual or the physical similarities without necessarily also correlating with the other.The average correlation between the physical and perceptual similarity measures across participants was t(7) = 8.11, p < 0.001), and stimuli 1 and 2  = 2.88, p < 0.05). To assess the effect of this difference, we used the reaction time differences among the four stimuli during the task in the scanner as a new behavioral similarity measure within each participant; e.g., if a particular participant took an average of 635 ms to respond to stimulus 1, and 649 ms for stimulus 2, the new behavioral similarity of these stimuli would be the negative reaction time difference, i.e. \u221214 ms (negative to turn the measure from a \u201cdifference\u201d into a \u201csimilarity\u201d measure). We then re-computed the correlations between the neural similarities and this new behavioral similarity measure. None of the resulting correlations were significant in any of our ROIs, although there was a nonsignificant trend towards a correlation between neural similarities and the new reaction time similarities in LO  = 1.75, p = 0.09).The one-back task participants performed in the scanner was designed purely to keep participants' attention focused on the stimuli and was very easy to perform; none of our participants made a single mistake on this task. However, to nevertheless control for any possible effects of performance in the scanner, we analyzed the reaction times during the task in the scanner as follows. Since the order of stimulus presentation was randomized using m-sequences, each stimulus was preceded equally often by each other stimulus; therefore, each of the six possible discriminations among the four stimuli entered the neural pattern an equal number of times, and differential performance on any of these conditions could therefore not influence the neural pattern. However, it is possible that the task was easier for some stimuli in general; such a difference would enter the neural pattern of those stimuli and could thus potentially bias our results. Indeed, we observed a significant difference in the reaction times of stimuli 1 and 3  = 6.32, p < 0.001; t(5) = 0.56, p = 0.32); in contrast, LO exhibited a significant correlation between neural and physical similarities  = 2.33, p < 0.05; r = 0.06, t(5) = 0.56, p = 0.32). The interaction was again significant  = 12.00, p < 0.005). Similarly, the inter-participant reliability was again high in LO  = 2.82, p < 0.05), but low in pFs  = 0.60, p = 0.33; t(25) = 2.04, p = 0.05). Note, however, that subsampling reduced the inter-participant reliability in LO by a factor of one-half (mean r = 0.42 to mean r = 0.21). In light of this change, and the fact that the average sizes of pFs and LO differed by a factor greater than two, we repeated the subsampling for the inter-participant reliability analysis using not 50% of voxels, but instead equalizing voxel numbers across the two ROIs. Specifically, we excluded random subsets of voxels from the larger ROI, until its size matched that of the smaller ROI, again with 100-fold bootstrapping. The results were comparable to that of the initial analysis: the inter-participant reliability was high in LO  = 7.79, p < 0.0001), but low in pFs  = 0.91, p = 0.26), with a significant difference between LO and pFs (t(25) = 2.65, p < 0.05). Thus, our results are independent of the size of our ROIs.As pointed out above, it is conceivable in principle that differential numbers of voxels in different ROIs affect the likelihood of detecting correlations. Indeed, LO contained more voxels on average than pFs . We therefore wished to test whether the results described above depend on the number of voxels in each ROI. To this end, we conducted a control in which we randomly excluded 50% of the voxels of each ROI. This procedure was repeated 100 times, and an average correlation estimate was obtained by averaging over the 100 bootstrapping iterations. The results were the same as in the main analysis reported above  and 6: tr = 0.67) with the physical similarity measure reported above, i.e., closeness of the stimuli in parameter space. However, the neural similarities of pFs and LO showed no correlation with this V1-type physical similarity measure: in pFs, the mean correlation of the neural similarities with the mean Gabor similarity measure was r = 0.06 (not different from zero: t(5) = 0.42, p = 0.34); in LO, it was r = 0.07 (t(5) = 0.45, p = 0.34). In addition, we repeated this analysis for each individual Gabor filter ; none of the resulting 20 correlations between neural and physical similarities were significantly different from zero across participants in either pFs  or LO . Thus, the correlation of neural and physical similarities in LO appears to be specific to the case when the physical similarities are described in terms of aspect ratio and skew [The physical similarity measure reported above was based on the distances of the stimuli from each other in terms of aspect ratio and skew parameters. The high correlation of these aspect ratio/skew distances with neural similarities in LO is consistent with the proposal that LO encodes stimuli in terms of aspect ratio and skew, as has been reported previously for high-level visual cortex in monkeys ,24. Thisr = 0.15, which did not differ from zero across participants (t(5) = 0.76, p = 0.27). Moreover, none of the correlations were significant when the individual physical similarity matrices resulting from each of the 20 Gabor filters were correlated one-by-one with neural similarities . This result is probably due to the fact that the images were presented with a random jitter of \u223c2 degrees during scanning, which likely resulted in sufficiently nonoverlapping activations in retinotopic cortex to disrupt the neural similarity estimates in this region, and therefore also any correlation between neural and physical similarities. In support of this hypothesis, the stimuli were not distinguishable in retinotopic cortex using Haxby's pattern discrimination method [The neural patterns in retinotopic cortex only showed a weak correlation with the physical stimulus distances based on this V1-type similarity measure; the mean correlation between neural and Gabor similarities in retinotopic cortex was F = 3.67, p < 0.05, two-way ANOVA with ROI and within/between as factors). As a further test of pattern discriminability, we computed the \u201cHaxby Index\u201d [t(7) = 4.20, p < 0.005; LO: t(7) = 4.55, p < 0.005). Thus, the patterns in both ROIs were stable enough across the split halves to successfully discriminate between our stimuli.The results reported above indicate that the neural patterns in our regions of interest contain fine-grained information about perceptual and physical stimulus similarity. These analyses were based on correlations between the neural activation patterns of pairs of stimuli; this measure controls for noise in the data because the formula for the correlation coefficient includes a division by the standard deviations of the data vectors. However, we additionally wished to confirm with a conventional analysis method that these neural patterns were indeed stable and contained information about stimulus identity. To this end, we applied the widely used technique of Haxby et al. : we extry Index\u201d ,12,13,26In sum, we have found that distributed activation patterns in human object-selective cortex contain information about the subjective perceptual similarities between complex visual stimuli. Specifically, we show a dissociation between neural coding of perceptual versus physical similarities within LOC: using independent measures of neural, perceptual, and physical similarity on our set of novel artificial shapes, we find that the neural similarities of shapes in anterior LOC (pFs) correlate with their perceptual similarities. Conversely, the neural similarities in posterior LOC (LO) correlate with the physical similarity of the shapes in the stimulus space. Furthermore, the agreement across participants of the neural similarities is high in LO, but low in pFs, consistent with a physically based representation in LO and a representation based on observer-specific subjective shape experience in pFs.These results are specific to object-selective cortex, i.e., the regions LO and pFs; additional ROIs including retinotopic cortex, FFA, OFA, and PPA did not show significant correlations between either neural and perceptual or physical similarities. Moreover, the results did not depend on the number of voxels in each ROI.Our findings confirm previous studies showing that object representations in the ventral stream reflect subjective perception \u20139,11,27,Similarly, the finding that posterior LOC shows a correlation between neural and physical stimulus similarity confirms previous studies that have shown selectivity for physical shape features of moderate complexity in high-level visual cortex. In area V4, single-cell and fMRI studies have demonstrated tuning to contour curvature in monkeys ,28,29 anPutting the results from pFs and LO together, our findings are consistent with previous evidence regarding an anterior-posterior functional subdivision within LOC: Grill-Spector et al.  showed tHowever, it should be noted that other recent studies have found evidence highlighting the informativeness and behavioral relevance of neural activation patterns in LO: Eger et al.  showed tThree previous studies have shown correlations between pattern information and subjective perception in object-selective cortex. First, Edelman et al.  used mulSecond, Op de Beeck et al.  showed iThird, Williams et al.  used a pIn conclusion, our results indicate that object shape may be coded in terms of physical features in posterior LOC, and in terms of subjective shape experience in anterior LOC. Of course, this claim simply replaces one puzzle with another: \u201cWhat is the neural code for object shape?\u201d is transformed into the equally difficult question, \u201cWhat are the determinants of subjective shape experience?\u201d However, the advantage of this new question is that it has been the subject of intensive study since the time of the Gestalt psychologists , and theWe recruited eight participants from the MIT Human Subject Pool. Each participant was compensated US$60. The study was approved by the MIT Committee on the Use of Humans as Experimental Subjects (COUHES). All participants gave informed consent.Localizer scans: The LOC was localized as the region that responded more strongly to grayscale images of intact objects than to images of scrambled objects (p < 10\u22124), as described previously [p < 10\u22124). The PPA [p < 10\u22124). The retinotopic ROI was defined based on activation at the occipital pole in a contrast between all stimulus conditions versus baseline in the localizer scans .Experimental scans and behavioral experiment: Four novel stimuli, each measuring 10 degrees across, were used for the experimental scans. The use of novel stimuli ensured that correlations were not due to semantic associations with the stimuli; this was a potential confound in previous pattern similarity studies [y = na x. The parameters a and n could be used to vary the skew and aspect ratio of each protrusion parametrically. In doing so, the total area of the stimuli was always kept constant to avoid low-level confounds. We defined aspect ratio as the ratio of the height to the base width of each protrusion, and skew as the position of the vertex with respect to the center of the base; for instance, 0% skew indicates a vertex directly above the center of the base, skew of 100% indicates a vertex directly above the right end of the base, and skew of \u2212100% indicates a vertex directly above the left end of the base. From the left to the right end of the stimulus spectrum, the aspect ratio of the second and fourth protrusions  decreased by 1.4 and 1.6 on each morph step, respectively; the aspect ratio of the first and third protrusions was fixed. For skew, the first, second, and fourth protrusions moved counterclockwise by 60%, 24%, and 24% for each step, respectively (where a cumulative skew change greater than 100% simply meant moving the vertex of the protrusion beyond its base); the skew of the third protrusion changed in the clockwise direction by 25% on each step. Thus, the four stimuli used were equidistant in terms of aspect ratio and skew, forming a straight line in the stimulus space. The magnitude of the parametric distances between the stimuli was chosen based on informal testing to be at the same time discriminable and not too obvious. The stimuli were filled with random dots, with a mean luminance of 50%, to ensure activation throughout the ventral visual stream. In addition, a chair and a face were included in the stimulus set, to prevent adaptation in ventral visual cortex due to the high similarity among the novel shapes. studies . Further studies ; we therfMRI experiment: Each participant was run in one session of about 2 h, consisting of eight experimental scans and four LOC localizer scans. Stimuli were presented using the Psychophysics Toolbox [ Toolbox  and MatlThe localizer scans were run as described previously ,20,23.The experimental scans were event-related, and each scan contained 144 stimulus trials and 36 fixation trials. On each trial, one of the six possible stimuli  was presented at the center of the screen for 300 ms, followed by a 1,700-ms response period during which participants indicated whether the current stimulus was identical or different from the previous one. The purpose of this task was to keep participants' attention focused on the stimuli. The order of stimulus presentation was optimized using m-sequences (Optseq). Each stimulus occurred 24 times per scan, resulting in a total of 192 times for the whole experiment.Behavioral experiment: In a separate behavioral session that followed the fMRI experiment with a delay of at least 1 wk, each of the original participants performed a same-different task on pairs of the same four shapes, plus the face and the chair, that were presented in the fMRI experiment. On each trial, two stimuli were shown sequentially, for 17 ms each, with a forward and a backward mask (consisting of a full screen noise field of random letters with high density and overlap) of 50 ms each, followed by a 1,500-ms response period. Perceptual similarities were obtained by computing the proportion of trials on which a particular pair of different stimuli was erroneously considered \u201cidentical\u201d. Participants performed 630 trials total. Each stimulus appeared with equal probability on each trial, and with equal probability as the first and second stimulus of each pair.fMRI scanning was performed on a 3T Siemens Trio Scanner (Siemens) at the Athinoula A. Martinos Center for Biomedical Imaging at the McGovern Institute for Brain Research at MIT. A Gradient Echo single-shot pulse sequence was used . Twenty-five slices were collected with a 12-channel head coil. Slices were oriented roughly perpendicular to the calcarine sulcus and covered most of the occipital and posterior temporal lobes, as well as some of the inferior parietal lobes. Slices were 2 mm thick, with a 10% gap, and had an in-plane resolution of 1.6 \u00d7 1.6 mm.http://surfer.nmr.mgh.harvard.edu), fROI (http://froi.sourceforge.net), and custom-written software. Before statistical analysis, images were motion corrected [Data analysis was performed using FS-FAST  to intact objects than to scrambled versions of the same objects [p < 10\u22124); the PPA [p < 10\u22124); and a retinotopic ROI based on activation at the occipital pole in a contrast between all stimulus conditions versus baseline in the localizer scans . The FFFor the blocked localizer scans, statistical maps were calculated by correlating the signal time course with a gamma function  for each voxel convolved with the block timecourse. For the event-related scans, the hemodynamic response was extracted using a deconvolution analysis, without any assumptions about the shape of the response. The peaks of the fMRI responses of each of the four novel shapes were extracted from each ROI, for all voxels separately. This resulted in four patterns per ROI, each representing the distributed activation pattern to a particular stimulus in that ROI. Neural similarities were obtained by computing the Pearson correlation coefficient between these patterns, as described above.t-tests and ANOVAs. This transformation is necessary because correlation coefficients do not follow a normal distribution, and are therefore strictly not amenable to analysis of variance statistics [r, the Fisher z is given byTo assess the statistical significance of the correlation matrices results, we first applied the Fisher z transformation to the data and then performed atistics . The FisWe took care to use the standard error formula specific to the Fisher z:This formula has fewer degrees of freedom and is therefore more conservative than the conventional standard error.r = 0.67) with our other physical similarity measure, i.e. distance of the stimuli in parameter space.To obtain a V1-like physical similarity measure, we applied a set of Gabor filters to the images and then computed pixelwise similarities between the images. This analysis was motivated by the fact that V1 cells exhibit tuning profiles that are well-described by Gabor filters ,43. Each"}
{"text": "In this study we investigated tumour growth in relation to the immunohistochemical expression of p53 and bcl-2 and to patient survival data in 33 operated hepatocellular carcinomas (HCCs). In order to estimate the growth, a growth index, based on the degree of cell proliferation, apoptosis and necrosis, was calculated for each tumour. Cell proliferation was determined immunohistochemically by the number of proliferating cell nuclear antigen (PCNA)-positive cells in tumours, the extent of apoptosis was determined by counting the number of cells labelled by the in situ 3'-end labelling technique and tumour necrosis was estimated as the percentage of necrotic areas in haematoxylin--eosin-stained tissue sections. In our analysis we found that the survival of patients with HCCs showing a high growth index (i.e. tumours showing a high proliferation and simultaneously a low degree of apoptosis and necrosis) was significantly shorter than with other patients . When analysed separately, cell proliferation, apoptosis or necrosis did not show any significant association with survival. p53 positivity was found in 8/33 (24%) of tumours. There were significantly more p53-positive cases in tumours with a high growth index  suggesting that dysfunction of the p53 gene may affect tumour growth. p53-positive cases did not, however, have a significantly shorter survival time than p53-negative cases . bcl-2 positivity was found in only 1/33 (3%) of the HCCs. Thus bcl-2 overexpression does not seem to play an important role in hepatocellular carcinogenesis. In summary, our results suggest that in HCCs a compound score based on the evaluation of the degree of cell proliferation, apoptosis and necrosis is a biologically more relevant prognostic indicator than any of its composite parameters alone."}
{"text": "A total of 169 colorectal adenocarcinomas, obtained from patients with a median follow-up of 6.5 years, were studied with immunohistochemical staining on cryosections using a monoclonal anti-tenascin antibody to evaluate the possible association between the staining patterns and tumour stage, tumour differentiation and survival. We found two different staining patterns in the tumour stroma--a diffuse stromal fibrillar staining in 92 out of 169 (54%) tumours and a subglandular staining in the remaining 77 tumours. When the entire group of patients (P < 0.01) and the group of potentially cured patients (P < 0.03) were analysed univariately, it was found that diffuse stromal fibrillar staining was associated with a shorter survival time than subglandular staining. In a multivariate analysis, the Dukes' stage and age were independent prognostic factors, whereas the tenascin expression did not retain a clear independent relationship to survival (P = 0.06). Hence, it appears that the tumour expression of tenascin may be a potential prognostic marker in colorectal cancer, in so far as a diffuse stromal fibrillar staining pattern seems to indicate an increased risk of poor outcome. However, after adjustment for age and Dukes' stage, the additional prognostic value of tenascin remains to be established in further analyses."}
{"text": "The fine mapping and deciphering of specific cancer phenotypes is taking advantage of molecular-profiling studies based on genome-wide approaches. Currently, high-throughput methods such as array-based comparative genomic hybridization (CGH array), single nucleotide polymorphism array (SNP array), expression arrays and ChIP-on-chip arrays are available to study mutant p53-associated alterations in human cancers. Here we will mainly focus on the integration of the results raised through oncogenomic platforms that aim to shed light on the molecular mechanisms underlying mutant p53 gain of function activities and to provide useful information on the molecular stratification of tumor patients.Cancer is caused by the spatial and temporal accumulation of alterations in the genome of a given cell. This leads to the deregulation of key signalling pathways that play a pivotal role in the control of cell proliferation and cell fate. The p53 tumor suppressor gene is the most frequent target in genetic alterations in human cancers. The primary selective advantage of such mutations is the elimination of cellular wild type p53 activity. In addition, many evidences  Structural studies have allowed the classification of p53 mutant proteins in two major categories [in vitro and in vivo studies that show how mutant p53 proteins confer transformed properties to cell cultures and increased tumorigenicity in mice [The p53 tumor suppressor protein is a sequence-specific transcription factor that is present in a latent form under normal conditions and becomes activated by a variety of stress signals, such as DNA damage, oncogene activation and improper mitogenic stimulation. p53 acts as a potent transcriptional activator through the binding as a homotetramer to specific sequences, named p53 responsive elements (p53RE). Activated p53 modulates the expression of a large set of target genes involved in many cellular processes, including cell cycle arrest, DNA repair, apoptosis and senescence. Growth arrest or cell death prevents damaged DNA from being replicated; thus suggesting a role for p53 in the maintenance of genome integrity -5. p53 itegories ,12: a) D in mice ,14. It h in mice ,16. p53- in mice ,17. Inde(a) mutant p53 can bind to DNA through the association with DNA binding proteins and transcriptionally activate specific target genes using its functional transactivation domain (TAD). In support of this molecular mechanism, it has been reported that human tumor-derived p53 mutants, whose TAD was inactivated by site-directed mutagenesis, lost the ability to increase tumorigenicity in vitro and in vivo [(b) recent work by Di Agostino et al. has shown that mutant p53 proteins physically interact in vivo with the transcription factor NF-Y, whose DNA binding consensus are present in the regulatory regions of many key genes involved in the regulation of the cell cycle. Mutant p53 can be recruited in vivo onto the promoter of NF-Y target genes, as a member of a large transcriptional complex that also includes histone acetyltransferases (HAT). Of note, mutant p53 favours the selective recruitment of HAT in response to DNA damage, thereby dictating the transcriptional activation of its target genes [ (c) mutant p53 binds to and sequesters proteins whose function is required for anti-tumor effects such as apoptosis or growth inhibition. Interestingly, it has been reported that human tumor-derived p53 mutants can associate with p73 and p63 and interfere with their transcriptional activity and ability to induce apoptosis when co-expressed in transient transfection assays  [To date, we can depict the three following molecular scenarios to explain gain of function of mutant p53 proteins.  in vivo , 19-21; et genes . (c) mut -26.in vitro and knock-in models in vivo. However, as mentioned above, mutations can occur within the entire DBD of the protein. The activity of a large number of p53 mutants has been analyzed by Kato et al. [Many of the studies exploring the molecular mechanisms that underlie gain of function of mutant p53 proteins have mainly characterized the activities of p53 hot spot mutations. These mutants display an oncogenic potential by themselves, as demonstrated by transformation assays o et al. . This ano et al. ,17,28 ano et al. .Since p53 mutations of the DBD are selected in cancer, it is reasonable to hypothesize that hot spot mutations are directly selected because they represent the driving force during the neoplastic transformation. The remaining p53 mutations are selected following a second mutational event that might be permissive for mutant p53 gain of function activities. Cell-type specificity for gain of function of mutant p53 might explain why certain p53 mutants do not display any activity when exogenously overexpressed in cell systems different from those where the mutation was originated. et al. [Since the first description of a p53 mutation in human cancers in 1989, several thousands of papers have described clinical studies in which p53 has been tentatively linked to the response to treatment (predictive power) or patient survival (prognostic power). The usefulness of these studies was questioned in terms of strategy  and methodology ,31. Indeet al.  have shoet al. -36. Now in situ hybridization, Sigurdsson et al. [et al. [et al. [et al. [The genome of cancer cells is thought to be unstable as they often harbour many chromosomal abnormalities. Due to the role of TP53 in maintaining genomic stability, its inactivation profoundly impairs repair of DNA damage and could promote genomic alterations. Several research groups have reported a correlation between genomic instability and TP53 mutations in breast cancer tissues. Using fluorescence n et al.  have sho [et al.  combined [et al. . They fo [et al.  and Klei [et al.  performe3). Currently, high throughput methods such as array-based comparative genomic hybridization (CGH array) and single nucleotide polymorphism array (SNP array) are available to study mutant p53-associated alterations in a genome-wide fashion. Array-CGH is an established high-resolution method to study the whole genome for chromosomal amplifications and deletions . One lima) cell lines overexpressing different mutant forms of p53 have been generated to analyze the impact of mutant p53 on cell transcriptome; b) tumor samples with characterized p53 status (mutated or wild-type) have been examined by microarray analysis for their global gene expression. The latter has been compared with that of normal counterparts to define  clusters of genes associated with the mutated status of p53.Two different approaches have been followed to identify mutant p53-associated gene-expression signatures: et al. report that the most obvious difference related to the ectopic expression of two diverse p53 mutants (R175H and A138P) is that R175H retained less wild-type transcriptional regulatory events than A138P. A long list of mutant p53-regulated genes has been obtained from microarray analyses, suggesting the involvement of mutant p53 in several cellular processes ranging from transcriptional and translational regulation, signal transduction, cell motility, invasion and metastatization. This list obviously includes some of the genes previously characterized as mutant p53 targets. Importantly, all the identified genes provide evidence for the potential disclosure of new molecular targets and for substantial insights into the molecular mechanisms underlying gain of function activities of mutant p53.A number of studies employed microarrays to evaluate global gene expression in H1299 lung cancer cells expressing p53-175H, p53-273H and p53-281G -47, p53-in vitro models of cell lines overexpressing p53 mutants, the impact of a mutated p53 on the transcriptome has also been evaluated through in vivo microarray analyses. As mentioned above, the mutational status of p53 is prognostic in many malignancies. In breast cancer, p53 mutations are associated with worse overall and disease-free survival rates and have been implicated in resistance to anticancer therapies. Miller et al. [In addition to the r et al.  reportedr et al. ) where pin vivo analyses in identifying gene expression signatures associated with wild type or mutant p53, unfortunately these studies are unable to assess whether the characterized genes are really dependent on the presence of mutant p53 or whether they merely represent the result of a loss of wild-type p53 activity. The data sets derived from microarray analyses on cell lines overexpressing p53 mutants, enclosing the putative genes responsible for gain of function, need to be matched with those derived from in vivo analyses to define which of the transcripts are truly expressed in cancer tissues carrying p53 mutation. Up until now few p53 mutations have been analyzed in vitro for their ability to transcriptionally affect gene expression. Further microarray analyses employing additional p53 mutants to be compared with those performed on tumor specimens with known p53 status might unveil important molecular details on the gain of function properties of the different mutant p53 proteins present in human cancers.Despite the efficacy of these via interaction with other transcription factors is strongly supported by the work of Di Agostino et al. [Little is known about the mechanisms through which mutant p53 proteins, that have lost the ability to bind DNA in a sequence-specific manner, can achieve the specificity in gene regulation. Chromatin Immunoprecipitation experiments have shown that mutant p53 can be found onto the regulatory regions of putative target genes, but despite extensive efforts, the identification of the specific DNA binding consensus of mutant p53 has not yet been found. It has been proposed that mutant p53 can be recruited to its putative target promoters through the physical interaction with sequence specific transcription factors whose specific DNA binding consensus are present on those regulatory regions. Sp1 and Ets, which were shown to cooperate with mutant p53 in transcriptional regulation -57, are o et al.  which hain vivo binding sites are outside the predicted promoter regions of given genes. It has been shown that the binding locations of NFkB, cMyc, Sp1, p53, CREB are found within both coding and non-coding regions. More binding sites than those predicted were found and of those only a relatively small fraction of the sites occurred in genomic regions that would typically be considered \u201cpromoters\u201d. The use of tiling arrays in the study of mutant p53 transcriptional activity will overcome the difficulties existing in the prediction of the binding sites for this oncogenic transcription factor. In addition, mutant p53 has been reported to bind a wide range of DNA secondary structures. For example, mutant p53 was shown to preferentially bind matrix attachment regions (MARs) [Due to the absence of a canonical consensus sequence for mutant p53, the only approach that seems truly promising for the precise deciphering of its transcriptional code is the use of whole-genome tiling arrays, designed to interrogate an entire genome in an unbiased fashion, for ChIP-chip analysis. ChIP-chip (Chromatin Immunoprecipitation-chip) permits a simultaneous analysis of the occupancy of a specific transcription factor on thousands of its target genes. Briefly, crosslinked chromatin (DNA/protein) complexes are extracted from the analyzed cell culture or from a tissue and sheared down to relatively short fragments (500-1000 bp). The chromatin fragments containing the transcription factor of interest are then immunoprecipitated using a specific antibody and the target sequences are subsequently identified through hybridization to DNA microarrays. At a high probe tiling resolution multiple overlapping probes may contain the actual transcription factor binding motif and thus enable a fine mapping of the binding site to a resolution of less than 25 bp. Apart from the most recent generated tiling arrays, different kinds of microarrays have been generated for ChIP-chip analysis. Initial studies were performed using slides carrying spotted oligonucleotides or PCR products from selected predicted promoters. However these arrays are unable to interrogate the entire genome and are unable to address the possibility that transcription factors might bind to other locations. Recent studies using tiling arrays interrogating entire chromosomes show that a large fraction of s (MARs) . MARs ar4).The ChIP-chip analysis of chromatin initially immunoprecipitated with antibodies against mutant p53 and successively with antibodies directed to the various transcription factors that mediate mutant p53\u2019s binding to its target genes (Re-ChIP) is likely to lead to the identification of subgroups of target genes controlled by different mutant p53-TF protein complexes (Fig. Genome wide analyses are turning out to be powerful instruments in the molecular stratification of cancer patients. The complexity of the data raised through the oncogenomic approaches calls for the integration of multidisciplinary expertises and potentially accounts for the fine deciphering and molecular characterization of specific cancer patient phenotypes. An understanding of the molecular events regulating and/or governed by gain of function mutant p53 proteins might lead to the disclosure of cancer signalling pathways to be tackled with novel anticancer therapies."}
{"text": "Science magazine , p53 may also function in a                            sexually antagonistic manner, limiting the life span of adult female flies                            while promoting the longevity of adult males.                                        New insights into the roles that p53 plays in animal                            aging are provided by Waskar et al. in this issue of Drosophila life span                            studies have primarily been concerned with the effects of reducing p53 function                            using DN p53 alleles , to dietary restriction                            [reviewed in 20], to mitochondrial gene manipulations ,21.  WesThe slight elevation in p53 may help maintain genomic                            fidelity during early life, resulting in healthier, longer-lived adults, at the                            cost of developmental speed and elevated energy requirements.  In addition,                            increased p53 activity during development may have resulted in a decrease in                            body mass/size of the animals.   Although the relationship between body size                            and longevity is complicated , this maIt has also not escaped our attention that p53                            regulates mitochondrial electron transport chain (ETC) activity .  Our owA final thought worthy of consideration is that it is at least possible that the effects                            observed with neuronal expression of DN p53 transgenes could be due to the                            transgenic p53 having effects distinct from inhibition of endogenous p53.  The                            DN p53 has either a mutation in the DNA binding domain, or it is a truncated                            version of the protein, but these can presumably still carry out some of the                            functions of p53, such as localization to the mitochondria,                            or direct regulation of autophagy.  In addition, while the adult fly is usually                            referred to as a \u2018post-mitotic' system, there are dividing stem cells and                            progeny cells in the gonad, gut, and malphigian tubule -28.  Cha"}
{"text": "Survival analysis of 144 histologically confirmed cases of colon carcinoma diagnosed in a 12 year period (1971-82) at the Tampere University Hospital was performed to test the hypothesis that p53 overexpression is associated with a poor clinical outcome. Immunohistochemical staining of paraffin-embedded sections using a polyclonal antibody CM-1 against p53 protein was performed to identify aberrant expression of the p53 tumour-suppressor gene. Sixty-nine per cent of the tumours (100/144) showed overexpression of the p53 protein. The prevalence of p53 overexpression was independent of age and sex of the patient and subsite of the tumour, but was slightly, although not statistically significantly, higher in advanced than in localised tumours. Overexpression was associated with a higher S-phase fraction. Some indication of a larger proportion of aneuploid tumours among those with overexpression was also observed, although this finding did not reach statistical significance. Significantly reduced patient survival for tumours with p53 overexpression was found. Patients with p53-overexpressing tumours had a corrected 5 year survival rate of 37% compared with 58% among patients whose tumours had normal expression of p53. The corresponding 10 year rates were 34% and 54%. In the multivariate analysis using a Cox model, the survival difference was independent of age and sex of the patient, as well as of subsite and stage of the tumour. Furthermore, the effect of p53 overexpression remained after controlling for flow cytometric parameters in an analysis of a subset of tumours. Thus, p53 overexpression appears to be a useful prognostic indicator in colon carcinoma."}
{"text": "Rb gene deletion in concert with inactivation of Rb family members have recently been developed. These new Rb knockout models of retinoblastoma provide excellent tools for pre-clinical studies and for the exploration of the genetics of tumorigenesis driven by RB inactivation. This review focuses on the developmental consequences of Rb deletion in the retina and the genetic interactions between Rb and the two other members of the pocket protein family, p107 (Rbl1) and p130 (Rbl2). There is increasing appreciation that homozygous RB mutations are insufficient for human retinoblastoma. Identifying and understanding secondary gene alterations that cooperate with RB inactivation in tumorigenesis may be facilitated by mouse models. Recent investigation of the p53 pathway in retinoblastoma, and evidence of spatial topology to early murine retinoblastoma are also discussed in this review.Novel murine models of retinoblastoma based on  The RB gene was the first tumor suppressor gene cloned [RB is now known to be mutated not only in retinoblastoma but in many cancers, including osteosarcoma, soft tissue sarcoma, small cell lung, breast, brain, prostate, leukemia, and other sporadic cancers [RB mutation are at increased risk for many of these tumor types and for melanoma [RB is mutated widely in various human tumor types, the retina is exquisitely sensitive to cancer upon RB loss and the reason for this is tissue sensitivity is still poorly understood. Our current knowledge of RB function places this gene in pathways central to growth control not only in the retina, but in all tissues. RB is a prototypic tumor suppressor gene that encodes pRB, a nuclear phosphoprotein implicated in cell cycle control, differentiation, apoptosis, and many other biological processes. Despite intensive study of this tumor suppressor gene since its cloning, the critical functions of pRB important for tumor suppression are still being unraveled.Retinoblastoma is the most common pediatric primary intraocular tumor. Study of retinoblastoma, a malignant cancer that occurs unilateral or a bilateral form, led to the \"two-hit hypothesis\" now applicable to other inherited cancer syndromes . In bilamutation ,3. In spe cloned , and RB  cancers -9. Patiemelanoma -12. Theymelanoma ,14. EvenC elegans SynMuvB genes  and Drosophila melanogaster dREAM  complex members. There is also evidence that pRB may bind pro- differentiation factors, such as CBFA1, MITF, C/EPBs and MyoD to promote cell type specific differentiation [RB family members that contribute to retinoblastoma are not well understood. Here, we focus on insights that study of the murine Rb family function in the retina in vivo have for understanding human retinoblastoma.pRB is one member of the \"pocket proteins\", a family that also includes p107 and p130. These proteins are named owing to the presence of a conserved pocket domain, a region that binds E2F transcription factors and various critical protein interactors, many containing an LxCxE motif. The three pocket proteins exhibit similarities, particularly in the A and B pocket, but there are also clear differences. p107 and p130 have much closer similarity to each other than either to pRB. The pocket proteins differ in their expression patterns, with p107 highly expressed in cycling cells, and p130 expressed at higher levels in cells that have exited the cell cycle . Criticantiation -25. Datain vivo using plasmid electroporation, or, if stable expression over many cell divisions is needed, viral vectors. These properties make the retina an ideal system to study not only development, but also cancer initiation. For childhood cancers these processes are clearly intertwined.The neural retina is composed of seven classes of cell types derived from a common population of progenitors, with cell bodies organized into three nuclear layers. The outer nuclear layer contains cell bodies for rod and cone photoreceptors; the inner nuclear layer contains bipolar, amacrine, horizontal and M\u00fcller glia cell bodies, and the ganglion cell layer is made up of ganglion and amacrine cells Fig . DevelopRetinoblastoma is a pediatric cancer that is typically detected in the first few years of life but can arise during embryonic retinal development . The ideLineage tracing studies in the normal rodent retina do not support the existence of specific progenitors that generate only rod and cone photoreceptors . InsteadThere are limitations to using human tumor samples to identify the tumor cell of origin. In the establishment and long term passaging of cell lines, genetic alterations may occur that alter the cell's properties. Also, accessing early retinoblastomas is rarely possible, limiting analysis to late stage tumors that may have quite different characteristics compared to the earliest lesions. Use of model systems can help in assessing the early events in tumorigenesis.LH-beta T-Ag model expresses the SV40 early region under control of the Luteinizing hormone beta promotor [LH-beta T-Ag line was in part a consequence of the genomic site of integration directing transgene expression to cells in the retina, a tissue in which luteinizing hormone beta is not normally expressed. Both Large-T and small-t oncoproteins are encoded by the SV40 early region and may contribute to tumorigenesis. Large-T Ag binds the pRB family, p53, and other proteins, such as CBP/p300. Small-t Ag also contributes to transformation in cell culture, in part through binding the phosphatase pp2A [LH-beta T-Ag model, retinoblastoma with neuronal characteristics and histological similarities to human retinoblastomas emerged. Transgenic mice expressing T-Ag/t-Ag from the IRBP promoter have also been generated and develop retinoblastoma [PNMT-T-Ag) transgenic mice develop retinal tumors that arise at the retina periphery. This spatial localization of retinal tumors in mice occurred despite T-Ag/t-Ag being expressed in amacrine and horizontal cells in both central and peripheral retina [A number of models of retinoblastoma have been developed that have made use of transgenic expression of specific viral oncoproteins. The most widely used such model, the promotor . The devase pp2A . In the blastoma -51. IRBPblastoma , raisingl retina . The autl retina  but it iIRBP-E7 mice underwent photoreceptor degeneration, while IRBP-E7; p53-/- animals were prone to tumors of the retina that arise in the photoreceptor layer [p53 loss suppressed E7-dependent apoptosis, leading to retinoblastoma, p53 loss may contribute in other ways, as these tumors are now recognized to emerge in the midst of high levels of apoptosis and photoreceptor degeneration even upon p53 deficiency [p53 independent of apoptosis are critical for tumor suppressor ability [p53 in retinoblastoma discussed further on in this review. Whether the tumors in the IRBP-E7;p53-/-, LH-beta T-Ag and PNMT-Tag models exhibit a similar cell of origin or if these models represent distinct tumors of different cell types remains to be determined.Papillomavirus E7 protein binds to the pocket protein family, and other effectors, but does not inactivate p53. or layer . While ificiency . This di ability . This isRb but not other effectors of viral oncoproteins has been a clear goal for improvement of mouse retinoblastoma models. The main disadvantage of transgenic models expressing viral oncoproteins concerns the pleiotropic effects of the oncoproteins, which impact upon multiple signaling pathways. These oncoproteins may also have important targets not yet identified. Furthermore, tumors in which p53 is functionally ablated through T-Ag expression are not ideal for testing therapies, whose effectiveness may depend on the p53 pathway being intact [Use of transgenic retinoblastoma models has been valuable for the testing of preclinical therapeutics; however, the development of models that target g intact ,59.Rb deletion took a number of years. Three groups inactivated Rb in the mouse germline, resulting in embryonic lethality in Rb-/- animals and the development of pituitary and thyroid tumors in Rb+/- animals [Rb heterozygotes, in stark contrast to the high retinoblastoma incidence in humans with germline RB mutation. The Jacks and the Berns groups both generated chimeric animals in which Rb-/- cells contributed to the retina, but retinoblastomas still did not form [Rb and p107 came the realization that mice were capable of developing retinoblastoma dependent on Rb deletion [Rb-/-; p107-/- cells to the developing embryo.The generation of mouse models that, like human retinoblastoma, exhibit  animals -62. Retinot form ,64. Withdeletion . HoweverCre-lox technology and Cre-expressing transgenic animals with expression in the developing retina paved the way for the generation of breedable Rb-knockout models of retinoblastoma. The first attempt, based upon the previous descriptions of photoreceptor characteristics in retinoblastoma used Cre regulated by the IRBP promoter together with RbLox alleles to drive Rb deletion in the photoreceptor compartment and in other tissues [p107 and p53 deficiency, indicating that the tumor-initiating cells in Rb/p107 DKO chimeras were not being targeted when these genes were deleted in developing photoreceptors. Breedable Rb-knockout models of retinoblastoma were independently generated using Cre transgenics with expression driven by Nestin, Chx10 and Pax-6 promoter in progenitor and other cells [p107 first revealed in chimera studies. Mouse models have now revealed that inactivation of the third pocket protein, p130, also cooperates very strongly with Rb deletion to promote retinoblastomas in both Rb knockout and chimeric mice [Rb-knockout and chimeric retinoblastoma models is provided in Table Rb knockout models of retinoblastoma now provide excellent tools to hone in on the cell of origin and interrogate the molecular genetics of retinoblastoma progression.Nevertheless, the advent of  tissues . These mer cells -70. New ric mice ,69,71. ARb in the retina. Homozygous germline Rb deletion in mice leads to embryonic lethality in midgestation, caused by placental deficiency [Rb has been inactivated in the retina using a number of different systems that bypass the lethality associated with systemic Rb deletion. Rb-/-;+/+ chimeric animals and various Cre-expressing transgenics, including Chx10-cre, Pax6 \u03b1-enhancer Cre (\u03b1-Cre) and NesCre1, in concert with an Rb lox/lox allele exhibit Rb deletion in the retina [Rb at different times and in different cells. Chx10-Cre is expressed in a mosaic pattern in progenitor cells, and in adults is expressed in most bipolar cells and in some M\u00fcller cells [NesCre1 is active in the optic vesicle by E9.5 and exhibits different expression upon paternal vs. maternal inheritance of the transgene [Cre transmission and is accompanied by lethality at birth in Rblox/lox animals. A mosaic pattern of Cre expression is observed with maternal NesCre1 inheritance allowing for the survival of some animals on an Rblox/lox background. \u03b1-Cre is expressed by E10.5 in distal progenitor cells, with a dorsal gap in expression [\u03b1-Cre at late stages both in central and peripheral retina. A synthesis of the data from many groups using different systems has revealed common phenotypes that have implicated Rb in control of proliferation, apoptosis and differentiation in the developing retina  to proliferate inappropriately [Rb loss [Rb-deficient retina ultimately exited the cell cycle or died such that by PND21, Rb-/- cells no longer proliferate, and retinoblastoma never develops [Rb deletion differentially affects specific cell types. Bipolar cells, ganglion cells as well as the majority of rod photoreceptors are lost in \u03b1-Cre Rblox/lox mice. Photoreceptor apoptosis is specific to rods, as cells expressing cone markers were present in normal numbers, revealing specificity to rod cell death [Rb -/-; +/+ chimeras, in which there was a low, but not null, contribution of Rb-/- cells to the adult retina [In embryonic stages of retinal development, the first indication that there were cell cycle defects upon to E18.5 . Ectopic mutants . Ectopicre1 mice ,69. Normextended -69. Notapriately . However[Rb loss . Importadevelops ,68. Intell death . The celt retina .Rb loss has been associated with differentiation defects. Rod photoreceptors in Chx10Cre Rblox/loxmice have been reported to exhibit an altered pattern of heterochromatin suggestive of rod maturation defects [Rb loss, but it is difficult to distinguish between downstream effects of Rb loss on cell cycle control vs. direct effects on differentiation. Interestingly, defects in rod development were not found when IRBP-Cre was used to drive Rb deletion in photoreceptor transition cells [Rb-/- explant cultures or in Chx10Cre Rblox/lox retinas [Chx10Cre Rblox/lox animals [NesCre1, Chx10Cre and \u03b1-Cre Rblox/lox mice all exhibit the presence of ectopic cells in the outer plexiform layer that extend to the photoreceptor layer [Rb-mutant retinas have also been noted. Rb-deficient starburst amacrine cells were found to lack the expression of markers associated with their maturation [Rb-deficient horizontal cells exhibit enlarged abnormally shaped nuclei, reminiscent of polyploid cells that have undergone endoreduplication in other Rb-deficient cell types, such as skeletal muscle and Purkinje cells [Rb-deficient horizontal cells involving ectopic processes that extend into the outer nuclear layer have also been described [Finally,  defects ,77,78. Ion cells . One expor layer ,78, and turation . Also, aje cells ,69. DefeRb deletion using chimera systems and different Cre-transgenic lines . A simiRb loss are quite similar across studies of chimeras, \u03b1-Cre and NesCre1 mice, with milder phenotypes observed upon Chx10Cre-mediated Rb inactivation. Differences highlight the need to assess the consequences of Rb deletion at different times across different cell types, and the need to better understand the extent to which phenotypes are cell autonomous. Some of these aims may be achieved by expressing Cre in a temporally and dosage controllable fashion using either viral delivery or tamoxifen-inducible Cre-ER transgenics with Cre-ER expression driven by cell-type specific promoters.The phenotypes upon E2f family members in mediating Rb dependent phenotypes is of great debate. Analyses of E2f contribution to Rb-mutant developmental phenotypes have been performed in the whole animal through compound mutant studies, but the non-cell autonomous consequences of placental dysfunction in Rb-/- embryos complicate the interpretation of these results. Compound mutant analysis specifically in the developing retina provides an excellent system to probe these interactions. The importance of E2f1 in mediating the effects of Rb loss was nicely shown by the Bremner group as compound mutation of Rb and E2f1 rescued much of the proliferation and apoptosis associated with Rb deletion using the \u03b1-Cre Rblox/lox system, and even restored response on the electroretinogram, a measure of retinal electrical activity in response to light [E2f1 loss did not rescue differentiation defects in Rb-deficient starburst amacrine cells. In contrast, compound mutation of Rb and E2F3a led to a rescue in starburst amacrine cell development suggesting that Rb/E2F3a interactions promote the differentiation of these cells. While pro-differentiation effects of pRB are often thought E2f-independent, this is one example of pRB promoting the differentiation of a cell type mediated through E2F.The importance of specific to light . InteresRb loss and apoptosis in the developing retina. The above study showed that apoptosis is E2f-dependent, but the E2f effectors that contribute to apoptosis remain to be identified. We were interested in whether p53 was downstream of Rb inactivation in the retina [NesCre1 Rblox/lox p53-/- double mutants did not exhibit rescue of apoptosis in the embryonic retinal ganglion cell layer. Also, the loss of photoreceptors was not rescued in NesCre1 Rblox/lox p53-/- animals exhibiting deletion of Rb in a mosaic pattern. Rb loss has been linked to apoptosis as pRB is an anti-apoptotic protein and many E2F target genes are apoptosis effectors [Rb deletion is abrogated [p53-independent apoptosis downstream of Rb loss in the retina will be an important goal for broadly understanding the events that synergize with Rb loss to promote cancer.It will be critical to dissect the pathway between e retina . Compounffectors . Whetherbrogated . ElucidaRb as well as p107 or p130 loss clearly illustrate tumor suppressive activity of p107 or p130 in the context of Rb deficiency. It is not clear why retinoblastoma manifests itself in mice only if p107 or p130 is inactivated in addition to pRB. p107 expression is upregulated in murine retinas lacking Rb but not in human retina explants with knockdown of RB expression, raising the possibility that p107 upregulation prevents retinoblastoma in mice. [Rb-/- murine retinal cells has been reported only at postnatal stages of retinal development, or in explant cultures differentiated in vitro to recapitulate postnatal stages of retinal development and not in embryonic Rb-/- retina [Rb-/- retinas at late time points in retinal development [p107 is an E2f target gene typically expressed in proliferating but not post-mitotic cells, the p107 increase has not been uncoupled from the cell cycle phenotypes associated with Rb loss at late stages in retinal development. Other Rb targets, such as Cyclin E are found strongly upregulated at both embryonic and postnatal stages of retinal develoment in Rb-deficient retinas (D.M. unpublished). It is also possible that functional overlap among the pocket proteins without increased p107 or p130 expression is critical for suppressing retinoblastoma in mice. Indeed, p130 is a stronger suppressor of retinoblastoma than p107 despite no alterations in p130 overall levels upon Rb loss [Rb loss) and many upstream regulators of pocket proteins are known to be important for cell cycle control in the retina. Cyclin D1-/- retinas are hypoplastic, corresponding to altered pocket protein phosphorylation [Rb family inactivation and this decrease may contribute to retinoblastoma suppression in mice through regulation of pocket protein activity [Rb or both Rb and p130 compared to controls. p27-/- retinas exhibit an extension in the period of retinogenesis, implicating p27 in cell cycle exit in a subset of cells [(Ink4d) loss also extends the normal period