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Mendelian randomization (MR) is attracting considerable critical attention. This paper aimed to explore the characteristics of the publications of MR, to reach an insight in this field and prospect the future trend. A bibliometric analysis was performed to identify published MR-related research. The articles were selected from the Web of Science Core Collection database. Excel 2019, VOSviewer 1.6.9, and CiteSpace 5.7.R3 were used to analyze the information. A total of 1783 papers of MR were identified, and the first included literature appeared in 2003. A total of 2829 institutions from 72 countries participated in the relevant research, while the UK contributed to 852 articles and were in a leading position. The most productive institution was the University of Bristol, and Smith GD who has posted the most articles (n=202) was also from there. The Int J Epidemiol (100 publications, 6861 citations) was the most prolific and high citation journal. Related topics of frontiers will still focus on coronary heart disease, diabetes, cancer, psychiatric disorder, body mass index, and lifestyle factors. We summarized the publication information of MR-related literature from 2003 to 2020, including country and institution of origin, authors, and publication journal. We analyzed former research hotspots in the field of MR and predicted future areas of interest. Exposures and outcomes detected in this paper will be the hotspots and frontiers of research in the next few years.

Objective: Several systematic reviews (SRs) have assessed the diagnostic accuracy of microRNAs (miRNAs) for NSCLC, and this overview aimed to assess the relationship between diagnostic accuracy of miRNAs for NSCLC and number of microRNAs combinations. Methods: Embase.com, PubMed, the Cochrane Library, and Web of Science were searched. The PRISMA-DTA was used for reporting quality evaluation. Meta-analysis was conducted to assess the pooled diagnostic accuracy of different miRNAs combinations, and subgroup analyses were performed based on the source of miRNA. Results: Fourteen SRs with 91 original studies were included. Three SRs had minimal reporting flaws, and 11 SRs had medium flaws. The pooled sensitivity and specificity were 0.76 and 0.80 for single miRNA, 0.80 and 0.81 for two miRNAs combined, 0.82 and 0.88 for three miRNAs combined, 0.88 and 0.92 for four miRNAs combined, 0.87 and 0.87 for five miRNAs combined, and 0.87 and 0.89 for six or more miRNAs combined. And miR-21 was mostly appeared. Subgroup analyses suggested that the serum-derived miRNA had the relatively best diagnostic value compared to other sources. Conclusions: Future studies should explore specific and serum-derived miRNAs in NSCLC and combine them to improve the diagnosis accuracy of NSCLC, which had great significance in economic efficiency.

The prevalence of health misinformation on social media could significantly influence individuals’ health behaviors. To examine the prevalent topics, propagation, and correction of coronavirus disease 2019 (COVID-19) misinformation, automated content analyses were conducted for posts on Sina Weibo, which is China’s largest microblogging site. In total, 177,816 posts related to COVID-19 misinformation during the COVID-19 outbreak in China were analyzed. The structural topic modeling identified 23 valid topics regarding COVID-19 misinformation and its correction, which were further categorized into three general themes. Sentiment analysis was conducted to generate positive and negative sentiment scores for each post. The zero-inflated Poisson model indicated that only the negative sentiment was a significant predictor of the number of comments (β = 0.003, p < 0.001) but not reposts. Furthermore, users are more prone to repost and comment on information regarding prevention/treatment (e.g., traditional Chinese medicine preventing COVID) as well as potential threats of COVID-19 (e.g., COVID-19 was defined as an epidemic by World Health Organization). Health education and promotion implications are discussed.

Abstract Neoantigens are mutation-containing immunogenic peptides from tumor cells. Neoantigen intrinsic features are neoantigens’ sequence-associated features characterized by different amino acid descriptors and physical–chemical properties, which have a crucial function in prioritization of neoantigens with immunogenic potentials and predicting patients with better survival. Different intrinsic features might have functions to varying degrees in evaluating neoantigens’ potentials of immunogenicity. Identification and comparison of intrinsic features among neoantigens are particularly important for developing neoantigen-based personalized immunotherapy. However, there is still no public repository to host the intrinsic features of neoantigens. Therefore, we developed GNIFdb, a glioma neoantigen intrinsic feature database specifically designed for hosting, exploring and visualizing neoantigen and intrinsic features. The database provides a comprehensive repository of computationally predicted Human leukocyte antigen class I (HLA-I) restricted neoantigens and their intrinsic features; a systematic annotation of neoantigens including sequence, neoantigen-associated mutation, gene expression, glioma prognosis, HLA-I subtype and binding affinity between neoantigens and HLA-I; and a genome browser to visualize them in an interactive manner. It represents a valuable resource for the neoantigen research community and is publicly available at http://www.oncoimmunobank.cn/index.php. Database URL http://www.oncoimmunobank.cn/index.php

