The p97/VCP segregase is essential for arsenic-induced degradation of PML and PML-RARA.

Acute Promyelocytic Leukemia is caused by expression of the oncogenic Promyelocytic Leukemia (PML)-Retinoic Acid Receptor Alpha (RARA) fusion protein. Therapy with arsenic trioxide results in degradation of PML-RARA and PML and cures the disease. Modification of PML and PML-RARA with SUMO and ubiquitin precedes ubiquitin-mediated proteolysis. To identify additional components of this pathway, we performed proteomics on PML bodies. This revealed that association of p97/VCP segregase with PML bodies is increased after arsenic treatment. Pharmacological inhibition of p97 altered the number, morphology, and size of PML bodies, accumulated SUMO and ubiquitin modified PML and blocked arsenic-induced degradation of PML-RARA and PML. p97 localized to PML bodies in response to arsenic, and siRNA-mediated depletion showed that p97 cofactors UFD1 and NPLOC4 were critical for PML degradation. Thus, the UFD1-NPLOC4-p97 segregase complex is required to extract poly-ubiquitinated, poly-SUMOylated PML from PML bodies, prior to degradation by the proteasome.

Grading of clear cell renal cell carcinoma using diffusion MRI with a fractional order calculus model.

BACKGROUND: The fractional order calculus (FROC) model has been developed to describe restrained motion of water molecules as well as microstructural heterogeneity, providing a novel tool for non-invasive tumor grading. PURPOSE: To evaluate the role of the FROC model in characterizing clear cell renal cell carcinoma (ccRCC) grades. MATERIAL AND METHODS: A total of 59 patients diagnosed with ccRCC were included in this prospective study. The diffusion metrics derived from the mono-exponential model (apparent diffusion coefficient [ADC]), intra-voxel incoherent motion [IVIM] model [D, D*, f], and FROC model [Dfroc, ß, µ]) were calculated and compared between low- and high-grade ccRCCs. Binary logistic regression analysis was performed to establish the diagnostic models. Receiver operating characteristic (ROC) analysis and DeLong test were performed to evaluate and compare the diagnostic performance of metrics in grading ccRCC. RESULTS: All the metrics except D* and f exhibited statistical differences between low- and high-grade ccRCCs. ROC analysis showed individual FROC parameters, µ, Dfroc, and ß, outperformed ADC and IVIM parameters in grading ccRCC. For single parameter, µ demonstrated the highest AUC value, sensitivity, and diagnostic accuracy in discriminating the two ccRCC groups while ß exhibited the optimal specificity. Importantly, the combination of Dfroc, µ, and ß could further improve the diagnostic performance. CONCLUSION: The FROC parameters were superior to ADC and IVIM parameters in grading ccRCC, indicating the great potential of the FROC model in distinguishing low- and high-grade ccRCCs.

Combination of Hematoma Volume and Perihematoma Radiomics Analysis on Baseline CT Scan Predicts the Growth of Perihematomal Edema.

PURPOSE: The aim is to explore the potential value of CT-based radiomics in predicting perihematomal edema (PHE) volumes after acute intracerebral hemorrhage (ICH) from admission to 24 h. METHODS: A total of 231 patients newly diagnosed with acute ICH at two institutes were analyzed retrospectively. The patients were randomly divided into training (N = 117) and internal validation cohort (N = 45) from institute 1 with a ratio of 7:3. According to radiomics features extracted from baseline CT, the radiomics signatures were constructed. Multiple logistic regression analysis was used for clinical radiological factors and then the nomogram model was generated to predict the extent of PHE according to the optimal radiomics signature and the clinical radiological factors. The receiver operating characteristic (ROC) curve was used to evaluate the discrimination performance. The calibration curve and Hosmer-Lemeshow test were used to evaluate the consistency between the predicted and actual probability. The support vector regression (SVR) model was constructed to predict the overall value of follow-up PHE. The performance of the models was evaluated on the internal and independent validation cohorts. RESULTS: The perihematoma 5 mm radiomics signature (AUC: 0.875) showed good ability to discriminate the small relative PHE(rPHE) from large rPHE volumes, comparing to intrahematoma radiomics signature (AUC: 0.711) or perihematoma 10 mm radiomics signature (AUC: 0.692) on the training cohort. The AUC of the combined nomogram model was 0.922 for the training cohort, 0.945 and 0.902 for the internal and independent validation cohorts, respectively. The calibration curves and Hosmer-Lemeshow test of the nomogram model suggested that the predictive performance and actual outcome were in favorable agreement. The SVR model also predicted the overall value of follow-up rPHE (root mean squared error, 0.60 and 0.45; Pearson correlation coefficient, 0.73 and 0.68; P < 0.001). CONCLUSION: Among patients with acute ICH, the established nomogram and SVR model with favorable performance can offer a noninvasive tool for the prediction of PHE after ICH.