of retinal proliferation in the retina [p107 or p130 being intact will be important to elucidate. It is likely that complex differences between mice and humans in the levels and activity of multiple regulators of the RB family contributes to the species difference in retinoblastoma susceptibility. Thus, a broader understanding of the regulation of pRB, p107 and p130 in the cell from which retinoblastoma is derived is needed.Data from chimeras and conditional mutants exhibiting inactivation of in mice. . However- retina ,84. Impoelopment -69. As p Rb loss ,69. Factrylation . High cyof cells . p19 that must be overcome.Long latency of tumorigenesis and incomplete penetrance suggests that Rb and p130 in the retina have revealed roles for p130 in tumor suppression and retinal development in \u03b1-Cre Rblox/lox p130-/-, NesCre1 Rblox/lox p130-/- and in Rb-/-p130-/- chimeras [NesCre1 Rb/p107 DKO retinas, histological defects were not found in NesCre1 Rb/p130 DKO embryonic retinas [\u03b1-Cre Rb/p130DKOs, relative to \u03b1-Cre Rblox/lox single mutants, revealing cooperation between Rb and p130 in controlling cell cycle exit in late stages of retinal development. By PND21, Rb-/- cells have all exited the cell cycle, but cells in \u03b1-Cre Rblox/lox p130-/- retinas continue to inappropriately undergo S-phase entry and exhibit the presence of cells with large aberrant nuclei [\u03b1-Cre Rblox/lox p130-/- retina. The extent to which this cell death is cell autonomous has not yet been determined and it is possible that effects of Rb and p130 loss on certain cell types may lead to the death of other cells through secondary effects. Cell death is more pronounced in these animals than \u03b1-Cre Rblox/lox p107-/- retinas, although some cells, including horizontal cells, M\u00fcller cells and rare amacrine cells survive at PND21. The survival and increase in horizontal cells at this stage is a specific effect of Rb and p130 loss not observed upon Rb and p107 deletion.Interactions between chimeras ,69,71. It nuclei . ProlifeRb and p130, NesCre1 Rblox/lox p130-/- and \u03b1-Cre Rblox/lox p130-/- mice all develop retinoblastomas in which marker expression is heterogeneous and includes the expression of amacrine markers [\u03b1-Cre Rblox/lox p130-/- and NesCre1 Rblox/lox p130-/- retinoblastomas . Finally, secondary alteration analysis reveals that tumors in both models undergo N-myc amplification, suggestive of a common pathway to transformation [Despite differences in kinetics and cell type composition, abundant data supports a model of a common cell of origin in rosettes . Rb/p107ormation .Rb and p130 deletion leads to an increase in Calbindin-positive horizontal cells [Chx10Cre drives Rblox/lox deletion in p130-/-, p107+/- animals, termed \"p107 single\" [PNMT-Tag mice, functional pRB family ablation in horizontal cells led to distinct effects depending on retinal topology [PNMT-Tag expression led to tumors in peripheral retina but not in central retina, where horizontal cell death eventually occurred; it will be important to carefully examine whether peripheral vs. central horizontal cells also respond differently in p107-single mice. While Dyer et al (2007) conclude that the cell of origin is a horizontal cell, the possibility of another cell type that generates the late-stage tumors cannot be ruled out. The metastatic cells in p107-single retinoblastomas did not resemble horizontal cells either in ultrastructure or in cell marker expression, suggesting that the retinoblastomas could derive from cells that can produce both differentiated horizontal cells and non-horizontal metastatic cells or that there may be a mixed tumor phenotype with differing cells of origin. This issue is also complicated by the labeling of amacrine cell subsets by horizontal cell markers such as Calbindin and Prox1 [p107-single studies clearly show that horizontal cells can divide and raise a very interesting hypothesis regarding a possible cell of origin to the tumors, deletion of these genes specifically in differentiated horizontal cells will be necessary to prove this hypothesis.While al cells , the hor single\" . The auttopology . PNMT-Tand Prox1 . OverallRB is much more likely to occur in a proliferating cell than a post-mitotic cell as DNA replication and cell division can promote an initial gene mutation or loss of heterozygosity. Despite differences in the cell type composition of murine vs. human retinoblastomas, the cell of origin in human and mouse retinoblastoma may both be a similar multipotent cell. That is, the requirement to inactivate p107 or p130 in addition to Rb in the mouse would be expected to mask potential for the murine retinoblastoma cells to exhibit photoreceptor character, as there is widespread death of both rods and cones in the absence of Rb and p107 or Rb and p130 when deleted in early progenitors using Pax6 alpha Cre [Rb and p107 or Rb and p130 deletion, i.e. amacrine, M\u00fcller and horizontal cells. The specific composition of tumor cell types may be a result of the effects of p107 or p130 on the survival, proliferation and differentiation of the retinoblastoma cell of origin and its daughters.Differentiated cells are not strong candidates for the cell of origin of human retinoblastoma, given the evidence of multipotent potential to human retinoblastoma cells , althouglpha Cre ,68. Instpatched heterozygous mice, persistent proliferation of cells expressing the progenitor marker nestin was found at the extreme retina periphery in adults, suggesting that the hedgehog pathway may be important for the late proliferating cells [\u03b1-Cre Rblox/lox p130-/- or \u03b1-Cre Rblox/lox p107-/- retinas [Observations of early lesions at the extreme retina periphery in mouse retinoblastoma models raised the question of whether spatial differences in the localization of human retinoblastomas occur. Careful examination of retinoblastoma topology has revealed that retinoblastomas that arise in older children tend to occur at the periphery of the retina, while those that occur in younger children are typically central ,98. In hng cells . Furtherng cells -105. The retinas . Potenti retinas ). It wilRb family loss in mouse models suggests a model in which inactivation of RB allows the proliferative period of retinal development to be extended in the mutant cells. Upon mutation of both Rb and p107 or Rb and p130, this phenotype is exacerbated in mice. This model proposes that homozygous inactivation of RB must occur during the normal proliferative period of retinal development, and that this event increases the chance of subsequent mutations by creating a proliferative lesion balanced by cell cycle exit and differentiation, or cell death. The loss of both copies of RB may increase the rate of subsequent mutations, as was found in embryonic stem (ES) cells, in which loss of Rb led to a dramatic increase in the rate of chromosomal alterations that result in loss of a selectable marker [Rb can lead to chromosome segregation defects through misregulation of the expression of genes important for processes such as centrosome duplication [RB deletion during development undergoes the secondary mutations needed for tumor formation. In some cases, retinoblastoma may form from a benign precursor lesion, retinoma that exhibits some features of senescence and has recently been shown to exhibit less complex patterns of genetic alteration than adjacent retinoblastoma [RB inactivation. Specifically, is this a differentiation block, or must cell death or other processes be overcome? Examination of the nature of secondary alterations will be needed to shed light on this question.An examination of the effects of lication ,109, mitlication  or throulication . In humablastoma ,113. Intblastoma . One of RB mutation [RB inactivation is not sufficient for retinoblastoma, as cytogenetic studies have revealed consistent chromosomal abnormalities in addition to RB deletion [Rb and p130, array CGH revealed recurrent whole chromosomal gains at chromosome 1  and 12  [\u03b1-Cre Rb/p130 DKO mouse model. While alterations in regions syntenic to human 6p and 16q were not observed in \u03b1-Cre Rb/p130 DKO retinoblastomas, larger studies are needed, and it is possible that loss of p130 in the murine model may negate selection for 6p gain or 16q loss. Indeed, RBL2 (p130) lies at 16q12 and is a good candidate tumor suppressor in this region in humans [DEK as well as an interesting candidate, E2F3, which conceivably could antagonize p130 upon overexpression. Use of mouse models will certainly help in defining the critical genes in these regions, as mouse/human comparisons have revealed highly concordant genomic alterations in other tumor types and have allowed for the identification and validation critical cancer-contributing genes [The two-hit hypothesis suggests that the first few years following birth reflects the time-interval for homozygous mutation . Howeverdeletion . Early cdeletion -120, revdeletion . The vasan 2p24) . Such fin humans . Genes ing genes ,123. As ng genes , here, IN-MYC locus [N-MYC is the gene in the amplicon selected for increased copy number, as other genes are co-amplified, including the DEAD box gene DDX1. However, in \u03b1-Cre Rblox/lox p130-/- metastatic retinoblastomas (3 of 16 metastases), amplicons containing N-myc were also observed, ranging from 450 kb to 3.3 MB, and the only known gene in the minimal region of amplification was N-myc [N-myc/N-MYC is found more frequently that high level amplification. The fact that N-Myc amplification is enriched in metastatic \u03b1-Cre Rblox/lox p130-/- retinoblastoma suggests that this event contributes to formation of more aggressive tumors but how this is mediated is unknown. Genetic inactivation of N-myc in the developing retina has revealed a critical role for this gene in progenitor cell proliferation; this is in part mediated by regulation of p27 levels [Rb and N-myc in the developing retina and in retinoblastoma models should help us to understand why these mutations synergize in tumorigenesis and to identify the critical N-myc target genes.The most well characterized secondary alteration in human retinoblastoma is gain or amplification of a region of 2p that includes the YC locus ,124-126.as N-myc . This re7 levels . ExaminaMDM4 and GAC1, and they also identified a second common minimal region of gain at 1q22 [MDM4 is related to MDM2 and both genes cooperate to inhibit p53 function, with MDM4 an inhibitor of p53 transcriptional activity [MDM4 undergoes copy number increase and is overexpressed in retinoblastomas. They report that 65% of human retinoblastomas had extra copies of MDM4 and 10% exhibited extra copies of MDM2. Furthermore, transfection of MDM4 plasmid DNA into newborn retinas in vivo led to more rapid tumorigenesis in the \u03b1-Cre Rb/p107 DKO model. p53 is a good candidate effector downstream of MDM4 as homozygous p53 loss accelerated retinoblastoma development in vivo in Chx10-Cre; Rblox/lox p53-/-; p107-/- animals compared to animals with wild-type p53 [Rb on a p53-/- background, (NesCre1 Rb lox/lox p53-/- mice) retinoblastomas did not result [p53 loss can impact upon retinoblastomas that have already been initiated, but that inactivation of a second pocket protein in addition to Rb is still critical. Based on experiments in culture using retinal explants, the authors suggest that suppression of p53-dependent apoptosis is crucial [Rb exhibit very low levels of apoptosis [Rb-/- retinas in vivo . Importse model ,59. It ipatients . The lonRb and p130 have revealed secondary alterations similar to human retinoblastoma, suggesting that the steps to tumorigenesis in the murine and human retina are similar. Mouse models will be particularly powerful for testing which candidate genes are important for retinoblastoma progression. The novel models provide valuable tools to help us understand the in vivo function of pRB, to elucidate the properties of a cancer cell of origin and to test preclinical therapies. We are now poised to gain important insights into the steps between RB inactivation in a developing retinal cell and the progression towards retinoblastoma.Research into retinoblastoma has revealed a great deal about a malignant eye cancer, and has also led to many general insights into the general mechanisms of tumor suppression. Early genomic analyses of murine retinoblastoma lacking"}
{"text": "We have previously shown that mutations in the N-ras gene occur more frequently in melanomas originating from sun-exposed body sites, indicating that these mutations are UV induced. To investigate whether sun exposure also affects p53 in melanoma, we analysed 81 melanoma specimens for mutations in the p53 gene. The mutation frequency is higher than thus far reported: 17 specimens (21%) harbour one or more p53 mutations. Strikingly, 17 out of 22 mutations in p53 are of the C:G to T:A or CC:GG to TT:AA transitional type, strongly suggesting an aetiology involving UV exposure. Interestingly, the p53 mutation frequency in metastases was much lower than in primary tumours. In the case of metastases, a role for sun exposure was indicated by the finding that the mutations are present exclusively in skin metastases and not in internal metastases. Together with a relatively frequent occurrence of silent third-base pair mutations in primary melanomas, this indicates that the p53 mutations, at least in these tumours, have not contributed to melanomagenesis and may have originated after establishment of the primary tumour. 1999 Cancer Research CampaignIn melanoma, the relationship between sun exposure and the origin of mutations in either the N-"}
{"text": "Gene lesions of both ras(0%) and p53 (3%) were rare. While bcl-2 expression was not observed in any sample, bax expression was noted in 76% of samples and was associated with a significantly worse patient prognosis both overall (P< 0.005) and specifically in Chinese patients (P< 0.02). Apoptotic cells were found in 61% of samples, and were significantly associated with the presence of bax expression (P= 0.002), but not patient survival. These results suggest that prostate tumours from non-Japanese Asians are genetically distinct from prostate tumour found in both Japanese and Caucasian patients, and that treatment modalities may need to be tailored for specific population groups. \u00a9 2000 Cancer Research CampaignMolecular studies have suggested that ethnicity may play a significant role in prostate tumorigenesis, but no information exists for groups other than Caucasian or Japanese patients. We examined 62 archival samples of prostate tumours from Asians of non-Japanese origin for the over-expression of p53, for the possible presence of mutated"}
{"text": "Gadd45 genes have been implicated in stress signaling in response to physiological or environmental stressors, which results in cell cycle arrest, DNA repair, cell survival and senescence, or apoptosis. Evidence accumulated implies that Gadd45 proteins function as stress sensors is mediated by a complex interplay of physical interactions with other cellular proteins that are implicated in cell cycle regulation and the response of cells to stress. These include PCNA, p21, cdc2/cyclinB1, and the p38 and JNK stress response kinases. What deterministic factors dictate whether Gadd45 and partner proteins function in either cell survival or apoptosis remains to be determined. An attractive working model to consider is that the extent of cellular/DNA damage, in a given cell type, dictates the association of different Gadd45 proteins with particular partner proteins, which determines the outcome. The cellular response to environmental and physiological stress is very complex, encompassing a myriad of molecular pathways, with a plethora of regulators and effectors. Both environment agents  and physAberrations in DNA repair pathways are the major molecular pathology in several cancer prone hereditary diseases . DefectsGrowth Arrest DNA Damage (Gadd) 45 genes play as sensors of cellular stress.Mammalian cells have evolved an elaborate defense mechanism to maintain genomic integrity by preventing the fixation of permanent damage from genotoxic stress. This includes activation of cell cycle arrest checkpoints at the G1/S and G2/M transitions -14 and aGadd45 genes were cloned in this laboratory ,25, in tEvidence accumulated in recent years implies that Gadd45 proteins function as stress sensors, is mediated by a complex interplay of physical interactions with other cellular proteins that are implicated in cell cycle regulation and the response of cells to stress Fig. .It was shown that all three Gadd45 proteins interact with PCNA -43, a nuAll three Gadd45 proteins were observed to interact with p21 ,43, a p5In normal cells p21 and PCNA are found in quaternary complexes that also include cyclins and cyclin dependent kinases (cdks) -52. It wInteraction of Gadd45 proteins with MEKK4, an upstream activator of the stress induced p38/JNK kinases, and the resulting stimulation of its kinase activity, has been implicated in stress induced p38 and JNK activation . EvidencGadd45 family members are rapidly induced by genotoxic stress agents ,64,65, acell cycle arrest[DNA repair[cell survival[apoptosis[senescence[susceptibility of cells for transformation in vitro and in tumor development in vivo[As detailed below, gadd45 genes have been implicated in the control of le arrest,28,38,58NA repair-41, cell survival,76-79, aapoptosis,43,80-82enescence, and sust in vivo,83.Inhibiting endogenous expression of gadd45a, gadd45b, or gadd45g in human cells by antisense gadd45 constructs was found to impair the G2/M checkpoint following exposure to UV radiation or MMS ,38,58, aEvidence was obtained that Gadd45a and Gadd45b function in DNA excision repair through their interactions with PCNA -41. FurtAmple evidence exists that Gadd45 proteins have a pro-apoptotic function. For example, it was observed that blocking MyD118 (Gadd45b) by antisense expression in M1 myeloblastic leukemia cells impaired TGFb-induced cell death, thereby implicating Gadd45b as a positive modulator of TGFb-induced apoptosis ,69. ConsIntriguingly, in apparent contradiction to the role Gadd45 proteins play in cell death, many observations are consistent with a role in cell survival. Clonogenic survival assays with gadd45a-/- MEFs  and RKO ARF, which is encoded by a partially overlapping alternative reading frame at the p16INK4A locus, has been implicated as a major mediator of replicative senescence. p19ARF binds directly to and sequesters MDM2, thereby inhibiting the ability of MDM2 to induce degradation of the p53 tumor suppressor protein [INK4A/pRB and p19ARF/p53 pathways is essential for oncogene-induced senescence. Studies with MEFs deficient for one or more gadd45 genes, notably gadd45a, have provided evidence that gadd45 genes play important roles in both replicative and oncogene mediated senescence [INK4A/pRB and p19ARF/p53 pathways remains to be determined.Senescence represents a physiological stress response associated with cellular aging (90). When primary mammalian cells are cultured in vitro they undergo a limited number of cell divisions and then arrest in a state known as replicative senescence. Such cells are irreversibly arrested in the G1 phase of the cell cycle and are no longer sensitive to growth factor stimulation ,86 & the & the86 nescence ,83. The The complex role of stress sensors in monitoring oncogenic stress in tumor development is not fully understood, the best example being the multiple functions attributed to p53 in tumor development and suppression. Recent observations have implicated Gadd45 proteins as important sensors of oncogenic stress, both in vitro and in vivo.It is known that whereas primary mouse cells require introduction of two activated oncogenes for transformation, disruption of certain key growth control genes allows single oncogene transformation ,86. It wEvidence was obtained that Gadd45 proteins also play a role in modulation of tumor development in vivo. Gadd45a-/- and gadd45b-/- mice were observed to display increased mutation frequency, and susceptibility to ionizing radiation (IR) and chemical carcinogenesis. Also, it has been documented that NF-kB-mediated repression of gadd45a and gadd45g is essential for cancer cell survival. More recently, evidence was obtained that loss of Gadd45a accelerates ras-driven mammary tumor formation. Ras-driven tumor formation in the absence of Gadd45a resulted in both a decrease in apoptosis, linked to a decrease in JNK activation, and a decrease in senescence, correlated with a decrease in p38 kinase activation . AltogetGadd45 genes have been implicated in stress signaling, which results in cell cycle arrest, DNA repair and cell survival, or apoptosis Fig. . What deThe authors declare that they have no competing interests.DAL participated in the sequence alignment and drafted the manuscript. BH participated in drafting the manuscript. All authors read and approved the final manuscript."}
{"text": "The aim of the study was to determine whether the combination of p53 over-expression and p53 low-expression associated with Ki67 over-expression (high Ki67/p53 ratio) could lead to a more sensitive parameter. Immunohistochemical expression of Ki67 and p53 was measured in 54 specimens from OSCC; 27 specimens from moderate/severe epithelial dysplasia; 32 specimens from oral leukoplakias without epithelial dysplasia, and 13 specimens with normal epithelium. p53 over-expression was found in 31 (53%) samples from OSCC, in 10 (37%) samples from severe dysplasias, and in 5 (15%) samples from non-dysplastic lesions, while the combination of high p53 values with high Ki67/p53 ratio was observed in 93% of OSCC, in 81% of dysplastic lesions, and in 50% of non-dysplastic lesions. This parameter may have a clinical implication to detect early lesions with an impairment of p53 pathway, and probably at risk of progress to OSCC. It has been widely demonstrated that most oral squamous cell carcinomas (OSCC) develop through their precancerous steps which are defined as potentially malignant lesions and are histologically characterized by increasing grades of epithelial dysplasia -4.So far, the only accepted method to quantify the risk of progression to OSCC of a potentially malignant lesion, is the presence/absence of histological findings of dysplasia -6, althoThese considerations have stimulated intense research in this area, and many studies have been conducted so far, in an attempt to find out molecular markers that are associated with OSCC and that can predict malignant transformation when found in epithelial precursor lesions, especially in cases without signs of dysplasia -13.The immunohistochemical analysis of p53 protein is a simple and inexpensive method that has been widely used for this purpose, and many studies have shown that it is involved in oral carcinogenesis and its alteration occurs early in the process of neoplastic transformation, often preceding recognizable histological alterations -14-16.p53 over-expression has been widely demonstrated to be a reliable predictor of progression to OSCC, either if we consider the over-expression as a physiologic response to induce cell cycle arrest in genetically altered hyper-proliferating cells or as a primary genetic defect leading to the production of high levels of mutated non-functional p53 protein; this is why the most utilized antibodies in the literature stain the total form of p53 protein and can not discriminate between wild or mutated form -18.However, p53 over-expression as a marker of progression to OSCC has not been considered highly sensitive, probably because only about 50% of OSCC are associated with over-expression of p53 protein -22.It follows that if the occurrence of p53 over-expression can be considered specific in detecting oral lesions at risk of developing p53-positive tumours, the presence of low p53 values should be considered as non-informative for prognostic purposes, since it can not distinguish between lesions with a genetically reduced production of p53 at high risk of developing p53-negative tumours, and lesions with a physiological low p53 activity or lesions that follow different carcinogenic pathways in which the p53 abrogation does not play a role ,11-22.It is well known that in non-neoplastic cells the expression of p53 is usually related to the cell proliferating rate, since p53 is considered as having an \u201concogene checkpoint\u201d function that guards cells against hyperproliferative signals .It follows that in non-neoplastic cells that respond to proliferative signals from inflammatory changes, traumas or hypoxia, a proportional increase in p53 activity should be expected -25.Thus, we may assume that the finding of an increased proliferating rate associated to a depressed p53 expression (increased mitotic/apoptosis ratio) in potentially malignant lesions might possibly identify genetically damaged lesions lacking of p53 protective role during cell cycling and may be at risk of progressing to p53-negative cancer.In the present study, the Ki67 protein has been utilized as a immunohistochemical marker of proliferating cells , and samA total of 126 biopsy samples from 126 consecutive patients who have referred to the Department of Oral Sciences of the University of Bologna, Bologna  between January 2004 to September 2006 were analyzed: 54 specimens from infiltrating OSCC, 27 from moderate/severe epithelial dysplasias , 32 from oral leukoplakias with epithelial hyperplasias and no signs of dysplasia, and 13 from normal epithelium oral mucosa obtained during third molar removal or as part of the excision of benign conditions. All samples underwent histological and immunohistochemical analysis.Histological diagnoses were performed at the Department of Pathology of the University of Bologna, at Bellaria Hospital , following the criteria describe in the WHO book. Specifically moderate\\severe dysplasia  was characterized by increasing atypia, loss of polarity, and frequent mitoses, involving more than two-third of the epithelium while lacking infiltrative growth. Squamous cell Hyperplasia  was characterized by increased basal-parabasal layers, acanthosis, in absence of architectural alterations . No case1) shows an example of positive and negative staining of p53 protein.Immunostaining was performed on 2-\u00b5m thick sections serially cut from the selected blocks. The following antibodies were employed: monoclonal anti-Ki67  and monoclonal anti-p53 . The processing was performed in an automatic stainer . All the cases contained an internal control as basal cells of the oral epithelium show nuclear positivity for the two markers under study. Negative controls consisted in omitting the primary antibody. Counting the percentage of positive nuclei in 400 consecutive epithelial cells of selected areas representative of the lesion gave a semi-quantitative evaluation of the immunohistochemical results. Fig.  were considered when equal or more than 20%, while high Ki67/p53 ratio was considered when equal or more than 3.A multiple logistic regression model was fitted to describe the relationship between the presence/absence of dysplasia (dependent variables) or between dysplasia and OSCC (dependent variables)and 2 independent variables: p53 over-expression versus p53 low expression, and the combination of samples with high p53 and samples with high Ki67/p53 ratio versus low p53 and low Ki67/p53 ratio.One-way ANOVA was used to compare p53 mean values (continuous dependent variable) between samples from OSCC, epithelial hyperplasia, epithelial dysplasia and controls . Fisher\u2019s least significant difference procedure (LSD) was the method to discriminate among the means at 95.0% confidence level.2). The wide Standard Deviation in samples from OSCC and oral lesions with dysplasia is the consequence of the contemporary presence of high and low p53 values in these oral lesions as documented by the density trace diagrams in Fig. (3).One-way ANOVA showed that the mean p53 value in OSCC (38.4\u00b134.9) was significantly higher  than those found in controls and in oral lesions either with or without dysplasia . Fisher\u2019s least significant difference procedure did not show any difference between the last three groups .Normal mucosa: the density trace diagram from normal samples showed a shape of distribution of p53 values characterized by a single peak with a median of 10% (range 5-15), and values always less or equal to 15%. The sample come from a normal distribution with a standardized skewness (.74) and kurtosis (-.17) values within a range expected from a normal distribution .OSCC: the density trace diagram from OSCC showed a shape of distribution of p53 values characterized by two well defined peaks that distinguished two very different population where high or low p53 values were expressed respectively in 31 (57%) and 23 (43%) samples . All samples with high p53 values had low Ki67/p53 ratios.A Ki67/p53 ratio equal or more than 3 was found in 19 patients out of 23 (83%) showing low p53 values (Table 3c). High Ki67/p53 ratio was found in 12 out of 17 (71%) samples with low p53 values of which 9 (82%) from margins of OSCC (Table 1). All samples with high p53 values had low Ki67/p53 ratios.Epithelial Dysplasia: similarly to OSCC the shape of distribution of p53 values showed two well defined peaks where high values are expressed respectively in 10 (37%) samples of which 8 (42%) from non-tumoral areas adjacent to OSCC , and high Ki67/p53 ratio was found in 11 out of 27 (37%) samples with low p53 values (Table 1). Again, all samples with high p53 values had low Ki67/p53 ratios.Epithelial Hyperplasia: high p53 values were found in 5 (15%) samples  of OSCC and in 81% (22 out of 27) of epithelial dysplasias, of which 89% (17 out of 19) from non-tumoral areas adjacent to OSCC, and in 50% (16 out of 32) of epithelial hyperplasias, while it was never observed in any sample from normal mucosa. Table The results of fitting a multiple logistic regression model to describe the relationship between the presence/absence of dysplasia and the 2 independent variables  showed that the only parameter related to the presence/absence of dysplasia and reaching the significance  was the presence/absence of samples with high p53 associated with samples with high Ki67/p53 ratio.No significant difference was found between OSCC and epithelial dysplasia when this combination was utilized .It is widely accepted that OSCC develops as a result of accumulation of genetic errors in the same tissue ,29, and TP53 mutations may result in a over-production of p53 inactive proteins which accumulate in the epithelium either due to blocking by another protein or due to partial degradation , or instThe shape of distribution of p53 values from OSCC in the present study, is in line with this statement, clearly showing the presence of two well defined peaks that distinguish two very different OSCC population, where high or low p53 values were expressed respectively in about 57% and 43% of cases. The prevalence of higher p53 values in the entire population explains the statistical difference in the mean p53 values between malignant samples and samples from normal mucosa.The evidence that the p53 pathway is very important in the biology of oral cancer has led to the application of such immunohistochemical analysis as a simple rapid and inexpensive method to potentially malignant lesions in an attempt to find a useful marker to predict progression to OSCC.Higher mean p53 values with respect to normal mucosa has been demonstrated in lesions with and without signs of dysplasia and it has been documented that lesions progressed to OSCC showed p53 over-expression early during the process, although most of the studies in the literature have utilized antibodies that can not discriminate between wild or mutated form ,35-36. NIn our study we did not find significantly higher p53 mean values in potentially malignant lesions with respect to normal mucosa although samples from lesions with severe dysplastic alterations showed higher mean p53 value than normal mucosa, but did not reach statistical significance. The limited number of controls may have contributed to hide a significant difference between p53 mean values from potentially malignant lesions and normal mucosa, although all p53 values from normal mucosa kept lower than the cut-off considered in the present and in a previously published study .However, p53 analysis did reveal differences between our potentially malignant lesions and normal mucosa since about 40% of sample in the group of dysplastic lesions and 15% in the group of leukoplakias without dysplastic alterations showed p53 values higher than the maximum value found in normal mucosa. We suppose that the prevalence of normal or low values in dysplastic lesions can explain the lack of statistical difference in the mean p53 values between pre-malignant lesions and normal mucosa.When concerning lesions with low p53 expression, the present data showed that low staining was present in more than 40% of OSCC, and even in about 60% of lesions with severe dysplasia, most of them at margins of OSCC and probably precursors of p53-negative tumours .If we take the p53 over-expression as a predictive marker, the occurrence of positive samples in our population is probably too low to discriminate lesions at \u201chigh risk\u201d of malignancy, also considering that not all p53-positive lesions will progress to cancer .Furthermore, if we consider samples with low p53 expression as lesions \u201cnot at risk\u201d of developing cancer, the occurrence of p53-negative lesions in our population of severe epithelial dysplasias is too high, considering that most of them were situated at margins of OSCC and thus at very high risk of malignant changes -39.These data underline the low predictive value of p53 when used as a single marker, since the high occurrence of low p53 staining in pre-malignant lesions is very difficult to be interpreted and not enough to rule out the progression to OSCC.This is in agreement with many reports that have stated that p53 has a good specificity, i.e the likelihood of malignant transformation is high when it is over-expressed, but a low sensitivity since the absence of p53 staining did not preclude malignant transformation in a considerable proportion of pre-malignant lesions ,40-41.Instead, in the presence of a lesion in predicate to develop cancer, the avoidance of false-negative results takes priority, so as not to miss patients who actually have a disease at risk of developing OSCC; in this situation, the sensitivity is most important to maximize negative-predictive accuracy, i.e. the likelihood that a negative test relates to the absence of any risk of malignancy .To this direction have gone our results obtained by the combination of samples with high p53 values and samples with low p53 values but high Ki67 values, i.e. a high Ki67/p53 ratio.We utilized this approach to see whether the presence of an increased mitotic activity not associated to an increased p53 activity might discriminate lesions with high proliferative activity where the presence of a genetic damage prevents a reactive stimulation of p53.The rationale was that in non-neoplastic cells, an increased cell proliferation due to a series of factors such inflammation, trauma etc, usually elicits a p53 protein accumulation that is considered to be a physiologic response to the increased proliferation rate of keratinocytes as a guardian of epithelial growth -44.In the present study, high Ki67/p53 ratios were found in 83% of OSCC with low p53 expression and even in 71% of severe epithelial dysplasias with low p53 values, and the combination of samples with p53 over-expression and samples with low p53 values but high Ki67/p53 ratio, led to a very high association with OSCC and epithelial dysplasias. About 90% of both OSCC and epithelial dysplasias were positive to this combination, while normal epithelium never was.Dysplasia is still now the most important predictive factor providing a surgical or non-surgical therapeutic approach. However histological assessment of dysplasia is extremely subjective , and somIn the present study, the combination of p53-over-expression and high Ki67/p53 ratio was present in about 50% of leukoplakias without signs of dysplasia.This percentage surely overestimates the risk of developing cancer, but could be useful to increase the predictive sensitivity and to rule out at least 50% of lesions with a presumably very low risk of developing OSCC.p53 immunostaining as a single marker can not be considered a sensitive marker that would help to assess the degree of cell alteration in oral lesions at risk of developing cancer.While it could be accepted that p53 over-expression in potentially malignant lesions may indicate a risk of developing cancer, the occurrence of low p53 staining is very difficult to be interpreted and does not rule out a possible link to oral carcinogenesis.In the present study the combination of p53 over-expression and high Ki67/p53 ratio seemed to better associate with the occurrence of OSCC and severe epithelial dysplasia, and may have a routine implication as an easy method to early detect oral lesions with an impairment of p53 pathway, and probably at risk of developing OSCC."}
{"text": "Overexpression of p53 correlates with neoplasia in many cytological specimens. To test the specificity of overexpressed p53 as a tumour marker for the detection of pancreatic cancer, we analysed cytological specimens of pancreatic juice samples from patients with pancreatitis or pancreatic carcinoma (n = 42) for p53 protein overexpression. p53 protein overexpression was found in 59% of patients with pancreatitis and 67% of patients with pancreatic carcinoma. Thus, the assessment of p53 protein overexpression is not useful in the diagnosis of pancreatic cancer. Overexpressed p53 during pancreatitis appears to be wild-type p53. Overexpression of p53 may result from DNA damage occurring during chronic inflammation. It is well established that p53 can induce apoptosis upon DNA damage. Consequently, we found apoptotic cell death in five out of five tested cytological preparations from patients with pancreatitis as well as in one out of one pancreatic carcinoma specimen."}
{"text": "Loss of p53 function because of mutation of the p53 gene is associated with over half all human cancers. Certain human p53 mutants are conformationally flexible in vitro and are temperature sensitive, with partial or complete recovery of wild-type (wt) properties at 32 degrees C. We have now tested the functional capacities of selected p53 mutants in vivo, by transfection into established human cell lines. Unexpectedly, we found that wt p53 can be temperature sensitive for transactivation of a co-transfected target gene in vivo. Flexible mutants retained varying degrees of functional capacity in transfected cells, and the recipient cell line appeared to be a significant determinant of both wt and mutant p53 function; importantly, two p53 null cell lines commonly used to study p53 function (Saos-2 and Hep3B) differed markedly in this latter respect. We also show that the p53 mutant V272M, which exhibits sequence-specific DNA binding in vitro, is nonetheless defective for transactivation and is unable to induce apoptosis in vivo. The valine 272 residue may thus be crucial for properties (other than sequence-specific DNA binding) that are important for p53 function(s) in vivo."}
{"text": "Understanding                        the molecular mechanisms and biological consequences of genetic changes                        occurring during bypass of cellular senescence spans a broad area of                        medical research from the cancer field to regenerative medicine. Senescence                        escape and immortalisation have been intensively studied in murine                        embryonic fibroblasts as a model system, and are known to occur when the                        p53/ARF tumour suppressor pathway is disrupted. We showed recently that murine                        fibroblasts with a humanised p53 gene  first senesce, and then become immortalised in the same way as their                        homologues with normal murine p53. In both cell types, immortalised cultures                        frequently sustain either a p53 gene mutation matching a human tumour mutation and                        resulting in loss of p53 transcriptional transactivation, or a biallelic deletion                        at the p19/ARF locus.  Whilst these genetic events were not unexpected, we were                        surprised to find that a significant proportion of immortalised cell cultures                        apparently had neither a p53 mutation nor loss of p19/ARF.  Here we consider                        various routes to p53/ARF disruption in senescence bypass, and dysfunction of                        other tumour suppressor networks that may contribute to release from tenacious                        cell cycle arrest in senescent cultures.                  Cellular senescence is an important                            defence mechanism against tumour metastasis, growth and progression ,2.     Our                            MEF cell line library comprises immortalised cell lines from cultures of                            fibroblasts derived from (a) embryos of a standard laboratory wild-type mouse                            strain (129/Sv), and (b) embryos from human p53 knock-in (Hupki) mice, which we                            constructed for our research on p53 biology -17.  TheWhilst                            the prevalence of cell lines immortalised by p19/ARF biallelic deletion or p53                            mutation was not unexpected , we were surprised to find that a significant                            fraction of cell lines (derived both from WT and Hupki primary cells) appeared                            to have retained WT p53 and p19/ARF expression, as examined by DNA sequencing                            or PCR amplification and promoter methylation analyses,  respectively ( and    Some                            of the MEF cell lines from (homozygous) WT or Hupki mice that we have examined                            and tentatively classified as p53 and p19/ARF wild-type thus may in fact have                            only one normal allele of these suppressor genes, having suffered loss of one                            copy in vitro allowing unlimited growth in culture.                                        Given                            that at least half of the spontaneously arising >100 MEF cell lines we                            examined for p53 and p19/ARF aberrations appeared to have overcome senescence                            block upon explanting in vitro by mechanisms other than the 2 canonical genetic                            events , there could well be various                            alternative pathways to immortalisation not directly involving damage to the immediate                            p53/p19ARF axis.                                        Considerable effort is underway to                            identify the key regulators of senescence and immortalisation. Since the                            process of senescence bypass provides an intrinsic phenotypic readout of                            functionality and automatically generates cell lines amenable to subsequent                            analysis, reverse genetics is a powerful approach to deciphering the important                            molecular events involved in senescence control . SeveralThe                            role of p53 mutation/p19ARF deletion in senescence bypass in MEFs reflects the                            importance of the p53/p19ARF axis as a master regulatory pathway of senescence                            in these cells.  What is apparent from genetic screening, as well as from other                            complementary work, is that many of the novel senescence-associated genes                            identified can also impact on this key pathway, both upstream and downstream of                            p53 ,26,28-31An entirely new aspect to the importance of cellular                            senescence has recently surfaced from experiments to produce iPS (induced                            pluripotent stem) cells from embryonic fibroblasts.  It has been shown that                            senescence provides a progressive barrier to conversion of primary MEFs (and                            indeed other types of differentiated cells) to pluripotency.  Crucially,                            disruption of the p53/p19ARF signalling axis greatly increased efficiency of                            their conversion ,41.  Fur"}
{"text": "The p53 tumor suppressor protein is arguably the most                        important guardian of the mammalian genome. p53 promotes longevity by reducing                        somatic mutations or the survival and proliferation of mutant cells. Under                        normal conditions, this proteins role is inconsequential because of the                        rapidity of its degradation. But stress signals of almost any form halt the                        degradation of p53, unleashing an active protein that triggers transient cell                        cycle arrest, apoptosis, or cellular senescence . Both apAlmost all human cancers contain                        impairments in the p53 signaling pathway . MDM2  Senescence is a complex genetic program and a cell                        fate decision that establishes permanent growth arrest, which is a theurapeutic                        goal in cancer treatment. Although there is no direct link between cellular                        senescence and aging, a correlation does exist, as the number of senescent                        cells increases in mammals as they age . ActivatSenescent cells are characterized by highly compacted                        heterochromatin, termed \"senescence-associated hetero-chromatin foci\", or SAHF                        . These SHuang et al. conclude that the decline in                        p53 functionality in senescent cells is due to changes in upstream signaling                        that ultimately leads to destabilization of p53 and abrogated signaling.  They                        point to the possibility that the pro-apoptotic functions of p53 may be                        redundant in senescent cells, which have already lost their ability to                        proliferate and become cancerous.  These findings add to a recent publication                        by this group revealing that nutlin-3 can induce a senescence-like state in                        several epithelial cancer cell lines , but it The study by Huang and coworkers highlights several                        important issues in cancer therapy development. In addition to p53-mediated                        apoptosis, nutlins may induce and maintain a permanent dormancy in cancer                        cells. The ability for nutlin-3 to activate p53 without the genotoxic stresses                        incurred by most chemotherapeutic agents is of tremendous clinical importance.                        It may be of value to note that acetylation of p53 also inhibits p53-MDM2                        interactions ,12. Seve"}
{"text": "The p53 signalling pathway has hundreds of inputs and outputs. It can trigger cellular senescence, cell-cycle arrest and apoptosis in response to diverse stress conditions, including DNA damage, hypoxia and nutrient deprivation. Signals from all these inputs are channeled through a single node, the transcription factor p53. Yet, the pathway is flexible enough to produce different downstream gene expression patterns in response to different stresses.We construct a mathematical model of the negative feedback loop involving p53 and its inhibitor, Mdm2, at the core of this pathway, and use it to examine the effect of different stresses that trigger p53. In response to DNA damage, hypoxia, etc., the model exhibits a wide variety of specific output behaviour - steady states with low or high levels of p53 and Mdm2, as well as spiky oscillations with low or high average p53 levels.We show that even a simple negative feedback loop is capable of exhibiting the kind of flexible stress-specific response observed in the p53 system. Further, our model provides a framework for predicting the differences in p53 response to different stresses and single nucleotide polymorphisms. The tumor suppressor protein, p53, is a transcription factor that regulates the activity of hundreds of genes involved in cell growth and death ,2. Over both activity and stability of p53. Mdm2 is an E3 ligase that binds to p53. Mono-ubiquitination of p53 by Mdm2 inhibits its transcriptional activity, while poly-ubiquitination triggers its degradation [mdm2 gene is activated by p53, thus forming a negative feedback loop [mdm2) on the p53 response.How does the regulatory network around p53 retain this exibility even though all inputs converge at a single node? We argue in this paper that the particular design of the p53-Mdm2 feedback loop at the core of this network could be the source of this flexibility. p53 is regulated by other proteins at two levels: its stability , and radation . In turnack loop . We use p; Mdm2, m; Mdm2 mRNA, mm; and the p53-Mdm2 complex, c. The temporal dynamics of these components of the model are specified by four differential equations:Our model focuses on the following four concentrations: nuclear-p53, http://www.ebi.ac.uk/biomodels-main, model number 1006280000.The model is provided in SBML format as additional file \u03b4), activation of mdm2 by p53 (kt)). Others are \"effective\" parameters which model the combined action of several proteins that all affect p53 or Mdm2. For example, \u03b1 models all Mdm2-independent processes which result in the reduction of the active p53 concentration in the nucleus: spontaneous degradation, export out of the nucleus, physical interaction with other proteins, sequestration in the cytoplasm (e.g. by Pirh2), modifications which prevent activity, etc. We also assume that activation of mdm2 by p53 has an associated Hill coefficient of 2 , and thpromotor  shows thMost previous models have used an explicit time delay to model transcription and translation . In This model system of a negative feedback loop shows plenty of variety in output behaviour. Depending on the values of parameters, the system is capable of steady state solutions with any combination of high or low p53 and Mdm2, as well as oscillations with high or low average p53. Figure Ideally, we would like to correlate each of these output states of the pathway to specific physiological responses like cell cycle arrest or apoptosis. Clearly, the level of p53 is an important determinant of the response ,22,23, aIn the absence of stress, p53 levels are typically maintained quite low. For this, a sufficient Mdm2 level is required to keep the half-life of p53 short. Thus, in a typical \"resting\" state there is a fairly high turnover of p53. The area shaded in green and blue in Figures \u03b4 and kt than to the p53-Mdm2 dissociation constant kD = kb/kf. Most stresses, however, affect more than one parameter.The system can be triggered by numerous stresses. We model different stresses as affecting different parameter combinations, as shown in Table Nutlin. Nutlin reduces the binding of Mdm2 to p53, while leaving its other properties unchanged. Nutlin treatment can trigger cell cycle arrest, but not apoptosis [kD (weakening the binding) alone causes a very modest increase in p53 levels  increased auto-ubiquitination of Mdm2, (b) decreased ubiquitination of p53 by Mdm2 and (c) weaker binding of p53-Mdm2 [\u03b3, decreasing \u03b4, and increasing kD. Single-cell experiments have found that irradiation of various types triggers oscillations in p53 levels with a period of 5-6 hours. The parameter changes used to mimic DNA damage stress were chosen such that the response matches the observations of Ref. [kD and \u03b3, while lowering \u03b4, corresponding to the molecular processes described above, our model produces an oscillatory solution in accordance with the experimental observations  when we apply a hypoxic stress when compared to other stresses with similar fold-changes in parameter values  results in an increased expression of mdm2 compared to the T allele [kt , as illustrated in Figure Our model can also be used to examine the behaviour of certain mutant cell lines. For instance, the G allele of the T allele . This conM, corresponds to 50,000 molecules per cell (assuming the cell is a sphere of radius around 6 \u03bcm). Such high numbers mean that the noise due to stochasticity in production and degradation of molecules is very small. Thus, the result of stochastic simulations of our model using the standard Gillespie algorithm [Single-cell measurements of p53 oscillations in response to various types of irradiation exhibit a fair amount of variability in the response across different cells ,31. The lgorithm  are indilgorithm  also see1. The variability in p53 response observed in ,31 must 2. Increasing amounts of noise are likely to introduce more variability in the position of later p53 peaks than in earlier peaks, as observed.However, a proper analysis of these hypotheses requires a better knowledge of which sources of noise underly the variability observed, so that they can be modelled accurately.\u03b3 result in large changes in peak p53 levels but hardly any change in average p53 levels.The sensitivity analysis in Figure mdm2 SNP309 - a prediction that is confirmed by observations. In addition, if the increase in kt due to the SNP is sufficient, then although p53 will be upregulated in response to stress, oscillations will not occur  between the first and second peaks.Our model could eventually be extended to cover other stresses that trigger a p53 response as more data becomes available. Nitric oxide (NO) is a free radical produced in inflamed tissue which can trigger the p53 pathway by phosphorylating p53 and thereby inhibiting its Mdm2-mediated degradation . Another\u03baB signalling in the immune system [\u03baB is a transcription factor that controls hundreds of downstream genes. It activates production of I\u03baB\u03b1, which binds to and inhibits the action of NF-\u03baB [Overall, we have shown that this kind of negative feedback loop, consisting of a relatively slow transciptional activation on one leg of the loop, and an inhibition based on fast complex formation on the other, can be designed to respond specifically to a number of different input triggers. This kind of negative feedback loop also occurs in another important signalling pathway that is triggered by hundreds of input signals, namely NF-e system . NF-\u03baB iof NF-\u03baB . The resof NF-\u03baB ,47 similAH, SK and MHJ designed, implemented and analyzed the model, and wrote the paper. All authors read and approved the final manuscript.Model in SBML format. This file provides the p53-Mdm2 model we use in SBML format.Click here for fileSupplementary information. This file provides additional information and figures to supplement the main text.Click here for file"}
{"text": "The p73 protein shows structural and functional homology to p53. For these reasons, p73 was considered as a positional and functional candidate tumour suppressor gene. Thus far, mutation analysis has provided no evidence for involvement of p73 in oligodendrogliomas, lung carcinoma, oesophageal carcinoma, prostatic carcinoma and hepatocellular carcinoma. In neuroblastoma, two mutations have been observed in a series of 140 tumours. In view of the occurrence of 1p deletions in Merkel cell carcinoma (MCC) and the location of p73 we decided to search for mutations in the p73 gene in five MCC cell lines and ten MCC tumours to test potential tumour suppressor function for this gene in MCC. In view of the possible complementary functions of p73 and TP53 we also examined the status of the TP53 gene. Sequence analysis of the entire coding region of the p73 gene revealed previously reported polymorphisms in four MCCs. In one MCC tumour, a mis-sense mutation located in the NH2-terminal transactivation region of the p73 gene was found. These results show that p73, analogous to neuroblastoma, is infrequently mutated in MCC. This is also the first report in which the role of TP53 in MCC has been investigated by sequencing the entire coding region of TP53. TP53 mis-sense mutations and one non-sense mutation were detected in three of 15 examined MCCs, suggesting that TP53 mutations may play a role in the pathogenesis or progression of a subset of MCCs. Moreover, typical UVB induced C to T mutations were found in one MCC cell line thus providing further evidence for sun-exposure in the aetiology of this rare skin cancer. \u00a9 2000 Cancer Research CampaignThe"}