The prevalence of nonalcoholic fatty liver disease (NAFLD) has been steadily increasing, and it has become one of the most prevalent chronic liver diseases worldwide. Recent studies have shown that dihydromyricetin (DHM) is influential in treating NAFLD. The purpose of this review was to describe how DHM prevents and treats NAFLD and its potential mechanisms through an in-depth summary of preclinical in vitro and in vivo studies. A brief overview of DHM's potential role in NAFLD involves regulation of lipid/glucose metabolism, possibly via anti-inflammatory or sirtuin-dependent mechanisms. For NAFLD, there is currently no effective and approved medication for therapy. DHM has the characteristics of liver protection, antioxidation, anti-inflammatory and apoptosis-regulatory benefits, which provides a new idea for the treatment of NAFLD. With the increasing interest in utilizing natural products to prevent and control liver diseases, our work aims to provide new ideas for the treatment of NAFLD and accelerate its translation from bench to bedside.

This review compared the efficacy and acceptability of different delivery formats for cognitive behavioral therapy for insomnia (CBT-I) in insomnia. We searched five databases for randomized clinical trials that compared one CBT-I delivery format against another format or control conditions for insomnia in adults. We used pairwise meta-analyses and frequentist network meta-analyses with the random-effects model to synthesize data. A total of 61 unique trials including 11,571 participants compared six CBT-I delivery formats with four control conditions. At post-intervention, with low to high certainty evidence, individual, group, guided self-help, digital assisted, and unguided self-help CBT-I could significantly increase sleep efficiency and total sleep time (TST) and reduce sleep onset latency (SOL), wake after sleep onset (WASO), and insomnia severity compared with treatment as usual (MD range for sleep efficiency: 7.81%-12.45%; MD range for TST: 16.14-33.96 min; MD range for SOL: -22.42 to -13.81 min; MD range for WASO: -40.84 to -19.48 min; MD range for insomnia severity: -6.40 to -3.93) and waitlist (MD range for sleep efficiency: 7.68%-12.32%; MD range for TST: 12.67-30.49 min; MD range for SOL: -19.07 to -10.46 min; MD range for WASO: -47.10 to -19.15 min; MD range for insomnia severity: -7.59 to -5.07). The effects of different CBT-I formats persisted at short-term follow-up (4 wk-6 mo). Individual, group, and digital assisted CBT-I delivery formats would be the more appropriate choices for insomnia in adults, based on post-intervention and short-term effects. Further trials are needed to investigate the long-term effects of different CBT-I formats.

IntroductionLaryngeal mask airway (LMA), an alternative to traditional tracheal intubation, is widely used in clinical practice and is considered to be an effective device for airway management. LMA and i-gel have been widely used in anaesthesia and emergency situations in children. Some systematic reviews have evaluated the efficacy of LMA and i-gel in children, but they have not shown consistent results in clinical performance. This study aims to evaluate the airway complications of all subtypes of LMA and i-gel in child patients under general anaesthesia using a Bayesian network meta-analysis (NMA).Methods and analysisPubMed, EMBASE.com, the Cochrane library, Web of Science and Chinese Biomedical Literature Database will be searched from inception to January 2019. We will include prospective randomised controlled trials (RCTs) that reported the subtypes of LMA and i-gel regardless of sample size. The risk of bias assessment of the included RCTs will be conducted according to the Cochrane Handbook V.5.1.0. A Bayesian NMA will be performed using WinBUGS V.1.4.3. Grading of Recommendations Assessment, Development and Evaluation will be used to explore the quality of evidence.Ethics and disseminationEthics approval and patient consent are not required as this study is an NMA based on published trials. The results of this NMA will be submitted to a peer-reviewed journal for publication.PROSPERO registration numberCRD42019127668.