A Radiomics Nomogram for Classifying Hematoma Entities in Acute Spontaneous Intracerebral Hemorrhage on Non-contrast-Enhanced Computed Tomography.

Aim: To develop and validate a radiomics nomogram on non-contrast-enhanced computed tomography (NECT) for classifying hematoma entities in patients with acute spontaneous intracerebral hemorrhage (ICH). Materials and Methods: One hundred and thirty-five patients with acute intraparenchymal hematomas and baseline NECT scans were retrospectively analyzed, i.e., 52 patients with vascular malformation-related hemorrhage (VMH) and 83 patients with primary intracerebral hemorrhage (PICH). The patients were divided into training and validation cohorts in a 7:3 ratio with a random seed. After extracting the radiomics features of hematomas from baseline NECT, the least absolute shrinkage and selection operator (LASSO) regression was applied to select features and construct the radiomics signature. Multivariate logistic regression analysis was used to determine the independent clinical-radiological risk factors, and a clinical model was constructed. A predictive radiomics nomogram was generated by incorporating radiomics signature and clinical-radiological risk factors. Nomogram performance was assessed in the training cohort and tested in the validation cohort. The capability of models was compared by calibration, discrimination, and clinical benefit. Results: Six features were selected to establish radiomics signature via LASSO regression. The clinical model was constructed with the combination of age [odds ratio (OR): 6.731; 95% confidence interval (CI): 2.209-20.508] and hemorrhage location (OR: 0.089; 95% CI: 0.028-0.281). Radiomics nomogram [area under the curve (AUC), 0.912 and 0.919] that incorporated age, location, and radiomics signature outperformed the clinical model (AUC, 0.816 and 0.779) and signature (AUC, 0.857 and 0.810) in the training cohort and validation cohorts, respectively. Good calibration and clinical benefit of nomogram were achieved in the training and validation cohorts. Conclusion: Non-contrast-enhanced computed tomography-based radiomics nomogram can predict the individualized risk of VMH in patients with acute ICH.

A comparison between deep learning convolutional neural networks and radiologists in the differentiation of benign and malignant thyroid nodules on CT images.

INTRODUCTION: We designed 5 convolutional neural network (CNN) models and ensemble models to differentiate malignant and benign thyroid nodules on CT, and compared the diagnostic performance of CNN models with that of radiologists. MATERIAL AND METHODS: We retrospectively included CT images of 880 patients with 986 thyroid nodules confirmed by surgical pathology between July 2017 and December 2019. Two radiologists retrospectively diagnosed benign and malignant thyroid nodules on CT images in a test set. Five CNNs (ResNet50, DenseNet121, DenseNet169, SE-ResNeXt50, and Xception) were trained-validated and tested using 788 and 198 thyroid nodule CT images, respectively. Then, we selected the 3 models with the best diagnostic performance on the test set for the model ensemble. We then compared the diagnostic performance of 2 radiologists with 5 CNN models and the integrated model. RESULTS: Of the 986 thyroid nodules, 541 were malignant, and 445 were benign. The area under the curves (AUCs) for diagnosing thyroid malignancy was 0.587-0.754 for 2 radiologists. The AUCs for diagnosing thyroid malignancy for the 5 CNN models and ensemble model was 0.901-0.947. There were significant differences in AUC between the radiologists' models and the CNN models (p < 0.05). The ensemble model had the highest AUC value. CONCLUSIONS: Five CNN models and an ensemble model performed better than radiologists in distinguishing malignant thyroid nodules from benign nodules on CT. The diagnostic performance of the ensemble model improved and showed good potential.

[Study of resting state functional connectivity of the red nucleus and substantia nigra were in normal adult].