{"text": "Purpose. Rhabdomyosarcoma (RMS) is an embryonal tumor thought to arise from skeletal muscle cells that fail todifferentiate terminally. The majority of RMSs express MyoD, a protein essential to the differentiation of skeletal muscle.It was recently shown that during myogenesis, MyoD activates the expression of the cyclin-dependent kinase inhibitor(CDKi), p21, which itself plays a critical role in normal muscle development. To investigate the integrity of the MyoD/p21pathway in RMS, we analyzed p21 and its relationship to MyoD expression in RMS.            Methods. A panel of RMS samples was assembled from primary biopsies and from cell lines. Integrity of p21 was analyzedby single-strand conformation polymorphism (SSCP) and sequencing. Expression of p21 and MyoD was determined byNorthern blot analysis, and the ability of exogenous p21 to arrest the cell cycle of RMS cell line was determined bytransfection studies.            Results. Our analysis indicates that although p21 is wild type in RMS, there is an inverse correlation between the levelsof p21 and MyoD in these tumors. Tumors that express significant amounts of MyoD fail to express p21. This does notappear to be the result of mutations within the potential CACGTG sites present in the p21 promoter region or in thecoding region of p21. An additional group of RMSs express very high levels of p21 but express little, if any, MyoD.Furthermore, RD, a RMS cell line which expresses high levels of endogenous p21, undergoes withdrawal from the cellcycle following forced expression of p21, suggesting that the pathway which would lead to G1 arrest from endogenous p21 activity is defective.            Discussion. These data suggest that the interaction between p21 and MyoD is defective in RMS although the precisenature of the defect remains to be elucidated."}
{"text": "BMC Genomics has uncovered the early transcriptional responses to three types of stress and has demonstrated a central role for p38 MAPK in mediating these responses.The mitogen-activated protein kinase p38 (p38 MAPK) is activated by a number of stresses. A recent study in http://www.biomedcentral.com/1471-2164/11/144See research article  BMC Genomics by Ferreiro et al. [When cells are subjected to stress, such as osmotic stress, heat shock or infection, a number of intracellular signaling pathways are activated, leading to a coordinated cellular response. This may include the cessation of the cell cycle, commitment to apoptosis, the activation of DNA-repair pathways, regulation of protein translation, and the initiation of immune responses. A large body of evidence over the past 15 years has demonstrated that the p38 mitogen-activated protein kinase (MAPK) pathway is an important mediator of these stress responses . What isThe p38 pathway, like other MAPK pathways, features a cascade of protein kinases, culminating in the phosphorylation of p38 MAPK on specific threonine and tyrosine residues within its activation loop by the dual-specificity MAPK kinases MKK3 and MKK6, thereby leading to its activation ,2. ThereSaccharomyces cerevisiae p38 homolog Hog1 is primarily activated by osmotic stress and mediates a specific program of gene expression to combat and adapt to high osmolarity [Caenorhabditis elegans, genetic experiments have established a role for p38 in innate immunity and transcription profiling has demonstrated that p38 is required for upregulation of pathogen-response genes [Studies in yeast and nematodes, where the p38 pathway is conserved, have shed some light on how it regulates the transcriptional response to stress. The molarity . This ismolarity . In the se genes . In mammse genes ,8).et al. [To uncover the immediate p38-dependent transcriptional response to stress, Ferreiro et al. [Which genes are upregulated by the stimuli and how do they function in the cellular response to stress? To begin to address this important question, Ferreiro et al.  analyzedet al. [et al. and there is little overlap in the pool of genes upregulated [To obtain a fuller picture of how changes in transcription direct the cellular response to stress, Ferreiro et al.  analyzedegulated ,7,8. ThiThe temporal changes in the transcriptional profiles in response to osmotic stress can be linked to specific cellular responses. For example, short exposures to osmotic stress (45 minutes) led to the upregulation of solute carriers and ion transporters to allow cells to rapidly counteract the differences in osmotic pressure between the inside of the cell and the environment. Longer exposures (2 hours) results in the upregulation of genes encoding transcription factors and those involved in ribosome biogenesis, indicating a requirement for new protein synthesis, whereas 8 hours of exposure promotes the expression of genes involved in regulating cell survival and cell growth as well as cytokine and chemokine genes, suggesting a strong defense response.jun transcription via MEF2 and ATF2, two direct substrates of p38, whereas fos and egr-1 can be regulated by other p38 substrates, such as ternary complex factors (TCFs) [One of the significant findings of the study was that the most highly enriched functional grouping across all three stresses was for genes encoding transcription factors Figure . This was (TCFs) . The enret al. [et al. [For the future it will be important to determine how the p38-mediated gene-expression profiles differ between cell types and in response to other types of stress. The prediction, based on the study by Ferreiro ChIP: chromatin immunoprecipitation; GO: Gene Ontology; IPA: Ingenuity Pathway Analysis; MAPK: mitogen-activated protein kinase; MEFs: mouse embryonic fibroblasts; MKK: MAPK kinase; TNF\u03b1: tumor necrosis factor-\u03b1."}
{"text": "Carcinoma is more commonly found within the confines of benign PTs; whereas it is more often found surrounding the PT or in the contralateral breast in malignant PTs. Previous radiotherapy treatment is reported in only two cases. The aetiology of co-existing carcinoma is unclear but the rarity of previous radiotherapy treatment suggests that it is incidental. This case highlights the diverse pathology that can occur with PTs, which should be considered when evaluating pathology specimens as they may impact on patient management.A 43-year-old woman presented with a right breast lump that had enlarged over 5 months. She had chemoradiotherapy for non-Hodgkin's lymphoma in 1989. Histology revealed a malignant phyllodes tumour (PT) with liposarcomatous differentiation and ductal carcinoma in situ (DCIS) within the tumour with invasive tubular carcinoma, DCIS, and lobular carcinoma in situ in the surrounding breast. She had surgery and adjuvant radiotherapy. One year follow-up showed no recurrence or metastatic disease. Liposarcomatous differentiation is uncommon in PTs, and coexisting carcinoma is rare with 38 cases in 31 reports in the literature. Carcinoma is reported in malignant ( Phyllodes tumors (PTs) of the breast are uncommon biphasic fibroepithelial neoplasms that account for <1% of all breast tumours. Most PTs are benign and carry a risk of local recurrence whereas malignant PTs have a 13% risk of haematogenous metastasis .PTs are believed to arise from intralobular or periductal stroma and may arise de novo or from pre-existing fibroadenomas . Up to 3A 43-year-old woman presented with a palpable lump in the central aspect of the left breast below the nipple. The lump was present for five months. It had increased in size over that time and was tender. Mammography showed a relatively well-circumscribed 3.4\u2009cm mass in the lower central aspect of the breast . An ultrA wide local excision of the left breast was performed which was followed by a wider cavity excision and latissimus dorsi flap reconstruction. The patient was given adjuvant radiotherapy. The breast received 50.4\u2009Gy at 1.8\u2009Gy per fraction with a cone-down boost to the tumour bed, giving a total dose of 63\u2009Gy to the area considered at the greatest risk of recurrence. Follow-up after one year showed no evidence of recurrence or metastasis. Axillary sampling was not performed but no axillary lymphadenopathy was identifiedat one year follow-up.per 10 high power fields) were seen  included skeletal muscle and showed no residual PT. A single focus of DCIS, high-nuclear grade with comedo-necrosis and microcalcification, 3\u2009mm in size, was present within 1\u2009mm to the medial margin . MultiplMetaplastic change within PTs is uncommon. In the largest series of PTs reported, stromal metaplasia was present in only 11 of 335 cases, and malignant transformation of epithelium in the form of DCIS and LCIS was seen in only two cases . Stromaln = 19) in malignant PTs. In the malignant PT category, carcinoma occurred more frequently in the ipsilateral [n = 7) [n = 6; DCIS, n = 10) than lobular phenotype  [n = 8, 47%) [Malignant epithelial elements are reported in all categories of PT. In situ and invasive carcinoma may involve the tumour itself and/or coexist with a PT elsewhere in the same or contralateral breast. Carcinoma is most commonly reported  , 28, 33., n = 4) , 19\u201333.  8, 47%) , 30, 33. 8, 47%) , 25, 27,n = 10) [n = 5) [n = 1) [n = 10) were invasive, with or without an in situ component and most were ductal NST type (n = 6) [Carcinoma is less common in benign PTs compared with malignant PTs, given the higher prevalence of the former. Coexistent carcinoma is reported in 16 benign PTs  , 13, 17 n = 10) , 14, 17  [n = 5) , 15\u201317 o (n = 6) , 13, 15  (n = 6) , and tub (n = 6) , 15, oneThere are only three reports of carcinoma (DCIS) arising in association with borderline PT. Two patients had DCIS both within the PT and in the ipsilateral breast , 18, oneThe molecular events involved in transformation and progression of PTs are largely unknown, and the rarity of coexisting epithelial malignancy makes it difficult to draw conclusions about the aetiological association between the carcinoma and PT in these cases. Genetic aberrations have been consistently demonstrated in PTs with increasing frequency from benign to borderline to malignant PTs. Most studies to date focused on stromal alterations and showed recurrent copy number gains and losses at +1q, \u221213q, \u22126q, +5, and \u221210p \u201339. WherThe present case taken together with those in the literature demonstrates the diverse pathology that can be found with PTs, both within the tumour and in the surrounding and/or contralateral breast. The distinction between a malignant PT with coexisting carcinoma and a metaplastic carcinoma or carcinosarcoma should be considered in the diagnosis as these entities are managed differently and the distinction affects patient outcome. Carcinosarcomas have mixed malignant epithelial and stromal components with the latter showing no reactivity for epithelial immunohistochemical markers. While the present case fulfils these criteria, the characteristic leaf-like structure with malignant heterologous differentiation favours a diagnosis of malignant PT with coexisting carcinoma rather than a true carcinosarcoma. Carcinomas coexisting with PTs are generally diagnosed incidentally on the wide local excision, and the prognosis is dictated by the category of PT. In the present case, the malignant PT and not the carcinomatous elements will have the greatest impact on prognosis. However, the presence of coexistent carcinoma must be taken into account in management decisions, especially for benign and borderline PTs. Wider adequacy of excision of carcinoma, as in this case, and lymph node sampling should be considered."}
{"text": "Bronchial epithelial dysplasia is thought to be a premalignant stage in the evolution of lung cancers. Using the CM-1 polyclonal antibody, we have examined the expression of the p53 protein in a larger series of bronchial dysplasias (n = 60) than hitherto investigated. The p53 protein was detected in 14% of mild, 25% of moderate and 59% of severe dysplasias; increased p53 expression correlated with the severity of dysplasia. p53-positive dysplasias had greater PCNA indices than p53-negative dysplasias. p53 expression in dysplastic tissues was compared with that in two groups of histologically normal epithelium: 14 bronchial biopsies from non-cancer patients of which all but one were negative and 32 bronchial margins from resected carcinomas, of which 17 showed infrequent solitary cells with p53-positive nuclei in predominantly basal locations scattered throughout the epithelium. These results for resection margins were confirmed by use of a second antibody, DO-1. Sixty-nine per cent of the corresponding carcinomas were p53 positive, but in 15 cases the p53 reactivity differed from resection margins. No correlation between p53 expression and any of the clinicopathological characteristics of these tumours was found. This study supports the observation that abnormal p53 expression may be an early but not obligatory event in malignant transformation in lung."}
{"text": "Many cancer patients develop antibodies that recognize the overexpressed p53 protein. The presence of these antibodies is, in some tumour types, associated with poor prognosis. Gastric cancer is a highly prevalent disease associated with a high rate of mortality, there is a need for improved clinical and biological markers for disease behaviour. To investigate the clinical relevance of serum anti-p53 antibodies in patients with gastric adenocarcinoma, we have examined the sera of 501 gastric cancer patients for the presence of serum antibodies against the p53 protein. By immunoblotting analysis using a cell lysate containing overexpressed p53 protein as well as affinity-purified recombinant p53 protein as antigens, we have detected anti-p53 antibodies in 11.2% (61 of 501) of gastric cancer patients, but in none of 46 cancer-free individuals. The presence of anti-p53 antibodies was tightly associated with tumours of higher nuclear grade and lymph node metastasis, and a negative association was found between the presence of anti-p53 antibodies and survival. These results suggest that a preoperative test of serum anti-p53 antibodies in gastric cancer patients can be useful to identify subset of patients who may need gastrectomy with lymph node dissection and post-operative adjuvant therapy. \u00a9 1999 Cancer Research CampaignMutation of the"}
{"text": "Malignant pleural mesothelioma (MPM) is a rare, aggressive tumor for which no effective therapy exists despite the discovery of many possible molecular and genetic targets. Many risk factors for MPM development have been recognized including environmental exposures, genetic susceptibility, viral contamination, and radiation. However, the late stage of MPM diagnosis and the long latency that exists between some exposures and diagnosis have made it difficult to comprehensively evaluate the role of risk factors and their downstream molecular effects. In this review, we discuss the current molecular and genetic contributors in MPM pathogenesis and the risk factors associated with these carcinogenic processes. Malignant pleural mesothelioma (MPM) is a solid, locally aggressive tumor of the pleura that encases and invades the lung parenchyma in late stages of the disease  at 22q12 are frequently altered TSGs that account for some of these deleted sites in MPMs [p16/CDKN2A in MPM tumor samples and cell lines, and for another TSG candidate RASSF1A located at 3p21 in cell lines [Tumor suppressor genes (TSGs) play vital roles in regulating the cell cycle in response to DNA damage and other stressors. The loss of TGS function is one of the fundamental events in tumorigenesis . Loss of in MPMs -22. Epigll lines ,24. Wilm+/- mice with crocidolite asbestos fibers, the mice that had lost the functioning allele of p53 had shorter latent periods and more aggressive tumors than the mice that still maintained one copy. Outside of this model, p53 function is more commonly affected by mutations in upstream and downstream members of the p53 pathways [A surprising finding in mesothelioma research is the lack of frequent mutations in the two most notorious TSGs: p53 and pRb. Although p53 mutations have been found in MPM cell lines ,25, therpathways ,25. The pathways . With thp16/CDKN2A is mutated. In brief, p16/CDKN2A can no longer inhibit cyclin-dependent kinases 4 and 6, which are responsible for the G1-S phase transition of the cell cycle. Left unchecked, these kinases can phosphorylate and thereby inactivate pRb, which allows an uncontrolled entry into S phase [p16/CDKN2A has been reported in >70% of malignant mesotheliomas, and has been associated with poor prognosis [The loss of pRb function occurs in a similar manner to p53. This time  S phase . Homozygrognosis ,31. In arognosis -37. HoweNF2, may help mesothelioma cells acquire this ability. Through the use of in vitro assays, Poulikakos et al. [in vivo.Not only must malignant tumors continue their growth, but they must also be able to move and invade. A recent paper by Poulikakos et al.  proposess et al.  found thWhile the loss of TSGs allows the cell to grow in light of aberrant changes in cellular DNA and function, it is the activation of oncogenes that inspires cell growth and proliferation. AP-1 and \u03b2-catenin transcription factors are implicated in MPM pathogenesis. The AP-1 family of transcription factors is known for mediating a wide range of processes including proliferation, apoptosis and transformation in response to a variety of stimuli . The actThe transcription factor \u03b2-catenin is regulated by an ubiquitin ligase pathway. In the absence of Wnt signaling, glycogen synthase kinase 3B (GSK3\u03b2) phosphorylates adenomatous polyposis coli (APC) and axin, which increases their affinity for \u03b2-catenin. GSK3\u03b2 can then phosphorylate \u03b2-catenin, marking it for destruction. When Wnt is present, GSK3\u03b2 phosphorylation is inhibited, allowing \u03b2-catenin to escape ubiquination, and enter the nucleus where it can bind to T-cell factor/lymphoid-enhancer factor (Tcf/Lef) transcription factor proteins thereby activating transcription of downstream effectors . The rolThe role of growth factors in carcinogenesis has already been suggested above in the case of MAPK pathway activation by EGF. Growth factors can stimulate proliferative pathways through their contact with membrane receptors. They may also play a role in the process of tumor invasion and metastasis, which has been demonstrated by their ability to stimulate chemotactic and/or chemokinetic motility in mesothelioma cell lines . MoreoveMany growth factors, such as insulin-like growth factor-1 (IGF-1) ,59, hepain vitro [VEGF is an angiogenic peptide, which is an independent prognostic factor in MPM . Higher in vitro . The usein vitro . One mecin vitro . It appein vitro .HGF has many possible roles in MPM pathogenesis. It may stimulate morphological changes , promotein vitro that asbestos induces the expression of TNF-\u03b1 and its receptor in human mesothelial cells, and that cell survival may be mediated by the NF-\u03baB pathway downstream of receptor activation. They also propose that other cells, macrophages in particular, may be important contributors of TNF-\u03b1 in vivo [TNF-\u03b1 may help to explain the survival of mesothelial cells after exposure to asbestos . Human m in vivo . More reApoptosis frequently occurs in response to signals from outside the cell. For example, TNF-related apoptosis-inducing ligand (TRAIL), Fas ligand and a lack of growth factor stimulation may result in programmed cell death . HoweverMany risk factors have been identified as contributors to MPM pathogenesis. Above, we began the discussion surrounding the contribution of genetics, asbestos, and SV40 infection. Here we will complete our discussion of these factors and further introduce concerns surrounding the relationship of MPM to radiation and to the pediatric population.Asbestos' contribution to the pathogenesis of MPM is multifaceted with effects ranging from direct to indirect, genetic to molecular. Asbestos induces mutations in mesothelial cells. The more direct mechanism of injury includes deposition of asbestos fiber in the pleura. Longer fibers can penetrate deeply into parietal pleura and have a high likelihood of causing cancer . AsbestoThe response of mesothelial cells to asbestos and ROS is an important factor in MPM pathogenesis. Above, we described the possible role of TNF-\u03b1 in protecting mesothelial cells from death after asbestos exposure . The abiThe use of the single term \"asbestos\" to describe at least five unique fibrous silicate minerals . A causal relationship between malignant mesothelioma and asbestos exposure, radiation, and/or isoniazid could not be established. As far as asbestos is concerned, the long latency of asbestos-related mesothelioma makes a role for this risk factor in pediatric MPM unlikely [Mesothelioma is very rare in childhood with an estimated 2%\u20135% of all cases occurring in the first two decades of life. The diagnosis may be challenging because of its rarity and its pathologic similarities to other papillary or spindle cell neoplasms in the pediatric age group ,138. To unlikely ,141 unleunlikely .WT1 as a TSG in MPM pathogenesis. However, this picture is complicated by data that reports a lack of inactivating mutations in MPM [WT1 in a 45-year old woman with a perotineal mesothelioma [Pediatric and familial cases may open an avenue for the study of genetic contributions to MPM. Small case studies have found that pediatric patients with Wilm's tumor who were treated with radiation may be at an increased risk for mesothelioma ,142. This in MPM -145. Alsthelioma . It may Familial cases also support the role of genetic predisposition to MPM ,147. Howin vitro studies, and epidemiology, we may be able to gain a better understanding of which risk factors and molecular targets are the most important for future preventative and therapeutic measures.MPM has a complex etiology in which asbestos, ionizing radiation, viruses, genetic factors, and even diet  may act The authors declare that they have no competing interests.SJW and SNM both contributed to the research and writing. All authors read and approved the final manuscript."}
{"text": "Recent reports suggest that the product of hepatitis B virus (HBV) interacts with p53 and that the hepatitis C virus (HCV) core protein reduces p53 expression. A novel p73 gene, which is related to p53, has recently been identified and mapped to chromosome 1p36.3, which is a locus of multiple tumour-suppressor genes for many cancers, including hepatocellular carcinoma (HCC) and neuroblastoma. Here, we investigated mRNA expression, allelotype and mutation of p73 in 48 HCCs obtained from untreated patients. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that p73 mRNA was expressed ubiquitously at low levels in all the tumour tissues, as well as in the adjacent normal liver tissues. The frequency of p73 loss of heterozygosity was observed in 20% of HCCs, but PCR-single strand conformation polymorphism (SSCP) analysis showed no mutations in the 48 tumours except for three types of polymorphisms. These results suggest that p73 may play a role in hepatocellular carcinogenesis in a different manner from a Knudson two-hit model. The regulatory mechanism of interaction between p73 and hepatitis viruses remains to be determined. \u00a9 1999 Cancer Research CampaignAccumulating evidence has demonstrated that aberration of the"}
{"text": "This review is intended to summarize the risk factors, classification, diagnosis, and treatment of heterotopic ossification (HO) of previously published studies. Heterotopic ossification is a common complication of total hip arthroplasty. Its prevalence is not the same in all of the patient groups. Frequency of HO varies from 15 to 90%. Hip ankylosis, male gender, and previous history of HO are said to be risk factors with a significant level. Diagnosis is based on a single AP radiograph: the Brooker classification that divides HO into four grades is the most commonly used. The confirmation test that can be used is a bone scan. A great amount of bone metabolic turnover markers have been tested, but none of them seems to be relevant in case of prevention or diagnosis of HO. The most effective prophylactic treatment is radiotherapy or administration of nonsteroidal anti-inflammatory drugs. Over the years a lot of different RT protocols have been tested. Nowadays the most often used regimen is 7 Gy given postoperatively in a single dose. The most commonly prescribed drug in prophylaxis of HO is indomethacin. Also, the efficacy of ibuprofen and diclofenac was proven. Recently researchers focused on selective COX-2 inhibitors. They appear to be as effective as nonselective NSAIDs having less side effects. The one and only treatment of HO is a revision arthroplasty. Heterotopic ossification (HO)  is a relThis is an extremely important challenge, as ossifications may destroy the healing effect of surgery, limiting to a large extent of range of motions in the hip joint and additionally exposing the patient to subsequent surgical treatment, revision arthroplasty.A search was performed on PubMed, Embase, and Cochrane Central Register. It was conducted using key word \u201cheterotopic ossification\u201d combined with either \u201cTHR\u201d, \u201ctotal hip replacement\u201d, \u201cTHA\u201d, \u201ctotal hip arthroplasty\u201d, \u201cNSAIDs\u201d, \u201crisk factors\u201d, \u201cradiotherapy\u201d, \u201cradiation\u201d, \u201cmarker\u201d, or \u201cmarkers\u201d. Also, the search of references in included studies was performed. There were no restrictions in language or date of publication. Studies were excluded from our analysis if there were abstracts, letters, case reports, case series, guidelines, or irrelevant significance for our study topic. We identified 4628 articles. Of these articles, 836 were removed due to duplicate reportage, and 3675 were excluded based on the titles and abstracts. The remaining 117 articles were accessed for the full text and screened for further assessment. Finally, we included 63 articles in our review.A number of studies demonstrating the existence of potential risk factors for heterotopic ossifications after hip arthroplasty have been found in the literature , 19\u201324.In the recent meta-analysis it was estimated that male sex, cemented prosthesis, bilateral hip joint arthroplasty procedure, ankylosing spondylitis, and hip joint ankylosis are associated with increased risk of nonarticular ossification . In addiAccording to the same study, the only factor that is said to decrease HO is rheumatoid arthritis .In addition, it has been demonstrated that surgeries due to femoral neck fracture, degenerative disease, previous hip fracture, hypertrophic type of osteoarthritis, and age are not associated with an increased risk of heterotopic ossification.There have been studies evaluating the assessment of the impact of revision surgery, lateral approach in hip arthroplasty, BMI, bone grafting, and trochanteric osteotomy on the potential occurrence of ossification but even if they reported a positive correlation they require further research .The most commonly used method of classification is Brooker classification based only on a single X-ray image in the anteroposterior (AP) projection , 25, 26.Usually, ossifications that develop after surgery are visible in the radiological examination after 4-6 weeks , 27\u201330.The Brooker classification divides the heterotopic ossifications into four grades :I - described as islands of bone within the soft tissues about the hip;II - consisting of bone spurs originating from the pelvis or proximal end of the femur, leaving at least 1 cm between opposing bone surfaces;III - consisting of bone spurs originating from the pelvis or proximal end of the femur, reducing the space between opposing bone surfaces to less than 1 cm;IV - hip joint ankylosis , 26.Types III and IV are referred to as clinically relevant , 30, 31.The level of alkaline phosphatase in serum increases during tissue injuries; therefore its predictive value in heterotopic ossifications after hip arthroplasty is doubtful . HoweverWilkinson et al. tried to find alternative bone metabolic turnover markers that could be used in the diagnosis of heterotopic ossifications in patients after hip arthroplasty. According to their study, C telopeptide of type I-collagen (CTX-1) is potentially useful marker, which increases its concentration in the first postoperative week by 42% and shows a sensitivity of 89% and specificity of 82% in the prediction of the diagnosis of ossifications in the 26th week after surgery . UnfortuBased on Wilkinson's research, we can reject N-terminal propeptide of type I-procollagen (PINP) and osteocalcin (OC) from potential testings, as their increased correlation with HO occur too late to implement effective prophylaxis. Moreover, the levels of N-telopeptide of type I-collagen (NTX-1) and deoxypyridinoline (DPD) were not correlated at all with the presence of ossifications .Bone metabolic turnover markers have also general diagnostic limitations. Their level fluctuates in relation to gender, coexisting diseases and some are prone to daily or seasonal fluctuations, which makes them difficult to use as a simple diagnostic tool .In papers heterotopic ossification symptoms include swelling, redness, and pain as subjective symptoms. Therefore, symptoms of ossification may imitate deep vein thrombosis in the lower limbs or cellulitis. Unfortunately, none of these studies are supported by the relevant figures , 39\u201341.Scintigraphy is the most sensitive method in diagnosing HO , 36, 42.The formerly used radiopharmaceutical was strontium, currently 99mTc; pyrophosphate is used , 41, 42.Scintigraphy is based on the administration of a radiopharmaceutical and the subsequent production of a series of scans that will provide information during three phases: a dynamic blood flow, blood accumulation, and the accumulation of a radiopharmaceutical in the bone .Scintigraphy is particularly effective in determining the maturity of heterotopic ossifications and planning their operational removal , 42. TheSchurch et al. conducted a study to evaluate the usefulness of prostaglandin E2 (PGE-2) measurements in urine to predict the formation of HO. They reported increased level of PGE-2 in urine in patients with developing ossification. The authors recommended examining 24-hour PGE-2 level once a week for a period of 3 to 4 months. The increase in this excretion was expected to indicate the development of heterotopic ossification .Orzel et al., in their retrospective analysis of 24 patients' ossifications, showed that calcium levels in serum had decreased below the reference level in 23 cases. The reduction occurred on average during fifth day and lasted on average ten days . It seemProphylaxis of Heterotopic Ossification: Methods and Their Effectiveness(1) Radiotherapy (RT). It is estimated that the prevalence of HO should not be greater than 10% after the radiation prophylactic treatment [reatment , 44.Over the years many RT regimens have been tested. The first observation was published by a team from Mayo Clinic. They used fractionated RT (2 x 10 Gy) . Then reIn a meta-analysis Milakovic et al. found no significant differences in a HO prevalence between the patients treated with banana equivalent dose (BED)>2500 cGy and the patients treated with BED doses <2500 cGy .The difference in HO occurrence was so significant that the researchers advised not to use that protocol again. It seems that Finegorth et al. found the boundary of an effective radiation protocol with a 6 Gy given postoperatively .Nowadays the most often prescribed is a single dose radiation of 700 Gy .Also, the effectiveness of preoperative radiation has been measured , 27, 43.The most important factor in a prophylactic radiation treatment is time. Seegenschmiedt concluded that it must not be given earlier than 8 hours before the procedure and later than 72 hours after the procedure . The othFinally, the fractionated radiation has been compared with a single dose radiation. Konsi et al. in a randomized study showed the same efficacy of a single dose radiation compared to a fractioned one . MilakovRT is not only effective, but safe. Sheybani et al. analyzed records of over 3500 patients who underwent a THR [(2) Relevance of Using Medications(a) Nonsteroidal Anti-Inflammatory Drugs (NSAIDs). The most commonly used drug in HO prophylaxis is indomethacin. It is probable mechanism of stopping the bone formation is by inhibiting the inflammation process [ process .Schmidt et al. in a prospective, double-blind, randomized study proved the effectiveness of indomethacin in HO prevention . It was Another drug whose efficacy has been proved is ibuprofen. Elmsted et al. showed its effectiveness compared to a placebo . SodemanIt appears that the shortest possible treatment time with nonselective oral NSAIDs is one week . It shouPritchett studied the efficacy of ketorolac in a prospective, double-blind, randomized study . It was It appears that there are no big differences between the two most studied prophylactic treatments. Kienapfel et al. conducted a study that compared indomethacin given in a dose of 2x 50 mg for 42 days with a single dose 600 Gy postoperative radiation. The treatments were equally effective in preventing HO [Knells et al. conducted prospective study comparing indomethacin with acetylsalicylic acid and radiotherapy. They concluded that the most effective prophylactic treatment for the general population is indomethacin prescribed in a dose of 250 mg daily for 14 days starting on the first postoperative day. The patients with a higher chance of developing HO or with the contradictions for nonselective NSAIDs should be qualified for RT .Kolbl et al. compared preoperative radiation (7 Gy 16-20h before the procedure) and diclofenac (2x75mg for 14 days starting at the 1st postoperative day). They showed no difference in lowering the clinically significant HO occurrence, although the overall prevalence of HO was lower in diclofenac group .The only prospective study evaluating efficacy of combined therapy conducted Pakos et al. . They coSelective cyclooxygenase-2 (COX-2) inhibitors are a very attractive form of treatment, because they show less gastrointestinal side effects and lack interaction with platelets aggregation , 23, 57.The elevated cardiovascular risk was observed after 6 months of treatment , period way longer than prophylactic treatment time after total hip replacement (THR) .Rofecoxib and celecoxib has been the most studied agents and proved reliable .Lavernia et al. proved effectiveness of etoricoxib compared to historical group that did not take any prophylaxis at all . PatientFinally, the very conclusive results came with both Grohs and van der Heide studies , 58. TheOni et al. conducted prospective study with celecoxib . PatientSaudan et al. compared celecoxib (2x200 mg) and ibuprofen (3x400 mg). Celecoxib turned out to be more effective in preventing HO than ibuprofen .Xue et al. conducted a meta-analysis of randomized studies that compared NSAIDs with selective COX-2 inhibitors . They foAnother meta-analysis by Shun Lin et al. showed better outcomes in HO prevention after NSAIDs prophylaxis treatment compared with placebo. In this study, researchers also measured the discontinuation of the therapy due to gastrointestinal side effects and compared it between groups treated with nonselective NSAIDs and selective COX-2 inhibitors. The discontinuation was more frequent in the nonselective NSAIDs group.(b) Diphosphonates. The prophylaxis of HO that was proven to be ineffective is treatment with diphosphonates [phonates , 47, 49.phonates , 47, 59.phonates , 47, 59.phonates . The firBijovet et al. also proved inefficacy of diphosphonates in HO prophylaxis by conducting a prospective study . PatientGrades I and II HO may not need any treatment unless they become symptomatic or functionally disabling. The gold standard therapy for grades III and IV HO is a revision arthroplasty and surgical resection of the ossification , 42, 46.HO is one of the most common complications after THA. Until now, no algorithm for detection and treatment has been developed. A series of papers agree on risk factors and we collected those in"}
{"text": "LPA, which encodes for apolipoprotein(a) [apo(a)], is causally associated with CAVD. Apo(a) is the protein component of Lp(a), a LDL-like particle, which transports oxidized phospholipids (OxPLs). Autotaxin (ATX), which is encoded by ENPP2, is a member of the ecto-nucleotidase family of enzymes, which is, however, a lysophospholipase. As such, ATX converts phospholipids into lysophosphatidic acid (LysoPA), a metabolite with potent and diverse biological properties. Studies have recently underlined that ATX is enriched in the Lp(a) lipid fraction. Functional experiments and data obtained in mouse models suggest that ATX mediates inflammation and mineralization of the aortic valve. Recent findings also indicate that epigenetically-driven processes lower the expression of phospholipid phosphatase 3 (PLPP3) and increased LysoPA signaling and inflammation in the aortic valve during CAVD. These recent data thus provide novel insights about how lipoproteins mediate the development of CAVD. Herein, we review the implication of lipoproteins in CAVD and examine the role of ATX in promoting the osteogenic transition of valve interstitial cells (VICs).Calcific aortic valve disease (CAVD) is a complex trait disorder characterized by calcific remodeling of leaflets. Genome-wide association (GWA) study and Mendelian randomization (MR) have highlighted that  Il1rn), a natural circulating inhibitor of interleukin 1 beta (Il1b), develop a thickening of the aortic valve and a rise of transaortic velocities  is a prevalent cardiovascular disorder. Fibro-calcific remodeling is one key process involved in the development of CAVD. During CAVD, fibrosis and mineralization of the aortic valve (AV) cusps progressively lead to a narrowing of the aortic orifice, a clinical condition referred to as aortic valve stenosis . Differelocities . VIC, a locities . As suchLPA, which encodes for apoliprotein(a) [apo(a)], is causally associated with CAVD  and traits/disorders, have underlined the genetic architecture of these traits and have fuelled the development of novel therapies. Though the genetic architecture of CAVD is just emerging, recent findings have transformed the field and have helped to pinpoint causal pathways  in the region encoding for kringle IV type 2 domain (KIV2), which is inversely related to the circulating level of Lp(a) (LPA locus rs10455872 is associated with the number of KIV2 repeats and with plasma level of Lp(a). It is worth highlighting that plasma level of Lp(a) is largely determined by genetic factors and heritability may explain up to 90% of the variance  . The indvariance . In a MR in CAVD . However in CAVD . In a la in CAVD . Also, g in CAVD . Among s in CAVD . These f10 domain of apo(a) is attached covalently to OxPL (Apo(a) is a highly polymorphic lipoprotein, which is linked to apolipoprotein B(apoB) moiety of low-density lipoprotein (LDL) by a disulfide bridge . Lp(a) i to OxPL . Of note to OxPL . Also, t to OxPL . OxPL is to OxPL , 30.ACTA2) and secrete ECM components such as collagen and remodeling enzymes (TGFB1) is expressed during the early stages of CAVD and promotes the transformation of quiescent cells into activated VICs . ALPL along with other ecto-nucleotidases, such as ENPP1, are overexpressed in surgically explanted mineralized aortic valves  . Of intePLPP3 was decreased by 49% in mineralized AVs. These data were corroborated by measuring PLPP activity, which was also reduced by 31% in mineralized AVs  was increased by 1.5-fold. Mapping provided evidence that a dysregulated epigenetic process was impinging on the expression of PLPP3 during the mineralization of cusps. DNA methylation mapping showed that CpG methylation in intron 1 (cg02468627) of PLPP3 was inversely associated with the expression of this gene and was increased in mineralized portion of explanted aortic valves  are membrane-associated enzymes involved in the degradation of LysoPA. As such, PLPPs, which reside at the cell membrane along with LysoPA receptors (LPARs), are key modulator of LysoPA signaling in cells. Transcriptomic analysis of control non-mineralized and mineralized aortic valves showed that among the PLPP enzymes PLPP3 was significantly downregulated during CAVD . Analysiized AVs . Of notec valves . This Cpof PLPP3 . Epigenen by 38% . These fCOL1A1 and the mineralization of VIC cultures through a nuclear factor kappa B (NF\u03baB) pathway. In VICs, LPAR1 activated RhoA and Rho kinase (ROCK), which promoted a robust expression of NF\u03baB-dependent genes such as IL6 and IL8. In addition, we found that BMP2, a morphogenetic protein that drives osteogenesis, was overexpressed downstream of LPAR1-NF\u03baB. Analysis showed that promoter region of BMP2 contained \u03baB responsive elements and that LysoPA promoted the phosphorylation of p65 on serine 536 (p65 S536) (BMP2 in chromatin immunoprecipitation (ChIP) assay. During NF\u03baB signaling the inhibitor of \u03baB (I\u03baB) is rapidly induced and transits to the nucleus where it competes with p65 for \u03baB sites. This process ensures a negative feedback loop to control the inflammatory response mediated by the NF\u03baB pathway. However, phosphorylation of p65 S536 is not sensitive to the nuclear inhibition provided by I\u03baB. Hence, genes dependent on phosphorylated p65 S536 are less sensitive to negative retro-feedback and experienced sustained expression. Experiments carried out with constitutively active mutant I\u03baB super repressor (I\u03baB\u03b1 SS32-36AA) showed that LysoPA-induced expression of BMP2 was not sensitive to the repression  . Of intepression . These dve cusps . In thisLPA in CAVD, which has been validated in different cohorts, has provided considerable interest to understand the role of lipoproteins in CAVD. MR studies showed that Lp(a) is causally associated with CAVD. Thus, these data strongly suggest that interventions aimed to modify the level of Lp(a) could provide therapeutic benefit   conducted with statins have been negative in CAVD and have questioned the role of lipoproteins as causal factors in CAVD. However, the strong genetic signal for  benefit . The fai benefit . For ins benefit . Recentl benefit . Also, i benefit . In addiPL (EO6) . FurtherPL (EO6) .LPAR1 and PLPP3 respectively. LPAR1 promotes a signaling cascade that culminate in the phosphorylation of p65 S536 and a sustained expression of BMP2, a key driver of osteogenic transformation in VICs. On the other hand, epigenetically reprogrammed VICs at a transposon-based enhancer is a proximal event that contributes to lower PLPP3 and to promote/exacerbate LysoPA signaling in VICs. Together, these data suggest that lipoprotein metabolism during CAVD is perturbed and promotes inflammation and mineralization. Further work and elucidation of key processes in lipoprotein metabolism during CAVD could lead to the development of novel therapies.Genetic studies have underscored that Lp(a) is causally associated with CAVD. Molecular analyses have provided evidence that interaction between ATX and Lp(a) is promoting inflammation and osteogenic reprograming in VICs. Dysregulation of LysoPA signaling in mineralized aortic valve includes a higher and a lower expression of The datasets for this manuscript are not publicly available. Requests to access the datasets should be directed to Patrick Mathieu.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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."}
{"text": "Alzheimer\u2019s Disease (AD) is a neurodegenerative disorder related with the increase of age and it is the main cause of dementia in the world. AD affects cognitive functions, such as memory, with an intensity that leads to several functional losses. The continuous increase of AD incidence demands for an urgent development of effective therapeutic strategies. Despite the extensive research on this disease, only a few drugs able to delay the progression of the disease are currently available. In the last years, several compounds with pharmacological activities isolated from plants, animals and microorganisms, revealed to have beneficial effects for the treatment of AD, targeting different pathological mechanisms. Thus, a wide range of natural compounds may play a relevant role in the prevention of AD and have proven to be efficient in different preclinical and clinical studies. This work aims to review the natural compounds that until this date were described as having significant benefits for this neurological disease, focusing on studies that present clinical trials. Neurodegenerative diseases induce alterations in the central nervous system with psychological and physiological negative effects . AlzheimA\u03b2 derives from the amyloid precursor protein (APP), proteolytic cleavage of amyloid precursor protein (APP), an integral membrane protein that possesses the general properties of a cell surface receptor , by the Therefore, it is fundamental to seek for new strategies for AD therapy ,20,21,22In this review, the natural compounds already in clinical trials phase are described and the reported results are presented and discussed. Other natural compounds with known potentially beneficial effects in AD in a preclinical development stage with in vitro and in vivo studies are also described. For preclinical studies, only the most recent reported works are cited. The systematic literature search was conducted using PubMed, Science direct, Google Scholar, Scopus and Web of Science as online databases until April 2019. Only papers written in English were considered with unlimited publication date.Natural compounds are an emerging approach for AD therapy. For the assessment of their therapeutic efficiency and potential side effects, human trials have been performed in the last years. The first natural product studied in a clinical trial was nicotine in 1992. However, no clinical trials were performed in the last two decades for this molecule. During the 90s, several other compounds were studied in clinical trials for AD therapy, such as vitamins. These molecules are still being tested in human trials up until this date. In the last years, other natural compounds are gaining interest by the scientific community and have achieved the clinical trials phase, such as bryostatin, which effects started to be evaluated in humans in 2017. A detailed report of these findings is described below. The natural compounds were divided into two groups: bioactive compounds and natural extracts, and they are summarized in Vitamins have been described as therapeutic compounds for AD. Among them, vitamin C, E and D have aroused great interest. Vitamin C A is founOn the other hand, vitamin E, which is present in several fruits and vegetables B, also s3, 2, Other vitamin with reported beneficial effects for AD, is vitamin D. Adding to several benefits of vitamin D , its theClinical trials revealed that vitamin D increased plasma A\u03b2 in mild cognitive impairment patients, suggesting a reduction in A\u03b2 levels in the brain. In fact, Miller et al. (2016) studied the effect of vitamin D supplementation on the plasma levels of A\u03b2 in eight patients over 60 years old in a pilot study. Patients were randomly divided in two groups, treatment and placebo groups. Patients from the treatment group were administered with 50,000 IU per week for eight weeks. The obtained results showed that vitamin D intake increased plasma A\u03b2 levels, suggesting a decrease in A\u03b2 brain levels .n = 47) were orally supplemented with vitamin D at 50,000 IU once a week for six weeks, followed by 1500\u20132000 IU daily for 18 months. The obtained results showed that lymphocyte susceptibility to death, A\u03b2 plasma levels and cognitive status improved after six months of vitamin D supplementation in cognitive impairment patients, but not in very early AD patients. Thus, supplementation with vitamin D proved to be beneficial in cognitive impairment patients. The lack of effects in very early AD patients suggest that vitamin D intake is not able to delay the progression of the disease in a more advanced stage [SanMartin et al. (2017) evaluated the role of vitamin D in the A\u03b2 clearance from the brain. Patients with mild cognitive impairment and very early AD (ed stage .n = 8), or memantine alone (n = 18), or vitamin D alone (n = 17). Patients were administered with drugs for 24 weeks. Memantine was administered orally at 5 mg per week for the first four weeks and then 20 mg per day for the rest of the trial. Patients received a drinking solution of vitamin D at 100,000 IU every four weeks. After the study, patients co-treated with memantine and vitamin D showed better cognitive performance than patients treated with vitamin D or memantine alone [Co-therapy with vitamin D and other molecules for AD therapy has also been explored in clinical trials. In fact, Annweiler et al. (2012) conducted a double-blind, placebo-controlled pilot trial with 43 white patients over 60 years with moderate AD symptoms . The maine alone .n = 78) received placebo or daily supplement containing 800 IU of vitamin E, 500 mg of vitamin D, 900 mg of \u03b1-lipoic acid and 400 mg of coenzyme Q for 16 weeks. The attained results showed that the co-supplementation did not affect amyloid or tau levels, but a reduction on levels of an oxidative stress biomarker, the cerebrospinal fluid F2-isoprostane, was verified.Co-supplementation with vitamin D and other natural compounds was also studied in clinical trials. In fact, Galasko et al. (2012) conducted a double-blind, placebo-controlled clinical trial to evaluate what antioxidant supplementation affected the levels of AD\u2019s histopathological marks, such as A\u03b2 peptide and tau protein . PatientAlso, co-supplementation with multivitamins was evaluated in clinical trials. In fact, Kontush et al. (2001) evaluated the efficiency of supplementation with both vitamin E and vitamin C to decrease oxidation of lipoproteins in AD patients . Lipid oCo-therapy of different drugs with vitamin E was also studied in clinical trials. Sano et al. (1997) evaluated the effects of vitamin E and selegiline co-administration . Selegiln = 769) were randomly divided in three groups, placebo, vitamin E alone or donepezil alone. The daily dose of vitamin E was 1000 IU, and after six weeks the dose was increased to 2000 IU, for five years. Vitamin E proved to not be able to delay the disease progression. The combined effect of donepezil and vitamin E was also studied. Donepezil is a drug used for AD therapy to control the symptoms. To compare the effects of this drug with vitamin E on the outcome effects on patients with mild cognitive impairment, a double-blind, placebo-controlled clinical trial was conducted by Petersen et al. (2005) . PatientDysken et al. (2014) studied the combination effects of vitamin E and memantine . For thaKryscio et al. (2017) intended to assess if vitamin E and selenium intake could prevent dementia in healthy men over 60 . AlthougDocosahexaenoic acid (DHA) is a polyunsaturated fatty acid from marine fish and algae  and its The effects of supplementation with DHA in AD patients were studied in different clinical trials. In fact, Freund-Levi et al. (2006) conducted a double-blind, placebo-controlled clinical trial with 204 AD patients . The maiQuinn et al. (2010) conducted a double-blind, placebo-controlled clinical trial to evaluate the efficacy of supplementation with DHA on the cognitive and functional decline in AD patients . A dailyThe same group conducted a double-blind, placebo-controlled, clinical study in the same year to evaluate the ability of DHA to improve the cognitive functions of 485 participants with age-related cognitive decline . The subn = 36) were randomly divided in two groups, placebo and treatment group. The treatment group was orally administered with 430 g of DHA three times a day, for one year. No significant side effects were verified, suggesting the potential of DHA to improve memory functions. However, studies with more patients and longer intervention periods, are necessary to define the optimal dosage.Lee et al. (2013) studied the effects of DHA administration using fish oil on the cognitive function in patients over 60 diagnosed with mild cognitive impairment . The parGrateloupia livida and its structural formula is presented in Homotaurine, also known as tramiprosate, is an aminosulfonate metabolite extracted from marine red alga Aisen et al. (2011) conducted a phase III double-blind, placebo-controlled trial with 1052 patients with mild to moderate AD symptoms to evaluate the effect of homotaurine in slowing AD progression ,49. ThisThe safety and tolerability of this compound administered to 58 patients with mild to moderate AD symptoms, were studied previously in a phase II clinical trial conducted by the same group . PatientMartorana et al. (2014) conducted a study with 10 patients with mild cognitive impairment with ages between 59 and 74 . The parHuperzia serrata (Thunb.) Trevis. (Lycopodiaceae) and its structural formula is presented in Huperzine A is isolated from Xu et al. (1995) evaluated the efficacy and safety of huperzine A in AD patients. Four tablets of huperzine A (200 \u00b5g) or placebo were administered orally to 103 patients, twice a day, for eight weeks . The resTo further compare the efficacy and safety of huperzine A administered into capsules and tablets in AD patients, the same group conducted a new trial four years later . In thisLater, Rafii et al. (2011) studied the safety and efficacy of two concentrations of huperzine A, 200 and 400 \u00b5g twice a day, in patients with mild to moderate AD in a phase II clinical trial . Placebobryozoan Bugula neritina [Bryostatin is a macrolide lactone extracted from neritina . The strneritina . Also, bneritina .2 was administered to six patients, while three patients received placebo. Bryostatin proved to improve cognitive functions and to be safe and well tolerated. Recently, Nelson et al. (2017) evaluated the safety, tolerability and effects on cognitive function of bryostatin on AD patients in a phase II clinical trial . A singlAnother phase II clinical trial was performed with the same goals . Farlow Melatonin is collected from animals, plants, fungi and bacteria and its structural formula is presented in Brusco et al. (1998) evaluated the efficacy of melatonin in monozygotic twins with AD, with similar cognitive and neuropsychologic impairments . Only onLater, the same group studied the effect of melatonin in cognitive dysfunctions of 14 AD patients . The patThe same results were obtained by Furio et al. (2007) that performed a clinical trial with 50 outpatients diagnosed with mild cognitive impairment, where half of patients received 3 to 9 mg of melatonin for 9 to 18 months . Wade et al. (2014) investigated the ability of 2 mg of melatonin to improve the cognitive functions of patients with mild to moderate AD . MelatonVitis vinifera L. (Vitaceae)) and red wine and its structural formula is presented in Resveratrol is a naturally occurring non-flavonoid polyphenol present in grapes  performed a phase 2 clinical trial for 52 weeks in mild to moderate AD patients. The group studied the safety, tolerability and the ability of resveratrol to reduce the biomarkers of the disease (A\u03b2 and tau). Here, 119 individuals