The purpose of this work is to propose a simple, portable, and sensitive biosensor structure based on singlemode fiber-multicore fiber-multimode fiber-singlemode fiber (SMF-MCF-MMF-SMF) for the detection of creatinine in the human body. Chemical etching has been used to modify the diameter of the sensing probe to approximately 90 μm in order to generate strong evanescent waves (EWs). The sensor probe is functionalized with graphene oxide (GO), gold nanoparticles (AuNPs), molybdenum disulfide nanoparticles (MoS2-NPs), and creatininase (CA) enzyme. The concentration of creatinine is determined using fiber optic localized surface plasmon resonance (LSPR). While EWs are used to enhance the LSPR effect of AuNPs, two-dimensional (2D) materials (GO and MoS2-NPs) are used to increase biocompatibility, and CA is used to increase probe specificity. Additionally, HR-TEM and UV-visible spectroscopy are used to characterize and measure the nanoparticle (NP) morphology and absorption spectrum, respectively. SEM is used to characterize the NPs immobilized on the surface of the fiber probe. The sensor probe's reusability, reproducibility, stability, selectivity, and pH test results are also tested to verify the sensor performance. The sensitivity of proposed sensor is 0.0025 nm/μM, has a standard deviation of 0.107, and has a limit of detection of 128.4 μM over a linear detection range of 0 - 2000 μM.

Several meta-analyses have evaluated the value of biomarkers in diagnosing breast cancer, but which biomarker has the optimal diagnostic value remains unclear. This overview aimed to compare the accuracy of different biomarkers in diagnosing breast cancer. PubMed, Embase.com, the Cochrane Library of Systematic Reviews, and Web of Science were searched. The assessment of multiple systematic reviews-2 (AMSTAR-2) was used to assess the methodological quality and preferred reporting items for a systematic review and meta-analysis of diagnostic test accuracy (PRISMA-DTA) for reporting quality. Pairwise meta-analyses were performed to estimate the pooled results for each biomarker, and indirect comparisons were conducted to compare diagnostic accuracy between biomarkers. Eleven systematic reviews (SRs) involving 218 original studies were included. All SRs were of critically low methodological quality, 3 SRs had minimal reporting flaws and 8 SRs had minor flaws. The pooled sensitivity and specificity were 0.77 and 0.87 for miRNA, 0.70 and 0.87 for circulating cell-free DNA, 0.29 and 0.96 for APC gene promoter methylation, 0.69 and 0.99 for 14-3-3σ promoter methylation, 0.63 and 0.82 for CA153, 0.58 and 0.87 for CEA, and 0.73 and 0.56 for PSA. Compared with CA153 and PSA, miRNA had a higher sensitivity and specificity. The sensitivity of miRNA was higher than circulating cell-free DNA and CEA, although they had the same specificities. APC gene promoter methylation and 14-3-3σ promoter methylation were more specific than miRNA, but they had unacceptably low sensitivity. In conclusion, miRNA had better diagnostic accuracy than the other six biomarkers. But due to the low quality of included SRs, the results need to be interpreted with caution. Further study should investigate the diagnostic accuracy of different biomarkers in direct comparisons and focus on the value of combined biomarkers.

Background: Among females, breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death over 100 countries. Generally, the prognosis of early-stage breast cancer is good. However, the prognosis is very poor when the disease is diagnosed at an advanced stage. Many screening methods have been used for early detection of breast cancer, but there are some limitations of these methods. Recently, some systematic reviews have evaluated the value of biomarkers for detecting breast cancer. However, most of the systematic reviews (SRs) only evaluated the diagnostic value of 1 biomarker, and it is unclear which biomarker is the best diagnostic test for breast cancer. This overview aims to assess the methodological and reporting quality of available systematic reviews and to compare the diagnostic value of different biomarkers. Methods: PubMed, Embase.com, the Cochrane Library of Systematic Reviews, and Web of Science were searched to identify published systematic reviews reporting the value of biomarkers for detecting breast cancer. Title and abstracts, as well as full texts, were screened in duplicate based on inclusion and exclusion criteria. The Assessment of Multiple Systematic Reviews-2 (AMSTAR-2) tool and Preferred Reporting Items for Systematic Reviews and Meta-analysis diagnostic test accuracy (PRISMA-DTA) checklist will be used to assess the methodological and reporting quality, respectively. We will conduct the pairwise meta-analysis and indirect comparisons using STATA 13.0. Results: The results of this study will be published in a peer-reviewed journal Conclusion: This overview will provide comprehensive evidence of different biomarkers for the diagnosis of breast cancer. PROSPERO registration number: CRD42019125880.

