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 –10.α1, α2, and α3 [α1 and α2 domains are highly conserved [α3 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, andβ3-adrenoreceptor in adipocytes by which it amplifies the induction of ZAG expression [β3-adrenoreceptor leads to an increase in ZAG expression is not known [ Glucocorticoids are able to enhance ZAG expression , 22, 23.pression . Furtherot known , 21.β3-adrenoreceptor agonist BRL37344, suggesting that the sympathetic system can play a role in the regulation of ZAG expression [β3-adrenoreceptor [ It has been shown that the expression of ZAG is increased by the selective pression  and thatreceptor , 20.γ (IFN-γ) is a potential mediator of the increased ZAG expression in cachexia. It has been shown that IFN-γ can strongly upregulate expression of ZAG in human epithelial cell lines [ Interferon-ll lines , 24.α (TNF-α), a major product of macrophages [α and the PPAR-gamma nuclear receptor [α can decrease ZAG expression and secretion [α 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-α 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 , 27–29. β3-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 α 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 .α1-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 α (TNF-α) 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 .β2-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.α3 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 cachβ3-adrenoreceptor [β3-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 β2-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 melα 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 10–20% 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 knowα is high [α 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.


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 7–13% 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 × 3 × 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 = “No vision” (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) = −4.15, p < 0.001), not different from zero in the P1-P1  condition (t(37) = −0.54, 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’s 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, Benoît G. Bardy and Stéphane Raffard conceived and designed the experiment. Manuel Varlet, Delphine Capdevielle, Jonathan Del-Monte, Jean-Philippe Boulenger, and Stéphane Raffard performed the experiment. Manuel Varlet, Ludovic Marin, R. C. Schmidt, Robin N. Salesse, Benoît G. Bardy and Stéphane 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.


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.


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 11. 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 A 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 B 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 years 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 Δ11/Δ11) is not compatible with life. Crossing Brca1Δ11/Δ11 mice with p53 heterozygous or null mice completely rescue the embryonic lethality of Brca1Δ11/Δ11 . 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 There are at least two non-exclusive models for p53 in development (Figure ES 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.


The characterization and cellular localization of transcription factors like NF-κB 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-κB activity in the CNS, antibodies detecting NF-κB proteins have been used to support the finding that NF-κB 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-κB are based on data garnered with antibodies that are not selective for the NF-κB subunit proteins p65 and p50. The data urge caution in interpreting studies of neuronal NF-κB activity in the brain. NF-κB 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-κB 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-κB 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-κB 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-κB in brain reveals that most of the claims of constitutive or induced NF-κB 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 μl 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°C 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°C 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-μm-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 α-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-κB 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-1β 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-κB 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-κB activity in neurons ,31. VeryFollowing peripheral lipopolysaccharide (LPS) administration eliciting an inflammatory response in the brain, ISHH showed strongly induced IκBα 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-κB 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-κB 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-κB in neuronal function and injury. The lack of neuronal NF-κB response observed with the authentic p65 antibodies is a finding consistent with studies employing in situ hybridization histochemistry showing non-neuronal localization of induced IκBα 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.


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 p66shс 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 – 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 “young” 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 PKCβ, 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’s 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 – 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.


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 –12. 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 –15. 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°C), annealing of complimentary oligonucleotide primers at a lower temperature (~55°C), and DNA replication at an intermediate temperature (~72°C) 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−/HPV−) or Class II (p16−/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−). 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.


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 6–94 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 8×2.1 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 (1×1.5 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 10×6×2.5 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 “pre-elastofibroma changes” 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 “checkerboard” 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


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 → 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 → 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