were orally administered once a day with placebo or 500 mg of resveratrol, with an increase of 500 mg each 13 weeks. Although this study suggests that resveratrol can cross the blood-brain barrier (BBB), the results were not satisfactory. Besides inducing some side effects like nausea, diarrhea, and weight loss, the brain volume and biomarkers levels were lower in the placebo group than resveratrol group .Recently, Zhu et al. (2018) evaluated the safety, tolerability and efficacy of a mixture containing 5 mg of resveratrol, 5 g dextrose and 5 g of malate. Fifteen mL of the mixture or placebo were orally administered twice a day to 39 patients with mild to moderate AD for one year. The administration was done together with an 8 oz glass of commercial grape juice. The results revealed that the preparation was safe and well tolerated. However, no evidence was observed concerning the efficacy of the product for AD therapy .Nicotiana tabacum L., Solanaceae) and its structural formula is presented Nicotine is extracted from the tobacco plant leaves  studied the effect of nicotine on AD patients . Three aThe effect of nicotine on behaviour, cognition, and physiology of six AD patients was evaluated in a pilot study proposed by Wilson et al. (1995) . PlaceboThe clinical and neuropsychological effects of nicotine was evaluated in eight AD patients by White et al. (1999) . TransdeCurcuma longa L. (Zingiberaceae) and its structural formula is presented in Curcumin is an active component founded in the root of Baum et al. (2008) performed a clinical trial to study the safety of curcumin on AD patients . For sixGinkgo biloba L., Ginkgoaceae) has been studied as therapeutic drug for AD and other neurological diseases therapy. In vitro evidence revealed that ginkgo biloba extract can prevent A\u03b2 aggregation, decrease A\u03b2 fibrillogenesis and destabilize preformed fibril [Ginkgo biloba  were considered in this study. Patients received 240 mg of extract or placebo once daily for 24 weeks. The treatment with gingko biloba was safe and improved the neuropsychiatric symptoms, which include apathy, irritability, depression, among others. Several clinical trials have been carried out in the last 10 years to test the viability of the compound in treating patients with dementia. Bachinskaya et al. (2011) examined the effect of gingko biloba extract EGb 761dementia ,68. OutpAlso, with the same conditions, Herrschaft et al. (2012) revealed that the treatment with gingko biloba improved the cognition and the life quality of patients . n = 333) or vascular dementia (n = 71). In addition to confirming the improvement of neuropsychiatric symptoms observed in the previous trial, the extract improved the cognitive functions and functional abilities of patients. Ihl et al. (2012) performed a similar 24-week randomised controlled trial involving 404 outpatients . Patient\u00ae or placebo for 24 weeks. The trial proved that the extract improved the neuropsychiatric symptoms and cognitive functions of patients. Also, the extract was safe and well tolerated. Taking together, the last clinical trials proved that a 240 mg daily dose of ginkgo biloba extract is safe in the treatment of dementia.Gavrilova et al. (2014) also conducted a clinical trial to study the effects of gingko biloba in neuropsychiatric symptoms and cognition in 160 patients with mild cognitive impairment . The patCrocus sativus L., Iridaceae) is a stem-less herb with antioxidant [Saffron  evaluated the efficacy of 30 mg saffron in the treatment of mild to moderate AD . SaffronMelissa officinalis L., Lamiaceae) from the mint family that is native to Europe with antioxidant activity in vitro [Melissa officinalis extract on patients with mild to moderate AD, Akhondzadeh et al. (2013) administered to 40 patients 60 drops of extract or placebo, for four months [Melissa officinalis extract ameliorated the cognition and agitation of AD patients. Lemon balm (in vitro . In vivoin vitro . To asser months . The resCamellia sinensis (L.) Kuntze, Theaceae) from steaming and drying of leaves of the Camellia sinensis plant proved to be a rich source of antioxidants in in vivo studies [Green tea  developed a clinical trial with 30 patients to study the antioxidant activity of green tea in patients with severe AD and its ability to improve cognitive functions . PatientCarica papaya L., Caricaceae) is a fruit often used in medicine that has amino acids, \u03b2-carotene, oligosaccharides and vitamins, with benefits in AD. Papaya (n = 20) received 4.5 g of extract daily for six months, while the 12 controls did not receive any treatment. The results showed that the supplementation with fermented papaya powder reduced the ROS generation and nitric oxide production in AD patients, with no significant changes in controls. Thus, the papaya can be used as antioxidant in the AD therapy.A clinical trial performed by Barbagallo et al. (2015) studied the antioxidant activity of fermented papaya powder extract in AD patients . AD patiSalvia officinalis L., Lamiaceae) is a medicinal plant with a long-standing reputation in European medical herbalism due to its anti-inflammatory and antioxidant properties observed in vivo [Sage  developed a clinical trial to evaluate the efficacy and safety of erate AD . PatientCocos nucifera L., Arecaceae) demonstrated to be able to reduce the A\u03b2 deposition and aggregation and the oxidative stress in a transgenic Caenorhabditis elegans AD model [Coconut  performed a clinical trial with 44 AD patients . Half ofMalus domestica Borkh., Rosaceae) showed to be a promising approach to prevent AD. In vivo evidence demonstrated that the apple extract prevents the oxidative stress and reduces the A\u03b2 levels, improving the memory of AD rats [Apple  performed an open-label pilot clinical trial with 21 patients with moderate to severe AD . The autVaccinium myrtillus L., Ericaceae) is a fruit composed by several polyphenols named anthocyanins, with antioxidant [Blueberry  evaluated the effects of daily administration of wild blueberry juice in a group of nine elderly subjects with early memory failures . The daiColostrinin, a milk form produced by mammary glands , presentThe effect of colostrinin on AD patients was studied in a clinical trial conducted by Szaniszlo et al. (2009) . PatientBesides the natural compounds that have been studied in clinical trials, several other products have proved to have a potential beneficial effect in AD therapy in a preclinical stage, namely in in vivo studies. The preclinical phase involving in vivo studies is conducted to assess if the new compounds are safe and effective, before they can proceed to the clinical trials phase. A detailed report of animal studies results is described below. The natural compounds were divided into two groups: bioactive compounds and natural extracts and organized by the number of mechanisms associated with AD therapy, from the highest to the least.Epigallocatechin gallate (EGCG) is a polyphenol found in green tea with several neuroprotective effects in AD. In vivo evidence suggests that EGCG decreased \u03b2- and \u03b3-secretase actions and enhanced the \u03b1-secretase activity, leading to the decrease of A\u03b2 levels improving the memory . BesidesRetinoic acid is a terpenoid and a metabolite of vitamin A. In vitro studies revealed that retinoic acid inhibited A\u03b2 fibrils formation and their extension and destabilized A\u03b2 fibrils . In vitrCaffeine is perhaps the most consumed psychoactive compound. It is present in the coffee bean, but it can be also found in some teas, cocoa drinks, candy bars, among other herbs. In vivo studies suggest that caffeine reduced the \u03b2-secretase and \u03b3-secretase levels, decreasing the A\u03b2 production . An in vScutellaria baicalensis Georgi (Lamiaceae). In vitro studies suggested that baicalein inhibits the ROS production, reducing the oxidative stress [Baicalein is a naturally occurring flavonoid from the roots of e stress . In vitre stress . In vivoe stress ,183. AlsBerberine is an isoquinoline alkaloid found in rhizoma coptidis, an herb frequently used in Chinese herbal medicine. In vivo evidence suggests that berberine inhibited the \u03b2-secretase expression, reducing the A\u03b2 production. Also, berberine stimulated the A\u03b2 clearance and inhibited the A\u03b2 plaque deposition and hyperphosphorylation of APP and tau . BerberiKaempferol is a polyphenolic flavonoid found in fruits, vegetables and herbs. In vivo studies proved its antioxidant effect, improving the learning and memory of a transgenic drosophila AD model . Also, iQuercetin is a flavonol, naturally occurring polyphenolic compounds present in fruits, vegetables and herbs. In vivo studies showed that quercetin improved the memory and cognitive impairments of an AD model and reduced the oxidative stress . MoreoveRhus succedanea L. (Anacardiaceae) and also found in some fruits and vegetables. Fisetin proved to inhibit A\u03b2 aggregation in vivo [Fisetin is a flavonoid extracted from  in vivo  and fibr in vivo , reducin in vivo . Also, a in vivo . Additio in vivo  and redu in vivo .Oleuropein is a polyphenol present in extra virgin olive oil with antioxidant  and antiTannic acid is a polyphenol found in herbs and fruits. An in vivo experiment showed that tannic acid is a natural inhibitor of \u03b2-secretase with anti-inflammatory properties, preventing the cognitive impairment of AD mice . One in Crocin is a carotenoid mainly found in the stigma of saffron flower. In vitro experiments showed that crocin inhibits the A\u03b2 fibril formation  through Epicatechin represents one of the antioxidants from the flavonoids family. High amounts of this compound can be found in cocoa beans, green tea and grapes. In vivo data showed that epicatechin has antioxidant  and antiGallic acid is a phenolic acid present in fruits, vegetables and herbs. Gallic acid proved to have antioxidant  and antiFerulic acid is a phenolic compound naturally present in numerous fruits and vegetables. In vivo results revealed that ferulic acid is an antioxidant  and antiRutin is a bioflavonoid extracted from some vegetables and fruits. This product is a glycoside of the flavonoid quercetin with antioxidant and anti-inflammatory properties in vivo . The samSalvia miltiorrhiza Bunge (Lamiaceae) root. In vivo experiments showed a strong antioxidant and anti-inflammatory activities, improving the memory and learning of an AD mouse model [Salvianolic acid B is a phenylpropanol founded in the se model . Also, sse model . Anotherse model .Myricetin is a flavonoid extracted from several fruits, vegetables and herbs. In vitro proofs showed that myricetin prevents A\u03b2 aggregation and consequent fibrillation ,230 due Citrus junos Siebold ex Tanaka, Rutaceae. An in vitro study revealed that naringenin inhibited the APP and \u03b2-secretase activity and reduced the levels of phosphorylated tau [Naringenin is a natural compound present in citrus fruits and tomatoes. It is the major flavanone constituent found in ated tau . As resuated tau . In vivoated tau  and antiated tau .Luteolin, a polyphenol flavonoid found in fruits, vegetables and herbs, exhibits potent anti-inflammatory activity in vitro  and antiAsiatic acid is a pentacyclic triterpene found in plants. Asiatic acid demonstrates an ability to inhibit the \u03b2-secretase and increase the \u03b1-secretase activity in vitro. Also, it demonstrates an ability to activate A\u03b2 clearance , which ePueraria lobata (Willd.) Ohwi (Leguminosae) used to treat some diseases. In vivo studies found that puerarin inhibited the tau phosphorylation and reduced A\u03b2 levels, ameliorating the spatial learning and memory in an AD mice model [Puerarin is an isoflavanone glycoside isolated from ce model . The bence model  and neurce model . Oleocanthal is one of the main active components of extra virgin olive oil. In vitro evidence suggests that this compound changes the structure of tau protein, inhibiting its aggregation  and fibrVitis vinifera L., Vitaceae. In vitro evidence proved the anti-inflammatory [Viniferin (trans \u03b5-viniferin) is a polyphenol present in a variety of vines, including ammatory  and antiammatory  activitiammatory  and inhiammatory .Cornus florida L. (Cornaceae) and coconut palm Cocos nucifera L. (Arecaceae). An in vivo study showed that this compound decreases the A\u03b2 levels and inhibits the A\u03b2 aggregation, improving the memory of AD rat model [Scyllo-inositol, also known as scyllo-cyclohexanehexol, is one of the stereoisomers of inositol, found in dogwood at model . In vitrat model .Magnolia officinalis Rehder & E.H.Wilson, Magnoliaceae. In vivo evidence suggested that honokiol is an antioxidant [Honokiol is a poly-phenolic product found in ioxidant  and antiioxidant . In vivoioxidant . As resuioxidant .Apigenin is a flavonoid found in plants, fruits and vegetables. Numerous in vitro and in vivo works showed its anti-inflammatory  and antiCaffeic acid is a phenolic acid present in food, beverages and Chinese herbal medicines with antioxidant and anti-inflammatory properties in vivo. This compound improved the learning of AD rat models . In vitr\u03b2-carotene belongs to the carotenoid family. One in vitro study reported that \u03b2-carotene has an anti-aggregation activity and destabilizes A\u03b2 . AnotherRosmarinic acid is a phenolic carboxylic acid found in rosemary, lemon balm and peppermint, among others. An in vivo study proved that this compound has antioxidant properties, protecting an AD mouse model against memory deficits . Also, rLarrea divaricata Cav. (Zygophyllaceae) with in vivo antioxidant properties [Nordihydroguaiaretic acid (NDGA) is a compound found in operties . An in voperties .Cnidium monnieri (L.) Cusson (Apiaceae). An in vivo study showed that this compound significantly enhanced the memory of an AD rat model, that can be linked to its antioxidant activity [Osthole is a coumarin isolated from activity  and withactivity . Also, iactivity .Punica granatum L. (Lythraceae). An in vitro study proved that this compound inhibits of \u03b2-secretase activity preventing neurotoxicity [Ellagic acid is a polyphenol extracted from toxicity . Ellagictoxicity .Glycine betaine is an organic osmolyte, which could be isolated from vegetables and marine products. In vivo evidence revealed that glycine betaine reduces tau hyperphosphorylation and A\u03b2 production, improving memory deficits . Also, gHydroxytyrosol is a phenolic compound extracted from the olive leaf and oil. In vivo studies demonstrated that it is a compound with antioxidant and anti-inflammatory properties . Also, hl-theanine is an amino acid present in green tea. An in vivo work showed that l-theanine decreased the oxidative stress and the A\u03b2 levels [\u03b2 levels . Also, t\u03b2 levels .Alexandrium ostenfeldii and accumulate in shellfish. An in vitro study revealed that 13-desmethyl spirolide C is a spirolide that can reduce intracellular A\u03b2 accumulation and hyperphosphorylated tau levels [13-Desmethyl spirolide C is a marine compound belonging to the cyclic imine group produced by the dinoflagellate u levels . The redu levels .Hibiscus vitifolius L.  and has been reported in in vivo experiments to exhibit anti-inflammatory [Gossypin is a flavonoid found in ammatory  and antiammatory .Gynostemma pentaphyllum (Thunb.) Makino (Cucurbitaceae) and they are reported in an in vivo study to be products with antioxidant and anti-inflammatory activities, improving the cognitive impairment [Gypenosides are triterpenoid saponins extracted from pairment .O-galloyl-\u03b2-d-glucopyranose (PGG) is a polyphenol and the main constituent of the Paeonia x suffruticosa Andrews (Paeoniaceae) root, a tree peony native to China and used in traditional medicine practices. In vivo experiments proved that PGG inhibits the A\u03b2 oligomerization, which prevents A\u03b2 fibril formation, resulting in the decrease of A\u03b2 levels and improvement of memory. PGG is also able to promote the destabilization of A\u03b2 fibrils [1,2,3,4,6-Penta- fibrils . Enoxaparin is a low molecular weight heparin present in the intestinal mucosa of pigs. Enoxaparin reduced the A\u03b2 load through the decreasing of \u03b2-secretase activity . Also, eMaclura pomifera (Raf.) C. K. Schneid. (Moraceae), Maclura tinctoria (L.) D. Don ex Steud. (Moraceae) and leaves of Psidium guajava L. (Myrtaceae), promoted the inhibition of \u03b2-secretase activity in vitro [Morin, a natural flavonoid mainly found in in vitro . Besidesin vitro . Naringin is a flavonoid present in citrus fruits, namely in grapefruit. In vivo studies suggested that the antioxidant and anti-inflammatory activities of this compound improved the learning and memory of AD rats .Angelica sinensis (Oliv.) Diels (apiaceae) with antioxidant and anti-inflammatory activities in vivo. As a result, an improvement in learning and memory of AD rats was observed [Vanillic acid is a phenolic acid extracted from the plant observed .Punica granatum L. (Lythraceae)). In vivo studies suggest that punicalagin has potential as a nutritional preventive strategy in AD due to its anti-inflammatory activity. This natural product favors the anti-amylogenic route through the inhibition of \u03b2-secretase, reducing A\u03b2 levels [Punicalagin is an ellagitannin found in the fruit peel of pomegranate  Scop. (Crassulaceae) that improved the learning and memory injuries in an AD rat model due to its antioxidant activity [Rhodosin is a flavonol extracted from the root of activity . Allium sativum L., Amaryllidaceae) is frequently used in culinary and medicine. Several studies showed that the administration of aged garlic extract significantly improves the memory deficit by several pathways. In vitro studies demonstrated that aged garlic extract has antioxidant properties [Garlic  is one of the most used spices and has been traditionally applied in the treatment of some diseases and their symptoms. Cinnamon extract is found to inhibit in vitro tau aggregation and promote the disassembly of tau filaments [Cinnamon  is the source of olive oil, one of the most important ingredients in the Mediterranean diet. In vivo studies showed that extra virgin olive oil ameliorated behavioural impairments. Also, the oil reduced the A\u03b2 and phosphorylated tau levels [Olive  is a dried fruit composed by fatty acids, vitamins, alpha tocopherol, and polyphenols, in particular ellagic acid. An in vitro study showed that walnut extract inhibited the A\u03b2 fibril formation through the inhibition of A\u03b2 fibrillation, and also defibrillated A\u03b2 fibrils [Walnut  are composed by several polyphenols including catechin, epicatechin, epigallocatechin and epicatechin gallate. In vivo studies have revealed that grape seed extract increases the memory performance and reduces ROS production, thereby protecting the central nervous system [Grapes  is a fruit with a variety of antioxidant polyphenols. Pomegranate juice reduced the A\u03b2 levels and amyloid plaques in an AD mouse model, improving spatial learning and cognitive performance [Pomegranate  is a native American plant commonly used in traditional Chinese medicine. An in vivo study found that skullcap was able to protect hippocampal neurons against A\u03b2-induced damage through the attenuation of oxidative stress and neuroinflammation [Skullcap (ammation . Fragaria x ananassa (Weston) Duchesne, Rosaceae) is known to contain high phenolic contents. In vivo studies showed that strawberries have anti-inflammatory [Strawberry , an Asian and African plant, presents several nutrients, including \u03b2-carotene, vitamin C and E and phenols, including quercetin and kaempferol. In vivo studies showed that this plant improved the memory and learning due to its antioxidant activity [Moringa (activity .Besides the aforementioned natural compounds studied in human and animal studies, several other products have gained an increasing interest in scientific community for AD therapy. In fact, different compounds were tested in vitro and showed promising results. Some compounds proved to be efficient in preventing the formation of A\u03b2 aggregates and disassembling A\u03b2 fibrils, such as the case of tetracycline , methyl The reduction of A\u03b2 levels can occur through changes in the structure of A\u03b2 aggregates induced by natural compounds such as piceatannol . This prBesides these mechanisms, natural compounds can prevent AD progression by other mechanisms. For example, yessotoxin , gambierDespite the verified good outcomes, the study of some of these compounds was abandoned. For example, tetracycline was studied in 2001 but no more studies were reported for this compound. Also, for epicatechin gallate no studies were reported since 2003, and for retinal and retinol since 2004.Several bioactive compounds and natural extracts that were described herein to treat and prevent AD were revised and discussed. Until this date, most of the studied natural compounds are mainly derived from vegetable sources, with just a few molecules isolated from animals and marine organisms. Since AD is a multifactorial disorder, different therapeutic mechanisms were associated with these natural compounds. The approval process for a new compound to become clinically available is an extremely lengthy process, and it is divided into different phases. Before tests on humans, new compounds must be evaluated in preclinical studies. Several natural compounds proved to be promising for AD therapy in in vitro and in vivo studies, as discussed in this work. However, due to physiological differences between tested animals and humans, clinical trials are still necessary to validate the safety and efficacy of these compounds. Clinical studies are of outmost importance for the development of new therapeutic compounds, drugs and devices. Human studies allow to assess safety, tolerance and effective therapeutic doses for treating diseases. Some of the performed clinical trials described in this review did not show significant improvement in the delay or treatment of the symptoms. However, even if the trials do not exhibit positive outcomes, the obtained results can be still used to guide the scientists in the right path for drug discovery. Also, some of the conducted clinical trials with natural compounds for AD therapy, showed no conclusive results due to the limited size of samples. However, several compounds proved to be safe in human studies and were allowed to proceed to subsequent phases. To this date, homotaurine is the only compound that reached phase III of clinical trials for AD therapy.\u00ae, demonstrates beneficial effects in the patients. This product is already commercially available in some countries being partially financially supported by the public health care systems.Despite only a few natural products having been studied in clinical trials, numerous compounds proved to have beneficial properties in preclinical studies, as shown in Still, the neuroprotective effects of natural compounds depend of their ability to cross BBB. The low bioavailability of drugs and the difficulty to cross the BBB remains the major obstacles for the development of new therapies . Drug deAmong the studied natural compounds, only a small percentage have been encapsulated in DDS for brain targeting. Only the encapsulation of curcumin ,347,348,AD is a disabling disorder with a major negative impact on our current society. At this moment, no drugs have been developed to prevent or treat AD. The existing molecules only aim to control the symptoms. With the increase of average life expectancy, it is fundamental to discover and develop new molecules able to prevent and treat AD. Several natural products have proven to be promising for AD therapy in clinical and preclinical studies. Clinical trials have shown that several compounds appear to be effective for AD therapy, whereas others have failed in human trials. Natural compounds in earlier phases of research need further studies to uncover their therapeutic potential for AD."}
{"text": "While most studies addressed postsynaptic defects in the absence of MeCP2, we took advantage of an in vivo activity-paradigm (seizures), two models of MeCP2 deficiency, and neurobiological assays to reveal novel defects in presynaptic structural plasticity in the hippocampus in RTT rodent models. These approaches allowed us to determine that MeCP2 mutations alter presynaptic components, i.e., disrupts the plastic response of mossy fibers to synaptic activity and results in reduced axonal growth which is correlated with imbalanced trophic and guidance support, associated with aberrant expression of brain-derived neurotrophic factor and semaphorin 3F. Our results also revealed that adult-born granule cells recapitulate maturational defects that have been only shown at early postnatal ages. As these cells do not mature timely, they may not integrate properly into the adult hippocampal circuitry. Finally, we performed a hippocampal-dependent test that revealed defective spatial memory in these mice. Altogether, our studies establish a model that allows us to evaluate the effect of the manipulation of specific pathways involved in axonal guidance, synaptogenesis, or maturation in specific circuits and correlate it with changes in behavior. Understanding the mechanisms underlying the neuronal compromise caused by mutations in MeCP2 could provide information on the pathogenic mechanism of autistic spectrum disorders and improve our understanding of brain development and molecular basis of behavior.Methyl cytosine binding protein 2 (MeCP2) is a structural chromosomal protein involved in the regulation of gene expression. Mutations in the gene encoding MeCP2 result in Rett Syndrome (RTT), a pervasive neurodevelopmental disorder. RTT is one of few autism spectrum disorders whose cause was identified as a single gene mutation. Remarkably, abnormal levels of MeCP2 have been associated to other neurodevelopmental disorders, as well as neuropsychiatric disorders. Therefore, many studies have been oriented to investigate the role of MeCP2 in the nervous system. In the present work, we explore cellular and molecular mechanisms affecting synaptic plasticity events  Rett syndrome (OMIM #312750) is one of the few ASDs of monogenic origin that results in mental retardation, motor dysfunction, seizures, and features of autism. In 1999, the main cause of RTT was shown to be mutations in the MeCP2 . This prNumerous studies in animal models of RTT indicate that MeCP2 contributes to the formation and maintenance of neuronal connectivity; MeCP2 deficiency affects neuronal maturation , axonal in vitro MeCP2 regulates gene expression induced by neuronal activity; activity increases MeCP2 phosphorylation .in vivo, in real-time studies for understanding the molecular basis of behavior in several neurodevelopmental or neurocognitive disorders.We propose that this is a model to study the causal relationships between structural alterations in the hippocampus and cognitive alterations present in mouse models of RTT. These studies may lay the foundations to establish a system that allows evaluating the effect of manipulating specific pathways involved in neural connectivity in a specific circuit and correlating it with changes in behavior. Moreover, these results may serve useful for approaching We used two RTT mouse models for these studies. (1) MeCP2-308 model (MUT), these animals carry a premature stop codon at amino acid 308, generating a truncated MeCP2 protein that lacks the C-terminal region   and (2) in vivo stimulation paradigm. In the case of the KO, we used 6-weeks-old males, since the response to KA was already reported in the literature  injection of kainic acid . As a control, the same volume of sterile PBS was injected . Consideterature . It is iterature  while materature .After KA injection, seizure response was recorded, which became evident after 20 min of injection. From the moment of KA injection, the animals were individually housed in separate boxes and monitored for several hours. The severity of the seizure response was graded according to the following scale: 0, similar to control; 1, increased respiratory rate; 2, in a state of \u201cfreezing\u201d and with erratic contractions of the body; 3, straight and stiff tail with or without shaking; 4, the front legs begin to tremble; 5, straight and stiff tail along with tremor of front legs (once); 6, continuously shows straight and stiff tail along with front leg tremor (more than twice); 7, complete clonic tonic seizures, jumps; 8, death .Two weeks after the KA injection, all animals were euthanized and perfused for morphometric analysis, according to F(1.12) = 1.228; p = 0.2895] and BDNF  in response to KA stimulation. These results demonstrate that in MUT MeCP2 animals an increase in neuronal activity was induced in response to KA administration, similar in magnitude to that induced in WT animals.To determine whether the lack of IPT growth in response to seizure activity in MeCP2 MUT mice was caused by defective neural activation in response to KA, the expression of activity-induced neuronal genes (Arc and BDNF) was evaluated. For this, new groups of mice from both RTT models were injected with KA and 6 h later, whole hippocampus were dissected from control and KA-injected MUT mice and WT littermates. The expression levels of Arc and BDNF were quantified by real-time RT-PCR. As shown in p < 0.05); and the levels of KA-induced Arc and BDNF expression in these animals were similar to the WTs  . KO mice the WTs . Neverth the WTs .F = 0.2665; p = 0.6197]. A significant increase in the number of dividing cells was observed in both WT and MUT KA-treated mice in comparison with their respective controls (p = 0.8169). To confirm that DG dividing cells were neurons, we performed double labeling IHC for BrdU combined with the neuronal marker tubulin \u03b2-III (Tuj1) or with an astroglial cell marker (GFAP). Most BrdU+ cells co-localized with the neuronal marker Tuj1, but not with GFAP. +) that remained 5 weeks after the induction of seizure activity. + cells per DG 8 days and 35 days after KA-induced seizures, when the new DG cells become mature neurons. At 35 days, BrdU+ cells from WT mice decrease about 60% and in MUT 71% respect to day 8; therefore, no significant differences were detected between the genotypes in terms of BrdU+ cell loss . Also, no differences were detected in the absolute number of BrdU+/DG cells between both genotypes at either 8 (p = 0.9987) or 35 (p = 0.8383) days after KA.It has been shown that the axons of adult-born neurons in DG contribute mainly to the plasticity of the IPT tract . Likewiscontrols . In addiThese observations suggest that the survival of the new DG neurons is not affected by MeCP2 mutation; therefore, we conclude that the lack of increase in the size of the IPT, registered 2 weeks after KA injection, was not due to a loss of new DG cells in MUT mice.+ NeuN+), the percentage of immature cells (BrdU+ DCX+) and at intermediate stage of maturation (BrdU+ DCX+ NeuN+). F = 13.73; p < 0.001]; the percentage of new DG cells that reached maturity was significantly lower in MUT mice . In addition, MUT mice showed a higher proportion of both immature cells  and cells in transit to maturation . Thus, MUT adult-born granule cells show abnormal maturation; immature cells accumulate at expense of lower numbers of mature cells.Previous work in the olfactory system and in the hippocampus from MeCP2-KO mice has reported a delay in neuronal maturation during early postnatal development, which is apparently compensated later on . Given tThese results indicate that mice carrying a mutated MeCP2 protein (MUT) show defective neuronal maturation in the adult hippocampus, similar to what was observed during early development in a different mouse model .Using the olfactory system and MeCP2-deficient mice, we have demonstrated that axonal guidance defects were accompanied by alterations in the expression of Sema 3F and its receptor complex (PlexinA3 and Neuropilin-2). It has been demonstrated that this axonal guidance molecule regulates axonal growth, guidance, and targeting of hippocampal MF, acting as an inhibitor of axonal growth when combined with its receptor complex present in axons and inducing growth cone collapse . ConsideMethyl-CpG 2 binding protein 2 MUT mice and WT littermates were injected with KA or PBS and 2 weeks later the hippocampus of the different experimental groups were dissected. mRNA expression levels of Sema 3F gene and their two receptors (PlxnA3 and Npn-2) were quantified by real-time RT-PCR. F = 4.7; p < 0.05]. A significant decrease of Sema3F expression was observed in WT mice + KA compared to WT controls, while in MUT mice no change was registered between controls and KA-treated mice. Likewise, the WT + KA mice presented significantly lower levels of Sema 3F compared to the MUT KA. For PlxnA3  = 1.327; p = 0.2687]. Although there were no changes in the expression of Npn-2 in response to KA treatment  = 8.072; p < 0.05].Regarding Sema 3F , consider PlxnA3 , no signreatment , we founSummarizing these observations, the lack of increase in IPT size after KA treatment, in the context of MeCP2 mutations, was accompanied by a deregulation in the expression levels of Sema 3F which, in concert with the receptor complex, exerts an important chemo-repulsive effect on hippocampal axons .in vitro, extends innervations of MF into CA3, and regulates its synaptic plasticity  = 10.61; p < 0.01]. WT animals showed a moderate but significant increase in BDNF expression, 2 weeks after injection with KA. In contrast, in MUT mice BDNF expression remained unchanged between the control and the KA-treated group. Also, BDNF levels in MUT+KA animals were markedly lower than those of WT + KA mice  = 11.58; p < 0.01]. While WT + KA showed a significant increase of pTrkB-positive cells in comparison with the controls, no significant differences were observed between treatments in MUT mice  in the dorsal region of MeCP2 MUT hippocampus and considering that this circuitry is mainly involved in the processing of spatial memory , we deciF = 0.744; p = 0.6150] ; \u201cnon-learners\u201d were those animals that did not enter the escape box in any of the three training sessions. However, when we evaluated the escape latency of the animals that did learn, no significant differences were found in the acquisition between the groups [ 0.6150] . We founF = 14.81; p < 0.001]  = 22.7; p < 0.0001]. WT mice explored the target quadrant and the escape hole significantly more than MUT mice .in vivo, as a way to provide controlled stimulation conditions, leading to more homogeneous activity. We based our experimental design on several reports using KA injections in different Rett mouse models, considering that those studies have established most of the activity-dependent responses described in the field  model which better resembles the human pathology; these mice express a mutated protein, their clinical evolution is progressive, and they survive for over a year. This is important because it permits experiments at more advanced ages and also avoids the results may be masked by near-death physiological conditions, as occurs with most of the studies performed in MeCP2 KO models . The original characterization of this mouse model revealed that MeCP2 MUT animals develop spontaneous seizures from 5 months of age . HoweverHowever, we observed no change in the IPT volume in 9-week-old MUT mice , even th+) that remain 5 weeks in the DG after the induction of seizures activity was similar in WT and MUT mice  . HoweverOn the other hand, a body of evidence suggests that semaphorins are critical determinants of axonal growth and targeting during the development of the nervous system . Mice deIt has been suggested that the process of structural plasticity shown by MF in response to synaptic activity involves a tightly regulated balance between growth factors and axonal guidance molecules . Here weIn this study, we did not analyze the dendritic components of granule DG cells. However, in contrast to postmortem studies in RTT patients , the braper se is altered in patients, cognitive abnormalities are present in individuals with MeCP2 mutations, supporting the concept that the results obtained with the MeCP2-308 animal model would represent a correlate of cognitive deficits in RTT , which follows guidelines from the National Institute of Health.MB, MZ, and MF contributed to the acquisition, analysis, or interpretation of data for the work. SA, GVR, and GAR contributed to key techniques and critically revised the work for important intellectual content. AD contributed to the conception and design of the study, and wrote the first draft of the manuscript. All authors contributed to the manuscript revision, read, and approved the submitted version.GVR is currently employed by company Janssen Research & Development, LLC. The remaining 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."}
{"text": "Diverticulosis of the colon is the most common condition in Western societies and it is the most common anatomic alteration of the human colon. Recurrent abdominal pain is experienced by about 20% of patients with diverticulosis, but the pathophysiologic mechanisms of its occurrence are not completely understood. In the last years, several fine papers have showed clearly the role of low-grade inflammation both in the occurrence of symptoms in people having diverticulosis, both in symptom persistence following acute diverticulitis, even if the evidence available is not so strong. We do not know yet what the trigger of this low-grade inflammation occurrence is. However, some preliminary evidence found colonic dysbiosis linked to low-grade inflammation and therefore to symptom occurrence in those patients. The aim of this paper is to summarize current evidences about the role of inflammation in symptom occurrence in symptomatic uncomplicated diverticular disease and in symptom persistence after an episode of acute diverticulitis. Diverticulosis of the colon is the most frequent anatomic alteration that can be detected during colonic exploration, and its incidence is increasing worldwide. It is a common finding especially in developed countries: by the age of 85, the large majority of the population living in developed countries will have developed colonic diverticula . Recent Most people with colonic diverticulosis remain asymptomatic. Just about one-fourth to one-fifth of those people will develop symptoms without complications, experiencing the so-called \u201cSymptomatic Uncomplicated Diverticular Disease\u201d (SUDD), and about 20-25% of them will ultimately develop diverticulitis (uncomplicated or complicated) \u20136.Although advancing age is obviously associated with diverticulosis, the occurrence of symptoms is probably not only linked to age but also to the prolonged time course, during which the colonic wall is exposed and makes the colon more susceptible to other pathogenetic factors.The pathogenesis of the disease is poorly understood and several etiological factors may play a role in its onset. In this paper, we focused on the roles of inflammation as part of the pathophysiology of diverticular disease.vasa recta [Diverticulosis is characterized by the presence of sac-like protrusions of mucosa and submucosa, called diverticula, which occur in the weakest point of the muscle layer. This occurs due to age-related degeneration of the mucosal wall and segmental increases in colonic pressure resulting in bulging at points of weakness, typically at the insertion of the sa recta .Although the underlying pathological mechanisms that cause the formation of colonic diverticula remain unclear, itis likely to be the result of complex interactions between genetic factors, age, diet, changes in colonic structure and colonic motility, and therefore colonic microbial imbalance .Inflammation probably does not play any role in the occurrence of colonic diverticulosis. Two European papers, published at the beginning of this decade, found that no signs of histological inflammation were found in people having colonic diverticulosis , 8. ThesAs stated, just one-fourth to one-fifth of patients with diverticulosis develop symptoms, the so-called Symptomatic Uncomplicated Diverticular Disease (SUDD). The pathogenetic mechanisms that lead to the occurrence of symptoms are still under debate, probably because there is still no consensus about the definition forms of the disease.p = 0.04) [SUDD is characterized by nonspecific attacks of abdominal pain without macroscopic evidence of an inflammatory process, with abdominal pain, bloating, and changes in bowel habits that may resemble IBS. As a consequence, several authors still talk about \u201cIBS with diverticulosis.\u201d For example, Jung et al. found that IBS occurs 4.7-fold more likely in patients after an episode of acute diverticulitis than controls , and mor = 0.04) .It is likely that those patients are suffering from SUDD than from \u201cIBS with diverticulosis,\u201d even when we describe symptoms' occurrence/persistence following an episode of acute diverticulitis. This is because IBS and SUDD are not the same clinical entity, as showed by clinical data currently available. IBS and SUDD do not share the same epidemiology  but onlyAs it is well known, acute diverticulitis (AD) shows significant sign of inflammation . However2-related enzyme in patients with AD. In particular, they observed a downregulation of cyclooxygenase-2/PGE2 and upregulation of 15-PGDH in the human colonic mucosa that may be predisposing factors for the development of acute DD [2 may predispose to AD occurrence. Moreover, we found that AD and Crohn's disease (CD) have a similar pattern of expression of basic fibroblast growth factor (bFGF), syndecan 1 (SD1), and tumor necrosis factor \u03b1 (TNF \u03b1), supporting the hypothesis that inflammation may also activate the fibrosis process [Also, inflammation may play a role in the pathogenesis of the disease. Dai et al. showed a different expression of prostaglandin Eacute DD . Based o process .\u03b1, its overexpression in AD as well as CD is not regulated by the same gene superfamily 15 (TNFSF15), which is an immunoregulatory, antiangiogenic gene [p = 0.03) in all DD versus all control comparison. A second haplotype, rs6478108 (A), rs6478109 (G), rs7869487 (A), and rs4263839 (G), was also associated with DD in that cohort (p = 0.025). A third haplotype, rs6478108 (A), rs6478109 (G), rs7848647 (G), rs7869487 (A), and rs4263839 (G), was demonstrated in the DD < 55 years versus control > 55 years' comparison (p = 0.045). This study demonstrates clearly that distinct but overlapping TNFSF15 haplotypes can be found in diverticulitis patients versus healthy controls when compared with the known CD haplotype suggesting similar but distinct genetic predispositions [About the role of TNFnic gene , 20. A rositions . This stositions , trigger\u03b1, interleukin- (IL-) 6, and IL-1\u03b2 levels were significantly higher and histology was significantly worse in patients having symptom persistence following severe AUD  [We know that up to 20% of patients complain of persistent abdominal pain after surgical treatment of diverticulitis , and thearisons) .p = 0.0004) [Persistent low-grade inflammation following an episode of AD may also explain the recurrence of AD. At the end of a 24-month follow-up period, we found that endoscopic inflammation was still detected in 27.67% of patients and active histological inflammation in 36.6% of patients following an episode of AD. Only the detection of endoscopic and of histological inflammation during the follow-up was a predictor of diverticulitis recurrence ( 0.0004) .Inflammation persistence may be therefore a risk factor for both symptoms' persistence and disease recurrence. Despite this, a recent review found mesalazine, an anti-inflammatory drug widely used in the treatment of Inflammatory Bowel Diseases, not superior to placebo in preventing diverticulitis recurrence . It is pAs stated, diverticulosis is merely the presence of colonic diverticula without symptoms or macroscopic/microscopic signs of inflammation. In this way, no signs of inflammation should be detected, both at endoscopic and also at histological assessment.A different scenario may be observed when we analyze patients with SUDD. In those patients, low-grade inflammation seems to really play a significant role in determining symptom occurrence and complication. The current evidences are as follows.p < 0.02).SUDD shows a significant microscopic inflammatory infiltrate. More than ten years ago, Narayan and Floch found a significant inflammatory infiltrate in SUDD patients compared with healthy controls . We asse\u03b1, a 17\u2009kDa polypeptide produced by macrophages, lymphocytes, and natural killer cells, has been shown to play a major role in the inflammatory process, with high levels being found both in Inflammatory Bowel Disease (IBD) and in rheumatoid arthritis patients [\u03b1 was assessed in those patients yet.TNF-patients \u201333. Hypo\u03b1 expression was significantly higher not only in AUD than in HC (p = 0.0007) but also in SUDD than in HC (p = 0.0007) and in asymptomatic diverticulosis (p = 0.0001). Moreover, TNF\u03b1 expression in AUD did not differ significantly from that of UC (p = 0.0678) and SCAD (p = 0.0610). Finally, it was significantly higher in UC, SCAD, and AUD than in SUDD  [\u03b1 expression was significantly higher in SUDD than in asymptomatic diverticulosis (p = 0.04) [Assessing its mucosal expression on biopsy samples by reverse transcription polymerase chain reaction, we found that TNFctively) . The samp = 0.067 and SCADp < 0.05). Moreover, they found a slight increase of IL-10 release in SUDD and a significant IL-10 release in SUDD-AD than HC (p < 0.05) [p = 0.326) [IL-10 is an important anti-inflammatory mediator that suppresses the production of proinflammatory cytokines . Surpris < 0.05) . We rece= 0.326) .2 in colonic mucosa between acute diverticulitis and IBD [2 is the dominant prostaglandin in the colon and it is associated with colonic inflammation, but we also know that it has a protective mechanism in the mucosa of the gastrointestinal tract. Lower prostaglandin E2 level may decrease the normal protection of the mucosa, which is made more susceptible to luminal insults, thus creating a permissive environment for the development of acute diverticulitis.Thus, we can speculate that IL-10 expression in SUDD may be an attempt of the immune system to control a low-grade inflammation. This hypothesis seems to be confirmed by Dai et al., who found an inverse expression of prostaglandin E and IBD . We knowFecal calprotectin (FC) is a cytoplasmic antimicrobial compound prominent in granulocytes, monocytes, and macrophages, and it accounts for approximately 60% of the total cytosolic protein. It is released from cells during cell activation or death, and it is stable in feces for several days after excretion . This hap = n.s.). Higher FC values were found in AUD (p < 0.0005) and SUDD (p < 0.005) than in HC and IBS patients. Moreover, FC values correlated significantly with inflammatory infiltrate (p < 0.0005) and decreased to normal values after treatment both in AUD (p < 0.0005) and in SUDD (p < 0.005) [After a preliminary report describing an increase of FC expression in patients with \u201csymptomatic diverticular disease\u201d (no more information were provided by authors) , we perf< 0.005) . Althoug< 0.005)  and doesSeveral clinical trials in SUDD patients found 5-aminosalicilic acid  \u201345 effecSome studies tried to show that these are flaw evidences, since patients having diverticulosis and chronic abdominal symptoms do not show any evidence of inflammation . HoweverLow-grade inflammation may also interact with other pathogenetic mechanisms to lead symptom occurrence.p = 0.01); a higher but not significant expression of galanin 1 receptor (GALR1) in SUDD than asymptomatic patients was found [\u03b1, and an upregulation of the neuropeptide receptor NK1, suggests that SUDD patients may exhibit visceral hypersensitivity due to peripheral sensitization with both inflammatory and neurochemical factors playing a role.For example, visceral hypersensitivity has been suggested as a possible mechanism involved in causing symptoms in those patients , 49. Humas found  as well.\u03b2, Cossais et al. found very recently that S100\u03b2 expression was increased in the submucosal and myenteric plexus of patients with DD compared with that in controls, whereas the expression of other glial factors remained unchanged. Moreover, this S100\u03b2 overexpression was correlated to CD3+ lymphocytic infiltrates in patients with DD, whereas no correlation was observed in controls [Recent studies demonstrated that DD is associated with peculiar alterations of the enteric nervous system (ENS), encompassing myenteric oligoneuronal hypoganglionosis, decreased intramuscular nerve fibers, and altered neurochemical coding \u201353. Analcontrols .Proteobacteria than controls [Microbacteriaceae were the most significant species associated with the disease [Akkermansia muciniphila, a mucolytic bacteria living in the mucus barrier that degrades mucin for utilization as a carbon and nitrogen energy source [Roseburia hominis [A. muciniphila and R. hominis have a significant abundance in SUDD [It has been hypothesized that microbial imbalance, which is known as dysbiosis, may be the trigger of inflammation (and therefore symptoms) in people having diverticulosis . Danielscontrols , and Sch disease . Microbiy source  and prody source , is signy source . The sam hominis . In SUDD in SUDD \u201365, and  in SUDD \u201365.Diverticulosis is a common condition that sometimes becomes symptomatic and may lead to severe complications.The pathophysiologic hypotheses behind symptom occurrence in those patients have changed in the last years. Although most of the mechanisms leading to its occurrence remain to be elucidated, the role of low-grade inflammation seems to be a proven fact (Why inflammation occurs in only a minority of people having diverticulosis is still under debate, and it has to be further elucidated."}