Background: The arrival of transcatheter mitral valve therapies has provided feasible and safe alternatives to medical and surgical treatments for mitral regurgitation. The aim of this study is to estimate the relative efficacy and safety of different transcatheter mitral valve therapies for mitral regurgitation patients through network meta-analysis. Methods: A systematic search will be performed using PubMed, EMBASE, the Cochrane Library, Web of Science, Chinese Biomedical Literature Database, and China National Knowledge Infrastructure to include random controlled trials and nonrandom controlled trials comparing the efficacy and safety of different transcatheter mitral valve techniques. The risk of bias for the included nonrandom controlled studies will be evaluated according to Risk of Bias in Non-randomized Studies - of Interventions. For random controlled trials, we will use Cochrane Handbook version 5.1.0 as the risk of bias tool. A Bayesian network meta-analysis will be conducted using R-4.0.3 software. Grading of recommendations assessment, development, and evaluation will be used to assess the quality of evidence. Results: The results of this network meta-analysis will be submitted to a peer-reviewed journal for publication. Conclusion: This study will provide broad evidence of efficacy and safety of different transcatheter mitral valve therapies for treatment of mitral regurgitation and provide suggestions for clinical practice and future research. Protocol registration number: INPLASY2020110034.

The blockade of immune checkpoints by anti-receptor and/or anti-ligand mAb is one of the most promising approaches to cancer immunotherapy. The interaction between Ig-like transcript 3 (ILT3), a marker of tolerogenic dendritic cells, also known as LILRB4/LIR5/CD85k, and its still unidentified ligand on the surface of activated human T cells is potentially important for immune checkpoint blockade. To identify the ILT3 ligand, we generated mAb by immunizing mice with Jurkat acute T cell leukemia, which binds ILT3.Fc to its membrane. Flow cytometry, mass spectrometry, and Biacore studies demonstrated that the ILT3 ligand is a CD166/activated leukocyte cell adhesion molecule. Knockdown of CD166 in primary human T cells by nucleofection abolished the capacity of ILT3.Fc to inhibit CD4+ Th cell proliferation and to induce the generation of CD8+CD28− T suppressor cells. CD166 displays strong heterophilic interaction with CD6 and weaker homophilic CD166–CD166 cell adhesion interaction. ILT3.Fc inhibited the growth of CD166+ tumor cell lines (TCL) derived from lymphoid malignancies in vitro and in vivo. CRISPR-Cas9–based knockout of CD166 from TCL abrogated ILT3.Fc binding and its tumor-inhibitory effect. The mechanism underlying the effect of ILT3.Fc on tumor cell growth involves inhibition of the p70S6K signaling pathway. Blockade of CD166 by ILT3.Fc inhibited progression of human TCL in NOD.Cg-Prkdc Il-2rg/SzJ mice, suggesting its potential immunotherapeutic value.

Immune activation needs to be tightly regulated to control immune-mediated tissue damage. Inhibitory pathways serve to terminate an immune response and resolve inflammation. Persistent exposure to antigens can drive development of adaptive regulatory cells. Similarly exposure of activated T cells to the recombinant ILT3-Fc molecule during priming triggers the differentiation of CD8 T suppressor cells and the induction of CD4 T helper anergy. Ts express high levels of immunoregulatory signature genes together with low levels of microRNA which control their function. Analysis of microRNA contained by exosomes from cultures in which T cells were alloactivated in the presence or absence of ILT3.Fc, demonstrated that this agent inhibits the release of inflammatory microRNA. The source of such inflammatory microRNA was found to reside in alloactivated CD4 T cells, since exosomes from MLC primed CD4 T cells were shown to diminish the suppressive activity of ILT3-Fc-induced CD8(+) Ts at high effector to suppressor T cell ratios. This indicates that inflammatory exosomes can swing the balance between effector and regulatory T cells in favor of immunity. These data suggest that isolation and characterization of micro-RNA containing exosomes in patients' circulation may be of use for treatment, prevention and monitoring of immune activation.

Supplemental Digital Content is available in the text, Background: Prostate cancer (PCa) is common, with it being the 2nd most prevalent cancer in men worldwide and the 6th leading cause of death in men. Screening for any type of cancer aims to increase the chances of successful treatment through early detection of the disease. There were some systematic reviews (SRs) evaluated the diagnostic value of biomarkers for the diagnosis of PCa and no studies have been conducted to analyze the quality of these SRs. We are not clear which kind of marker is the best choice. Thus, this study aims to assess the methodologic quality of the SRs and reanalyze the published data based on SRs for the biomarkers to find the optimal biomarker for the early diagnosis of PCa. Methods: We performed a systematic literature search of PubMed, Embase, Web of Science, and Cochrane Library and to identify relevant SRs from inception to April 2019. Diagnostic accuracy studies included any type of single biomarker or combined biomarkers aimed at evaluating the diagnostic value is considered eligible for this overview. The Assessment of Multiple Systematic Reviews-2 (AMSTAR-2) instrument will be used to evaluate the risk of bias of the included SRs. Standard pairwise meta-analysis and adjusted indirect comparison will be used to compare the diagnostic value of different biomarkers. Results: The results of this study will be submitted to a peer-reviewed journal for publication. Conclusion: This study will reanalyze the published data based on SRs. We hope that the results will help find a biomarker with the superior diagnostic performance for the diagnosis of PCa. PROSPERO registration number: CRD42019125880.

Supplemental Digital Content is available in the text, Background: Ovarian cancer is one of the deadliest gynecological diseases and the annual mortality of ovarian cancer continues to rise. The prognosis of ovarian cancer is poor because it is prone to early metastasis during progression. Therefore, early diagnosis of ovarian cancer is very important. Some systematic reviews have evaluated the diagnostic value of different biomarkers for ovarian cancer. However, there is no consensus in the conclusions, and some are even contradictory. This study aims to assess the methodological and reporting quality of available systematic reviews and to find an optimal biomarker for diagnosing ovarian cancer. Methods: The PubMed, Embase.com, the Cochrane Library of Systematic Reviews, and Web of Science were searched to identify relevant systematic reviews from inception to February 2019. We included systematic reviews that include randomized controlled trials, cross-sectional studies, case-control studies, or cohort studies as long as the systematic reviews evaluated the diagnostic performance of biomarkers for ovarian cancer. The methodological quality will be assessed using assessment of multiple systematic reviews-2 checklist, and the reporting quality will be assessed using preferred reporting items for systematic reviews and meta-analysis diagnostic test accuracy (PRISMA-DTA) checklist. The pairwise meta-analysis and indirect comparisons will be performed using STATA (13.0; Stata Corporation, College Station, TX). Results: The results of this overview will be submitted to a peer-reviewed journal for publication. Conclusion: This overview will provide comprehensive evidence of different biomarkers for diagnosing ovarian cancer. PROSPERO registration number: CRD42019125880.

As a central regulator for cell cycle arrest, apoptosis, and cellular senescence, p53 requires multiple layers of regulatory control to ensure correct temporal and spatial functions. It is well accepted that Mdm2-mediated ubiquitination plays a crucial role in p53 regulation. In addition to proteasome-mediated degradation, ubiquitination of p53 by Mdm2 acts a key signal for its nuclear export. Nuclear export has previously been thought to require the disassociation of the p53 tetramer and exposure of the intrinsic nuclear export signal. To elucidate the molecular mechanism of degradation-independent repression on p53 by Mdm2, we have developed a two-step approach to purify ubiquitinated forms of p53 induced by Mdm2 from human cells. Surprisingly, however, we found that ubiquitination has no effect on the tetramerization/oligomerization of p53, arguing against this seemingly well accepted model. Moreover, nuclear export of p53 alone is not sufficient to completely abolish p53 activity. Ubiquitination-mediated repression of p53 by Mdm2 acts at least, in part, through inhibiting the sequence-specific DNA binding activity. Thus, our results have important implications regarding the mechanisms by which Mdm2 acts on p53.

p53 promotes tumor suppression through its ability to function as a transcriptional factor and is activated by posttranslational modifications that include acetylation. Our earlier study demonstrated that p53 acetylation can enhance its sequence-specific DNA binding in vitro , and this notion was later confirmed in several other studies. However, a recent study has reported that in vitro acetylation of p53 fails to stimulate its DNA binding to large DNA fragments, raising an important issue that requires further investigation. Here, we show that unacetylated p53 is able to bind weakly to its consensus site within the context of large DNA fragments, although it completely fails to bind the same site within short oligonucleotide probes. Strikingly, by using highly purified and fully acetylated p53 proteins obtained from cells, we show that acetylation of the C-terminal domain can dramatically enhance site-specific DNA binding on both short oligonucleotide probes and long DNA fragments. Moreover, endogenous p53 apparently can be fully acetylated in response to DNA damage when both histone deacetylase complex 1 (HDAC1)- and Sir2-mediated deacetylation are inhibited, indicating dynamic p53 acetylation and deacetylation events during the DNA damage response. Finally, we also show that acetylation of endogenous p53 indeed significantly augments its ability to bind an endogenous target gene and that p53 acetylation levels correlate well with p53-mediated transcriptional activation in vivo . Thus, our results clarify some of the confusion surrounding acetylation-mediated effects on p53 binding to DNA and suggest that acetylation of p53 in vivo may contribute, at least in part, to its transcriptional activation functions.