{"text": "Lactation is at one point perilously near becoming a cancerous process if it is at all arrested\u201d, Beatson, 1896. Most breast cancers arise from the milk-producing cells that are characterized by aberrant cellular, molecular, and epigenetic translation. By understanding the underlying molecular disruptions leading to the origin of cancer, we might be able to design novel strategies for more efficacious treatments or, ambitiously, divert the cancerous process. It is an established reality that full-term pregnancy in a young woman provides a lifetime reduction in breast cancer risk, whereas delay in full-term pregnancy increases short-term breast cancer risk and the probability of latent breast cancer development. Hormonal activation of the p53 protein (encode by the TP53 gene) in the mammary gland at a critical time in pregnancy has been identified as one of the most important determinants of whether the mammary gland develops latent breast cancer. This review discusses what is known about the protective influence of female hormones in young parous women, with a specific focus on the opportune role of wild-type p53 reprogramming in mammary cell differentiation. The importance of p53 as a protector or perpetrator in hormone-dependent breast cancer, resistance to treatment, and recurrence is also explored.\u201c Breast cancer is endemic worldwide, with a cumulative lifetime risk of 1:8 by age 70 ,3,4. AboTP53 gene) and female hormone regulation during the differentiation of the mammary gland in early and late pregnancies, which influences a woman\u2019s susceptibility to breast cancer later in life [There is a very strong association between the function of the p53 tumor suppressor  and post-genetic alterations  during pregnancy are strongly implicated in breast cancer development and protection .Late-onset breast cancers are more likely to originate from ER\u03b1-positive luminal epithelial cells, or milk-producing cells, which have been exposed to years of fluctuating hormonal changes from puberty to pregnancy and menopause . As mentN-methylnitrosurea (MNU) or oral 7,12-dimethylbenz(a) anthracene (DMBA), with a near 100% latency period between 8 and 12 weeks [There is irrefutable, reproducible evidence from human studies and in rodent models that full-term and multiple pregnancies have a lifetime protective effect against breast cancer ,31,32. R12 weeks . Pioneer12 weeks ,39). Dur12 weeks . In preg12 weeks . An inte12 weeks , found t12 weeks ,42,43. A12 weeks . This ob12 weeks . ChromatFrom early studies in rodents, it was unclear whether completion of full differentiation per se (in the absence of estrogen and progesterone) reduced breast cancer risk. A few studies tested whether induction of full differentiation of the mammary gland was indeed the basis of protection against carcinogenicity ,14,45. PEarly studies, discussed in Breast tumors in rodents possess high similarity to ER\u03b1-positive and progesterone receptor (PR)-positive human breast tumors. Thus, rodent models have been key in our understanding of breast cancer development ,43. As eStudies, dating back to 1962, demonstrated that the female hormones estradiol-17\u03b2 and progesterone administered to rats and mice in doses that mimicked pregnancy reduced the incidence of carcinogenicity and conferred long-term cancer protective effects; however, in these studies neither hormone individually was protective ,16,17,18In a study treating rats with estradiol-17\u03b2 and progesterone for 21 days to mimic the parous protective effects against MNU, a set of 100 markers were identified, displaying persistent changes (upregulated) in the mammary gland compared to virgin rats . One of In general, the consensus from these studies, and many more not cited, is that short-term administration of estrogen and progesterone, that mimic pregnancy, can reduce malignant tumor formation and breast cancer latency. These changes are accompanied by persistent changes in the cellular and molecular profiles of the mammary cells, making the mammary gland less vulnerable to carcinogens or carcinogenic insults.Early studies by Russo and others suggested that human placental hormone chorionic gonadotropin (hCG) provided an inhibitory action, leading to reduction in breast cancer risk, as reviewed in reference . This loOverall, these studies are illustrative of many other works that explore the influence of the female hormone flux in pregnancy on breast cancer risk. They demonstrate that short-term administration of female hormones, whether it be estrogen and progesterone or hCG to induce mammary gland differentiation, has promise as a therapeutic protective intervention for breast cancer latency. Nevertheless, the challenge is when and how to administer preventative hormones. One of the major questions which still remains unanswered is: what are the molecular changes that govern persistent alterations in hormone-dependent signaling pathways that occur in young parous females and reduce lifetime breast cancer risk? The answer may be in p53\u2019s function, as p53 is believed to be implicated in a number of steps relating to the risk and development of breast cancer.TP53 gene) has stereotypically been widely researched as the ultimate tumor suppressor protein important in driving cancer cell death through apoptosis or permanent irreversible cell senescence [For decades, the p53 protein , the homolog of the human p14ARF protein, led to immortalization of mammary epithelial cells . p19ARF A major breakthrough in supporting the role of p53 in governing parity-related changes in the mammary gland came from p53-null mammary gland transplantation studies . By tranA quote from Beatson\u2019s seminal paper on carcinoma of the mamma (1896) states: \u201ca reasonable ground for thinking the active processes seen in a cancerous tumor are best explained by regarding the epithelium of the part as having taken on the powers of the germinal epithelium\u201d . A likelThe mammary cancer stem cell theory is based on a model whereby transformation of early progenitor cells or stem cells eventuate in latent tumorigenesis. A central event in the development of cancer is the deregulation of normal stem cell self-renewal, eventually leading to uncontrolled proliferation, aberrant differentiation, and generation of a heterogeneous breast cancer cell population ,136. AltWild-type p53 protein regulates p53 transcriptional programs in the repair of genetic alterations and also by preventing epigenetic abnormalities ,137. TovA major difference in the molecular characteristics of normal breast epithelial cells and breast cancer cells is that the majority (approximately 75\u201385%) of normal breast cells do not express ER\u03b1 and PR, whereas the majority of breast cancer cells contain high levels of both hormone receptors (ER\u03b1+PR+ cells) . Normal In this section, we review the mounting evidence that p53 and ER activities are mutually regulated in breast cancer. Conceptually, a question worth asking is: is the loss of, or change in, p53 function associated with the signature of \u201cparacrine-to-autocrine\u201d response in the transition from normal to breast cancer cells? In This review would be amiss if we did not comment on the seminal findings of the epic Million Women Study by Beral and colleagues, investigating the use of hormone replacement therapy (HRT) in postmenopausal women . In thisTP53 gene that disrupt cell cycle inhibition are associated with the onset of more than 80% of all cancers [TP53 gene are not the main culprits in breast cancer development and progression. The majority of breast cancers occurring later in life  are hormone-responsive (ER\u03b1-positive) and, interestingly, harbor functional wild-type TP53 [The p53 tumor suppressor is intimately associated with inhibition of cancer and, in general, mutations in the  cancers ,148,149. cancers . This obype TP53 . In manyype TP53 ,153. Resype TP53 ,156,157.As mentioned in Although reactivation of the p53 signaling pathway has been considered as a good breast cancer treatment strategy, there is growing evidence at the molecular level to suggest an association between ER\u03b1 and p53, which protects breast cancer cells from dying ,162,163.Our laboratory has demonstrated that activation of the p53 pathway in ER\u03b1-positive breast cancer cells rapidly, within hours, shuts down DNA replication, downregulates proteins involved in apoptosis, and increases the levels of proteins that are involved in normal breast cell metabolism ,97,98. OER\u03b2 [\u03b1 and ER\u03b2 knockout (KO) mouse models [\u03b1 and ER\u03b2 KO mouse models, ER\u03b1 was shown to be essential for normal reproductive development [\u03b2-KO mice had decreased side branching and partial alveolar development, the mammary glands secreted milk normally, and mothers were able to nurse their young [In 1997, a second ER was identified, named ER\u03b2 ,169. TheER\u03b2 . The majER\u03b2 . Each ofe models . In seleelopment . In contir young . Unlike ir young . Conventir young . Alternair young . In two ir young ,178. Thuir young . Thus, tIn essence, the role of p53 as a protector or perpetrator in breast cancer may very well be dependent on the ratio of ER\u03b1 and ER\u03b2 in breast cancer. In turn, the crosstalk between ER\u03b1, ER\u03b2, and p53 has the potential to affect hormone-dependent breast cancers responsiveness to endocrine and other breast cancer therapies ,178. Beatson\u2019s theory from his doctorate dissertation was that lactation was very close to being a cancerous process and, if arrested in its progression, could become carcinogenic, as cited in reference . In otheMCF-7 cells are derived from the luminal breast epithelial, which consists of cells that differentiate to become milk-producing cells. This cell line has provided vital information on hormone regulation of breast cancer and treatments to combat this disease. We have recently demonstrated that activation of p53 in MCF-7 breast epithelial cells initiated changes in cell morphology and cellular metabolism consistent with a differentiated phenotype and was most likely to be important in cell survival and recurrence in ER\u03b1-positive breast cancer cells ,97,98. TOur studies and the work of other groups have strongly suggested that re-activation of the p53 signaling pathway is strongly implicated in chemotherapy- and endocrine therapy-resistant breast cancers, especially in the most prevalent breast cancer sub-type (ER\u03b1+/p53+) ,98. ActiThe theory that the p53 protein suppresses cell cycle progression, induces resistance to apoptosis, and maintains the breast cancer cells viable through metabolic reprogramming ,98 is anIn summary, we do not know the extent of the function of p53 in the origin of latent hormone-responsive breast cancers, and this review does not delve into the many complexities of the normal p53 function. What is emerging from these cited reports and others is that p53 expression at a critical timing in young age pregnancy is pivotal in preventing mammary gland tumorigenesis. The underlying associated molecular changes could be permanent reprogramming of cellular events and/or DNA methylation, imprinting a protective signature and maintaining normal cell homeostasis. With older age pregnancies or in nulliparous women, accumulation of DNA mutations or other aberrations in the mammary gland abrogate this protective signature, and the cells are more susceptible to carcinogenic agents. Hormonal activation of the p53 in the mammary gland is implicated in the development of latent breast cancer. Confidence in the protective effect of p53 comes from p53null rodents, where, in the absence of p53, spontaneous tumors are found in the mammary glands, independent of age, as summarized in How can we utilize this knowledge for more efficacious treatments and to prevent recurrence and metastasis? While the tumor suppressor role of p53 in breast cancer treatment is well recognized, the evidence supporting an opposite action of p53 in treatment resistance and recurrence in breast cancer is not as clear. Breast cancer recurrence may well be a consequence of the heterogeneous nature of the initial tumor, confounded by the change in hormone signaling from a paracrine to an autocrine proliferative response upon estrogen exposure. Breast cancer cells have a \u201cmemory of self\u201d, and investigations in hormone-responsive breast cancer cells after p53 activation show characteristics of aberrant mammary differentiation, followed by dormancy, mediated by p53 activation. Further studies into how p53 protects breast cancer cells from cell death by switching cellular metabolism, downregulating apoptotic proteins, and initiating a cellular differentiation phenotype reminiscent of the normal mammary function is key to our understanding of recurrence in latent breast cancer development and a basis for the design of more efficacious treatments. We need to look to the past to move on to the future and we now have the technology to do so."}
{"text": "CDK2NA protein (p16) immunostaining has been found to be prognostic for human breast cancers, this marker has never been evaluated as a prognostic marker for canine neoplasms. In the present study, the prognostic utility of p53 and p16 was evaluated in 35 canine malignant MGTs. It was observed that 19 (54%) dogs died due to their MGTs with an overall mean survival time (MST) of 882 days. Seven MGTs showed p53 immunostaining, but this was not significantly associated with death  or MST . Five dogs had MGTs with no p16 immunostaining, 28 MGTs had intermediate p16 immunostaining, and two MGTs had increased p16 immunostaining. Neither death due to MGT  nor MST  were significantly associated with p16 immunostaining. Interestingly, p53 immunostaining was significantly associated with an increase or loss of p16 immunostaining. This is the first time that p16 has been evaluated as a prognostic marker for canine neoplasms. While these results suggest that a proportion of canine MGTs develop by cellular mechanisms that alter both p53 and p16 expression, there was no evidence that defects in p53 or p16 alter the behavior of a MGT. Neither p53 nor p16 was found to significantly predict prognosis, although this could reflect the limited number of MGTs included in the study.Mammary gland tumors (MGTs) are common in dogs and show a variable clinical behavior that is difficult to predict. Currently, few immunohistochemical markers have been established to predict the prognosis of a canine MGT. However, p53 immunostaining has been variably reported to be prognostic for canine MGTs. Additionally, while p16 Mammary gland tumors (MGTs) are common in intact female dogs, and 40%\u201350% of these neoplasms are malignant . The bioOne marker that has previously been evaluated in human and canine MGTs is p53. The p53 protein is considered to be the \u2018guardian of the genome\u2019 as it is responsible for detecting DNA damage and triggering apoptosis of damaged cells. Despite the critical role of this protein in cancer biology, studies in humans have typically found only weak associations between prognosis and p53 expression within breast cancers . FurtherCDKN2A protein (p16) is a tumor suppressor protein that prevents cell cycling by regulating the function of the retinoblastoma (pRb) protein [The p16 protein . Cells n protein . Mutatio protein . While l protein ,12. In d protein , althougDue to current uncertainty regarding the prognostic significance of p53 immunostaining for canine MGTs, the ability of p53 immunostaining to predict prognosis was evaluated in a series of canine malignant MGTs. Additionally, the association between p16 immunostaining and prognosis was also investigated in these canine MGTs. This is the first time that p16 immunostaining has been evaluated as a prognostic marker for a canine neoplasm.The databases of two diagnostic laboratories  were searched for cases of canine malignant MGTs for which a diagnostic sample had been submitted for histology. Cases that had been received during 2012\u20132015 were selected, and surveys were sent to the submitting veterinarians in January 2018. Information requested on the surveys included the treatments that had been used, whether or not the dog had died due to the MGT, and the survival time of the dog. Cases were excluded from the study if the dog had received additional treatments aimed at curing the MGT, although dogs that received palliative treatments, such as anti-inflammatories or antibiotics, were included.Histological sections were evaluated from all cases to confirm the diagnosis of malignant MGTs, and neoplasms were subclassified according to the classification scheme proposed by Goldschmidt et al. .Immunohistochemistry was performed to detect p53 and p16 as previously described ,16. A moCanine MGTs were classified as p53-positive if 20% or more of the cells contained nuclear immunostaining and as p53-negative if less than 20% of the neoplastic cells demonstrated p53 immunostaining. The MGTs were classified as p16-positive, p16-negative, or p16-intermediate. Neoplasms that were p16-positive had intense nuclear and cytoplasmic immunostaining in over 80% of the neoplastic cells. Neoplastic cells within p16-negative MGTs did not contain any significant p16 immunostaining, while p16-intermediate MGTs had low to moderate intensity of p16 immunostaining that was confined to the cytoplasm in over 10% of the cells. The thresholds used to classify the immunostaining for both antibodies were based on the thresholds used most frequently in humans, and more limitedly, in veterinary literature.Differences between groups were investigated by Pearson chi-squared or Fisher\u2019s exact tests and survival times were investigated using Kaplan\u2013Meier and Cox regression analyses using IBM SBSS Statistics v25 .A total of 35 malignant MGTs were included in this study. These included five that were diagnosed in 2012, 12 in 2013, 8 in 2014, and 10 in 2015. The MGTs were subclassified into 8 different histological types. The tumors included 17 simple carcinomas, which were further subclassified into tubular, tubulopapillary, cribriform, and cystic papillary carcinomas .p = 0.73).A total of 28 of 35 (80%) MGTs were p53-negative a, while p = 0.16).Of the 35 canine malignant MGTs, 28 (80%) were p16-intermediate and were characterized by showing a pattern and intensity of immunostaining that was similar to the immunostaining within surrounding non-neoplastic epithelium. In contrast to the p16-positive MGTs, immunostaining in the p16-intermediate MGTs was strictly cytoplasmic. Five (14%) MGTs were p16-negative c, and twp = 0.001).Canine MGTs that were p53 positive were significantly more likely to have altered p16 immunostaining, with only 2 of 7 (29%) p53-positive MGTs being classified as p16-intermediate. In contrast, 26 of 28 (93%) of p53-negative MGTs were classified as p16-intermediate (p = 0.17). Of the dogs with p53-positive MGTs, 4 of 7 (57%) died of their MGTs. This was not significantly different from the dogs with p53-negative MGTs of which 15 of 28  died due to their MGTs. A total of 50% of dogs with p16-intermediate and p16-positive MGTs died due to their MGTs. While 4 of 5 (80%) of dogs with p16-negative MGTs died due to mammary cancer, this difference was not statistically significant (p = 0.46). When MGTs with altered p16 immunostaining (either increased or decreased) were considered as a group, 5 of 7 (71%) of these dogs died due to their MGTs, which was not significantly higher than dogs with p16-intermediate MGTs .A total of 19 (54%) dogs died of their MGTs in this study. Seven dogs died of other causes, and nine dogs were still alive in January 2018. Of the dogs that remained alive at the end of the study, the minimum follow-up time was 807 days after diagnosis. There were no significant differences in survival rates between the different histological types of mammary gland neoplasms (p = 0.57). There were no significant differences in the MST of dogs with p16-positive MGTs , p16-negative MGTs , or p16-intermediate MGTs . When dogs with p16-positive or p16-negative MGTs were considered as a single group, these dogs had a MST of 620 days (95% CI 372\u2013864), which was not significantly shorter than dogs with p16-intermediate MGTs .The overall estimated mean survival time (MST) of the 35 dogs with malignant MGTs in this study was 882 days . Likewise, the MST of the dog with the p16-positive simple carcinoma (131 days) was not significantly different from the MST of the three dogs with p16-negative  or the 9 dogs with p16-intermediate carcinomas .To reduce the potential confounding effect of comparing immunostaining in types of carcinomas that have previously been shown to have different survival times, associations between immunostaining and survival were evaluated in just the 13 dogs with simple carcinomas. In these dogs, the MST of the three dogs with p53-positive simple carcinomas  was not significantly different from the MST of the 10 dogs with p53-negative carcinomas  , and 12 There were no significant differences in the survival rates or the MSTs between dogs with MGTs that were p16-negative, p16-intermediate, or p16-positive. However, a potential role of p16 as a prognostic marker cannot definitively be excluded, as dogs with p16-negative MGTs had the lowest survival rates and there appeared to be a trend for dogs with either p16-negative or p16-positive MGTs to survive for a shorter time than dogs with p16-intermediate MGTs. Similarly, in humans, breast cancers that were p16-positive were reported to have a less favorable prognosis. It is possible that significant differences between the groups were not detected in the present study because of the small number of either p16-negative or p16-positive MGTs that were included. The small number of p16-negative MGTs in the present study (14%) was unexpected, as a previous study had detected loss of p16 immunostaining in 24 of 79 (30%) of canine MGTs . In the In the present study, three distinct patterns of p16 immunostaining were recognized in canine MGTs. The most common pattern consisted of faint to moderate immunostaining that was confined to the cytoplasm. As this p16-immunostaining pattern is similar to that seen in normal epithelium, this staining pattern was considered to represent an intact pRb-p16 pathway. The high proportion of MGTs with this immunostaining pattern suggests that most neoplasms develop without developing mutations within the pRb-p16 pathway. The second pattern was seen in five MGTs and consisted of an absence of p16 immunostaining within the neoplastic cells. This pattern is consistent with the presence of mutations within the p16 gene preventing protein production. Finally, the least common pattern was intense cytoplasmic and nuclear immunostaining in almost all neoplastic cells. Evidence from human oral SCCs suggests that these neoplasms had mutations that prevented the production of pRb . As p16-Canine MGTs with missense mutations in p53 were significantly more likely to also have mutations that disrupted the normal pRb-p16 pathway. It is uncertain why mutations in p53 would be predisposed to additional mutations in pRb or p16; however, the results of the immunostaining in this study suggest that most MGTs develop without missense mutations in p53 or a disrupted pRb-p16 pathway. A smaller subset of MGTs develop as a result of disruption to both pRb-p16 and p53.In the present study of 35 canine malignant MGTs, the histological type was not found to be prognostic. This is in contrast with an earlier study of 229 canine MGTs that revealed a prognostic significance of histological type [As significant differences in survival times between MGTs of different types have been reported , the proFor a prognostic marker to be clinically useful, it has to be shown to consistently predict prognosis when used by multiple different pathologists in multiple different diagnostic laboratories. In veterinary pathology, many prognostic grading schemes and other markers have been proposed, but few have been shown to consistently predict prognosis when widely used . A commoThe results of this study show that canine MGTs demonstrate variable p53 and p16 immunostaining. However, these variations do not appear to influence the biological behavior of the neoplasm sufficiently to significantly alter survival rates or MSTs of the affected dogs. This study is the third to investigate p53 immunostaining as a prognostic marker in canine MGTs and the second to suggest that p53 may not be strongly predictive of prognosis for these neoplasms. This is the first time that p16 has been evaluated as a prognostic marker for canine MGTs."}
{"text": "In recent years, great interest has been devoted to finding alternative sources for human stem cells which can be easily isolated, ideally without raising ethical objections. These stem cells should furthermore have a high proliferation rate and the ability to differentiate into all three germ layers. Amniotic fluid, ordinarily discarded as medical waste, is potentially such a novel source of stem cells, and these amniotic fluid derived stem cells are currently gaining a lot of attention. However, further information will be required about the properties of these cells before they can be used for therapeutic purposes. For example, the risk of tumor formation after cell transplantation needs to be explored. The tumor suppressor protein p53, well known for its activity in controlling Cell Prolif.eration and cell death in differentiated cells, has more recently been found to be also active in amniotic fluid stem cells. In this review, we summarize the major findings about human amniotic fluid stem cells since their discovery, followed by a brief overview of the important role played by p53 in embryonic and adult stem cells. In addition, we explore what is known about p53 in amniotic fluid stem cells to date, and emphasize the need to investigate its role, particularly in the context of cell tumorigenicity. Oct4, Nanog, c-Myc, and Klf4 [Stem cells are present throughout the embryonic development and in adult organs. A coordinated control of stem cell self-renewal and differentiation is essential for maintaining tissue and organ homeostasis. Since 1980, a huge amount of work has been invested into the analysis of embryonic stem (ES) cells. These cells show incredible features like i) the ability to be expanded in vitro while maintaining an undifferentiated state, and ii) pluripotency, which means that the cells possess the capability to differentiate into every cell type of the adult body [and Klf4 . They deand Klf4 . A seconand Klf4 . To overPerinatal stem cells can be derived from postembryonic tissues, which include the tissues sourced at the time of birth, but also comprise the time period from the 16th week of gestation through the neonatal period ,5. TheseThe amnion encloses the amniotic cavity containing the amniotic fluid, a protective and nutrient-containing liquid for the developing fetus . It is mOct4, Klf4, Sox2, Lin28, and c-Myc to induce a pluripotent state, and then differentiated into functional cardiomyocytes using inhibitors of glycogen synthase kinase 3 (GSK3) and Wnt [By using various factors and methods, AFS cells can also be reprogrammed into iPS cells ,24,25. HThe lack of tumorigenesis after transplantation is an interesting feature of AFS cells, although no information is available regarding the underlying mechanisms. Important clues could be gathered by investigating in AFS cells the activities and functions of crucial cell cycle regulators, like the tumor suppressor gene p53.p53 is one of the most well-known and most intensively investigated tumor suppressor proteins. A lot of work has already been done on investigating the role of p53 in ES cells and other adult stem cells and it has been established that apart from its traditional tumor suppressor function, p53 is also reported to be involved in controlling Cell Prolif.eration, self-renewal, and differentiation of stem cells . Amniocentesis is routinely performed between 16\u201318 weeks of pregnancy. The collected amniotic fluid is used for prenatal genetic testing and as a source of AFS cells. Different approaches and protocols have been used to isolate AFS cells ,31. ThesBased on morphological and growth characteristics, human amniotic fluid cells are grouped into three main categories: i) amniocytes or amniotic fluid specific AF-type cells; this type of cell comprises about 60% of the amniotic fluid cells. ii) Epitheloid E-type cells; this type of cells make about 34% of the amniotic fluid cells. iii) Fibroblastic F-type cells; about 6% of the amniotic fluid cells belong to this type of cells ,40,41. TApproximately 1% of the AFS cells express the surface antigen c-kit CD117), a receptor tyrosine kinase type III, which plays a role in cell survival, proliferation, and differentiation . Some au, a recepAFS cells maintain a normal karyotype and stable telomeres when taken into culture, even after over 200 population doublings , yet othOne of the most important qualities of AFS cells is that they are non-tumorigenic when transplanted into immunocompromised mice . AlthougOct4, Nanog, and Sox2 [Likewise, AFS cells also do not form teratomas after in vivo transplantation . Even 11and Sox2 . Since the discovery of AFS cells, several groups have been exploring their differentiation potential. Even before it was known that the amniotic fluid contains Oct4-positive stem cell-like cells, Streubel and coworkers had demonstrated the expression of skeletal muscle proteins when amniotic fluid cells were cultured in the supernatant of rhabdomyosarcoma cell lines . After tGuan and colleagues showed that undifferentiated human AFS cells express several cardiac genes , acquire a cardiomyocyte-like phenotype after 24-h incubation with 5-aza-2\u2032-deoxycytidine (5-AZA-dC) and develop mechanical and electrical connections with neonatal rat cardiomyocytes when co-cultured . Rat AFSAFS cells have furthermore been demonstrated to form embryoid bodies when cultured in suspension, accompanied by a decrease in the expression of the pluripotency stem cell marker Oct4 and nodal, a marker for the self-renewal potential of ES cells, followed by the induction of differentiation markers like Flk1  for endothelial cells, E-cadherin for epithelial cells, Pax 6 (paired box protein 6) for ectodermal cells, TBXT (T-box transcription factor T) and HBE1 (hemoglobin subunit epsilon 1) for mesodermal cells, and GATA4 for endodermal cells . Similar to ES cells and iPS cells, AFS cells are capable of differentiating into primordial germ cells when cultured under appropriate culture conditions and through formation of embryoid bodies ,71. ThusIn summary, the enormous efforts in the past years by various groups to exploit and investigate differentiation of AFS cells have yielded positive results and demonstrate that amniotic fluid cells have a broad differentiation potential.mdm2 gene and activates its transcription, and the resultant Mdm2 binds to p53, blocks its activity, and mediates the proteolytic degradation of p53 [Functionally, p53 is a transcription factor that elicits its cellular activity mostly through transcriptional activation of target genes. However, besides its primary function as a transcription factor, p53 can also promote apoptosis independent of transcription via direct interaction with pro- and anti-apoptotic proteins . Pertainn of p53 . As a man of p53 , an Mdm2n of p53 . This Mdn of p53 . Addition of p53 . COP1 ann of p53 .p53 is part of a multiple gene family that also includes p63 and p73, as well as several splice variants of the three proteins ,83. p53 The role and effector functions of p53 however are context dependent and such context dependence is affected by cell type, genetic background of the cell, cell microenvironment, and nature of stress the cell is undergoing. Because p53 is such a critical cellular protein, multiple mechanisms have evolved to modulate its activity. The existing strategies consist of manipulating p53\u2019s transcriptional or translational control by altering the level of p53 mRNA, by changing its posttranslational modifications, by altering p53\u2019s half-life by freeing wild type p53 from MDM2, and by altering p53\u2019s intracellular localization . Bax and Puma resulting in apoptosis [Nanog and Oct4 after DNA damage, thus evoking differentiation of these cells [Similar to differentiated cells, p53 becomes activated in stem cells in response to DNA damage, where p53 activates the promoters of its canonical target genes including poptosis . In addise cells ,90,91. BHowever, p53 is not only active in stem cells after DNA damage. A few years ago, it was discovered that p53 is also active in non-stressed stem cells. Yet, the anti-proliferative activity of p53 is compromised in these cells and p53 instead directs a transcriptional program that is highly reminiscent to that of tumor-derived mutant p53 . More recent studies implicate p53 also into the plasticity of stem cells. Here, p53 seems to be involved in the regulation of the self-renewal of stem cells as well as for the onset of differentiation . The ememiR-34a and miR-145. The latter one operates as a direct repressor of the pluripotency factors Oct4, Klf4, Lin28A, and Sox2 upon retinoic acid-mediated differentiation of ES cells [\u2212/\u2212 human ES cells further confirmed the suppressive activity of p53 for stem cell pluripotency [The first evidence for a role of p53 in embryos dates back to the 1980\u2019s, when this protein was found highly expressed in primary cell cultures obtained from 12\u201314 day old mouse embryos but not in 16-day-old mouse embryos . A year ES cells . LikewisES cells . Maimetsipotency ,105,106.ipotency . Silenciipotency . A very ipotency . They shipotency . miRNA-34 family [In adult stem cells including neural and hematopoietic stem cells, p53 negatively regulates proliferation and self-renewal and helps to maintain the quiescent state ,110. Mor4 family . Further4 family . While the role of p53 has been investigated in ES cells and adult stem cells as described above, only little was known about the regulation and function of p53 in AFS cells in the past. Recently, our group investigated p53 protein levels, p53 localization, and p53 activity during Cell Prolif.eration, differentiation, and during the DNA damage response in human AFS cells . Earlier reports showed both constant stable expression and also downregulation of p53 mRNA during long term passaging of AFS cells and AF mesenchymal stromal/stem cells ,113,114.p21 and mdm2 [When we treated AFS cells with retinoic acid to induce neural differentiation, we observed an increase in p53 protein levels  which isand mdm2 . In addiand mdm2 .AFS cells are emerging as an alternative source of stem cells with promising advantages for disease modeling and applications in regenerative medicine, although there are still questions to address regarding their safety and potential, and p53 may play important roles in these processes. As the amniotic fluid contains a heterogeneous population of cells, in many studies, mixtures of unselected, non-clonal cells have been used, causing conflicting results and uncertainty regarding the identity of the cell population employed. However, stem cells within the amniotic fluid do possess a high proliferation rate, clonogenicity, and a broad differentiation potential ranging from multipotent cells to unipotent committed progenitors. The therapeutic potential of AFS cells has been shown in experimental models of diseases of the lung, heart, skeletal muscle, bone, cartilage, kidney, blood and nervous system. Since AFS cells are primary cells of a very early stage of human development, these cells are also interesting as a model system for basic research. Finally, although AFS cells have profiles similar to ES and adult stem cells, it is quite surprising why these cells are not tumorigenic when transplanted into immune-compromised animals. p53, which is already known to be an important tumor suppressor protein in pluripotent stem cells and differentiated cells, may be considered as a crucial control point in AFS cells as well. Therefore, it is essential to identify if p53 is involved not only in the processes underlying the \u2018stemness\u2019 of AFS cells but also in their tumorigenicity.p53\u2019s role in tumorigenesis is widely known with thousands of publications and an increasing list of p53 targets and effector functions, making it even more difficult to understand the complexity and reach of this single protein. Interest is also driven to focus on potential mechanistic links between the loss of p53 and the stem cell\u2013like cellular plasticity which has been suggested to contribute to tumor cell heterogeneity and to drive tumor progression. We now know that p53 is actively present and involved in a variety of cellular processes in AFS cells, but its involvement is less well understood. Does p53 play a different cellular function in AFS cells? Is p53 essential for tumor suppressive functions in AFS cells? Does the loss of p53 in AFS cells lead to tumor formation? These are yet unanswered questions, the knowledge of which could potentially improve the usage of AFS cells for therapy in the future."}
{"text": "Under stressful conditions, p53 tightly regulates cell growth by promoting apoptosis and DNA repair. When p53 becomes mutated, it loses its function, resulting in abnormal cell proliferation and tumor progression. Depending on the p53 mutation, it has been shown to form aggregates leading to negative gain of function of the protein. p53 mutant associated aggregation has been observed in several cancer tissues and has been shown to promote tumor growth. Recent studies show correlation between p53 mutant aggregation, functional loss, and tumor growth. Moreover, p53 aggregation has been observed in biopsies, patient tissues, and in vivo studies. Given the fact that over fifty percent of cancers have p53 mutation and several of them are prone to aggregation, therapeutic strategies are needed for treating p53 mutant aggregation associated cancers. Recent studies using polyarginine analogues and designer peptides for inhibiting p53 aggregation and tumor growth gives further encouragement in treating cancer as a protein aggregation disease. In this review, we highlight the recent efforts in targeting p53 aggregation in cancer and propose the use of small stress molecules as potential p53-antiaggregation drugs.  Many protein aggregation diseases are the result of protein misfolding/aggregation due to genetic mutations or environmental stress conditions . ProteinNormally, p53 proteins are switched off and are activated when cells experience stress and undergo uncontrolled division and proliferation ,10. WhenWhen p53 is mutated, it loses its function and becomes inactivated ,16,17. MSeveral methods have emerged to rescue p53 mutant function ,25. TherTreating cancer as a p53 aggregation disease is still in its early stages. Only a few studies have reported targeting cancer as a p53 mutant aggregation disease. We have shown that small stress molecules, such as polyarginine and its analogues, exhibit p53 aggregation inhibition both in vitro and in p53 mutant cancer cells ,28. AnotSeveral small stress molecules have been shown to stabilize proteins under harsh conditions. In particular, compatible solutes have been shown to stabilize protein aggregation. The molecules have shown inhibitory potential in neurodegeneration caused by peptide aggregation ,30,31,32Inspired by the above, we recently studied the ability of arginine molecules to modulate p53 aggregation and cancer cell proliferation, to identify therapeutic candidates for treating cancer due to p53 aggregation. We tested the inhibitory effects of polyarginine and its analogues on the R248Q p53 mutant mimetic peptide QRPILTIITL aggregation, and p53 mutant-bearing cancer cell growth in vitro. The study revealed that polyarginine and polyornithine significantly inhibit p53 mutant peptide aggregation in vitro and inhibit the growth of p53 mutant (R248Q) lung cancer cells H719 and p53 mutant (R175H) breast cancer cells SK-BR-3. The molecules showed no effect on p53 wildtype and p53 null cancer cell growth. In addition, the molecules enhanced p21 expression, one of the p53 target genes responsible for cell cycle arrest , indicatLactobacillus plantarum [In addition to arginine and analogues, in a separate study, we have also shown that the cationic small stress molecule acetylcholine chloride had the ability to inhibit aggregation of p53 mutant peptide WRPILTIITL (mimicking the p53 hot spot mutant R248W) in vitro . Acetylclantarum , and is lantarum . The aboRecently, Soragni et al.  developeHowever, the method has its limitations as well. The authors noted that if the wildtype p53 is partially unfolded and aggregated, the designer peptide would probably target the wildtype p53 structures as well. Hence, there can be systemic toxicity effects if this happens in normal cells. In addition, the inhibitory ability is limited to certain p53 mutant aggregations and may not be applicable to all p53 aggregation-associated cancer. Further, like all small molecule-based therapies, the peptides will have short half-life in vivo and will be cleared from circulation rapidly, and to realize the full potential, further modifications and optimization may be needed. The studies discussed in this review show that small molecules are capable of modulating p53 mutant aggregation and subsequent tumor progression. However due to their small molecular weight and size, they have a higher probability of getting rapidly cleared by the body, minimizing their potential as effective therapies. Hence, it would be ideal if the small stress molecules could be formulated in a stealth drug carrier, and to have target specificity to the tumor microenvironment to realize its full translational potential. Compared to conventional therapies which suffer from poor circulation, bioavailability, and efficacy, nanotechnology approaches can remedy some of the problems due to their tunable design . NanoparFinally, to further enhance the therapeutic potential, personalized medicine approaches could be utilized. To precisely target specific p53 mutant aggregation-associated cancer, mutant specific anti-aggregation targets could be developed. A similar concept to recent studies reported on p53 mutant antibodies to inhibit p53 mutants in cancer  could be"}
{"text": "Cervical cancer mainly occurs among women from the developing world, and women face unique challenges in terms of their disease and treatment. Most women present with advanced cervical cancer and receive the standard curative treatment with external beam radiotherapy and brachytherapy with or without chemotherapy.To describe the quality of life (QOL) of women treated for cervical cancer during treatment (M0), at 6 months after completing treatment (M6) and at 12 months after treatment (M12).n = 153) and convenience sampling were used. Data were collected through structured interviews, and the EORTC QLQ-C30 and EORTC QLQ CX24 served as data collection instruments. Descriptive statistics were used to analyse the data, and the Kruskal\u2013Wallis H test was used to compare the mean responses across the groups (p \u2264 0.05).A cross-sectional design, calculated sample size (The mean age of the respondents was 50.6 years (standard deviation [SD] 11.9). The global health status improved significantly in contrast with the functional scores. Financial difficulties were rampant, especially during the treatment phase. Insomnia and urinary frequency were the most cumbersome problems and remained so even after treatment.Despite an improvement in the global health, cervical cancer and its treatment had a negative influence on the QOL in all domains of lives of these women. Assessing the QOL of patients during treatment and follow-up visits would allow nurses to develop interventions to address distressing problems timeously. In addition, Africa\u2019s nurses should assess social functioning and develop programmes to prevent social dysfunction. The significance of this study derives from the fact that best practices focus on the prevention and treatment of cervical cancer and little attention has been given to the quality of life (QOL) of women living with this condition. Although it is known that cancer and its treatment influences QOL, studies focusing on this phenomenon in the African population appear to be sparse , most commonly type 16 and 18, which is found in 70% of all cervical cancers. Cervical cancer develops slowly, usually taking 10\u201320 years for mild dysplasia to develop into cancer , at 6 months after completing treatment (M6) and at 12 months after treatment (M12).A cross-sectional design was selected for the study as it allows researchers to investigate time-related phenomena by collecting data from different groups at one point in time and comparing the responses  was 157 and the target population (N) was 7735.The study population consisted of all women diagnosed with cervical cancer treated at the study setting. The inclusion criteria were 18 years and older and receiving the standard curative treatment for cervical cancer at the time of data collection, or 6 or 12 months after treatment. The sample size was calculated with the assistance of a statistician. A finite population correction was used where the initial sample size (The formula used was:n = 153), thus 51 (n = 51) in each of the groups. The total sample (n = 153) was determined to achieve a confidence level of 95%. Convenience sampling was used to select the sample as it allowed the researchers to select women who were readily available .n = 153) ranged from 30 to 79 years, with a mean age of 50.6 years standard deviation (SD) \u00b1 11.9. Nearly half  were single and unemployed , and 50.3% (n = 77) suffered from stage IIIA and more advanced disease  between study groups one (M0) and two (M6), between groups one (M0) and three (M12) (p = 0.000), and between groups two and three (p = 0.024).When calculating the global health status of the three groups, it was found that the M12 group had the better health status. A Kruskal\u2013Wallis H test showed that the global health status was statistically significant among the study groups . A post m = 82.0), while social functioning scored the lowest overall mean (m = 60.5). In addition, social functioning had the lowest mean score in M0 compared to all the other functioning scores across the different groups. Kruskal\u2013Wallis H tests showed there was no statistically significant difference between the role, physical, cognitive, emotional and social functioning scores among the three study groups. Financial difficulties had the highest mean score in M0 (m = 81.7), and a post hoc analysis indicated a statistically significant difference between groups one and two (p = 0.000) and between groups one and three (p = 0.001)  .m = 10.5) and insomnia had the highest overall mean (m = 33.6). Kruskal\u2013Wallis H tests showed there was a statistically significant difference in fatigue, insomnia, dyspnoea, swelling of the feet and financial difficulties, but no statistically significant differences were found in pain and tingling of the hands or feet. A post hoc analysis indicated there was a statistically significant difference in fatigue between groups one and two (p = 0.003) and between groups one and three (p = 0.000). There was a statistically significant difference in insomnia between groups one and two (p = 0.024) and between groups one and three (p = 0.002). With regard to dyspnoea, the study revealed a significant difference in groups one and three (p = 0.001). Groups one and three had significant differences (p = 0.000) in swelling of feet as well as groups two and three (p = 0.000).When investigating the general problems, consisting of fatigue, pain, insomnia, dyspnoea, swelling of the feet and tingling of the hands of feet, dyspnoea had the lowest overall mean (m = 24.8), while diarrhoea had the highest mean score (m = 22.7). A statistically significant difference was found in diarrhoea, appetite loss, nausea and vomiting. A post hoc analysis showed a statistically significant difference between groups one and three (p = 0.000) and between groups two and three (p = 0.007) in terms of diarrhoea and nausea and vomiting. There was also a significant difference in appetite loss between groups one and two (p = 0.000) and between groups one and three (p = 0.000).Gastrointestinal, urologic and gynaecologic symptoms were also investigated . With rem = 7.2) and urinary frequency the highest (m = 44.9). In addition, urinary frequency had the highest mean score of all the symptoms investigated across the groups. There was no statistically significant difference in burning sensation when urinating, urine leakage and difficulty emptying the bladder; however, there was a statistically significant difference in urine frequency. A post hoc test showed there was a significant difference between groups one and two (p = 0.008) and between groups two and three (p = 0.010).When examining the urological data, difficulty in emptying the bladder had the lowest mean score (m = 9.2), while menopausal symptoms, hot flushes and/or night sweats had the highest mean (m = 24.2). With regard to menopausal symptoms, there was no significant difference among the three groups; however, according to a post hoc analysis, there was a statistically significant difference in vaginal discharge and vaginal bleeding. Regarding vaginal discharge, there was a significant difference between groups one and two (p = 0.000) and groups one and three (p = 0.000). There was also a significant difference between groups one and two (p = 0.000) and between groups one and three (p = 0.000) in terms of vaginal bleeding.Vaginal bleeding was the gynaecological symptom with the lowest overall mean  and between groups one and three (p = 0.000). There were significant differences in sexual enjoyment between groups one and two (p = 0.000) and between groups one and three (p = 0.000). There were statistically significant differences in vaginal functioning between groups one and two (p = 0.000) and between groups one and three (p = 0.000)  .The age profile of the sample seems to be typical of women treated for cervical cancer. Snyman and Herbst  in a SouThe study provided evidence that slightly more than half of the women suffered from stage IIIA and more advanced cervical cancer. Although a similar trend was reported in a Polish study (Pasek, Suchocka & Urba\u0144ski It was positive to find that the global health status of the groups improved significantly from the time they received treatment to 12 months after treatment. Du Toit and Kidd , in a SoNone of the functioning scores showed a statistical significant difference between the groups. This is in contrast with the findings of Bjelic-Radisic et al.  who, in Financial difficulties, especially during the time of treatment, had a negative influence on QOL. This does not seem to be unique to Africa, as, although not to the same extent, studies conducted across Europe and in Turkey and Poland report financial difficulties in this group of women especially during the treatment phase (Bjelic-Radisic et al. It was interesting to find that insomnia was the most troublesome general symptom, followed by pain and fatigue. Goker and others  confirm What is of concern is the significant upward trend of swelling of the feet as lymphoedema is a well-known complication of pelvic radiotherapy. Heijkoop and others  report aUrinary frequency was the most troublesome cervical cancer-related symptom experienced accompanied by a burning sensation, most commonly occurring in the treatment phase. A study conducted in the Netherlands by Heijkoop and others  supportsThe study provided evidence that all the domains of sexual functioning, sexual activity and enjoyment and vaginal functioning declined after treatment. However, when the short-term and late effects of cervical cancer and its treatment are considered, this finding does not seem surprising. It was interesting to note that other studies found a similar trend except for sexual activity, for instance Rahman and others , in a stThe study has various limitations. Using a cross-sectional design did not allow for the investigation of changes in the same respondents over time. A longitudinal design may have resulted in results that were more accurate. In addition, respondents could have provided socially acceptable answers and not reflect what is true for them. However, the researchers believe the study provided base line data regarding the QOL of women treated for cervical cancer.Nurses practising in radiation oncology care settings should be cognisant of how cervical cancer and its treatment influence the QOL of women. Assessing the QOL during treatment and follow-up visits would allow nurses to develop interventions to address distressing problems timeously. In addition, nurses in Africa should assess social functioning before and after treatment and develop programmes to assist women to optimise their functioning as social beings despite having cervical cancer and prevent social dysfunction.Despite an improvement in the global health status, cervical cancer and its treatment had a negative influence on the QOL of the women and influenced all domains of their lives. Social functioning was the most affected and did not improve significantly over time. Financial difficulties were troublesome, especially during the time of treatment; urinary frequency and insomnia remained problematic; and swelling of the feet increased significantly over time."}
{"text": "Child mortality rate in sub-Saharan Africa is 29 times higher than that in industrialized countries. Anemia is one of the preventable causes of child morbidity. During a humanitarian medical mission in rural South-Eastern Nigeria, the prevalence and risk factors of anemia were determined in the region in order to identify strategies for reduction. A cross-sectional study was done on 96 children aged 1-7 years from 50 randomly selected families. A study questionnaire was used to collect information regarding socioeconomic status, family health practices, and nutrition. Anemia was diagnosed clinically or by point of care testing of hemoglobin (Hb) levels. 96 children were selected for the study; 90 completed surveys were analyzed . Anemia was the most prevalent clinical morbidity (69%), followed by intestinal worm infection (53%) and malnutrition (29%). Mean age (months) at which breastfeeding was stopped was 11.8 (\u00b12.2) in children with Hb <11mg/dl (severe anemia), 10.5\u00b12.8 in those with Hb = 11-11.9mg/dl (mild-moderate anemia), and 9.4\u00b13.9 in children with Hb >12mg/dl (no anemia) (P=0.0445). The longer the infant was breastfed, the worse the severity of childhood anemia was. Childhood anemia was likely influenced by the low iron content of breast milk in addition to maternal anemia and poor nutrition. A family-centered preventive intervention for both maternal and infant nutrition may be more effective in reducing childhood anemia and child mortality rate in the community. Childhood malnutrition and anemia remain significant public health concerns in Southern Nigeria and globally. While sub-Saharan Africa has made notable progress in combatting food insecurity, 25% of the population remains undernourished and below national goals . In NigeOverall, exclusive breastfeeding in Nigeria is declining, but the length of time mothers breastfeed has increased to a mean of 13 months in 2014 . The benWhile many studies have linked breastfeeding with infant anemia, few, if any, have examined risk factors for persistent anemia in childhood, despite the impact of childhood anemia on morbidity and mortality. This study sought to determine the risk factors and prevalence of childhood malnutrition and anemia in a rural community in Imo State, Nigeria, in sub-Saharan Africa.An institutional review board at the Federal University of Technology, Owerri (F.U.T.O), Imo State, Nigeria, completed a review of the planned research study and approved the study prior to study commencement.A survey of 38 questions addressing sociodemographics, agricultural diversity, and diet diversity was developed. Fifty children between the age of two and five were assessed for possible malnutrition and anemia and surveys administered to their parents or accompanying guardians. A single interviewer performed all interviews. A parent or household guardian for each of the selected children was asked to complete a demographic survey, with the help of interpreters as necessary. Completion of the questionnaire was not a requirement for medical treatment, and informed consent was obtained. Demographic information was collected for all fifty families including family size, age of parents, age of other children, and living situation. Families' socioeconomic status was scored using a series of eight questions adapted to the specific living conditions of the region using knowledge from past missions and local information. Families' agricultural and diet diversity were graded using recommendations from the United Nations Food and Agriculture Organization . QuestioNutritional status of each child was determined after measuring height, weight, and median upper arm circumference (MUAC) and compared to WHO arm circumference for age standards . Anemia  p-values were two-sided and p<0.05 was used to define statistical significance. Data analysis was conducted using SAS software , version 9.4.Categorical variables were summarized in terms of frequencies and percentages while continuous variables were summarized in terms of means \u00b1 standard deviations (SD). Logistic regression analysis, chi-square, or Fisher's exact test was used to compare categorical variables between cases and control. Odds ratios (OR) were reported along with the corresponding 95% confidence intervals. Continuous variables  were compared between cases and controls using a two-sample t-test. AllOut of 90 children tested, 57% were female (n=51) and 43% were male (n=39). The prevalence of chronic systemic disease was determined in the cohort of children. Anemia was the most prevalent (69%) followed closely by worms (53%), malnutrition (29%), upper respiratory infections (23%), cardiac disease (4%), and gastrointestinal illness (2%). The hemoglobin testing on all children revealed that 27% had a low hemoglobin, 58% had borderline hemoglobin, and 16% had normal hemoglobin.p=0.35 interaction effect), presence of upper respiratory infection (p=0.9946 interaction effect), or malnutrition (p=0.4949). There were not enough cases of gastrointestinal disease to examine the interaction between hemoglobin and gastrointestinal disease on clinical anemia. There was no correlation between median upper arm circumference and worm infestation (p=.79) or anemia diagnosis (p=.86).There was a significant association of hemoglobin levels with clinical anemia (p=0.0457), with lower levels of hemoglobin being associated with a higher rate of anemia, as expected,  p=0.25). There was no difference between hemoglobin levels and type of occupation of the head of household  (p=0.752). Example of skilled labor would be a tailor or mechanic; nonskilled labor would be a household servant or laborer; while professional could be a teacher, business manager, or profession requiring more than a secondary education. Although professionals were more likely to read to their children each week (an average of 3.7\u00b10.5 times a week) than head of households with skilled (3.1\u00b10.9) and nonskilled labor (2.8\u00b10.6) (p=0.0002), there was no difference in frequency of medical checkups (p=0.9411) and likelihood of child attending school, There was no association between hemoglobin levels and socioeconomic status score (SES) , There was no significant association between hemoglobin levels and people that both sell and eat farm products compared to those that do not sell or eat farm products (p=0.4257). the number of farm animals owned in a household did not correlate with hemoglobin level (p=0.25). The number of family meals a day (p=0.742) or number of meals the child has a day did not have any association with hemoglobin levels (p=0.069). There was no significant association between hemoglobin levels and food insecurity (p=0.21) or diet diversity score (p=0.39). Children with low hemoglobin levels were breastfed a mean of 2.4 months longer than those with normal hemoglobin levels. Longer breast feeding was associated with lower hemoglobin levels  without food or nutrient supplementation on anemia , 31, 34.The prevalence of anemia in our population 69%) is consistent with other studies in tropical Africa that found 63% prevalence in children under 3 years % is cons. In contOverall, the community's diet was homogenous between families, and it was difficult to accurately assess a child's dietary intake. This likely contributed to lack of association between dietary diversity and anemia, as it was difficult to stratify diet diversity in this community. Similarly, it was difficult to stratify socioeconomic status in the community\u2014despite using a targeted questionnaire build from a previous community needs assessment in the area. While there was no significant correlation between frequency of medical care and professional level, this is likely due to the deficiency of medical resources in the area. From our study, the importance of education appears to be understood in the community. Regardless of head of household's occupation, most children were likely to be enrolled in school. However, professional households seemed to be more consistent in promoting literacy as they were more likely to read to their children compared to nonprofessional households. However, this did not affect the healthcare choices made by both professional and nonprofessional households as both had infrequent medical care preventive visits for their children and had equally poor diet diversity or meal choices. This alludes to the fact that the overall poor financial status of both professional and nonprofessional household affected their healthcare and food choices and attitudes regardless of level of literacy , 40.A major limitation identified in this study is that it was primarily designed to assess modifiable risk factors for childhood anemia, and it did not specifically examine risk factors for prolonged breastfeeding or exclusive breastfeeding or maternal anemia status. Furthermore, it relied on field-testing kits to diagnose anemia and did not include diagnosis of possible etiologies of anemia like measures of iron deficiency using ferritin levels, for example, chronic disease, inflammation, or hemoglobinopathies. Further research is necessary in two primary areas. First, while the most likely cause of anemia in this community is iron deficiency based on large-scale population studies, further research should be done to elucidate the various causes of anemia in this community , 42. A rWhile iron supplementation would likely decrease anemia prevalence in this area, it is important to evaluate other causes of anemia, notably hemoglobinopathies, to guide public health and clinical interventions. Another limitation in this study is that the risk factors or interactions between prolonged breastfeeding and anemia were not explored or evaluated. Many studies have evaluated cultural, economic, and social determinants of breastfeeding in many settings with a wide range of conclusions \u201347. In NIn conclusion, persistent childhood anemia was likely influenced by the low iron content of breast milk, an indication of maternal anemia and poor nutrition. Although continuous breastfeeding is a known strategy to reduce child mortality, a family-centered preventive intervention to diagnose and treat childhood anemia could lead to reduced morbidity and mortality from anemia. Early identification of iron deficiency in children is vital to the prevention of anemia and long-term psychomotor and developmental consequences. A longitudinal study examining childhood anemia pathophysiology, determinants of exclusive breastfeeding, and treatment effects would both be cost effective given the presence of the annual medical missions and provide important data on the effects of infant anemia on childhood anemia."}
{"text": "Skin cancer is the most common type of cancer worldwide. Ozone depletion and climate changes might cause a further increase in the incidence rate in the future. Although the early detection of skin cancer enables it to be treated successfully, some tumours can evolve and become more aggressive, especially in the case of melanoma. Therefore, good diagnostic and prognostic markers are needed to ensure correct detection and treatment. Transcription factor p63, a member of the p53 family of proteins, plays an essential role in the development of stratified epithelia such as skin. In this paper, we conduct a comprehensive review of p63 expression in different types of skin cancer and discuss its possible use in the diagnosis and prognosis of cutaneous tumours. Trp53, Trp63, and Trp73 knockout mice [p63, like its sibling p73 , belongsTP63 gene gives rise to several protein isoforms resulting from the use of alternative transcription start sites and alternative C-terminus splicing events. Indeed, the TP63 gene harbours two different promoters that generate two N-terminal isoforms (TAp63 and \u0394Np63) (The d \u0394Np63) a. The TAd \u0394Np63) a. The \u2206Np63 isoform is mainly expressed in ectoderm-derived tissues, such as the epidermis, skin appendages, simple epithelia, and the thymus ,17,18,19Remarkably, no direct evidence has been provided using immunohistochemistry for p63 expression in Langerhans cells and melanocytes. However, p63 was found to be absent in normal melanocytes by polymerase chain reaction (PCR) . This obIn summary, p63 appears to be expressed in stem cells and highly-proliferating transit-amplifying cells of ectodermal origin. However, it is present at low levels or absent in differentiating cells as well as cells of mesenchymal origin. CDKN1A [HES1 [bona fide p63 target genes are components of hemidesmosomes such as BPAG1 [PERP [ITGA6 and ITGB4 [CDH3, a component of adherens junctions [FRAS1 codifying for an extracellular protein [KRT14, a component of keratin intermediate filaments [As a transcription factor, p63 can activate or repress expression of its target genes through direct binding to their promoter regions c. For inCDKN1A , a gene 1A [HES1 , one of as BPAG1  and PERPG1 [PERP ; integrind ITGB4 ; the P-cunctions ; FRAS1 c protein ; and KRTilaments . Furtherilaments  by direcilaments  and cytoilaments . Differeilaments ,40 and Kilaments  cooperatSATB1 [SMARCA4 (codifying for Brg1) [Conversely, p63 is closely involved in the process of keratinocyte differentiation in both early and late stages. ZNF750 is one of the earliest transcription factors activated during differentiation by p63 . It induSATB1  and SMARor Brg1) . Their aRecent investigations have unveiled the existence of pre-established epithelia-specific enhancers in undifferentiated keratinocytes. These might interact with the promoter regions of target genes to allow their transcription during differentiation . NotablyAltogether, p63 is well the established master regulator of epidermal homeostasis and differentiation. Its transcriptional network is an example of an extremely complex spatiotemporal system of positive and negative regulation, which allows for the proper development, self-renewal, and differentiation of the skin. p63 both impedes and triggers keratinocyte differentiation depending on the epithelial compartment, proliferative state, and the network of interacting proteins. Although mechanistic aspects of p63 function in the cutaneous basal cell carcinoma (cBCC) and rare skin tumours remain poorly investigated, the role of p63 in tumourigenesis has been intensively studied in squamous cell carcinoma (SCC). A transcriptional programme of p63 in cutaneous SCC is similar to SCC from other epithelial origins such as the head and neck . In The oncogenic properties of \u0394Np63 were established in in vivo experiments that showed overexpression of \u0394Np63 enhances mutant Ras-driven tumourigenesis of undifferentiated cSCC . Indeed,Skin cancer is the leading type of cancer worldwide with up to 3 million new cases every year. The most common risk factor for the development of skin cancer is ultraviolet (UV) exposure to the sun. This provokes multiple cytosine-to-thymine transitions that result in DNA mutations . HoweverTP53 and PTCH1. However, cBCC genomes are considered genetically stable compared to those from aggressive types of cancer [Cutaneous basal cell carcinoma (cBCC) is the most common type of cancer worldwide with almost 750,000 cases detected every year in the USA. cBCC accounts for up to 80% of all non-melanoma skin tumours. Basal cell carcinomas are slowly growing and rarely metastasise. Among the most frequently mutated genes are f cancer . Recent f cancer . Multiplf cancer . Moreovef cancer ,66,67,68RAS and TP53 are frequently mutated in cSCC. In contrast to cBCC, cSCC can originate from hair follicle bulge stem cells as well as interfollicular epidermal stem cells [The second most frequent type of skin cancer after cBCC is cutaneous squamous cell carcinoma. With a diagnosis rate of 15\u201330 per 100,000 people, cSCC accounts for 20% of all skin cancer cases. Notwithstanding a lower frequency with respect to cBCC, cSCC is characterised by a higher incidence of metastases and poorer prognoses . RAS andem cells . p63 is em cells ,67,68,71em cells  with higem cells . A rare variant of cSCC is cutaneous spindle cell squamous cell carcinoma (cSCSCC). The epidemiology of cSCSCC is associated with sun- or radiation-exposure of the skin. Radiation exposure is thought to be correlated with more aggressive phenotypes of the disease. Histologically, cSCSCC is characterised by bizarre and pleomorphic tumour cells that infiltrate deep into the dermis . SeveralAlthough several cutaneous lesions are of a benign nature, they can be precursors of skin tumours such as cSCC. The most frequent benign tumour of the skin is seborrheic keratosis (SK), which primarily affects people over the age of 50 and has low malignant potential . AnotherTakeuchi and colleagues performed a comprehensive analysis of p63 expression in several skin tumours of different origins, including very rare cases. As expected, p63 was found to be positive in epidermal tumours as well as most adnexal neoplasms (as will be discussed in the next section). In contrast, in all neural tissue tumours, mesenchymal tumours, lymphomas, and histiocytosis, p63 expression was found to be negative ,86. RemaAlthough malignant melanoma (MM) is less frequent than cBCC or cSCC, it is among the deadliest forms of skin cancer, leading to 50,000 deaths every year . MM arisTP63 gene in melanoma samples (14.7% of samples) [TP63 gene are common in epidermal dysplasia [TP63 mutation of melanomagenesis remains elusive. No consensus has been reached regarding the pattern of p63 expression in melanoma. Multiple lines of evidence suggest that p63 is virtually never expressed in MM samples  . In contysplasia . Therefop63 is used as a gold standard marker for highlighting myoepithelial cells in multiple types of cancer ,96,97. ICutaneous adnexal neoplasms (CANs) are a wide group of benign and malignant skin tumours that normally exhibit differentiation towards one of the cutaneous appendages: hair follicle, sebaceous gland, eccrine, or apocrine glands. Even though the precise origin of these neoplasms remains unknown, it has been hypothesised that CANs might arise from stem cells located within normal cutaneous adnexa . CANs arAtypical fibroxanthoma (AFX) is a rare benign tumour with a favourable prognosis that mainly occurs in sun-exposed skin on the head and neck regions of elderly patients, predominantly men. AFX is believed to be of fibroblastic origin and is characterised by myofibroblastic differentiation. The diagnosis of AFX is difficult and is performed by excluding other malignancies such as cSCSCC ,108. p63 was found to be negative in 188 out of 199 samples. Notably, in several reports the expression of p63 in AFX samples was assessed in parallel with its expression in cSCSCC. Indeed, 110 out of 130 cSCSCC samples stained positively for p63 ,112. ThuMerkel cell carcinoma (MCC) displays neuroendocrine features and is a rare but aggressive type of skin cancer. It mainly affects elderly patients (>75 years old) and can be caused by chronic UV exposure or Merkel cell polyomavirus. The origin of MCC remains elusive; however, it is speculated that this kind of cancer may arise from Merkel cell precursors as well as pre-B cells or fibroblasts, rather than mature post-mitotic Merkel cells. MCC is characterised by a high risk of metastases and death . Due to Since the discovery of p63 , researcThe reason for the discrepancy between 4A4 and p40 staining patterns remains unclear. One possible explanation might be that tumour cells express not only \u0394N but also TA isoforms of p63 that are recognised by 4A4 but not p40. Another, more plausible, hypothesis is that different staining results are due to the non-specific binding of 4A4 to other members of the p53 family or even other proteins. Indeed, it has been shown that the 4A4 antibody can readily recognise p73 isoforms in vitro . Several reports have also confirmed the utility of p40 in the diagnosis of skin tumours. Lee et al. demonstrated higher specificity of p40 with respect to pan-p63  in distinguishing adnexal carcinoma from metastases in skin . As descForty years ago, the master tumour suppressor p53 was discovered, heralding a new era of understanding regarding the ubiquitous molecular mechanisms of cellular defence against cancer initiation and progression . Almost Recent studies carried out with primary keratinocytes have elucidated a very complex and thorough transcriptional programme governed by p63. Via interaction with chromatin remodelers, p63 enables the establishment of a well-organised three-dimensional genomic architecture to ensure the proper development of the skin as well as its homeostasis. Not surprisingly, in skin cancer, p63 pathways can be altered, disrupting the balance between proliferation and differentiation.The paramount importance of p63 in epithelia development encouraged researchers and pathologists to explore its expression in epithelial cancer. Multiple groups in the early 2000s carried out an extremely comprehensive study of different types of skin cancer using immunohistochemistry, providing strong evidence for high expression of p63 in virtually all types of non-melanoma skin cancer, including pre-cancerous lesions as well as rare forms of squamous cell carcinoma. By contrast, several skin-related tumours of non-epithelial origin such as atypical fibroxanthoma, along with diverse metastases to skin from other organs, are in most cases p63 negative. Even though no decision has been made regarding the use of p63 as a gold standard in skin cancer, multiple reports have proposed its utility in differential diagnosis. Furthermore, p63 has emerged as a putative marker for outcomes in Merkel cell carcinoma, where high p63 expression is associated with a poorer prognosis. Based on the pre-clinical studies, summarised in this review, we can speculate that p63 might be an important and targetable molecule, although no clinical trials have been reported that address how the level of p63 expression could modify current therapies. Nevertheless, a growing body of evidence suggests that p63 could be a promising diagnostic and predictive marker for unusual cases of skin cancer."}
{"text": "Drugging the p53 pathway has been a goal for both academics and pharmaceutical companies since the designation of p53 as the \u2018guardian of the genome\u2019. Through growing understanding of p53 biology, we can see multiple routes for activation of both wild-type p53 function and restoration of mutant p53. In this review, we focus on small molecules that activate wild-type p53 and that do so in a non-genotoxic manner. In particular, we will describe potential approaches to targeting proteins that alter p53 stability and function through posttranslational modification, affect p53\u2019s subcellular localization, or target RNA synthesis or the synthesis of ribonucleotides. The plethora of pathways for exploitation of p53, as well as the wide-ranging response to p53 activation, makes it an attractive target for anti-cancer therapy. The tumour suppressor p53 in its role as the \u2018guardian of the genome\u2019 exerts a multitude of effects on cells in response to cellular stress . As a keTP53, making it one of the most consistently and frequently mutated genes in cancer . HDM2 acts as an E3 ubiquitin ligase, which binds to p53 and subsequently causes its ubiquitination and nuclear export, targeting p53 for proteasomal degradation . Interestivation . The HDMtivation .Escherichia coli thioredoxin  . The morp53 not only induces the expression of its E3 ligase HDM2 but also acts as a transcription factor for the expression of WIP1/PPM1D, another of its negative regulators. This p53 inducible phosphatase 1 not only destabilizes p53 by dephosphorylating serine 15 in p53  but alsoThe deacetylation of p53 at lysine residues has been implicated in enhancing its subsequent ubiquitination by HDM2 . The claIn 2002, it was reported that HDM2 is able to promote p53 deacetylation by recruiting a complex containing HDAC1, and therefore HDAC1 inhibition could promote p53 stability and function . TrichosAnother mechanism by which ubiquitination of p53 is altered is through the inhibition of deubiquitination. The deubiquitinase USP7 is responsible for the deubiquitination of both p53 and HDM2, though it displays a preferential activity towards HDM2 . A decreNEDDylation is another postranslational modification that can be modulated with small molecules. In the case of p53, NEDDylation is favoured by HDM2 and leads to inhibition of the transcription factor function of p53 . FBX011 MLN4924 (Pevonedistat)  is thougInhibitors of the proteasome, including the clinically approved bortezomib, rapidly increase p53 protein levels . Not onlFor p53 to exert its transcription factor function, it requires nuclear localization. HDM2-mediated ubiquitination of p53 causes its nuclear export and subsequent proteasomal degradation . Therefode novo pyrimidine synthesis pathway, has gained increasing attention following a seminal paper by Indirect activation of wild-type p53 by depleting cells of ribonucleotides is another possible strategy to activate p53 without causing DNA damage . In the in vivo would be to combine with a relevant therapy. Indeed, we have already shown that combination of a DHODH inhibitor, HZ00/HZ05, with an inhibitor of p53 degradation, nutlin-3, led to a synergistic tumour cell kill both in vitro and in vivo in tumour xenograft studies -L-aspartate (PALA) has been noted for its anti-tumour activity for many years having been tested in both in vivo tumour models as well as in patients  are also potent activators of the p53 response. For example R-roscovitine (Seliciclib), which targets multiple CDKs in the nanomolar range, induces the expression of functional p53 in cells . AccordiCDK4 and HDM2 amplification are frequently linked in liposarcoma and the combination of inhibitors to each of these enzymes slightly improves the in vivo antitumor effect (V600E and BRAFV600K inhibitor, to suppress melanoma growth in vivo, by inducing p53-dependent apoptosis and growth suppression.The inhibition of cell cycle progression through inhibition of CDK4 and CDK1 has been associated with the p53/HDM2 axis. r effect . Furtherr effect . FurtherThe exploitation of the p53 pathway therapeutically has been a formidable challenge, and one that has been under investigation for more than two decades. As summarized in Figure Multiple clinical trials where HDM2 inhibitors are combined with standard chemotherapeutics or radiation are ongoing Table . StudiesFinally, one must bear in mind that HDM2 inhibitors can protect normal cells from S-phase poisons, mitotic poisons, as well as other agents such as DHODH inhibitors. Therefore, the order in which these agents are administered could be postulated to be of great importance to mitigate an undesirable outcome and to be successful as treatments."}
{"text": "This perspective was written in celebration of Dr Arnold J. Levine\u2019s 80th birthday. I chose to write this prospective about restoration of p53 activity in tumors for two reasons: Arnie  has become interested in restoring p53 activity in tumor cells and such a review did not exist. Arnie was also my postdoctoral advisor and writing this perspective is just a small tribute for the huge impact he has had on my research career (and that of many others), from supporting me when I left his laboratory for a faculty position before I had even published a paper, to serving as a sounding board for numerous ideas. Arnie is an amazing thinker. He is able to almost effortlessly incorporate new knowledge into current thinking and collate a big picture. It has been my pleasure to interact with him scientifically on numerous occasions. I want to thank Arnie from the bottom of my heart for sharing with me his love of science, his critical thinking skills, and his friendship.p53 gene, should be treated with drugs that disrupt binding of these inhibitors to p53 to restore p53 function. Tumors lacking p53, on the other hand, need to reintroduce p53 through a virus encoding wild-type p53 or convert mutant p53 to wild type. Genetically modified mouse models have been used to examine p53 restoration in various contexts and will be reviewed here. Tumor responses have been heterogeneous suggesting that other factors contribute to the outcome. Resistance mechanisms will also likely emerge and need to be understood in more detail.The p53 tumor suppressor is inactivated in most  cancers via various mechanisms indicating a potent role in inhibiting tumor cell growth . The idep53 alleles that were not functional but could be reactivated in a Cre-dependent manner. The Jacks laboratory generated a wild-type p53 locus with a lox-stop-lox (LSL) cassette in the first intron effectively eliminating p53 expression but allowing p53 re-expression in the presence of Cre recombinase  of these tumors regressed and 20% showed tumor stasis (1 T-cell lymphoma and 1 osteosarcoma). One tumor lost the T2Cre-ER allele and thus grew like controls. In this context, p53 restoration led to apoptosis in lymphomas but decreased proliferation, cell cycle arrest, and senescence in sarcomas  that dramatically reduces expression of p53 to \u2009~7% of wild type. The p53 response upon restoration in autochthonous tumors that develop due to p53 loss was similar with lymphomas inducing apoptosis and angiosarcomas inducing senescence. Tumors regressed and in some cases completely disappeared, which coincided with increased survival of the mice. In both of the above studies, it should be noted that the p53 alleles are germline such that all cells of the mouse have less p53 than normal, and Cre recombinationrestores p53 not just in tumor cells , and the CAG-CreER Tamoxifen-inducibletransgene develop lymphomas and sarcomas. p53 was restored with the addition of Tamoxifen and in contrast to p53-null tumors that regressed in the two studies described above, lymphomas and sarcomas in this case showed tumor stasis but not tumor regression  was used , which include cytokines and chemokines with the potential to fuel tumor cell growth ; genetic reconstitution of p53 is permanent and does not mimic drug pharmacology. However, even a partial response appears to be effective in slowing tumor growth. In most of these studies as well, the tumor microenvironment (TME) has p53 alterations, distorting interpretation of the data. We also need to expand these studies beyond the tumor types (lymphomas and sarcomas) that occur with germline loss of p53.The caveats of the above studies are many: the studies were performed in mice not humans; most studies involved germline p53. This was observed in the Jacks study above  have been developed to restore p53 activity .]"}
{"text": "The p53 family of proteins has grown substantially over the last 40 years. It started with p53, then p63, p73, isoforms and mutants of these proteins. The function of p53 as a tumour suppressor has been thoroughly investigated, but the functions of all isoforms and mutants and the interplay between them are still poorly understood. Mutant p53 proteins lose p53 function, display dominant-negative (DN) activity and display gain-of-function (GOF) to varying degrees. GOF was originally attributed to mutant p53\u2032s inhibitory function over the p53 family members p63 and p73. It has become apparent that this is not the only way in which mutant p53 operates as a large number of transcription factors that are not related to p53 are activated on mutant p53 binding. This raises the question to what extent mutant p53 binding to p63 and p73 plays a role in mutant p53 GOF. In this review, we discuss the literature around the interaction between mutant p53 and family members, including other binding partners, the functional consequences and potential therapeutics. In 1979 the research into p53 started off with the finding of a 53kDa protein using immunoprecipitation with the SV40 T antigen in transformed cells ,3. FortyTP53 can cause loss of p53 expression or expression of mutated proteins. The mutant proteins are almost always caused by single nucleotide changes in the DNA, resulting in mutant proteins that differ in only one amino acid from the wildtype molecule. Unlike many other tumour suppressors, mutations are found throughout the amino acid sequence of p53, although more frequently in the DNA binding domain, giving rise to thousands of different mutant proteins [The importance of p53 in tumour suppression is apparent when looking at the frequency of p53 mutations in all cancers, the early onset and frequency of cancers in Li-Fraumeni patients and the many mouse models that display tumourigenesis when p53 is lost or mutated. Using data from the TCGA (The Cancer Genome Atlas), it has become apparent that more than 50% of all cancers carry p53 mutations, but in some cancers, such as small cell lung cancer and ovarian cancer, this percentage can go up to levels higher than 90% ,7. Mutatproteins . The mosproteins .To function as a transcription factor, p53 forms dimers and tetramers that interact with the DNA. The observation that mutant p53 is often impaired in wildtype function led to the observation that it can exert a dominant-negative role over any remaining wildtype p53 protein through direct binding ,12. In aTP53, the family members TP63 and TP73 were cloned and identified as homologues of p53 that share a similar DNA binding domain but differ in N- and C-termini. All family members can be transcribed from different promotors resulting in variants with long N-termini that contain transcription activation domains (TA) or shortened N-termini (\u0394N) (Shortly after ini (\u0394N) a. TA isoini (\u0394N) a. Spliceini (\u0394N) a,b.Tp63 KO mice and Tp73 KO mice that show lethality during late gestation or early after birth due to skin and craniofacial epithelial defects [Tp53 knockout embryos survive until early onset of tumourigenesis [p63 and p73 are thought to be expressed in a more tissue-specific manner than p53, playing important roles in the skin epithelium and in the central nervous system. This is reflected in the phenotypes of  defects ,32 or ne defects  Althoughigenesis , subtle igenesis , suggestTP53, mutations in TP63 and TP73 are rarely found in cancers. This raises the question to what extent p63 and p73 contribute to tumourigenesis. Although mice that are knockout for all p63 isoforms or all p73 isoforms show early lethality, loss of TAp63 or TAp73 expression specifically promoted tumourigenesis or metastasis [In comparison to tastasis ,37,38. Ftastasis ,40. Moretastasis . Loss oftastasis .Under normal conditions, \u0394N isoforms dominant-negatively interact with TA forms of their family counterparts, and isoforms can transcriptionally regulate each other ,41,42. AIn addition to inhibiting wildtype p53, many mutant p53 proteins interact with most isoforms of p63 and p73  43,44,4,444,45. Interactions between mutant p53 and p63 or p73 can be observed in vitro and are strong, indicating that mutant p53-p63 or mutant p53-p73 bind directly with each other ,45,48. FMuch work on mutant p53 interactions has so far focused on delineating how mutant p53 proteins inhibit TA variants. All TA isoforms of the p53 family contain an oligomerisation domain (OD) that is used for homo-dimerisation and tetramerisation. Remarkably, heterodimerisation between TA isoforms of the different family members is seldom observed under physiological circumstances. Mutant p53 R175H can dominant negatively inhibit wildtype p53 by binding through the OD . HoweverSome of the mouse models that were generated of N-terminally truncated p53 display a similar phenotype to mutant p53 (R172H or R270H) in promoting invasion and metastasis . As \u0394133To investigate how mutant p53 inhibits p63 and p73 function, several laboratories have studied potential binding partners of the mutant p53-family member complexes . These sSome of these binding partners were found to act in a similar manner in both the mutant p53-p63 and mutant p53-p73 complexes. The topoisomerase TopBP1, for instance, promotes the formation of both complexes by binding p53 and increasing the affinity for mutant p53 to bind to p63 or p73. This leads to loss of p63 or p73 mediated transcription and an increase in invasion and metastasis . ConversInterestingly, the binding partners of the complexes do not always act on p63 or p73 in the same way. MDM2 and HSP70 differentially regulate the two complexes by promoting the dissociation of the mutant p53-p63 complex but enabling the formation of the mutant p53-p73 complex. This results in a loss of p73-mediated transcription and a gain in p63-mediated transcription ,57. InteMost binding partners have been shown to regulate only p63 or p73. For instance, SMAD and Pin1 promoted the mutant p53-p63 complex formation, inhibiting p63 function ,59. The These findings show that the interacting proteins of mutant p53-family member complexes in cancer can easily modulate some aspects of mutant p53 GOF. This regulation, if appropriately targeted, could be used to reduce the GOF of mutant p53 that is related to p63 and p73 function. In particular, looking at proteins that affect both complexes in the same manner could be interesting. Inhibition of TopBP1 or restoration of ANKRD11 expression would both lead to wild type p63 and p73 signalling, which would suppress metastasis and induce apoptosis.Over the last few years, the interaction between mutant p53 and family members has gotten significant attention from researchers interested in prion-like aggregation. In vitro, the DBD domain was shown to form aggregates, and the OD and mutant forms of p53 were prone to form amyloid ,63. Xu eMost likely, aggregation cannot be fully responsible for all gain-of-function that we see on mutant p53 expression. By aggregating, the mutant p53 complex with all of its binding partners is rendered inactive. Although mutant p53 inhibits TAp63 in activating canonical p53 target genes, some reports describe that through mutant p53, TAp63 binds to a unique set of promotors to promote gene expression ,70. In akRAS mutation, tumours were found to frequently metastasise to the liver and adjacent tissues. Loss of p53 expression in combination with loss of TAp63 expression also led to metastasis, but with a lower prevalence, suggesting that loss of TAp63 does not exclusively contribute to mutant p53 GOF in metastasis in this model [TAp63 and TAp73 knockout animals have been reported to display additional phenotypic abnormalities in ageing, metabolism or spermatogenesis, respectively, that have so far not been reported to occur in any of the mutant p53 mouse models [As mutant p53 has been shown to exert gain-of-function in many ways, it will be important to determine to what extent the interaction between mutant p53 and p53 family members, as well as with other molecules, plays a role in the different aspects of tumourigenesis in vivo. Most mutant p53 mouse models have investigated hotspot mutations that are generally considered fully impaired in wildtype function, stabilised in tumours and capable of GOF ,19. Thesis model . In addie models ,78,79. TTp53 KO mice was shown to inhibit tumourigenesis in p53 knockout animals by upregulating TA isoforms [To fully elucidate how the interactions between mutant p53 and family members work together in promoting tumourigenesis and metastasis, it will be important to combine loss of TAp63 expression with mutant p53 in the same model system and determine whether the tumour burden is exacerbated. For this, it will also be important to fully characterise the expression of isoforms of the p53 family. Loss of \u0394N isoforms of p63 and p73 in isoforms . It willAlthough p53 has been studied for 40 years and is the most mutated gene in all cancers, hardly any therapies or clinical decisions are based on p53 status. This is not due to a lack of effort in trying to design clever ways to combat mutant p53 GOF or to increase wildtype function. Stabilisation of wildtype p53 has been thoroughly studied, with nutlin-mediated inhibition of MDM2 (the E3 ubiquitin ligase involved in p53 degradation) being one of the most famous protein\u2013protein interference drugs . TherapeBAX or GADD45 [Remarkably, there are quite a few drugs that prevent mutant p53 from binding to TAp73, but no therapeutics that directly and specifically interfere in the mutant p53-TAp63 interaction . This rar GADD45 . These dr GADD45 ,88,89. PTAp73\u2032s role is certainly not limited to apoptosis as it has been shown to play a role in invasion in pancreatic cancer cells via a protein\u2013protein interaction with NF-Y . It woulSeveral compounds have been found that restore the folding of mutant p53 to promote wildtype function, which are listed in Many of these compounds will, therefore, also abolish mutant p53 from binding to the family members, but this is not always specifically investigated. The most well-known example of a drug that restores p53 function is Prima-1, which completed a Phase 1 clinical trial ,119. ApaAn important consideration for this review is that only a handful of p53 mutants, predominantly the hotspot mutations, have been thoroughly investigated. Due to large cancer genome sequencing efforts, we now know that thousands of different mutant p53 proteins exist in cancers. Traditional classification into the DNA contact mutants and the structurally unfolded mutants has not been able to attribute specific functions or cancer characteristics to either one of these classes of mutants. Although unfolded p53 mutants were more able to interact with p63 and p73 than DNA binding mutants, both types of mutants have been shown to inhibit p63 and p73 function. In addition, RETRA and peptide aptamers were able to increase TAp73 function in cells expressing the folded mutant p53 R273H ,85. ImpoInstead of using the folding state of p53, perhaps we can classify mutants in a different way. Although the traditional six hotspots can be detected in almost all cancers with p53 mutations, certain cancers seem to have a predisposition for certain mutants. An example of this is hepatocellular carcinoma (HCC), in which there appears to be a selection for expression of one hotspot, mutant p53 R249S . Remarka"}
{"text": "CDKN1B plays a key role in many cellular events.3 p27 is a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, which functions to negatively regulate cell cycle progression at the G1/S boundary in response to antiproliferative stimuli. In addition, numerous p27 functions, not related to CDK inhibition, have been described. For instance, cytosolic p27 plays a role in the regulation of cytoskeleton assembly/disassembly, therefore, regulates the cell morphology and movement. In addition, p27 is involved in apoptosis and autophagy modulation.6The cell cycle regulation and tumor suppressor p27 encoded by CDKN1B) was cloned in 19947 and mapped to chromosome 12p13. Later on, p27 mutations were discovered in several types of human cancers including breast cancer, sporadic parathyroid adenomas, endocrine neoplasia, small intestine neuroendocrine tumors.14Mutations, abnormal expression and mislocalization of p27 have been found in many diseases suggesting the important role of p27 in the pathogenesis of diseases. Human p27 gene . Treatment with an AKT inhibitor rescues the mislocalization of p27 to the cytoplasm in endometrial carcinoma cells.18 Similar findings are observed in other types of human cancers.23 p27 is reduced in premalignant and non-invasive cancerous lesions, including ductal carcinoma in situ of the breast. The reduced p27 expression is prognostic for subsequent development of oral squamous carcinoma. In addition, in benign prostatic hypertrophy and low malignant potential of ovarian tumors, the p27 expression levels are decreased compared to normal tissues.Reduced expression and mislocalization of p27 have been identified as an early event in some types of diseases. A study by Mc-Campbell et al demonstrates that loss of p27 expression is an early event in the progression of endometrial carcinoma in the setting of obesity. p27 expression is severely reduced and/or mislocalized to the cytoplasm in histologically \u201cnormal\u201d endometrial glands and endometrial complex hyperplasia with atypia from obese women (CAH) as compared to normal weight women. In luteal phase endometrium, p27 expression is high and primarily nuclear. In contrast, in the majority of endometrial CAH, p27 expression is severely reduced or absent in >70% of these early lesions, and is harshly reduced or absent in 89% of primary endometrial carcinoma. These data indicate that loss of p27 is retained as a feature of early (CAH) and neoplastic endometrial lesions arising in the setting of obesity.Tsc2 tumor suppressor gene are a genetically-defined model for endometrial hyperplasia that processes to endometrial carcinoma by 16 months of age.18 At the early stage of this model, appearing \u201cpre-hyperplastic\u201d glands with activated mTORC1 signaling correlate with loss of the wild-type Tsc2 allele. Early life exposure to xenoestrogen accelerates the development of endometrial hyperplasia in adult female rats.24 Similar to human disease, loss of p27 occurs early in association with the development of obesity-associated endometrial hyperplasia. The energy balance intervention study by McCampbell et al demonstrates that caloric restriction is capable of reducing weight, providing a favorable to leptin/adiponectin ratio, and decreasing the circulating insulin levels in response to early life exposure to genistein. Importantly, caloric restriction also significantly decreases hyperplasia incidence with increased p27 expression levels and relocalization of p27 to the nucleus.18For animal study, Eker rats carrying a defect in the 25 and ovarian cancers,26 decreased expression of p27 correlates with reduced survival in response to platinum-based chemotherapy. In breast cancer,27 decreased expression of p27 is associated with poor outcome after adjuvant chemotherapy. In head and neck squamous cell carcinomas,28 p27 expression serves as a significant predictor of chemotherapy response in multivariate analysis.In human, the effect of chemotherapy can also be predicated according to the expression levels of p27 in some types of cancers. For instance, in non-small cell lung cancer30 Does this dietary intervention also work for uterine fibroids through the same mechanism? Further understanding the mechanism and role of p27 may lead to the development of novel treatment options against many challenging diseases.Although progresses have been made to understand the role of p27 in the pathogenesis of diseases, there remains a gap in our knowledge regarding the abnormal expression and subcellular localization of p27, which contribute to the pathogenies of varied diseases. How these events link to the processes of abnormal cell cycle and development of diseases related to the network of signaling pathways and epigenome? What is the role of p27 in favorable and unfavorable effects of chemotherapy? Also, more pre-clinical studies are needed to determine the effect of treatments in varied types of cancers and diseases. For instance, the energy balance intervention study shows a potent inhibitory effect on hyperplasia incidence in Eker rat model. In addition to endometrial hyperplasia, Eker rats are also a genetically-defined model for the development of uterine fibroids."}
{"text": "M2 gangliosides are a group of lysosomal lipid storage disorders that are due to mutations in HEXA, HEXB and GM2A. In our study, 10 patients with these diseases were enrolled, and Sanger sequencing was performed for the HEXA and HEXB genes. The results revealed one known splice site mutation  and three novel mutations  in HEXA. In HEXB, one known mutation (c.1597C>T [p.R533C]) and one variant of uncertain significance (c.619A>G [p.I207V]) were identified. Five patients had c.1597C>T in HEXB, indicating a common mutation in south Iran. In this study, a unique large deletion in HEXA was identified as a homozygous state. To predict the cause of the large deletion in HEXA, RepeatMasker was used to investigate the Alu elements. In addition, to identify the breakpoint of this deletion, PCR was performed around these elements. Using Repeat masker, different Alu elements were identified across HEXA, mainly in intron 5 and intron 10 adjacent to the deleted exons. PCR around the Alu elements and Sanger sequencing revealed the start point of a large deletion in AluSz6 in the intron 6 and the end of its breakpoint 73 nucleotides downstream of AluJo in intron 10. Our study showed that HEXA is an Alu-rich gene that predisposes individuals to disease-associated large deletions due to these elements.G HEXA), hexosaminidase subunit beta (HEXB) and GM2 ganglioside activator (GM2A). The HEXB gene produces a subunit of two related enzymes that include beta-hexosaminidase A and beta-hexosaminidase B. Both enzymes have two subunits. Beta-hexosaminidase A is made from one alpha subunit produced from the HEXA gene and one beta subunit (produced from the HEXB gene). However, beta-hexosaminidase B is only produced from two beta subunits encoded from the HEXB gene. Beta-hexosaminidase A and B, which are mainly found in lysosomes, break down sphingolipids, oligosaccharides and glycoproteins, and are critical in the central nervous system as they have very essential roles in breaking down the GM2 ganglioside in this system, preventing the accumulation of this product and its damage to the nervous system.1\u20133GM2 gangliosides are a group of lysosomal lipid storage disorders that resulted from disease-causing mutations in three autosomal recessive genes, including hexosaminidase A (http://www.hgmd.cf.ac.uk/ac/index.php), more than 182 disease-causing mutations of the HEXA gene have been identified in patients affected by Tay-Sachs disease (MIM #272800), including missense/nonsense, 96; splicing, 33; small deletions, 28; small insertions, 8; small indels, 3; and gross deletions, 1 (7.6\u2009kb incl. promoter+ex. 1). Regarding the HEXB gene, to date, more than 107 pathogenic mutations in this gene have been reported in patents affected by Sandhoff disease (MIM #268800), including missense/nonsense, 39; splicing, 18; small deletion, 18; small insertion, 3; and gross deletion, 6. These mutations result in the accumulation of GM2 ganglioside in the central nervous system, damaging this systems and presenting the signs and symptoms identified in patients with the corresponding disorders. On the basis of the fully nonfunctional HEXA and HEXB proteins or reduced activity of these proteins resulting from mutations in their genes, these disorders are presented as a severe form found in infancy or a less severe type with a later onset. Tay-Sachs and Sandhoff diseases  are autosomal recessive, progressive neurodegenerative disorders. Their classical infantile forms develop in infancy with developmental retardation, paralysis, dementia, blindness and death usually by 2 or 3 years of age.4\u20136 A study conducted by Smith et al.3 showed that the clinical findings in infantile-onset disease were generally as follow: hyperacusis with excessive startle response in 85% of individuals; increased myotatic reflexes in 85% of individuals ; a pattern of combined axial hypotonia with limb spasticity in 86% of individuals; visual deterioration in 89% of individuals; and seizures in 84% of individuals. Considering the importance of identifying genetic causes in our patients that are suggestive of Tay-Sachs and Sandhoff diseases, the aim of our study was to identify disease-causing mutations in these patients from the south of Iran and report their clinical findings.According to the Human Gene Mutation Database  according to the manufacturer\u2019s instructions. The genomic DNA concentration was measured by a NanoDrop  and stored at \u201320\u2009\u00b0C until use.Ten unrelated patients suspected of GM2 ganglioside diseases from the south of Iran were recruited in the current study. All patients gave informed consent before undergoing genetic analysis of the HEXA and HEXB genes were amplified by the PCR primers given in https://www.ensembl.org) and the use of NCBI BLAST(https://blast.ncbi.nlm.nih.gov), ENSEMBLE BLAST/BLAT (https://www.ensembl.org), UCSC  and Allele ID 7.5 (a bioinformatics software that helps design primers and probes). The total volume of the PCR reaction was 50\u2009\u03bcl, which contained 1\u2009\u03bcl of the forward and reverse primers (20\u2009pmol/\u03bcl), 3\u2009\u03bcl of DNA template (50\u2013200\u2009ng), 25\u2009\u03bcl of TEMPase Hot Start 2x Master Mix Blue  and 20\u2009\u03bcl of dH2O. PCR was carried out according to the Amplicon TEMPase Hot Start program. Ten microliters of PCR amplicons were visualized on a 2% agarose gel containing SYBR Safe . Then, the PCR products were sequenced using Sanger sequencing, and their results were analyzed using NCBI BLAST and Codon Code Aligner software .All of the exon and exon\u2013intron boundaries of the HEXA gene due to Alu elements, RepeatMasker (http://www.repeatmasker.org) was used with the RMBlast search engine (a version that was compatible with the NCBI Blast tool suite). The Repeat masker program screens DNA sequences of interspersed repeats and low complexity DNA sequences. Sequence comparisons in RepeatMasker are carried out using one of the different search engines, including nhmmer, cross_match, ABBlast/WUBlast, RMBlast and Decypher. RepeatMasker uses information from curated libraries of repeats, Dfam and Repbase. We investigated any Alu repeats  in the whole genomic structure of HEXA, covering a total of 34,695\u2009bp, starting 1.4\u2009kb upstream of exon 1 to 1.4\u2009kb downstream of exon 14. Moreover, to identify the breakpoint of the identified large deletion around exons 6 and 10 in our patient, we performed a second PCR around the predicted Alu elements using the primers given in AluJo and AluSx in intron 5, and the reverse primers downstream of AluJo in intron 10). The PCR products were then used for Sanger sequencing. In addition, a coding sequence translator (http://www.fr33.net/translator.php) was used to predict the possible effect of this deletion on the amino-acid sequence of the HEXA protein.In addition, to predict the cause of a new large deletion in the http://www.ebi.ac.uk/Tools/msa/tcoffee/) was applied to compare the amino-acid sequence of HEXA proteins across different species to obtain information about the conservation of amino acids at the site of mutation.To predict the functional effects and conservation status of a newly identified missense mutation in another patient, we also used different bioinformatics tools, including MutationTaster, MutationAssesor, Polyphen, Grantham, PhastCons, GERP, CADD, Fathmm and SIFT. The T-coffee multiple sequence alignment program : c.919G>A [p.E307K], the deletion of exons 6\u201310 in a homozygous state and NM_000520 (HEXA-201): c.622delG [p.D208Ifsx15], which are suspected as being homozygous, were identified. One variant of uncertain significance (NM_000521 (HEXB-201): c.619A>G [p.I207V])8 and one known mutation (NM_000521 (HEXB-201):c.1597C>T [p.R533C])9 in HEXB were found in a homozygous state  in HEXA was identified in our patient. Using the repeat masker tool, different Alu elements were found across the HEXA gene , one in intron 7, one in intron 10 flanking exon 10 (the last exon which was deleted in the deleted block) and three Alus at the 3\u02b9 untranslated region. The exact breakpoint of the deletion around exons 6 and 10 were determined using different PCR products flanking the Alu elements in their corresponding intron-flanked deleted exons. In addition, the consequence of this deletion was predicted to be an in-frame deletion of 192 amino acids of the HEXA protein, leading to its abnormal function , revealing the homozygous status. In her parents, all of the exons produced PCR bands and bands around Alu elements, indicating its heterozygous status  followed by Sanger sequencing, the results revealed that the breakpoints of the large deletion occurred in AluSz6  in the intron 6 and 73 nucleotides downstream of AluJo in intron 10 : c.346+1G>A, IVS2+1G>A)us state . From 10EXA gene , includifunction  and 3. Is status . Using PHEXA was also identified as homozygous in one patient and was confirmed as being heterozygous using Sanger sequencing in her parents . Alu elements are considered to be one of the most successful mobile elements.11 They are ~300 nucleotides in length and have a dimeric structure. The 3\u02b9-end of an Alu element has a poly A-like structure, which plays a key role in its amplification mechanism.12\u201315 There are different subfamilies of Alu sequences that share similar nucleotides in each group, and the most common Alu subfamily in the human genome is the Alu S class (the most common Alu is AluSx).16Alu sequences are responsible for the instability of the genome, which resulted from intrachromosomal and interchromosomal recombination events between these sequences.18 Deletions due to Alu repeats have been identified in different previously reported genes, such as the low-density lipoprotein receptor,19 NACHT, leucine-rich repeat, PYD-containing 720 and HEXB genes.21Alu elements, one of the deletion breakpoints in our study was observed 73\u2009bp downstream of the AluJo element in intron 10 . As shown in AluSz6 and AluJo as the breakpoint occurred in the last nucleotides of AluSz6 and 73\u2009bp downstream of AluJo. Although there is an AluJo in intron 6, its direction is opposite of AluJo located in intron 10 (Alu/Alu occurs). In addition, the similarity between AluSz6 and AluJo is 76% . In addition, in most of the observed events, Alu has a mismatch (homeologous) at the start of recombination.22\u201324It is worth noting that while most breakpoints of deletions occur in o is 76% , which iAluJo is that that some microhomology was observed at this breakpoint, such as with nucleotide adenine  to lysine, a positively charged, polar amino acid (p.E307K). This amino acid is predicted to be essential for the proper functions of this protein , which lacks all of the amino acids after position 373, caused nonsense-mediated decay. Therefore, all of these amino acids are vital for the normal function of the HEXA protein. Since the only identified damaging variation across HEXA and HEXB in both parents of the patient was this deletion and all of the phenotypes of the patient overlapped with the clinical abnormalities found in Tay-Sachs disease, this frameshift mutation can be reported to be the genetic cause of Tay-Sachs disease in this family.Regarding c.622delG and p.D208Ifsx15, it is obvious that this mutation causes a frameshift deletion that led to a premature translation termination. It is worth noting that one of the reported transcripts of http://exac.broadinstitute.org), was identified as homozygous in one of our patients  that has a high allele frequency (0.15148 for G) in the main database, ExAC (HEXA is an Alu-rich gene and is predisposed to disease-associated large deletions. We also found different mutations in the HEXA and HEXB genes and that the most common mutation in HEXB was c.1597C>T, which suggests that screening the HEXB gene for this prevalent mutation is a cost-effective test in individuals with suspicion of having Sandhoff disease. Such studies can help understand the different possible mechanisms of genetic changes in different diseases and help to investigate large deletions in HEXA and HEXB in individuals affected by Sandhoff and Tay-Sachs diseases who do not have any point mutations or small deletions and insertions. In addition, with identification of all of the Alu elements across HEXA in this study, other researchers can look for any possible deletions more easily in special cases of Sandhoff and Tay-Sachs diseases. Until now, no drugs have been used to treat Sandhoff and Tay-Sachs diseases, and a better understanding of their exact pathomechanism in the future can shed light on therapeutic approaches for these diseases using components involved in their biological processes.Current studies have shown that Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
{"text": "To evaluate the frequency of NIH category IV prostatitis, and the use of expressed prostatic secretions tests in an effort to improve the reliability of prostate specific antigen as an indicator, to avoid unnecessary prostate biopsy.178 expressed prostatic secretion positive patients with serum prostate specific antigen levels of \u2265 2.5 ng / mL were included in present prospective study. The diagnostic evaluation included detailed history and physical examination, digital rectal examination, urine analysis, urine culture, and expressed prostatic secretions tests. Transrectal ultrasonography was used both to measure prostate volume and conduct 12 core prostate biopsy.The prevalence of NIH category IV prostatitis was 36.9% (178 / 482) in our population of men. In our study patients (n: 178) prostate biopsy results were classified as; 66 prostatitis, 81 BPH, and 31 Pca. In asymptomatic prostatitis group, expressed prostatic secretion mean leucocyte ratio was higher compared to other two groups (p < 0.0001). The relation between number of expressed prostatic secretion leucocytes and prostatitis, benign prostate hyperplasia, and prostate cancer is analyzed. If 16 is taken as the cut of number for leucocyte presence, its sensitivity is 0.92 (AUC = 0.78 p = 0.01).The number of leucocytes in expressed prostatic secretion is higher in the chronic prostatitis group. If the leukocyte presence of 16 and above is taken as the cut off point, the sensitivity becomes 0.92 (AUC = 0.78). We firmly believe that our new cut off value may be used as to aid prostate specific antigen and derivates while giving biopsy decision. In 1979 Wang et al. discovered Prostate Specific Antigen (PSA) . Pca is National Institute of Health (NIH) classified prostatitis into 4 categories in 1995. NIH defined Category IV as asymptomatic chronic prostatitis due to the presence of inflammatory cells either in expressed prostate secretions (EPS) or during histopathological examination of prostatic biopsies from asymptomatic men , tissue  and urine based  prostate cancer biomarker candidates have been presented during the last ten years -15. ThesThe aim of this prospective study was to further investigate the predictive role of the number of leukocytes in expressed prostate secretions in differentiating histological inflammation from PCa and other non neoplastic lesions. Also, to evaluate the use of EPS tests in an effort to improve the reliability of PSA as an indicator, to avoid unnecessary prostate biopsy.After institutional review board  approval, all study participants provided informed written consent before enrolment and 482 men were recruited in the present prospective study. Patients underwent a four-specimen study according to Meares-Stamey method . After pThe prevalence of NIH category IV prostatitis was 36.9% (178 / 482) in our population of men. In our study patients (n: 178) prostate biopsy results were classified as; 66 prostatitis, 81 BPH, and 31 Pca . None ofWhen prostatitis, BPH, and Pca groups were evaluated according to tPSA levels, there was a statistically significant difference between BPH and Pca (p = 0.022). In terms of tPSA, there was no statistically significant difference between the BPH-prostatitis (p = 0.21) groups and Pca-prostatitis groups (p = 0.45) (p > 0.5). In terms of free PSA (fPSA), no statistically significant difference was seen between in the BPH-Prostatitis group (p = 0.99), and in BPH-Pca (p = 0.72), or Prostatitis-Pca (p = 0.84) groups.In the prostatitis group the number of leukocytes in the EPS was higher than in the other two groups (p < 0.0001). The relation between the number of leukocytes in the EPS and prostatitis, BPH, and Pca was also evaluated. The most appropriate cut off value for the amount of leucocytes in the prostate massage fluid was determined as \u2265 395 (in a high enhancement area with a mean of \u2265 16) as shown in The incidence of Pca patients who diagnosed at low stage, increased because of widespread using PSA testing. The rate of detecting Pca clinically in elevated levels of serum PSA ranges between 17.5% and 38% , 19. DetChronic prostatitis is a disease still without a clear etiology. Polymorphonuclear leukocyte invasion is determined in the intra-prostatic channels and peri-prostatic tissue during histopathological analyses , 21. ChrVarious studies about the prevalence of prostatitis subtypes determined by NIH have been conducted. In studies made with needle biopsy analyses, prostatitis incidence ranging on a large scale between 17.2% and 42% have been reported , 23. HowPSA, an important tumor marker, is organ but not cancer specific, studies about new methods verifying PSA results are in progress. To reduce unnecessary biopsies and to improve on PSA specificity, past research primarily investigated PSA derivatives. These have included PSA velocity, PSA density, age-specific PSA, fractionated PSA and percentage of free prostate-specific antigen (% fPSA) . ForexamUrologists often manage asymptomatic men with a high serum PSA level by observation after antibiotic treatment. Because inflammation can often result in elevated PSA at documented chronic bacterial prostatitis patients. But studies examining this subject in the asymptomatic population have yielded conflicting results. While some studies state that serum PSA can be reduced with a course of antibiotics , 6, otheProstatitis should be diagnosed if there are 10 and more leukocytes present in a high enhancement area (40 X). According to the findings of the present study, number of polymorphous nuclear leucocytes in prostatitis group is higher than in BPH and Pca groups (p < 0.0001). Normally, 10 or more leucocyte counts are sufficient for prostatitis diagnosis. However, the number of leukocytes at the prostate massage fluid in the prostatitis group was higher than the other two groups, so that have us an idea.We thought that, if a new cut off value for leucocyte presence in prostatic fluid is determined, the predictability of prostatitis will be increased and thus unnecessary biopsies may be avoided. The outcome of the Roc curve analysis revealed that average leucocyte presence of \u2265 16 or total leucocyte presence of \u2265 395 is most appropriate and should be taken as the new cut off point in a large enhancement area. The analyses revealed AUC = 0.78 and sensitivity for prostatitis as 0.92.In summary, if the number of leucocytes is 16 and above in the prostatic massage fluid, this may most probably an indicator of prostatitis driven PSA increase. Previous to a biopsy decision, this new cut off value may be applied in clinical practice when encountering a asymptomatic patient presenting for the first time with an elevated PSA and without clinical evidence of prostatitis. Also, we recommend in these patients, it may be more beneficial to utilize other tools such as PSA velocity, PSA density, complexed PSA or newer clinical tools such as the prostate health index. Our new cut off value may be used to lend assistance these tools before biopsy decision.Our study has some methodologic factors that might affect accuracy of our estimates. We didn't correlate the EPS results with histology results of our biopsy. Because, we classified chronic inflammation histologically as; available or unavailable. We didn't make detailed classification. Chronic inflammation of the prostate was defined as infiltration of prostate biopsy specimens by inflammatory cells, lymphocytes, plasma cells and / or histiocytes. Irani et al. classified inflammation with regard to the histological grade and aggressiveness of the inflammatory process: histological grades 0 and 1 were regarded as the \u201cnon-inflammation group\u201d, and histological grades 2 and 3 as the \u201cinflammation group\u201d . This clThe number of leucocytes in EPS is higher in the chronic prostatitis group. If the leukocyte presence of 16 and above is taken as the cut off point, the sensitivity becomes 0.92 (AUC = 0.78). We firmly believe that our new cut off value may be used as to aid PSA and derivates while giving biopsy decision. Further investigations will be necessary to evaluate our new cut-off value accuracy and determine exact cut-off points."}