The p53 tumor suppressor is maintained at low levels in normal cells by Mdm2-mediated degradation and strongly stabilized in response to various types of stress including hypoxia. Although hypoxia-inducible factor 1 alpha (HIF-1 alpha) has been implicated to be involved in p53 stabilization, the precise mechanism by which HIF-1 alpha regulates p53-mediated function remains unknown. Here, we found that HIF-1 alpha directly binds Mdm2 both in vitro and in vivo; in contrast, p53 fails to directly interact with HIF-1 alpha in vitro. Interestingly, Mdm2 expression can significantly enhance the in vivo association between p53 and HIF-1 alpha, indicating that Mdm2 may act as a bridge and mediate the indirect interaction between HIF-1 alpha and p53 in cells. Furthermore, HIF-1 alpha protects p53 degradation mediated by Mdm2, and leads to activation of p53-mediated transcription in cells. To elucidate the mechanism of HIF-1 alpha-mediated effect, we also found that HIF-1 alpha can significantly suppress Mdm2-mediated p53 ubiquitination in vitro and blocks Mdm2-mediated nuclear export of p53. These results have significant implications regarding the molecular mechanism by which p53 is activated by HIF-1 alpha in response to hypoxia.

The PTEN tumor suppressor is a lipid phosphatase that has a central role in regulating the phosphatidylinositol-3-kinase (PI3K) signal transduction cascade. Nevertheless, the mechanism by which the PTEN activity is regulated in cells needs further elucidation. Although previous studies have shown that ubiquitination of PTEN can modulate its stability and subcellular localization, the role of ubiquitination in the most critical aspect of PTEN function, its phosphatase activity, has not been fully addressed. Here, we identify a novel E3 ubiquitin ligase of PTEN, Ret finger protein (RFP), that is able to promote atypical polyubiquitinations of PTEN. These ubiquitinations do not lead to PTEN instability or relocalization, but rather significantly inhibit PTEN phosphatase activity and therefore modulate its ability to regulate the PI3K signal transduction cascade. Indeed, RFP overexpression relieves PTEN-mediated inhibitory effects on AKT activation; in contrast, RNAi-mediated knockdown of endogenous RFP enhances the ability of PTEN to suppress AKT activation. Moreover, RFP-mediated ubiquitination of PTEN inhibits PTEN-dependent activation of TRAIL expression and also suppresses its ability to induce apoptosis. Our findings demonstrate a crucial role of RFP-mediated ubiquitination in controlling PTEN activity.

The ubiquitin-proteasome pathway has become an increasingly important regulatory mechanism for protein function. Countless proteins are degraded by the addition of polymeric ubiquitin chains, but more recently, monoubiquitination has emerged as a mechanism for regulatory functions other than proteasomal degradation. Monoubiquitination acts as a signal in nuclear export for the tumor suppressor protein p53. Different levels of Mdm2 are capable of inducing both mono- and polyubiquitination in a dosage dependent manner, thus determining p53's fate. Our findings demonstrate monoubiquitin-mediated protein trafficking can be expanded to nuclear-cytoplasmic shuttling, and also imply similar scenarios may apply to other cellular factors.

Both p53 and Mdmx are ubiquitinated and degraded by the same E3 ligase Mdm2; interestingly, however, while p53 is rapidly degraded by Mdm2, Mdmx is a stable protein in most of cancer cells. Thus, the mechanism by which Mdmx is degraded by Mdm2 needs further elucidation. Here, we identified the noncoding 5S rRNA as a major component of Mdmx-associated complexes from human cells. We show that 5S rRNA acts as a natural inhibitor of Mdmx degradation by Mdm2. RNAi-mediated knockdown of endogenous 5S rRNA, while not affecting p53 levels, significantly induces Mdmx degradation and subsequently, activates p53-dependent growth arrest. Notably, 5S rRNA binds the RING domain of Mdmx and blocks its ubiquitination by Mdm2 whereas Mdm2-mediated p53 ubiquitination remains intact. These results provide insights into the differential effects on p53 and Mdmx by Mdm2 in vivo and reveal an critical role of noncoding 5S rRNA in modulating the p53-Mdmx axis.