{"text": "SDHB and SDHD) mutations represent the most frequent cause of hereditary pheochromocytoma and paraganglioma (PPGL). Although truncation of the succinate dehydrogenase complex is thought to be the disease causing mechanism in both disorders, SDHB and SDHD patients exihibit different phenotypes. These phenotypic differences are currently unexplained by molecular genetics. The aim of this study is to compare disease dynamics in these two conditions via a Markov chain model based on 4 clinically-defined steady states. Our model corroborates at the population level phenotypic observations in SDHB and SDHD carriers and suggests potential explanations associated with the probabilities of disease maintenance and regression. In SDHB-related syndrome, PPGL maintenance seems to be reduced compared to SDHD (p = 0.04 vs 0.95) due to higher probability of tumor cell regression in SDHB vs SDHD (p = 0.87 vs 0.00). However, when SDHB-tumors give rise to metastases, metastatic cells are able to thrive with decreased probability of regression compared with SDHD counterparts (p = 0.17 vs 0.89). By constrast, almost all SDHD patients develop PGL (mainly head and neck) that persist throughout their lifetime. However, compared to SDHB, maintenance of metastatic lesions seems to be less effective for SDHD (p = 0.83 vs 0.11). These findings align with data suggesting that SDHD-related PPGL require less genetic events for tumor initiation and maintenance compared to those related to SDHB, but fail to initiate biology that promotes metastatic spread and metastatic cell survival in host tissues. By contrast, the higher number of genetic abnormalities required for tumor initiation and maintenance in SDHB PPGL result in a lower penetrance of PGL, but when cells give rise to metastases they are assumed to be better adapted to sustain survival. These proposed differences in disease progression dynamics between SDHB and SDHD diseases provide new cues for future exploration of SDHx PPGL behavior, offering considerations for future specific therapeutic and prevention strategies.Succinate dehydrogenase subunit B and D ( Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that arise either in head and neck parasympathetic paraganglia or paraaortic chromaffin tissue, which comprise sympathetic adrenal medulla and extra-adrenal paraganglia. Tumors that derive from either parasympathetic or sympathetic paraganglia are collectively named paragangliomas (PGLs) with the term pheochromocytoma (P) being restricted to adrenal PGL .et al. described the first PGL syndrome related to deficiency in succinate dehydrogenase (SDH) enzyme activity due to mutations in SDH subunit D (SDHD), part of mitochondrial complex II and the tricarboxylic acid (TCA) cycle  (where the superscript \u201cb\u201d stands for SDHB); i.e: 70% will remain without disease corresponding to 30% penetrance, and among patients with active disease: 6% HNPGL and 14% sympathetic PPGL, 10% metastatic disease). For the particular value p11 = 0.75, a transition matrix Tsdhb was estimated and is shown in ijp). This choice is p11 is discussed in the Sensitivity Analysis section.For modeling disease dynamics in sdhb  was: P*sdhb = , describing exactly the clinical picture of the disease Pbobs. Our principal findings show that for SDHB sub-group, the stabilization into a clinical state with the development of PPGL is a process driven by high rate of regression to a state without a  tumor (low p21 and p31). However, when PPGL develops, it easily gives rise to metastasis and metastatic PPGL sustains survival (high p44).The model allowed us to simulate PPGL initiation and spontaneous regression of tumors and demonstrate how these two processes can contribute to the disease phenotype. In this case, the asymptotic probability distribution P*SDHD disease dynamics with the following set of Pdobsv = , where the superscript \u201cd\u201d stands for SDHD . The Tsdhd matrix is shown in sdhd = . These results accurately describe the observed probabilities.A first model was parameterized to simulate SDHD PPGL steady-state values were characterized by high values of p12, p11, p22 and p43 and low values of p21 and p31. Therefore, it seems that SDHD PPGL (mainly those from parasympathetic paraganglia) develop more easily than SDHB ones and persist throughout the life (high p12) of a patient. Furthermore, although SDHD primary PPGL can transition into metastatic disease, it mostly fails to develop a stable advanced disease state and are therefore not diagnosed as metastatic (low p44).The observed SDHB and SDHD carriers have different dynamics in PPGL occurrence, regression, and progression thus, providing new insights into the understanding of these tumors. Tables SDHB and SDHD regarding the probabilities for tumor maintenance or regression. First, tumor maintenance is reduced in SDHB patients (p22 and p33 = 0.04) compared to SDHD patients (p22 and p33 = 0.95). Second, the probability of staying in metastatic state is, however, higher for SDHB than SDHD patients (p44 = 0.83 vs 0.11).Our model corroborates clinical observations by showing that 11) had to be fixed prior to the estimation procedure, which was unavoidable to ensure that the number of free-parameters to estimate was equal to 4, the number of observations, as described in the Sensitivity Analysis section.The sensitivity analysis shows robustness of the model with respect to reasonable variations in the 4 percentages of disease, which supports the validity of the modeling method. One parameter  with a more complex pattern and a greater genomic instability compared to SDH with gains and losses confined to other chromosomes. It is, therefore, possible that chromosome 11p loss is necessary and sufficient to trigger SDHD and SDHAF2 tumorigenesis, whereas SDHB tumors require more complex changes with amplification or deletion of multiple driver genes located on different chromosomes, especially 1p. Assuming a constant rate of genetic instability, this could be in agreement with differences in penetrance of the SDHD and SDHB disease. A second assumption is that additional genetic events contribute to the transformation of PPGL cells into a biology that favours metastatic spread , to be in state i throughout life, which is closest to observed value Pobvs[i]. In others words, mathematically, these transition probabilities were estimated by using the method of the mean least squares, which select the pij, which minimizes the quantity: (P*[1]\u2014Pobvs[1])2 +(P*[2]\u2014Pobvs[2])2 +(P*[3]\u2014Pobvs[3])2 +(P*[4]\u2014Pobvs[4])2.Transition probabilities, which are \u201cinvisible\u201d for clinicians, were estimated in order to fit with the limits P* corresponding to the phenotype observed in The following assuptions were made in calculating transition probabilities:p11 was fixed; this choice being discussed below;the transition probability 21 = p31 and p22 = p33;to match the number of free parameters with the number of steady-states, it was assumed that transition probabilities do not depend on tumor location (i.e. head and neck PGL vs. sympathetic PGL): p23 and p32 were fixed at zero;since the coexistence of head and neck PGL and sympathetic PPGL is very rare condition, the transition probabilities p21, p22, p24, p44), the other parameters being either fixed (p11 = 0.75 for SDHB and p11 = 0.10 for SDHD), or set at zero , or constrained by the two assumed relationships (p21 = p31), (p22 = p33), and, obviously, by the fact that the transition probabilities from one state must sum to one. Then the 4 free-parameters were estimated to fit the 4 clinically-defined steady states.These assumptions have allowed reduction of free-parameters to 4: . Therefore, the stability of the model has been assessed by running many computations against different probabilities of stable states drawn at random: the 95% confidence interval for SDHB at state 1 was set to 60\u201380%  and to 0\u201310% for SDHD . The uncertainty on the proportion of patients in states  has been described with two independant Gaussian laws of distribution with a relative standard deviation of 20% . The proportion of patients in a metastatic state was logically chosen so that the sum of the four probabilities would be one. For each random observation generated by this procedure, a check was done to verify that the four probabilities were in , then the model was solved with the least-square method already described; the quality of the fit was verified by checking residuals and the output of the solving algorithm and the probabilities of transition were stored. Eventually (after 500 runs), the 95% confidence intervals on the transition probabilities were extracted.Since PPGL penetrance and the proportion of patients with either HNPGL, sympathetic PGL, or metastatic disease are 11, since this parameter has been fixed in the model. Simulations showed a good fit between clinical observations and model predictions in a wide range of p11 values:  for SDHB and  for SDHD. In SDHB-related PPGL, the quality of fit decreased rapidly for values of p11 below 0.70. However, the results remain qualitatively unchanged and lead to the same biological interpretations regarding transition probabilities. For simplicity, it was decided to provide the estimated results of transition probabilities for p11 = 0.75 for SDHB and p11 = 0.10 for SDHD, since these values are linked to the overall disease penetrance of both SDHB and SDHD diseases.The robustness of the estimation procedure has been evaluated for various values of p"}
{"text": "Restoration of p53 activity in mouse models leads to the suppression of established tumors of different origin. These findings provide a strong support to the anti-cancer strategy aimed for p53 reactivation. In this review, we summarize recent progress in the development of small molecules, which restore the tumor suppressor function of wild-type p53 and discuss their clinical advance. We discuss different aspects of p53-mediated response, which contribute to suppression of tumors, including non-canonical p53 activities, such as regulation of immune response. While targeting p53 inhibitors is a very promising approach, there are certain limitations and concerns that the intensive research and clinical evaluation of compounds will hopefully help to overcome. Since p53 discovery 40\u00a0years ago, it has been established that p53 is a transcriptional factor, which binds to the promoters of its target genes in a sequence-specific manner and regulates their expression, thereby controlling cell cycle and cell death. In unstressed cells, p53 has low activity; however, upon its activation by oncogenes, DNA damage, and other types of stresses, p53 blocks the proliferation of pre-malignant and malignant cells or eliminates them by inducing apoptosis  of hundreds of human cancers revealed an almost indefinite number of combinations of mutations, chromosomal aberrations, copy number changes, and epigenetic alterations. However, these very diverse cancer lesions converge on a few key pathways.To successfully fight cancer, we need to focus on these most crucial pathways and find the best targets within these. To identify the best targets, we should apply the following criteria: the factor is a critical player in essential pathways affected in many cancers; it is non-redundant and is involved in different aspects of tumor development; targeting this factor results in elimination of tumor cells, but does not kill normal cells.p53 fits these criteria very well; inactivation of the p53 tumor suppressor function is required for the development and maintenance of most human cancers. Importantly, p53 is negatively controlling most of the hallmarks of cancer: deregulated proliferation and cell death, replicative immortality, angiogenesis, invasion and metastasis, metabolism and genomic instability, as well as immune response . p53 is Due to the unique mode of p53 inactivation in cancer, restoration of p53 appears to be feasible. In contrast to other tumor suppressors such as Rb, p16, or PTEN, the p53 protein is usually expressed in tumors, although its function is ablated. However, the fact that p53 is a transcriptional factor have made the idea of p53 reactivation unpopular in the past, since transcriptional factors were deemed \u2018undruggable\u2019 until very recently. Latest advances have proven these views wrong and made it possible to develop different strategies for the restoration of p53 activity, depending on the type of p53 inactivation. Reactivation of mutant p53 protein by stabilizing its folding with small molecules appears to be a promising strategy, i.e. development of small molecule PRIMA-1MET/APR246 , which iMDM4 gene). MDM2 regulates p53 via different mechanisms. Due to its E3 ligase activity, MDM2 can either monoubiquitinate p53, promoting its nuclear export, or polyubiquitinate, inducing p53 degradation by the proteasome , or the absence of negative regulators  . A singlin vitro and in vivo  are another class of high-affinity compounds-antagonists of MDM2, designed specifically to mimic the three key hydrophobic residues  of p53, making contacts within the MDM2 pocket. MI-63 and MI-147 induce cell growth arrest in several cell lines due to p53 activation . Moreoveinotecan , while Minotecan . MI-773 inotecan . Howeverinotecan .De novo design, based on the binding mode of previously known MDM2 inhibitors, resulted in the development of a novel scaffold for inhibiting MDM2. Piperidinones AM-8553  induced apoptosis in osteosarcoma SJSA-1 cells overexpressing MDM2 by reactivating the p53 signalling pathway , a steroid fatty acid present in bile, as MDMX inhibitor with higher preference over MDM2. It induces apoptosis in HCT116 human cells, predominantly in p53-dependent manner. However, the induction of apoptosis was achieved only at high concentrations of LCA, limiting its use in clinics  . DHODH iin vivo .Compounds that prevent the interaction of human papilloma virus (HPV) oncogene E6 with p53, such as leptomycin B (LMB) and RITA, can be applied in HPV-positive cancers where proteasomal degradation of p53 by E6 is critical for the survival of cancer cells. LMB blocks nuclear export by inhibition of the export protein CRM1 , while RUpstream regulators of MDM2/X are attractive targets for the design of p53-reactivating compounds. For example, it has been shown that upon nucleolar stress, ribosomal proteins  are released from nucleoli and bind to MDM2, triggering p53 activation . Several\u03baB without causing detectable genotoxicity. The intercalation of curaxins into DNA cause the \u2018chromatin trapping\u2019 of the facilitates chromatin transcription (FACT) complex, which in turn leads to phosphorylation of the p53 Ser392 by casein kinase 2 and inhibition of NF-\u03baB-dependent transcription. Importantly, curaxins suppressed tumor growth in different types of human tumor xenografts grown in mice and are currently being tested in clinical trials  plays a critical role counteracting p53 and MDM2 degradation, making it an interesting target for the development of inhibitors . AlthougTP53 with elevated FLT3 and MDM2 expression are most sensitive to the combined treatment with cobimetinib and idasanutlin  models and ignature . A gene ignature . The preThe development of biomarkers and companion diagnostics is ongoing; it is crucial for the success of p53-based therapies. Clinical data from patients involved in trials would hopefully pave a way for the discovery of reliable biomarkers for p53-based therapies.A number of MDM2 inhibitors are being tested in solid tumors with promising preliminary results. Two clinical trials have been conducted and completed for the small molecule MI-773 in patients with advanced solid tumors. The first study was designed to evaluate the safety and the maximum tolerated dose, as well as pharmacokinetics, biomarkers, and biological effects in solid tumors with no other treatment available, as well as lymphomas. Recently published results from the completed study revealed an accepted safety but, although p53 pathway was activated, it displayed a limited activity as a single agent . TherefoRibosomal protein activators such as actinomycin D and roscovitine also have been or are being tested in several Phase I, II, or III clinical trials, either as monotherapy or in combination therapy with other drugs in several type of cancers .de novo resistance and increase the overall survival in melanoma patients  as the most common adverse effect . The datBesides the effects of p53 reactivation in normal cells, another concern of targeting negative regulators of p53 is the inhibition of their cellular functions other than p53 regulation. Both MDM2 and MDMX have several p53-independent functions, including gene expression regulation and chromatin modification , DNA repARF, amplification or overexpression of MDM2 positive regulators HAUSP, Wip1, and others , the possible selection of p53 somatic mutations in normal cells such as hematopoietic progenitors may lead to the development of new cancers and therefore should be carefully investigated. Systematic search for the mechanisms of resistance to MDM2 inhibitors using piggyBac transposon insertional mutagenesis in spontaneous tumors in p19ARE\u2212/\u2212 mice revealed several mechanisms of resistance. More than half of tumors acquired inactivating mutations in p53 (54%), while others obtained the gain-of-function alterations resulting in high expression of anti-apoptotic protein Bcl-xL, MDMX, and \u0394NTrp63 or \u0394NTrp73, which confer a dominant-negative effect on p53  will be beneficial to combine with p53-reactivating drugs. However, synergistic or synthetic lethality drug interactions remain a largely unexplored area"}
{"text": "A meta-analysis (n = 5) on total energy intake found no significant differences between groups. The BCTs with the highest effectiveness ratio were habit formation (100%), salience of consequences (83%) and adding objects to the environment (70%). The review highlights the potential of behavioural interventions to improve young adults\u2019 fruit and vegetable intake but was less convincing for other dietary outcomes. Due to the lack of studies including each BCT, the BCTs imperative to success could not be identified.Poor eating habits are common during young adulthood and influence chronic disease morbidity. This systematic review evaluates the effectiveness of interventions aiming to improve dietary intake among young adults and, identifies which behaviour change techniques (BCTs) are most effective. Six electronic databases were searched for RCTs published until October 2018, and evaluating behavioural interventions assessing change in dietary intake in young adults (17\u201335 years). Of the 18,779 articles identified, 54 were included. Forty studies focused on fruit and/or vegetable intake, of which 63% showed a significant between-group difference in favour of the intervention group. Meta-analysis ( Globally, young adults have some of the unhealthiest eating habits compared to other age groups. In a systematic assessment of diet quality among adults in 187 countries, those aged 20\u201329 years had the lowest diet quality score  compared to any other age group \u00bd and the sample size was as reported for the study. If SDs rather than SEs were reported, the SDs were used in place of the SEs and n1, n2 was set at 1. For studies reporting intake of fruit and vegetables separately, we converted the results as follows; the means for fruit and for vegetable were summed to give a total fruit and vegetable mean. For the standard deviation (SD) and sample size, a conservative approach was taken assuming the Standard Error\u2019s (SE) for each mean were different. SD combined score was determined as [n1.SE1For each study, the effect at baseline and each time point, expressed as months post baseline, was estimated as the mean difference (mean intervention group \u2212 mean control group) using the unbiased option for variance estimates. Uncertainty in the mean estimates was expressed as 95% confidence intervals. To account for multiple measures per study a series of multilevel models were investigated with nested random effects top level being study, then treatment type and time period using REML estimation. The final model contained study and time as nested random effects as the treatment type random effect had zero variance. Moderator variables examined as fixed effects in the model were treatment type (simplified into three groups for fruit and vegetables and two groups for TEI), and time was treated as a categorical variable in two versions  and a simplified grouped form with baseline, up to three months and greater than three months. The interaction between these two was tested. As the time random effect was similar in size to the study random effect there was substantial correlation between time periods, so the moderator effect for the three-group version of time was estimated at each of the two follow-up time periods as difference from baseline. Residual plots were used to examine homogeneity of variance and normality assumptions. To assess for publication bias, funnel plots were produced and visually inspected and supported with rank correlation tests for funnel plot asymmetry.In parallel with similar reviews ,30, we cOf the 18,779 manuscripts identified, a total of 54 individual studies  were included in the review . Study cn = 29, 54%). In addition, most studies were from the US , in a college/university setting  and predominantly in white/Caucasian populations . The included studies had a total of 16,383 participants  and the mean age of participants was 20.2 years with 57% (n = 31) within the age category of 17\u2013\u2264 25 years. Most studies included both male and female samples , and of these the average proportion of males was 32%. Over one-third of the interventions were multicomponent , and there was an equal proportion of studies which solely used either ehealth or face-to-face as the intervention delivery . Over half of the included studies had dietary change as a primary outcome , while dietary change was a secondary outcome for the remaining 22 (40.7%) studies. Studies where dietary change was a secondary outcome primarily focused on obesity/ weight gain prevention , obesity treatment  or PA . In the studies with dietary change as a primary outcome, most focused on fruit and/or vegetable intake , while the remaining focussed on diet quality score , micronutrients  or percent of energy coming from fat . Interventions to improve eating habits among young adults has escalated in recent years, with over half of the included studies published from 2014 to 2018 (Twenty-five 46%) studies used a food frequency questionnaire (FFQ) for assessment of dietary intake ,64,65,66% studiesn = 38, 70%). The mean intervention duration across the studies was 4.2 months (range: single session to 24-months), with the majority being short term between one single session and \u22643-months . The mean length of follow-up from end of intervention was 1.8 months (range: no follow-up to 23-months), with the majority having between no follow-up and \u22643-months . Finally, there was a mean retention rate of 78% (range 22% to 98%) at the end of the intervention and a mean retention rate of 66% (range 11% to 97%) at the longest follow-up point. The total number of study arms was 133 (range: 2 to 6 arms), with the majority being two-arm interventions  adequately described study attrition and any exclusions from the analysis. Almost half  of the included studies described suitable generation for the allocation sequence, yet most  failed to describe the method for allocation concealment. There was a high or unclear risk of bias for most studies for blinding of participants , blinding of those delivering the intervention  and blinding of outcome assessors . Most studies provided insufficient detail  to determine if they were free of selective outcome reporting.The risk of bias assessment is summarised in p = 0.014) with a reduction of 326 kcal/day in the intervention group (comprised of group-based face-to-face lecture and lab sessions) compared to +72 kcal/day in the usual-lifestyle control group. This effect was not significant at 16-months. When all studies were considered, there were a total of twelve studies which measured change in TEI (none as primary outcome), reported as either Calories or kilojoules per day ,77,79,83p < 0.790) showing a mean decrease in TEI relative to baseline \u2212132.9 kJ/day up to 3 months  and \u2212300.2 kJ/day for >3 months  (p = 0.4904). The funnel plot  in five studies ,50,51,52, 402.4) . There wnel plot  demonstrA total of 40 studies measured fruit and/or vegetable intakes ,78,79,81Fruit and vegetable intake were reported as (i) serves per day in 12 studies ,71,74,81There were 18 studies that reported fruit intake separately from vegetable intake ,77,78,792(5) = 16.96, p < 0.001) showing a mean increase in fruit and vegetable intake relative to baseline 68.6 g/day up to 3 months  and 65.8 g/day for >3 months  (2(2) = 0.42, p = 0.8091) nor an interaction between treatment type and time Wald \u03c72(4) = 0.318, p = 0.53). The funnel plot (p < 0.001. Removing the study by Chang (2010) with the two largest values in the funnel plot did not change the bias tau 0.30, p = 0.003. Plots of the means for the effects are in Meta-analyses of fruit and vegetables in 17 studies ,46,47,81, 104.0) . There wnel plot  demonstr 95% CI: .6, 100.5A total of 15 studies assessed the change in intakes of at least one nutrient dense food ,79,80,81p < 0.001) increased Diet Behaviour Score  when compared to usual lifestyle control group  at 1-month. Statistically significant improvements in diet quality score was evident in one study (out of five) . This stp < 0.001) increase in whole grain bread intake in the intervention group (+46g/day) compared to control (\u221212g/day) at 5-months. A significant between-group difference in individual food groups was evident in one study (out of five) . This nuehealth weight loss program  for young women on percentage energy from core foods found a significant improvement in the intervention group when compared to control  at six months. The one study which explored the effectiveness of an p = 0.049). Statistically significant improvements in specific micronutrient and dietary fibre rich foods were evident in two studies (out of seven) ,79. SpecEight studies assessed the change in energy-dense beverage intake ,67,69,76eHealth components  in young Australian adults for 12 weeks. The odds that the TXT2BFIT intervention group improved intake compared to the control group were significantly greater for SSBs at the end of the program (p = 0.024) and at 9-month follow-up (p = 0.018) [p = 0.03). The SSB intake decreased in the intervention group (\u22120.53 drinks/day) and increased in the control group (+0.50 drinks/day) [Two studies (out of seven) demonstrated a significant reduction in SSB intake when compared to control ,67. Specnks/day) .p = 0.001) [One study reported change in fruit juice intake and a small but significant between-group effect was evident (= 0.001) . AlthougA total of 12 studies assessed the change in intakes of at least one ED-NP food ,69,76,79ehealth  and face-to-face sessions. Those in the HEYMAN intervention had a reduction of 9.8%  in EDNP foods/day, compared to a reduction of 2.6%  in the control group. The \u2018Be Positive Be Healthe\u2019 weight loss program for young women was delivered via ehealth components . Those in the intervention group had a reduction of 8.3%  in EDNP/day, compared to an increase of 0.9%  in the control group. Two studies (out of three) demonstrated a significant reduction in percentage energy from ED-NP foods when compared to control ,57. Bothp = 0.013) and at 9-month follow-up (p = 0.010) [p = 0.007).Two studies (out of five) demonstrated a significant reduction in takeaway/fast food intake when compared to control ,67. The = 0.010) . Those aFour studies considered the effect of a healthy eating intervention on hot chips (serves/week) , sweets/One obesity treatment study explored the effect of an intervention consisting of a smartphone application combined with SMS from a health coach on sodium intake, added sugar intake and total sugar intake  in overwA total of 15 studies assessed change in macronutrient content  ,81,82,83Specifically 15 studies explored change in dietary fats ,81,82,83p < 0.01) and at 15-month follow-up  in percent energy from fat [p < 0.01) in percent energy from fat [eHealth intervention consisting of email and website to improve diet and physical activity had a significant linear time by treatment interaction for percent energy from saturated fat  at 12 weeks and 24 weeks [p = 0.02) [Four studies (out of 15) reported a significant intervention effect in dietary fat intake ,45,50,82from fat . Anotherp < 0.05), but this effect was not sustained at 16-month follow-up [One study (out of eight) reported a between-group decrease in total grams of carbohydrate at the end of the program at four months  but this effect was not maintained at 16-month follow-up [n = 6) which assessed change in protein intake did not establish any significant differences between groups.The same study which identified a change total grams of carbohydrate also reported a between-group decrease in total grams of protein at the end of the program at 4 months , \u2018Goal setting behaviour\u2018(n = 32), \u2018Information about health consequences\u2019 (n = 29), \u2018Action planning\u2019 (n = 27) and \u2018Social support (unspecified)\u2019 (n = 24). n = 24) were included in the analysis [The percentage effectiveness ratio for the coded BCTs is displayed in analysis .Thirteen BCTs had an effectiveness ratio >50%. The BCTs with the highest effectiveness ratio were \u2018Habit formation\u2019 (effective in five out of five or 100% of interventions), \u2018Salience of consequences\u2019 (effective in five out of six or 83% of interventions), \u2018Adding objects to the environment\u2019 (effective in seven out of 10 or 70% of interventions), \u2018Action planning\u2019 (effective in 16 out of 27 or 59% of interventions) and \u2018Prompts/cues\u2019 (effective in seven out of 12 or 58% of interventions). p = 0.85, indicating that none of the BCTs were different to the overall mean value. Although \u2018Habit formation\u2019 had the highest percentage effectiveness ratio, many more studies are required before this could be confirmed as having greater effectiveness than others.An overall test of significance was carried out using a contingency table between type of BCT and effectiveness, there was no significant relationship, Monte\u2013Carlo exact chi-squared test, \u03c72(23) = 16.7, p = 0.93. The percentage ratio of effective versus noneffective interventions by the number of BCTs was also explored . The resn = 54 RCTs) that has assessed the effects of interventions aiming to improving dietary intake outcomes in young adults. It is also the first to explore the effectiveness of BCTs for improving dietary intake among this group. Predominantly, studies targeted fruit and/or vegetable intake as the primary outcome and a meta-analysis (n = 17 studies) demonstrated intervention participants to significantly increase intake of fruit and vegetables by +68.6 g/day up to three months and +65.8 g/day for >3 months when compared to control. A meta-analysis exploring change in TEI (n = 5 studies) showed no significant differences between groups. The narrative synthesis showed that interventions targeting fruit and/or vegetable intake were most effective (significant between group differences evident in 25 out of 40 studies or 63%), followed by energy-dense beverages (three out of eight or 38%), nutrient dense foods (five out of 15 or 33%) and macronutrients (four out of 15 or 27%). The BCTs with the highest percentage effectiveness ratio were habit formation, salience of consequences and adding objects to the environment. Despite this, the overall test of significance demonstrated no significant relationship between type of BCT and effectiveness and many more studies are required before these BCTs could be confirmed as having greater impact on effectiveness than others.This is the largest systematic review to date (The modest increases in fruit and vegetable intake (mean difference of +68.6 g/day up to three months and +65.8 g/day for >3 months) is clinically important. In another meta-analysis among prospective cohort studies with 833,234 participants, the risk of all-cause mortality was decreased by 5% and cardiovascular disease by 4% for each additional serve a day of fruit and vegetables . This isWhen compared to other reviews in adults and children, the mean difference in fruit and vegetable intakes between intervention arms  is lower. A systematic review of 34 behaviour-based interventions found an average increase in fruit and vegetable intake of +1.13 and +0.39 serves per day in adults and children , while aAdditionally, the meta-analysis demonstrated no significant effect on TEI. Of the five studies in the meta-analysis, none had TEI change as a primary outcome, and only one study  aimed to+\u2009\u2248\u20090.45). Previous work has suggested that habits are behavioural patterns that are learned through context-dependent repetition [This review compared the individual BCTs in effective versus noneffective interventions to identify which specific BCTs may be agents of change. Thirteen BCTs had an effectiveness ratio >50% and \u2018habit formation\u2019 had the highest percentage effectiveness ratio . An example of this technique in an intervention includes; prompting participants to fill half of their plate with either salad or cooked vegetables each night at dinner. These results correspond with another meta-analysis which found habits to be consistently correlated with physical activity and nutrition behaviour ; with a petition ,93, and petition .Focusing on the natural consequences of performing a behaviour was found to be effective; with \u2018information about health consequences\u2019 demonstrating a percentage effectiveness ratio of 52% (effective in 15 out of 29 studies). An example of this technique in an intervention includes providing participants with morbidity and mortality statistics from related health conditions. Emphasising the consequence by making it more memorable had even greater effectiveness with \u2018salience of consequences\u2019 demonstrating an effectiveness ratio of 83% (effective in five out of six studies). An example of this techniques used in an intervention includes; presenting visual images of the negative consequences of eating unhealthily, i.e., blocked arteries. These results are comparable to a review of 45 brief nutrition interventions in adults, whereby accentuating the consequence of a behaviour had greater effectiveness than just providing information about a health consequence . Also thp < 0.04) predicted long-term effect on diet and physical activity behaviours [Adding objects to the environment was a technique that was shown to be effective with a percentage effectiveness ratio of 70% (effective in seven out of 10 studies). An example of this in an intervention was to provide participants with a free fruit and vegetable box to facilitate healthy eating. The effectiveness of this technique may coincide with known financial barriers associated with this age group . These rhaviours .p < 0.001) and long term (p < 0.01) [Furthermore, setting goals (effective in 18 out of 32 studies or 56%) and planning actions (effective in 16 out of 27 studies or 59%) in terms of performing a target behaviour were found to be effective BCTs. Examples of these techniques in interventions were (i) participants set a goal of eating two pieces of fruit per day and (ii) participants were instructed to write plans of when and where they would consume an extra piece of vegetable per day, for the next week. The effectiveness of goal setting of behaviour is comparable to a meta-regression analyses among 48 interventions in overweight and obese adults, which showed that this particular BCT was independently associated with better intervention effects on diet and physical activity behaviours at short ( < 0.01) . Similar < 0.01) .The current review established no difference in number of BCT\u2019s in relation to effectiveness on nutrition behaviours. This corresponds with several other systematic reviews in different population groups ,98. OverThe risk of bias assessment identified several strengths. Generally, studies provided adequate detail to describe study attrition, any exclusions from the analysis and suitable generation for the allocation sequence. Despite this, many studies either had high risk of bias or lacked the necessary detail to judge the quality for selective outcome reporting, allocation concealment and blinding of participants, personnel and/or outcome assessors. Additionally, publication bias was evident in the meta-analysis of fruit and vegetable intake. There was an over-representation of studies in primarily female populations, in Caucasian samples and within college/University settings, which may limit the generalisability. Studies primarily focused on a limited selection of food groups with most focusing on fruit and vegetable intake, but very few considered overall diet quality. Few studies measured long-term dietary outcomes; as such, there is limited evidence to determine the long-term effectiveness of dietary interventions in young adults. Coding of BCTs was problematic due to inconsistencies and differences in reporting of intervention components. A stringent coding strategy was applied, where BCTs were not coded as present if there was insufficient description. However, some of those BCTs coded as absent techniques may have been apparent but not adequately described. This issue has been identified in previous attempts to categorise intervention content , and theThe novelty of this review in being the first to explore which BCTs are associated with effective interventions in young adults is a strength, as is the use of a robust method to code the BCTs, using the most recent and comprehensive taxonomy of BCTs available . AlthougThis systematic review and meta-analysis highlights the potential of lifestyle behavioural interventions to improve young adults\u2019 fruit and vegetable intake, but the evidence was less convincing for other dietary outcomes. More high quality studies are needed to determine longer-term effectiveness, and with consideration of overall diet quality. This is especially important due to the poor eating habits of young adults worldwide. This review also demonstrated the potential of the following BCTs; \u2018habit formation\u2019, \u2018salience of consequences\u2019 and \u2018adding objects to the environment\u2019 in positively changing dietary behaviour. However, due to the lack of studies, including each BCT, this review could not identify which BCTs are imperative to success and more studies are required before confirming which BCTs can be considered as having greater effectiveness than others. To better understand which BCTs are linked to intervention efficacy, future research must describe interventions in detail to allow for identification and replication of information, for example, publishing intervention protocol papers and/or including a checklist of exact BCTs implemented with supplementary material of each publication. Identifying and utilising effective BCTs for dietary interventions in young adults will aid the development of potentially more effective and replicable interventions, policies and nutritional guidelines, thus building a strong evidence base to support healthy eating habits among young adults."}
{"text": "The 2-year overall survival (OS) and locoregional control (LRC) rates of all patients were 62% and 71%, respectively. The 2-year OS and LRC rates in patients who had received EBRT and concurrent chemotherapy with docetaxel, cisplatin and 5-fluorouracil  were 100%. These results were higher than the 2-year OS and LRC rates of 62% and 69%, respectively, in patients who had received radical surgery and EBRT (n = 13). The rates were 0% in those who had neither received TPF nor undergone surgery in addition to EBRT (n = 2). Grade 3 bone or soft tissue necrosis was observed in 2 patients who had undergone surgery and postoperative EBRT. Our data suggest that the combination therapy of EBRT and surgery and/or chemotherapy may be the most effective treatment options for advanced EACC, and EBRT with concurrent chemotherapy with TPF is potentially the most acceptable.External auditory canal cancer (EACC) is a rare malignant tumor. In the present study, we retrospectively evaluated the treatment results in patients with advanced EACC who were treated using external-beam radiotherapy (EBRT) combined with chemotherapy or radical surgery. Overall, 21 patients with Stage III ( External auditory canal cancer (EACC) is a rare malignant tumor that affects 1\u20136 per 1 million population-years . The lowet al.  and 71% , respectively Fig. .n = 13) and patients who did not receive surgery (n = 8), between patients who received chemotherapy (n = 12) and patients who did not receive chemotherapy (n = 9), or between patients who received an EBRT dose of >50 Gy (n = 11) and patients who received EBRT dose of \u226450 Gy (n = 10). Regarding the influence of the chemotherapy regimen used, the OS and LRC rates of patients who received TPF (n = 6) were longer than those who received chemotherapy with other chemotherapeutic regimens (n = 6) or no chemotherapy (n = 9), although the differences were not significant  and 80% , respectively, those of patients who received a combination of surgery and EBRT were 50%  and 63% , respectively, and those of patients who received a combination of chemotherapy and EBRT were 83%  and 86% , respectively. Patients who received EBRT alone showed local disease progression at 2 months after the start of the treatment.n = 6) were 100% and 100%, respectively. These results were better than those of patients who received surgery and EBRT with or without chemotherapy , and also better than those of patients who did not receive TPF or surgery . There was a significant difference in the OS and LRC between the three groups [OS, P < 0.001 . The EBRT dose differed significantly between the above two groups, and there was a borderline difference in the follow-up duration , stereotactic radiotherapy (n = 1), or stereotactic radiotherapy and additional surgery (n = 1). However, all patients died between 4.7 and 16  months after the find of recurrences.The two patients with in-field recurrences received chemotherapy, followed by best supportive care, and the patients with out-of-field recurrences received chemotherapy  was only 29% [i et al.  and Leon [et al.  showed tet al. [et al. [There was an impressive result in our study: the 2-year OS rate of the eight patients who did not receive surgery was 73% (which was higher than the rate found in past studies) , 6. Amonet al.  reportedet al. [Katori et al.  have comet al. , the sevet al. [et al. [Although brain injury has been seen with radical radiotherapy doses of 66 Gy in 2 Gy per fraction, Pemberton et al.  have rep [et al.  have repBecause this present study was retrospective and the treatment policy has changed with time, it appears that there could have been a bias in patient selection. Moreover, the observation period of patients who received EBRT and TPF was shorter than that of the patients who underwent EBRT and surgery, and further long-term follow-up may be necessary for a reasonable comparison between the two treatment strategies.In conclusion, our data suggest that the combination therapy of EBRT and surgery and/or chemotherapy may be the most effective treatment options for advanced EACC, and EBRT with concurrent chemotherapy with TPF is potentially the most acceptable. However, further investigation is needed to determine the optimal dose and the appropriate field for EBRT in patients with EACC. Furthermore, because of the limited number of patients with this cancer, a multicenter study would be desirable."}
{"text": "E. coli (STEC) can cause serious illnesses, including hemorrhagic colitis and hemolytic uremic syndrome. This is the first systematic review of STEC in Brazil, and will report the main serogroups detected in animals, food products and foodborne diseases. Data were obtained from online databases accessed in January 2019. Papers were selected from each database using the Mesh term entries. Although no human disease outbreaks in Brazil related to STEC has been reported, the presence of several serogroups such as O157 and O111 has been verified in animals, food, and humans. Moreover, other serogroups monitored by international federal agencies and involved in outbreak cases worldwide were detected, and other unusual strains were involved in some isolated individual cases of foodborne disease, such as serotype O118:H16 and serogroup O165. The epidemiological data presented herein indicates the presence of several pathogenic serogroups, including O157:H7, O26, O103, and O111, which have been linked to disease outbreaks worldwide. As available data are concentrated in the Sao Paulo state and almost completely lacking in outlying regions, epidemiological monitoring in Brazil for STEC needs to be expanded and food safety standards for this pathogen should be aligned to that of the food safety standards of international bodies.Shiga-toxin producing  Brazil is one of the largest producers and exporters of food in the world. Animal products, such as beef, poultry, pork, fish, and crops such as corn, soybeans, and rice represent Brazil\u2019s major exports . HoweverEnterobacteriaceae, with Escherichia coli representing a major species. This bacterium represents one of the most extensively studied and was one of the first to be sequenced [Escherichia coli is the main bacterium involved in food contamination in Brazil [The main microorganisms involved in food contamination belong to the equenced ,5. In adn Brazil . It possn Brazil .These pathogenic groups are involved in outbreaks related to food consumption, and STEC in particular are of importance to public health, potentially causing diarrhea, bloody diarrhea (hemorrhagic colitis), hemolytic uremic syndrome (HUS) and renal injury ,9,10. STEscherichia coli STEC with a focus on Brazil and to compare the presence of serogroups detected in food products, animals and humans.In Brazil, there are no reports of outbreaks involving STEC. The hypothesis is that: (i) Disease outbreaks are not being recorded due to lack of a centralized reporting system or (ii) disease outbreaks are not being recognized as there is no surveillance system for STEC. Farm animals have been shown to be carriers and contamination of food as well as sporadic STEC infections in humans have been reported ,14. In aEscherichia coli\u201d OR \u201cVerotoxigenic\u201d OR \u201cSTEC\u201d OR \u201cShiga Toxigenic E. coli\u201d OR \u201cShiga Toxigenic Escherichia coli\u201d OR \u201cShiga Toxin-Producing Escherichia coli\u201d OR \u201cVTEC\u201d OR \u201cVero Cytotoxin-Producing Escherichia coli\u201d OR \u201cVerotoxigenic E. coli\u201d OR \u201cVerotoxigenic Escherichia coli\u201d OR \u201cVerotoxin-Producing Escherichia coli\u201d AND \u201cBrazil\u201d. Papers in both English and Portuguese were included in this review. Duplicates were traced and excluded. A total of 161 papers were collected, independent of sample size or culture/detection methods. The full text for each paper was obtained and evaluated individually  and noted that stx1 and stx2 were present in 30.9% of the strains, indicating possible dispersion of the stx genes between STEC and APEC. In addition, stx1 and stx2 were detected in 4.7% of poultry litter samples in the south of Brazil  and USDA-FSIS (Food Safety and Inspection Service), which require their absence in meat products. In addition, STEC were isolated from 2.7% to 78.3% of sheep and O103:H2 was detected in sheep  and has f Brazil .Stomoxys calcitrans). Prevalence rates in dogs ranged between 0 and 48%, but to date there are no reports of STEC in cats in Brazil.In other species that could possibly act as vectors, STEC rates were 0.7% to 20.4% in wild birds and 15% in stable flies . Through whole genome sequencing, the O104:H4 strain in Brazil was found to be similar to a strain isolated from an American citizen diagnosed with hemolytic uremic syndrome (HUS) who had traveled to Germany during the 2011 HUS outbreak [The presence of STEC contamination in humans was evaluated in 22 scientific articles . In fiveoutbreak .In other studies of diarrhea or healthy subjects, the serogroups predominantly detected were O26:H11, O103:H2, O111 Not typeable (NT), O118:H16, O165:NT and O157:H7 .Some of these serogroups were reported in animal and food studies in Brazil. For instance, the O111 and O157 serogroups were verified in all three groups. Our hypothesis is that the dispersion of the strains has contaminated all stages of the food chain (pre and post-processing). Moreover, the O103 strains in animals were linked to those found in humans. In contrast, stains O165 and O118:H16 were only detected in human clinical cases.The most frequent serogroups described in peer-reviewed papers in samples collected in Brazil are represented in Serogroup O111 was also reported in animals, food and humans and has been linked to clinical human cases in Brazil ,66,77 anSerogroup O165 has been reported in cases of hemolytic uremic syndrome in Japan , and GerEscherichia coli strains, enabling the bacteria to survive during passage through the human stomach [Studies evaluating different cattle feeds indicate that different diets may increase the acid resistance of  stomach . Strain  stomach  identifistx2e) or other genetic factors including virulence [Foods showed the greatest diversity among serogroups, followed by animal sources . This diirulence ,108.Studies were concentrated mainly in Brazil\u2019s most developed areas and especially in the state of Sao Paulo , with thE. coli STEC tests for O157 and the \u201cbig six\u201d non-O157 in beef destined for export [Currently, the main legislation in force in Brazil concerning food production is Resolution 12 of 2001 , which ar export . HoweverThe analysis procedure determined by MAPA is based on the methodology used by the USDA FSIS , comprisMoreover, the improvement and cost reduction of molecular tools such as whole genomic sequencing (WGS), metagenomics, and others will facilitate the understanding of serotype epidemiology and the dispersion of these strains in neighboring countries and in other continents. Epigenetics advances will also improve the understanding of gene expression and the impact of good animal management practices, as well as possible genome mutations that may influence virulence and antimicrobial resistance profiles .The epidemiological data presented in this review indicate that O157:H7, O26, O103 and O111 strains, classified as foodborne pathogens and monitored by the USDA-FSIS (USA), U.S. FDA, EFSA (European Food Safety Authority - EU) and MAPA , are currently circulating in different regions of Brazil. Although no STEC outbreak cases in Brazil have been reported, several animal, food and human studies have indicated the presence of STEC in Brazil and it has been related to several foodborne outbreaks around the world. Thus, improved epidemiological monitoring and food production control is necessary. Novel studies should be financed in regions presenting significant agricultural production, such as Brazil\u2019s central region in order to better assess potential threats and prevent human STEC infections."}