In unstressed cells, the tumor suppressor p53 is maintained at low levels by ubiquitin-mediated proteolysis mainly through Mdm2. In response to DNA damage, p53 is stabilized and becomes activated to turn on transcriptional programs that are essential for cell cycle arrest and apoptosis. Activation of p53 leads to accumulation of Mdm2 protein, a direct transcriptional target of p53. It is not understood how p53 is protected from degradation when Mdm2 is up-regulated. Here we report that p53 stabilization in the late phase after ionizing radiation correlates with active ubiquitination. We found that an E3 ubiquitin ligase RFWD3 (RNF201/FLJ10520) forms a complex with Mdm2 and p53 to synergistically ubiquitinate p53 and is required to stabilize p53 in the late response to DNA damage. This process is regulated by the DNA damage checkpoint, because RFWD3 is phosphorylated by ATM/ATR kinases and the phosphorylation mutant fails to stimulate p53 ubiquitination. In vitro experiments suggest that RFWD3 is a p53 E3 ubiquitin ligase and that RFWD3–Mdm2 complex restricts the polyubiquitination of p53 by Mdm2. Our study identifies RFWD3 as a positive regulator of p53 stability when the G 1 cell cycle checkpoint is activated and provides an explanation for how p53 is protected from degradation in the presence of high levels of Mdm2.

The c-Myc proto-oncogene encodes a transcription factor that is essential for cell growth and proliferation and is broadly implicated in tumorigenesis. However, the biological functions required by c-Myc to induce oncogenesis remain elusive. Here we show that c-Myc has a direct role in the control of DNA replication. c-Myc interacts with the pre-replicative complex and localizes to early sites of DNA synthesis. Depletion of c-Myc from mammalian (human and mouse) cells as well as from Xenopus cell-free extracts, which are devoid of RNA transcription, demonstrates a non-transcriptional role for c-Myc in the initiation of DNA replication. Overexpression of c-Myc causes increased replication origin activity with subsequent DNA damage and checkpoint activation. These findings identify a critical function of c-Myc in DNA replication and suggest a novel mechanism for its normal and oncogenic functions.

SummaryAlthough the importance of the ARF tumor suppressor in p53 regulation is well established, numerous studies indicate that ARF also suppresses cell growth in a p53/Mdm2-independent manner. To understand the mechanism of ARF-mediated tumor suppression, we identified a ubiquitin ligase, ARF-BP1, as a key factor associated with ARF in vivo. ARF-BP1 harbors a signature HECT motif, and its ubiquitin ligase activity is inhibited by ARF. Notably, inactivation of ARF-BP1, but not Mdm2, suppresses the growth of p53 null cells in a manner reminiscent of ARF induction. Surprisingly, in p53 wild-type cells, ARF-BP1 directly binds and ubiquitinates p53, and inactivation of endogenous ARF-BP1 is crucial for ARF-mediated p53 stabilization. Thus, our study modifies the current view of ARF-mediated p53 activation and reveals that ARF-BP1 is a critical mediator of both the p53-independent and p53-dependent tumor suppressor functions of ARF. As such, ARF-BP1 may serve as a potential target for therapeutic intervention in tumors regardless of p53 status.

Repression of gene transcription is mediated by histone deacetylases containing repressor-co-repressor complexes, which are recruited to promoters of target genes via interactions with sequence-specific transcription factors. The mammalian Sin3A co-repressor complex contains a core of at least seven proteins including the pRb-interacting protein RBP1 and a putative tumor suppressor p33(ING1b). By biochemical purification and mass spectrometry, we have identified a novel component p40 from this complex. p40 bears homology to both yeast Sds3, a component of yeast histone deacetylase complexes, and its mammalian homologue mSds3. The p40-associated complex purified from human cells shows a strong histone deacetylase activity. When tethered to a Gal-DNA binding domain, the Gal-p40 is able to significantly repress transcription of a Gal-luciferase promoter. Interestingly, database analysis reveals that p40 is also highly homologous to BRMS1, a breast carcinoma metastasis suppressor, and overexpression of p40 in human cells can significantly inhibit cell growth. Thus, our data indicate that p40 may be critically involved in transcription repression of cell growth-associated gene expression by recruiting the HDAC1 deacetylase complex.