{"text": "CDKN1A gene. It is a major p53 target gene involved in cell cycle progression that has been extensively evaluated. To date, p21 has been reported to regulate various cell functions, both dependent and independent of p53. Besides regulating the cell cycle, p21 also modulates apoptosis, induces senescence, and maintains cellular quiescence in response to various stimuli. p21 transcription is induced in response to stresses, including those from oxidative and chemotherapeutic treatment. A recent study has shown that in response to metabolic stresses such as nutrient and energy depletion, p21 expression is induced to regulate various cell functions. Despite the biological significance, the mechanism of p21 regulation in cancer adaptation to metabolic stress is underexplored and thus represents an exciting field. This review focuses on the recent development of p21 regulation in response to metabolic stress and its impact in inducing cell cycle arrest and death in cancer cells.Cancer cells possess metabolic properties that are different from benign cells. These unique characteristics have become attractive targets that are being actively investigated for cancer therapy. p21cip1/waf1, also known as Cyclin-Dependent Kinase inhibitor 1A, is encoded by the  Eukaryotic cells utilize complicated mechanisms to regulate cell division and quiescence in response to both internal and external stimuli. These cells rapidly divide under certain conditions such as embryonic development and wound healing. Conversely, they stop proliferating in response to adverse conditions and enter cell cycle arrest and post-development quiescence. Hence, the cells develop a sophisticated braking system required for survival. Deregulation of this mechanism can result in loss of genome integrity and cancer development. p21 (Cip1/WAF1) was first identified as a Cyclin-Dependent Kinase (CDK) regulator that inhibits the retinoblastoma gene (Rb) phosphorylation and G1/S cell cycle progression ,4,5. p21p21 transcription can be regulated by either a p53-dependent or -independent manner. These regulations were described in the early years of p21 studies, where p21 induction in response to radiation-induced DNA damage was characterized as p53-dependent . In contDuring normal tissue development and serum-stimulated growth in vitro, p21 is primarily regulated in a p53-independent manner by a number of transcription factors . Under vEpigenetic processes can also regulate p21 transcriptional activation. They can be induced through the p53-independent pathway by chromatin remodeling following acetylation of histones H3 and H4 in the p21 promoter region ,38,39,40p21 is also subjected to post-translational modification. Most notably, it is regulated by serine-threonine kinases phosphorylation at Ser-130, Ser-146, Thr-57 and Thr-145. These phosphorylation events impact both the function and stability of p21. For example, Thr-145 phosphorylation by AKT kinase disrupts p21 binding to PCNA and enhances its cytoplasmic localization. This, in turn, promotes cell survival and transformation in breast cancers ,45,46,47Inhibition of cell cycle progression is one of the main cellular functions of p21. It mainly occurs by inhibiting cyclins A/CDK2, E/CDK2, and D1/CDK4 activities, thereby preventing the phosphorylation of Rb protein . The abip21 has been demonstrated to regulate apoptosis in a paradoxical manner, depending on the cellular context ,54,55. Ip21 is also a major regulator of cellular senescence, a complex program involving multiple signaling pathways. p21 interacts with p16INK4A tumor suppressor to inhibit cyclin-dependent kinases, suppressing retinoblastoma protein phosphorylation and the expression of cell proliferation-associated genes . SimilarA hallmark of stem cells is their ability for self-renewal. Most stem cell populations are in the quiescent state until they are stimulated. p21 has been reported to maintain cellular quiescence ,67 and pApart from its well-established roles, p21 may also directly or indirectly influence cancer metastasis. However, the association between p21 expression and metastatic ability is inconsistent in different cancers. In some cancers, p21 levels are inversely related to the metastatic phenotype, where a higher p21 expression predicts a favorable disease outcome ,73,74,75Both p21 inactivation and activation have been implicated in cancer therapy resistance. p21 inactivation is associated with paclitaxel and 5-Fluorouracil (5FU) resistance in noncancerous breast epithelial and colon cancer cells, respectively ,81. In cMetabolism is a cornerstone for various cellular functions. It is so important that Major metabolic enzymes and regulatory pathways are conserved from unicellular organisms to mammals. Metabolic stress can be triggered by either nutrient unavailability or malfunction of important proteins involved in the metabolic machinery. Adaption to metabolic stress is essential for cell proliferation and survival. p21 is involved in cellular responses to various stressors ,87,88. Rp21 plays an important role in cell response to nutrient deficiency. Studies have shown that p21 is transcriptionally upregulated in mice under short-term fasting ,90. A maCellular nutrient uptake can stimulate intracellular nutrient-sensing mechanisms. This, in turn, activates anabolic pathways aiming at increasing biosynthesis. Apart from nutrient availability, the stimulation of growth factor signaling also accelerates anabolism. Nutrient deprivation, particularly glucose, is sensed by the AMP-activated kinase (AMPK), a key regulator of the catabolic-anabolic balance. AMPK is an allosteric enzyme that is directly regulated by the AMP to ATP ratio, which is a readout for cellular energy status ,96. GiveWhile it is not the focus of the current review, it is important to note that AMPK can also sense DNA damage stress and induce p21 transcription in both p53-dependent and-independent manners ,100,101.Amino acids are important building blocks and signaling intermediators. Their deficiency can alter many cellular metabolic functions that, in turn, regulate cell cycle progression and survival through complex regulations, including those involving p21 ,107,108.In cancer, p21 is generally perceived as a tumor-suppressor protein. This hypothesis is supported by the observations that p21-null mice spontaneously developed tumors . AdditioCompared with their benign counterparts, cancer cells have distinct metabolic characteristics that present therapeutic opportunities. One of the most recognized signatures of cancer metabolism is the Warburg effect, or aerobic glycolysis, which describes the preference of cancer cells using glycolysis over oxidative phosphorylation to metabolize glucose and derivatives for energy production. As a result of incomplete oxidation, most cancer cells consume glucose to generate more metabolic intermediates and lactic acid than benign cells ,117. MulDespite their dependence on glycolysis in the metabolism, mitochondria respiration in cancer cells is important for generating energy and nucleic acid synthesis . As a maRAS mutation driven-oncogenesis accounts for one-third of human cancers. The relevance of the RAS mutation becomes even more apparent if the aberrancies in relevant pathways such as EGFR  mutations and MAPK (Mitogen-Activated Protein Kinases) signaling components are also taken into consideration ,136,137.To better understand the mechanisms underlying p21 elevation in response to ICMT inhibition and its role in cancer cell proliferation and survival, we evaluated a panel of cancer cells from diverse tissue origins. Cancer cells that are sensitive to ICMT inhibition show a constellation of changes related to metabolism that include the reduction of mitochondria respiration, the induction of autophagy, cell cycle arrest and apoptosis ,146,147.To better understand how p21 regulation leads to cell adaptation following metabolic distress, it is necessary to briefly discuss the cellular process of autophagy\u2014a fundamental eukaryotic cell adaptation in response to a nutrient-depleted state. The double membranous structure of autophagosomes forms as a self-digestive organelle in the lysosomal pathway to degrade damaged/aged organelles and protein aggregates; this maintains homeostasis in cells under normal conditions . AutophaIn evaluating the impacts of ICMT inhibition in cancer cells, we found that p21 is induced only in the sensitive cell lines upon ICMT suppression. Marked reduction in mitochondrial respiration and consistent depletion of cellular \u201cfuel molecules\u201d, such as ATP and other NTPs, were also noted following exposures to the ICMT inhibitor. The mitochondria targeting effect has been shown to be responsible for most of the anti-proliferative effects . The pheAs discussed earlier, p21 regulates cell cycle progression and apoptosis. Therefore, it makes physiological sense that p21 would be involved in sensing and transmitting the signal of metabolic stress. Indeed, when we further evaluated the role of p21 in response to ICMT inhibition, we found that p21 is an upstream regulator of cell cycle progression, autophagy and apoptosis, through the transcriptional regulation of downstream effectors that include BNIP3 (BCL2 Interacting Protein 3), LC3 (Microtubule-associated protein 1A/1B light chain 3B) and ULK1 (Unc-51 Like Autophagy Activating Kinase 1). Both ULK1 and LC3 are well-known proteins that promote the initiation and extension of autophagosomes. BNIP3 is a mitochondrial member of proapoptotic BCL2 family proteins containing a motif similar to the BH3 domain, which is a regulator of both autophagy and apoptosis ,165,166.In summary, we have discussed the evidence that supports p21 as a key regulator and signaling node in mediating cancer cells\u2019 response to nutrition and fuel depletion. Overall, the evidence supports that p21 functions to integrate catabolism activities, cell cycle progression, and ultimately programmed cell death B. While The field of p21 has grown tremendously in the last three decades. Since its discovery in the 1980s as a Cyclin-Dependent Kinase inhibitor, p21 has attracted significant attention from researchers in many fields, as evident from the thousands of research publications on this protein. To date, p21 is known not only as a cell cycle regulator, but also as a key player in various other cellular functions including senescence, apoptosis, and self-renewal. p21 has also been long regarded as a stress response protein. In this regard, p21 can function as a coordinator for DNA damage repair pathways. Adding to its portfolio, p21 has recently been reported to act as a central regulator in cell adaptation to metabolic stress, particularly energy depletion in response to either starvation or treatment with mitochondria respiration inhibitors. Although this aspect has only been recently investigated with relatively few publications, the impact of these findings in understanding basic cell biology and the development of potential new therapy is apparent. For example, although recent findings demonstrate that stress-induced levels of p21 primarily leads to cell cycle arrest and cell death, the exact outcomes of p21 activation are very much dependent on the type, intensity and duration of particular stress, as well as the cell type upon which the stress acts . AnotherThis review hopefully highlights (i) the significance of emerging p21 research as a promising therapeutic approach for biologists and oncologists, and (ii) the necessity to continue the effort in understanding p21\u2019s function as a coordinator of metabolic stress responses. As mentioned above, because of its dichotomy function, it is a challenging task to fully understand how p21 regulates various biological and cellular functions, particularly those associated with the cancer metabolism and its potential therapeutic implementation. Concerted explorations to understand p21 in adaptation to stress, metabolic and other stimuli, are essential for its translational application in cancer therapy."}
{"text": "Across various axis-1 disorders, the severity of dissociative symptoms is significantly related to a history of childhood traumatization. Thus, the question arises if coping with childhood trauma leads to neural adaptations that enhance the frequency of dissociative processing during adulthood. The aim of the two reported studies therefore was to identify and replicate gray matter alterations associated with dissociation.In a first study, whole-brain MRI data were acquired for 22 female in-patients with trauma-spectrum disorders and a history of severe childhood trauma. Voxel-based morphometry (VBM) was carried out to test for significant correlations between dissociation  severity and gray matter volume. Dissociation severity was positively associated with volume in the left angular gyrus. This result was diagnosis-invariant. The replication study involved 26 female in-patients with trauma-spectrum disorders and 25 healthy controls. No significant association between dissociation severity and brain volume in a left angular gyrus region of interest located at the peak identified in study 1 was identified and no significant group difference in this region could be established.trans-diagnostically with structural brain alterations in the left angular gyrus and independent replication in a larger patient sample is essential.The angular gyrus has previously been implicated in the processing of agency and vestibular integration as well as dissociative processing. The current attempt at a direct replication of brain volume alterations however, failed. The data thus only partially support the notion that dissociative processing is associated  Within the dissociative disorders, DPD is the most alike to the set of dissociative symptoms reported by patients suffering from other axis-1 disorders, the difference being that in DPD the symptoms are typically chronic, often present without any fluctuation, and include excessive emotional numbing. DPD is a highly disabling condition and constitutes a dissociative disorder in its own rights. Several large-scale investigations of DPD indicate that this disorder is not consistently associated with severe childhood trauma . The etitrans-diagnostically in subjects with a history of childhood trauma. Morphological patient studies have previously been criticized for both reporting biases  dissociative processing following childhood trauma is associated with specific neural alterations in structures such as the angular gyrus, superior frontal region, and temporal poles, and (b) those alterations exist Study 2 was conducted in order to replicate the results of Study 1 using a confirmatory analysis limited to the brain structures identified in Study 1. In addition, the patient data were compared to healthy controls.All patients were recruited from an in-patient treatment program at the Clinic of Psychotherapy and Psychosomatic Medicine of the University Hospital Dresden. The clinic is specialized in the treatment of patients with a history of severe childhood trauma who express symptoms of dissociation, emotion regulation difficulties, and PTSD \u2013 a disease pattern often described as complex PTSD. Clinical diagnostics as well as detailed anamnestic interviews were performed by trained psychotherapists in all patients. In addition, the Structured Clinical Interview for DSM-IV Diagnosis SCID;  was perfIn order to delineate the neural correlates of dissociative symptomatology from potential effects of childhood maltreatment, only patients with a clear history of significant childhood trauma were included. Criterion was a score of \u226511 on the childhood trauma questionnaire, indicating a value within or above the 95th percentile in the American  and Germ2014Both studies were conducted according to the Declaration of Helsinki and approved by the local ethical board of the Technical University of Dresden. After complete description of the study, written informed consent was obtained from all participants.In Study 1, data from 22 female in-patients aged 22\u201359 years  and 64% suffered from PTSD (F43.1: n = 14). Further, somatoform disorders (F45: n = 9), personality disorders (F60-61: n = 7), anxiety disorders (F40-41: n = 4), and a history of substance abuse (F1: n = 4) were frequent. 59% were diagnosed with other mental disorders (n = 13).In this in-patient sample, comorbidity was the norm rather than the exception. On average, each patient was diagnosed with 3.4 mental disorders. In total, 64% of the patients suffered from major depression . Furthermore, anxiety disorders (F40-41: n = 18), somatoform disorders (F45: n = 8), and personality disorders (F60-61: n = 2). 12% were diagnosed with other mental disorders (n = 3). Similar to Study 1, most patients were on medication, mainly antidepressants  = 0.5; ionnaire  served aAll participants completed the childhood trauma questionnaire CTQ;  and the \u22171 mm\u22171 mm). For Study 2, imaging data were collected on a 3 T Siemens Magnetom Verio scanner  with an 8-channel phased-array head coil. T1-weighted anatomical images of the whole head were acquired .All imaging data of Study 1 were collected using a 1.5 T MRI scanner  with the manufacturer\u2019s 8-channel phased array head coil. T1-weighted anatomical images of the whole head with 1mm isotropic resolution using a 3D magnetization prepared gradient rapid acquisition gradient echo (MPRAGE) sequence  = 352 mm \u00d7 352 mm \u00d7 384 mm; reconstructed voxel size: 1 mm1) and Matlab R2015b  with settings recommended by Separately for both studies, VBM analysis was performed using SPM12  was used for global normalization to account for differences in brain size.In Study 1, voxel-wise correlation of gray matter volume with dissociation severity (mean percentage score on the FDS) was performed using multiple regression analyses with absolute threshold masking at 0.2. Total gray matter volume , were added as nuisance effects in separate covariance analyses.post hoc added additional analyses controlling for these group differences. As different classes of medications are known to have different effects on brain activation , we tivation  and morptivation  in PTSD p < 0.001 in combination with a non-stationary threshold to balance the risks of Type-I and Type-II errors  for potential effects of medication by covarying the intake of each of the five types of medication , (2) covaried the severity of CM, and (3) covaried depression severity by using those variables as regressors of no interest in the analysis.In Study 2, we carried out confirmatory testing of the main result of Study 1 by extracting averaged VBM parameter estimation from an 8 mm sphere built around the angular gyrus peak obtained in Study 1 (-45 -56 24) for each individual. In addition, the data were analyzed for the group of patients and controls separately by replicating the exploratory whole-brain, voxel-wise correlation of gray matter volume with dissociation severity following procedures outlined above  in order to ensure that our null-finding was not due to a slight misplacement of the sphere used for confirmatory testing. In order to test whether the diverging results in the two patient groups might be due to differences in sample composition, we In a separate analysis, the gray matter volume of patients and controls were contrasted against each other under control of the total gray matter volume. In order to unmask the effect of dissociation, the group comparison was done with and without inclusion of the FDS value as a regressor of no interest.r = 0.52, p = 0.014), but not with depression levels .FDS scores assessing dissociation severity ranged from 3.2 to 61.4  and depression scores on the BDI ranged from 8 to 51 (mean 26.5 \u00b1 10.6 SD), and childhood trauma severity measured with the CTQ ranged from 11.2 to 22.4 , but missed statistical significance. An additional cluster was observed in the left inferior frontal gyrus, pars triangularis, however this did not hold for non-stationary cluster extent correction and is only reported in order to get a comprehensive picture  = 1.4; p = 0.18] and childhood maltreatment , but they reported significantly stronger depression .For patients, FDS scores assessing dissociation severity ranged from 5.5 to 79.0 , depression scores ranged from 23 to 62 (mean 39.2 \u00b1 9.5 SD) and childhood trauma severity ranged from 11.4 to 24.6  or depression levels .In this sample of patients, severity of dissociation did not correlate significantly with severity of childhood trauma (t(49) = 6.2; p < 0.001, d = 2.5], depression , and childhood maltreatment . Dissociation scores did not correlate significantly with childhood trauma  or with depression scores  in the control group. As dissociation scores deviated significantly from a normal distribution in controls, Spearman correlation was used for the latter statistics.Controls reported the expected low values for dissociation , depression , and childhood maltreatment . As compared to patients, controls did report significantly lower values for dissociation [r = 0.13, p = 0.53; r = 0.32, p = 0.12). To test whether the replication in the patient group failed due to the significantly higher depression scores, the correlation between dissociation severity and brain volume in the angular gyrus was repeated while covarying out depression scores. However, taking depression severity into account did not render this correlation significant  and right middle frontal gyrus  and in the thalamus  and no significant volume increases were observed in the group of patients. After inclusion of the FDS as covariate, the volumetric differences between groups were limited and only the left inferior frontal gyrus  was found to be significantly reduced in the group of patients.Compared to controls, patients exhibited a significantly reduced volume in the left  only marginally missed the extent threshold, indicating that it was mostly driven by the more proximal dissociation variable. Similarly, depression severity and medication status cannot fully account for this association, albeit the detected cluster were also smaller in size in these post hoc covariation analyses .In Study 1, a significant positive correlation between dissociation severity and gray matter volume in the left angular gyrus, extending into the middle temporal lobe, was established in female patients with a history of severe childhood trauma The angular gyri are involved in many different functions and are considered a cross-modal integration hub . No significant correlation between dissociation severity and volume of the left angular gyrus in the region of interest was established neither in the patient nor the control group. In addition, the group comparison in Study 2 did not result in a significant difference in angular gyrus volume. The explorative, whole brain analysis revealed that patients exhibited a significantly reduced volume bilaterally in the dorsolateral prefrontal gyrus and the thalamus as compared to controls. This group difference is likely related to differences in dissociation severity as it was rendered non-significant upon covariation of FDS scores. The dorsolateral prefrontal gyrus has been implicated in emotion regulation , and oneThere are some notable differences between the two data sets outlined below. However, whether these are significant enough to potentially explain the failed replication attempt remains unknown. While the two patient samples were both convenience samples from the same, specialized treatment center and their average target symptom scores were comparable , the sample of Study 2 exhibited significantly higher depression scores  than the sample of Study 1. However, covarying out the depression scores did not render the association between dissociation severity and regional gray matter volume significant.r = 0.82; in-treatment patients r = 0.80; r = 0.53 in an in-patient sample; In addition, no significant correlation between childhood trauma severity and dissociation scores was established in Study 2, while it was highly significant in Study 1. This is noteworthy as a large review found consistently significant associations between childhood trauma severity and dissociation scores across several samples of both healthy controls and patients for the dissociation instrument used in our study . There iPotentially, the observed alterations could also be due to differential effects of psychotropic medication . HoweverOn a more technical note, it might be worth mentioning that patients from Study 1 were scanned on a 1.5 Tesla scanner vs. a 3 Tesla scanner used in Study 2 and different reconstruction filter settings in the two scanners cannot be excluded.In short, we noted several limitations to our studies: small samples sizes, different clinical characteristics in terms of depression severity and the association of dissociation severity with childhood trauma severity, different medication profiles in the two groups, and differences in the technical instrumentation. Both studies were limited to female participants and hence no conclusions can be drawn regarding potential effects in males.However, it seems unlikely that the described differences in sample composition and data acquisition can fully account for the divergence of the results. It is possible that the selection of participants was biased in undetected ways, i.e., that not both samples are good representatives of the population of patients. Thus, it is possible that a true effect was detected in Study 1 but not Study 2. Vice versa, and taking into account that a severe publication bias in this field has previously been demonstrated convincingly , it alsoJD and IC drafted the manuscript. All authors contributed to revising the manuscript. JD, PH, KD, and IC were involved in the data analysis. AS, JS, and KW were involved in recruitment and clinical assessment of patients. AS and FR were involved in MRI data acquisition.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."}
{"text": "However, the crucial factors that are responsible for the initiation of this crosstalk or the \u2018recruitment\u2019 process remain poorly understood. We propose that oncogenes themselves may influence the \u2018recruitment\u2019 of the stromal cells, while focusing on mutant p53. Apart from losing its tumor-suppressing properties, mutant p53 gains novel oncogenic functions, a phenomenon dubbed mutant p53 gain of function (GOF). Here, we discuss possible ways in which mutant p53 may modulate the microenvironment in order to promote tumorigenesis. We thus propose that mutant p53 may serve as a key player in the modulation of the tumor\u2013stroma crosstalk in a way that benefits the tumor. Further elucidation of these \u2018recruitment\u2019 processes, dictated by mutant p53, may be utilized for tailoring personalized therapeutic approaches for patients with tumors that harbor p53 mutation.A plethora of studies suggest that the non-transformed cellular and non-cellular components of the tumor, collectively known as the tumor microenvironment, have a significant impact on the tumorigenic process. It was suggested that the microenvironment, which initially restricts tumor development, is recruited by the tumor and maintains a crosstalk that further promotes cancer progression. Indeed, many of the molecules that participate in the tumor However, the crucial factors that are responsible for the initiation of this crosstalk or the \u2018recruitment\u2019 process remain poorly understood. We propose that oncogenes themselves may influence the \u2018recruitment\u2019 of the stromal cells, while focusing on mutant p53. Apart from losing its tumor-suppressing properties, mutant p53 gains novel oncogenic functions, a phenomenon dubbed mutant p53 gain of function (GOF). Here, we discuss possible ways in which mutant p53 may modulate the microenvironment in order to promote tumorigenesis. We thus propose that mutant p53 may serve as a key player in the modulation of the tumor\u2013stroma crosstalk in a way that benefits the tumor. Further elucidation of these \u2018recruitment\u2019 processes, dictated by mutant p53, may be utilized for tailoring personalized therapeutic approaches for patients with tumors that harbor p53 mutation.A plethora of studies suggest that the non-transformed cellular and non-cellular components of the tumor, collectively known as the tumor microenvironment, have a significant impact on the tumorigenic process. It was suggested that the microenvironment, which initially restricts tumor development, is recruited by the tumor and maintains a crosstalk that further promotes cancer progression. Indeed, many of the molecules that participate in the tumorIt is now well accepted that cancer development does not depend solely on cancer cell-autonomous characteristics. Ample evidence suggests that various normal cellular and non-cellular components found adjacent to the tumor, known as the tumor microenvironment or tumor stroma, play a major modulating role in different stages of tumor development . The tumIt was suggested that initially, the microenvironment may serve as a barrier to tumor formation, even in the presence of potentially oncogenic genetic aberrations in precancerous cells . Howeverin vivo (Though wild-type (WT) p53 has been extensively studied, most studies focused on its cell-autonomous functions. However, accumulating evidence suggests that p53 has a non-cell autonomous tumor-suppressing role, particularly in the tumor stroma . One of in vivo . Conditiin vivo . Our recin vivo .Though the aforementioned studies are consistent with the role of WT p53 as a tumor suppressor, a recent paper described a confounding finding, in which WT p53 seems to promote tumor-promoting functions of CAFs, suggesting that WT p53 may be implicated in the \u2018education\u2019 of stromal cells in the microenvironment . AnotherWhile the non-cell autonomous role of WT p53 in stromal cells has been investigated, only few studies addressed how mutant p53, which is found predominantly in tumor cells, as well as being the most commonly mutated gene in cancer , affects\u03b2 and IL-6, and extracellular matrix (ECM)-related proteins, such as the ECM remodeler, matrix metalloproteinase 3. Intriguingly, we found that different p53 mutations promoted the CGS in different underlying mechanisms. While p53 DNA contact mutations, namely p53R248Q and p53R273H, promoted the CGS via nuclear factor \u03baB (NF-\u03baB) activation, p53 conformational mutants, namely p53R175H and p53H179R, promoted the CGS by inhibiting BTG2, thus alleviating its inhibition on mutant H-Ras , thus suggesting a possible dominant-negative effect of mutant p53 over WT p53  response elicited by the CAFs and triggered non-cell autonomously by the presence of mutant p53 in the cancer cells. Interestingly, the activation of the IFN-\u03b2 response seemed to be dependent on cell\u2013cell contact, since CM derived from mutant p53-harboring cells and CAFs caused increased activation of this response, while CM derived from tumor cells alone produced a much milder effect. In turn, mutant p53 caused an attenuation of the IFN-\u03b2 activation in the cancer cells themselves by upregulating SOCS1, a negative regulator of the IFN pathway, therefore alleviating a suppressing effect of IFN-\u03b2 on tumor cell migration. Thus, we demonstrated both non-cell autonomous function of cancer cell-expressed mutant p53, by inducing the IFN-\u03b2 response in adjacent stromal cells, as well as modulation of the IFN-\u03b2 signal by mutant p53, in a way that benefits the tumor cells.Finally, in our previous study, we were able to demonstrate an intricate crosstalk between patient-derived CAFs and lung cancer cells, in which mutant p53 induces the secretion of a factor from the neighboring cells and modulates the response to this factor in the cancer cells . We utilIn light of the aforementioned studies, it is tempting to speculate that mutant p53 in cancer cells may indeed serve as a potential stromal modulator. Mutant p53, as well as other oncogenes, may serve as both the initiators and the propagators of the tumor\u2013stroma vicious cycle, during which they promote the \u2018re-education\u2019 of the cells in the stromal compartment, as well as causing a modulation of the signals arriving from the stromal cells in a way that benefits the tumor cells."}
{"text": "Dear Editor-in-Chief,We read Mauro Cozzolino\u2019s criticism regarding vitamin D as a follicular marker of human oocyte quality and a serum marker of in vitro fertilization outcome with great interest , 2. In hFirstly, we did explain in the discussion that one of the limitations of our study was due to not measuring vitamin D binding globulin level (VDBP). Instead, we considered a total 25(OH)D level, as bioavailable 25(OH)D is not bound to VDBP. We also explained in detail that VDBP is a highly polymorphic protein. The most common VDBP phenotypes in the Caucasian population, which was our study population, have the lowest affinity for 25(OH)D. Furthermore, recent study from Fabris et al. on the oocyte donor population did not show any dependence or statistical association between the values of total 25(OH)D and bioavailable 25(OH)D regarding the ovarian reserve, response to ovarian stimulation, oocyte quality in egg donors, or ongoing pregnancy rates after fresh embryo transfer in oocyte recipients . Also, FSecondly, regarding sperm parameters and the blastocyst rate, we did describe male factor as an exclusion criterion mainly based on semen concentration. We agree with Cozzolino that men commonly present not just one alteration but a combination of sperm defects. However, some studies indicated that total motile sperm count is a better indicator for the severity of male factor infertility than the WHO sperm classification system . NeverthBut it was not the aim of our study. We would like to emphasize that the primary aim of our study was to determine whether intrafollicular 25(OH)D level correlates with ICSI/ET results. We did not provide data concerning blastocyst rate in the \u201cResults\u201d section of our study population, what we believe Cozzolino is referring to. We have focused on blastocyst rate in a group of oocytes with matched FF gathered from the first dominant follicle, which usually contains a good oocyte. That is why we think that comparing our blastocyst rate of oocytes gathered from the dominant follicle with other studies is like comparing apples with oranges. Nevertheless, in our study, 88 embryos out of 322 MII oocytes were transferred after ICSI on the third day of the culture. In this group, we had 28 clinical pregnancies (31.8%). Then, out of 164 remaining embryos, 78 developed blastocysts , which gave a blastocyst rate of 47.5%. In contrast, quoted Piccolomini et al. saw 10,925 blastocysts develop in 4205\u00a0cycles out of 32,031 MII oocytes with a blastocyst rate of 34.1% [To conclude, oogenesis is a complicated process, and it is impossible to have one definitive marker of oocyte competence in follicular fluid . We agreSincerely,Przemys\u0142aw Ciepiela, M.D. Ph.D."}
{"text": "Cryptococcus neoformans, the causative agent of cryptococcosis, causes ~\u2009181,000 deaths annually, with males having a higher incidence of disease than females (7M:3F). The reason for this sex bias remains unclear. We hypothesized that this disparity was due to biological differences between the male and female immune response.C. neoformans \u00b1 exogenous testosterone or 17-\u03b2-estradiol. C. neoformans, B, T, and NK cell proliferation was quantified by flow cytometry. Cytokine analysis was conducted via protein array or ELISA. Serological testing was conducted to determine previous exposure to C. neoformans.Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated and infected with C. neoformans proliferated more in male PBMCs. T cell percentages in both sexes were lower in infected versus uninfected cells. Male PBMCs had lower CD3+, CD4+, and CD8+ T cells percentages during infection compared to females. Cytokine profiles showed differences in uninfected male and female PBMCs, which subsided during infection. Only one donor was sero-negative for prior C. neoformans exposure. There was an effect of estrogen in one dataset.These results suggest that males show an inherent deficit in T cell response during infection, which may contribute to the increased incidence of disease in males.The online version of this article (10.1186/s13293-019-0258-2) contains supplementary material, which is available to authorized users. Cryptococcus neoformans is responsible for an estimated 220,000 cases of cryptococcosis, resulting in more than 181,000 deaths each year worldwide . Males also had lower percentages of CD4+ helper T cells compared to females . Amounts of CD8+ cytotoxic T cells, and B cells, as measured by CD19, a marker expressed on the surface of all B cells, were all approximately the same in uninfected control cultures from men and women (p > 0.05). Numbers of dead immune cells after 7 days of culture were also similar between sexes . The numbers of NK cells and dead immune cells after 7 days of culture were similar between sexes  cell CD6+, and CC. neoformans proliferation in the presence of immune cells from healthy men and women, we quantified yeast numbers via flow cytometry after seven days incubation with PBMCs. While there was no difference in proliferation in male or female PBMCs with a specific hormone treatment, when C. neoformans counts were compared between men and women, C. neoformans generally proliferated better in PBMCs from men compared to women, except when exogenous 17-\u03b2-estradiol was added. Male PBMCs had significantly higher cell counts in treatments without hormone  and with the addition of exogenous testosterone , but there was no difference in cell counts between males and females when exogenous 17 \u03b2-estradiol was added  and female cell populations . Like T cells, B cell percentages declined in male  and female  samples. Interestingly, compared to uninfected cells, the percentages of dead PBMCs decreased during infection in males  but not females .odulator , 39. ThuF = 17.9, df = 1476, p < 0.0001) and samples from women . Compared to uninfected PBMCs, the NK cell percentage remained the same in samples from men  and in samples from women  in the presence of C. neoformans. Percentages of dead cells also remained the same in men  and women . These results confirm that, overall, C. neoformans depresses the immune response, even in proliferating cells from healthy individuals. Similar results were seen in subsets of cells incubated with exogenous sex hormones (data not shown).Similar to the first dataset, the second dataset showed a depression of T cells measured during infection in samples from men . The original differences between CD3+ and CD4+ observed in PBMCs from males and females grew significantly during infection . There was no significant difference between male and female B cell percentages in the presence of C. neoformans   or in the immune response in cell subsets with exogenously added testosterone or 17 \u03b2-estradiol (data not shown). Like the first dataset, the second dataset showed significantly lower percentages of T cells in infected male compared to infected female samples . In this dataset as well, original differences between the percentages of CD3+ T cells observed in PBMCs from males and females grew significantly during infection (p < 0.0001). There were no differences in NK cells   or dead cells  between sexes (data not shown). This data reveals lower percentages of T cells in healthy male PBMCs during a C. neoformans infection.After establishing baseline differences in immune cells, coccosis , 40\u201344. F = 21.05, df = 738, p < 0.0001) in the second dataset. To determine if this was due to circulating levels of estrogen in the female donors, we tested whether there was a difference depending on the week of the menstrual cycle the donors were in when they donated blood. Of the 14 women volunteers in the second dataset, six of them were in the second week of their cycle, which is known to have higher levels of circulating estrogen [+  and CD56+  cells compared to donors that were in weeks 1, 3, and 4 of their cycle. When we looked for differences in cell percentages in uninfected female samples based on the week of their menstrual cycle, the pattern was different. Uninfected PBMCs from female donors in week 3 of their cycle had significantly higher percentages of CD3+  and CD56+  cells compared to donors that were in week 0 of their cycle (data not shown). These data suggest that circulating levels of estrogen can affect immune cell proliferation.Interestingly, there was a significant effect of estrogen between infected cell percentages for males and females . There were no differences in IL-8, IL-12, and GM-CSF levels between uninfected cell supernatants in males and females, nor were there any differences in cytokine levels in males and females in infected cell supernatants. Comparing cytokine profiles between uninfected and infected groups , we saw significant increases in the Th1 cytokines IL-2, GM-CSF, IFN-\u03b3, and TNF-\u03b1 , and IL-4 (p = 0.047), a Th2 cytokine, during a C. neoformans infection. There were no differences in IL-6, IL-12, and IL-10 levels between uninfected and infected cells . However, recent studies have shown relatively equal exposure rates between men and women [C. neoformans antigenic testing was performed on sera collected from subjects in both datasets to determine serology status of each, using an agglutination test (Immy) and confirmed by immunoblot. Only one donor had a history of working with C. neoformans. Of the 62 apparently healthy subjects tested (40 from the first dataset and 22 from the second), 35 were male (56.4%), and the mean age was 24.7 years. Only one sample was confirmed as sero-negative (1.61%) through immunoblotting. The sero-negative donor was a healthy female. Since previous C. neoformans exposure should generate an enhanced memory response, the immune response was correlated to serology status. There was a significantly higher percentage of CD3+/CD4+ T cells in the sero-negative donor compared to the sero-positive donors . There were no differences in CD3+/CD8+ T cell percentages, B cell percentages, or cytokine response (data not shown). As there was only one donor without previous exposure to C. neoformans, this data should be interpreted with caution.Historically, it has been postulated that males suffered greater incidences of cryptococcosis, due to more frequent environmental exposure of nd women , 48. To C. neoformans in the absence or presence of testosterone or 17 \u03b2-estradiol. The goal was to understand the differences in the host immune response between males and females to determine if those differences could contribute to the observed differences in cryptococcosis between the sexes. Our cohort of subjects is unique, in that, they are healthy and not typical candidates for cryptococcal disease. Given the nature of cryptococcosis, it is accepted that patients suffering from the disease have multiple comorbidities, or at the very least, a compromised immune system, making it difficult for researchers to discern how C. neoformans acts on the host devoid of other variables. Keeping this in mind, these experiments were designed with the aim of teasing apart differences that may be masked or accentuated when studying typical C. neoformans patients.This is the first study to look at the adaptive immune responses of healthy male and female PBMCs when exposed to C. neoformans. The cytokine data clearly suggest that male PBMCs, but not female PBMCs, were proliferating in response to infection. Additionally, levels of CD25+ cells increased in infected male and female PBMCs, but the increases were not significant, likely due to the small sample size. It is interesting that the cytokine data suggest that male PBMCs, but not female PBMCs, were proliferating, especially since we saw significantly lower percentages of all tested T cells in male PBMCs compared to female PBMCs after infection. We hypothesize that since females naturally have higher levels of CD3+ and CD4+ T cells [The in situ cytokine levels and amounts of the activation marker CD25 between uninfected and infected male and female PBMCs were measured to determine if the PBMCs were proliferating in response to infection with  T cells \u201337, perhC. neoformans proliferated better in PBMCs isolated from males compared to females, both without additional hormone and in the presence of testosterone. Interestingly, there was no difference in C. neoformans cell counts between males and females in the presence of added 17 \u03b2-estradiol, suggesting that 17 \u03b2-estradiol may either inhibit proliferation or boost the immune response, as observed by Mohr et al [C. neoformans in male macrophages ex vivo [C. neoformans proliferation in males is decreased in the presence of 17 \u03b2-estradiol. Since 70% of patients with cryptococcal disease are male [C. neoformans infection.Notably, hr et al . Given o ex vivo , we hypo ex vivo \u201352, we hare male , 53, thi+ and CD4+ counts are typically higher in females [+ and CD4+ population. During infection, T cell percentages in both males and females dropped significantly compared to uninfected controls. This is unsurprising given C. neoformans known immunomodulatory effects [C. neoformans. This finding could also be due to differences in the phosphorylation state of SAMHD1 during C. neoformans infection, as the authors found that sex influenced the phosphorylation state of SAMHD1 during HIV-1 infection, which translated into decreased macrophage susceptibility in females [This idea was reinforced by the T cell data in males. Reference ranges for healthy adults in the USA show that CD3 females , 37. Uni effects , 54\u201356.  females .+) response is needed to successfully mediate the immune response to a cryptococcal infection and has been shown to directly inhibit the growth of C. neoformans [+) response is more complex. Despite their ability to decrease fungal burden in the host, CD4+ cells have recently been linked to advanced clinical symptoms and mortality [+/cryptococcal meningitis+ patients and found that despite having higher CD4+ T cells, males had higher risk of death [+ patients, T cell depletion is the primary predisposing cause for development of cryptococcal meningitis [C. neoformans. This may begin to explain why males experience higher rates of disease and death from cryptococcosis.A robust cytotoxic T cell  percentages fell significantly in cell populations from both men and women, but there was no difference between the two groups in the presence of C. neoformans, suggesting that the significant drop in T cells observed in men may be more important than B cells in an immune response to C. neoformans. The role B cells play in a C. neoformans infection have not been as well elucidated as those of T cells, but multiple studies report the necessity of B cells, often serving as a first line of defense during C. neoformans infections and a correlation between increased levels of B cells (IgM+) and a decreased likelihood of immunocompromised patients developing cryptococcosis [+ subjects that already have T cell deficiencies and therefore may rely more heavily on an effective B cell response.While T cells represent one arm of the adaptive immune response, B cells represent the other. During infection, B cell  that levels of circulating sex hormones can actively affect an immune response, and (2) higher levels of 17 \u03b2-estradiol may boost the immune response. The known pro-inflammatory effects of 17 \u03b2-estradiol may explain both this data and the data of Mohr et al. [Many factors are thought to influence the differences between male and female immune responses. Environmental stimuli, microRNAs, and X-linked immunoregulatory genes are all evidenced to contribute to what is commonly considered the female immune advantage \u201374. The r et al. , 69.C. neoformans in men and women, thus increasing the reliability of our findings. There are limitations in this study worth noting. First, these experiments were done in healthy individuals who normally do not develop cryptococcal disease. Thus, in order for the observed differences in the male and female immune response to contribute to the future understanding of cryptococcal disease, the differences would have to result in important changes in immune function that would persist in relevant \u2018at risk\u2019 populations. Future studies will examine this hypothesis. Second, since these studies were conducted on ex vivo cells, they may not have contained the full cellular milieu found within an intact organism, which possibly limited the effect. However, the fact that we saw differences between the T cell responses in PBMCs from males vs. females ex vivo indicates that enough of the cellular milieu remained to have an effect. In addition, the cells were grown in media and FBS that did not have estrogens removed [C. neoformans, simultaneously. Fourth, we consistently saw a decrease in the percentages of the immune cells measured in the presence of C. neoformans. This is unsurprising given the immunomodulatory effects of C. neoformans; however, we did not see a corresponding increase in any of the cell markers measured. This is possibly due to a proliferation in unmeasured immune cells ; PBMCs damaged due to infection such that the flow cytometer was unable to properly detect them; or most likely, a combination of both. However, when quantifying B cells, T cells, NK cells, and C. neoformans during infection, we were able to account for approximately 88\u201395% of the total cell population collected, suggesting that any remaining unmeasured immune cells are a small percentage of the population. Finally, the age of our donor population was relatively young (24.7 years). We know that age plays an important role in the immune response of both men and women; therefore, results of similar testing may be different in both pre-pubescent and post-menopausal populations. Future studies should include immune cell analysis of different age groups as well as samples from immune compromised individuals and diseased in vivo models.The key strength of our study is the large number of healthy donors . To our knowledge, this study is the largest reported to date evaluating the healthy immune response to  removed , 79. ThuC. neoformans infection of healthy male and female PBMCs, all percentages of measured T cell markers were significantly lower, and C. neoformans proliferated in greater numbers in the presence of male immune cells compared to female immune cells. These results, in combination with the similar response of B cell and NK cell proliferation in this study and in intracellular fungal proliferation in macrophages as observed in another study [C. neoformans infection. Looking at the immune response of healthy individuals reveals differences that may otherwise be overlooked. At present, it is not clear what role testosterone and 17 \u03b2-estradiol play in the immune response to C. neoformans, but the data suggest that they do play a role. In total, these results indicate that the difference in outcomes of cryptococcosis between men and women is biologic, can be at least partially explained by levels of circulating sex hormones and the adaptive immune response, and cannot simply be chalked up to differences in exposure rates. These experiments are an important first step in elucidating the biological sex differences seen in response to a C. neoformans infection and hopefully lay the groundwork for future research that uncovers the mechanisms behind these differences.During a er study , betweenAdditional file 1:Table S1. Flow cytometry cell counts for first dataset (DOCX 20 kb)Additional file 2:Flow cytometry cell counts for second dataset (DOCX 1010 kb)"}
{"text": "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\u2014balancing 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 \u201cGuardian of the Genome,\u201d 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 \u2018balancing the pendulum between cell death and survival\u2019. 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\u201330% 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 \u2018guardian of metabolism\u2019 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 \u2018accidental\u2019 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 \u2018self-devouring\u2019. Autophagy is an evolutionarily conserved, genetically mapped process that forms the \u201crecycling facility\u201d 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\u2013fission 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\u2019s 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 (ER\u03b1) 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\u2019s 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."}
{"text": "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\u00a0mutation 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\u2019s Surveillance, Epidemiology, and End Results program (SEER data21) provides the comprehensive cancer incidence for the overall US population. This cannot be obtained from the\u00a0second 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\u2009\u2265\u20090.3) 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.\u00a0p-value \u22480; Supplementary Data\u00a032, which are largely pseudoautosomal\u00a0(reviewed in ref.\u00a033) 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.\u00a0ome Fig.\u00a0. In contVariant allele frequency (VAF) is a commonly used metric employed to identify\u00a0 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 \u2248 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.\u00a0These 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\u00a0.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.\u00a0.KDM6A, DDX3X\u2009\u2009and 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.\u00a0. This prers Fig.\u00a0. 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\u2009A), DDX3X\u2009\u2009(dead-box RNA helicase 3) and UBA1. Notably, KDM6A inhibits EMT by antagonising TGF-\u03b2 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\u00a0males, 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.\u00a0. The p53dom Fig.\u00a0. 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\u2013Meier 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\u00a0Firehose81; annotated mutations were analysed. Mutations classified as \u201c3\u2019UTR\u201d, \u201c5\u2019UTR\u201d, \u201cFrame_Shift_Del\u201d, \u201cFrame_Shift_Ins\u201d, \u201cMissense_Mutation\u201d, \u201cNonsense_Mutation\u201d, \u201cSplice_Region\u201d and \u201cSplice_Site\u201d 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\u2019 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\u2013Hochberg 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"}
{"text": "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\u2013p53-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\u2212Fraumeni 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 \u201318. In aant form \u201325. Finaant form \u201332. 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 \u201cundruggable\u201d 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+/\u2212 HCT and RKO cells, indicating specificity  . Transfeity Fig. . Concomills Fig. . Moreoveted Fig. , collect+/\u2212 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\u2009mmo 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\u22128). One siRNA against all BamHI at the 5\u2032 and HindIII at the 3\u2032). The coding region for each hairpin is nested within a single oligonucleotide (upper oligo: 5\u2032-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 \u201329 (sensAAGCTTTN \u201329 (antiAAGCTTTN \u201329 (sensAAGCTTTN \u201329 (antiSupplemental TextSupplemental Figure and Table"}
{"text": "Dear Sir,89Zr-immunoPET\u201d in EJNMMI Zr4+ chelators\u201d is no more correct.During the manuscript review process, DFO*-NCS was made commercially available at ABX . Therefore, the following statement in the introduction \u201cHowever, 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 \u201cDirect comparison of the in vitro and in vivo stability of DFO, DFO* and DFOcyclo* for n EJNMMI , readers"}
{"text": "Results: The response rate was 91%. There was no significant correlation between patients\u2019 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\u2019 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\u2019 future preference.Background: A number of factors  may influence patients\u2019 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\u25a1Would there be any significant difference between patients who would prefer placing a DD in a future visit and those who would not?\u25a1Could patient acceptance be influenced by factors such as DD application time and length of the treatment session with the DD in place?\u25a1Which 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\u201330 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\u201330 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\u2019s  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."}
{"text": "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\u20138 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\u20137 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 ) \u2212 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\u2019s 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\u2212 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 \u00b1 0.17, 4 h SD raw expression: 10.6 \u00b1 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."}