The p53 tumour suppressor exerts anti-proliferative effects, including growth arrest, apoptosis and cell senescence, in response to various types of stress. However, p53 is a short-lived protein and its activity is maintained at low levels in normal cells. Numerous studies indicate that CBP/p300-mediated acetyl-transferase activity is critical for its role in both catalysing p53 acetylation and activating p53-mediated function during stress response. Interestingly, two additional regulators have also been identified in the p53 acetylation pathway. PID/MTA2 is a p53-interacting protein that induces p53 deacetylation by recruiting the HDAC1 complex. Subsequent work has also identified Sir2alpha, a NAD-dependent histone deacetylase that can attenuate p53 transcriptional activity through deacetylation. The prominence of deacetylase activity on p53 certainly raises the defining question of its physiological purpose. It is likely that deacetylation proxides a quick acting mechanism to stop p53 function once transcriptional activation of target genes is no longer needed. We present data indicating that both HDAC1 and Sir2alpha are critical for p53-dependent stress response. Furthermore, we also try to define the functional consequence of p53 acetylation at the molecular level. Finally, we propose a model regarding the differential roles of HDAC1 and Sir2alpha in the regulation of p53 function.

Our previous study showed that ubiquitination of p53 is reversible and that the ubiquitin hydrolase HAUSP can stabilize p53 by deubiquitination. Here, we found that partial reduction of endogenous HAUSP levels by RNAi indeed destabilizes endogenous p53; surprisingly, however, nearly complete ablation of HAUSP stabilizes and activates p53. We further show that this phenomenon occurs because HAUSP stabilizes Mdm2 in a p53-independent manner, providing an interesting feedback loop in p53 regulation. Notably, HAUSP is required for Mdm2 stability in normal cells; in HAUSP-ablated cells, self-ubiquitinated-Mdm2 becomes extremely unstable, leading to indirect p53 activation. Furthermore, this feedback regulation is specific to Mdm2; in HeLa cells, where p53 is preferentially degraded by viral E6-dependent ubiquitination, depletion of HAUSP fails to activate p53. This study provides an example of an ubiquitin ligase (Mdm2) that is directly regulated by a deubiquitinase (HAUSP) and also reveals a dynamic role of HAUSP in the p53-Mdm2 pathway.

Although Mdm2-mediated ubiquitination is essential for both degradation and nuclear export of p53, the molecular basis for the differential effects of Mdm2 remains unknown. Here we show that low levels of Mdm2 activity induce monoubiquitination and nuclear export of p53, whereas high levels promote p53's polyubiquitination and nuclear degradation. A p53-ubiquitin fusion protein that mimics monoubiquitinated p53 was found to accumulate in the cytoplasm in an Mdm2-independent manner, indicating that monoubiquitination is critical for p53 trafficking. These results clarify the nature of ubiquitination-mediated p53 regulation and suggest that distinct mechanisms regulate p53 function in accordance with the levels of Mdm2 activity.

Nuclear localization of p53 is essential for its tumor suppressor function. Here, we have identified Parc, a Parkin-like ubiquitin ligase, as a cytoplasmic anchor protein in p53-associated protein complexes. Parc directly interacts and forms a approximately 1 MDa complex with p53 in the cytoplasm of unstressed cells. In the absence of stress, inactivation of Parc induces nuclear localization of endogenous p53 and activates p53-dependent apoptosis. Overexpression of Parc promotes cytoplasmic sequestration of ectopic p53. Furthermore, abnormal cytoplasmic localization of p53 was observed in a number of neuroblastoma cell lines; RNAi-mediated reduction of endogenous Parc significantly sensitizes these neuroblastoma cells in the DNA damage response. These results reveal that Parc is a critical regulator in controlling p53 subcellular localization and subsequent function.

In response to DNA damage, the activity of the p53 tumor suppressor is modulated by protein stabilization and post-translational modifications including acetylation. Interestingly, both acetylation and ubiquitination can modify the same lysine residues at the C terminus of p53, implicating a role of acetylation in the regulation of p53 stability. However, the direct effect of acetylation on Mdm2-mediated ubiquitination of p53 is still lacking because of technical difficulties. Here, we have developed a method to obtain pure acetylated p53 proteins from cells, and by using an in vitro purified system, we provide the direct evidence that acetylation of the C-terminal domain is sufficient to abrogate its ubiquitination by Mdm2. Importantly, even in the absence of DNA damage, acetylation of the p53 protein is capable of reducing the ubiquitination levels and extending its half-life in vivo. Moreover, we also show that acetylation of p53 can affect its ubiquitination through other mechanisms in addition to the site competition. This study has significant implications regarding a general mechanism by which protein acetylation modulates ubiquitination-dependent proteasome proteolysis.