OBJECTIVE: To detect the functional networks of the red nucleus and substantia nigra during the resting state in normal subjects with functional magnetic resonance imaging (fMRI). METHODS: Sixteen normal subjects were performed resting state fMRI scanning and susceptibility weighted imaging. The function connectivity networks base on seed regions of the red nucleus and substantia nigra were extracted from low frequency fluctuation signal in fMRI data by using a temporal correlation method. Individual functional maps were entered two-tailed one-sample t test to determine brain regions with significant positive correlation to the seeds. The statistic threshold was set at P < 0.001, cluster size>42 (336 mm(3)), cluster connectivity criterion 5 min with Alphasim correction. RESULTS: Brain regions involved in the functional connectivity network of the red nucleus include: dorsal anterior cingutate, supramarginal gyrus, the ventrolateral and the ventromedial nucleus of the thalamus, globus pallidus, dorsal thalamus, hippocampus, substantia nigra, red nucleus, pons, dentate nucleus, vermis; Brain regions involved in the functional connectivity network of the substantia nigra include: anterior cingutate, supramarginal gyrus, globus pallidus, dorsal thalamus, hippocampus, lobus insularis, substantia nigra, red nucleus, pons, dentate nucleus. The distribution of the networks of the red nucleus and substantia nigra presented symmetrical. Although the functional networks of the red nucleus and substantia nigra over lapped largely with each other, the rubral network was slightly different with the nigral network, witch showed strong correlations with more wide-spread striatum and thalamus areas. CONCLUSION: The functional networks of the red nucleus and substantia nigra reflected strong interplay within the extrapyramidal subcortical system, as well as correlations between some limited cerebral cortices; Functional magnetic resonance imaging is a potential powerful tool to explore the extrapyramidal system.

SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage.

Here we demonstrate that RNF4, a highly conserved small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, plays a critical role in the response of mammalian cells to DNA damage. Human cells in which RNF4 expression was ablated by siRNA or chicken DT40 cells with a homozygous deletion of the RNF4 gene displayed increased sensitivity to DNA-damaging agents. Recruitment of RNF4 to double-strand breaks required its RING and SUMO interaction motif (SIM) domains and DNA damage factors such as NBS1, mediator of DNA damage checkpoint 1 (MDC1), RNF8, 53BP1, and BRCA1. In the absence of RNF4, these factors were still recruited to sites of DNA damage, but 53BP1, RNF8, and RNF168 displayed delayed clearance from such foci. SILAC-based proteomics of SUMO substrates revealed that MDC1 was SUMO-modified in response to ionizing radiation. As a consequence of SUMO modification, MDC1 recruited RNF4, which mediated ubiquitylation at the DNA damage site. Failure to recruit RNF4 resulted in defective loading of replication protein A (RPA) and Rad51 onto ssDNA. This appeared to be a consequence of reduced recruitment of the CtIP nuclease, resulting in inefficient end resection. Thus, RNF4 is a novel DNA damage-responsive protein that plays a role in homologous recombination and integrates SUMO modification and ubiquitin signaling in the cellular response to genotoxic stress.

Identification of genes downstream of the Shh signalling in the developing chick wing and syn-expressed with Hoxd13 using microarray and 3D computational analysis.

Sonic hedgehog (Shh) signalling by the polarizing region at the posterior margin of the chick wing bud is pivotal in patterning the digits but apart from a few key downstream genes, such as Hoxd13, which is expressed in the posterior region of the wing that gives rise to the digits, the genes that mediate the response to Shh signalling are not known. To find genes that are co-expressed with Hoxd13 in the posterior of chick wing buds and regulated in the same way, we used microarrays to compare gene expression between anterior and posterior thirds of wing buds from normal chick embryos and from polydactylous talpid³ mutant chick embryos, which have defective Shh signalling due to lack of primary cilia. We identified 1070 differentially expressed gene transcripts, which were then clustered. Two clusters contained genes predominantly expressed in posterior thirds of normal wing buds; in one cluster, genes including Hoxd13, were expressed at high levels in anterior and posterior thirds in talpid³ wing buds, in the other cluster, genes including Ptc1, were expressed at low levels in anterior and posterior thirds in talpid³ wing buds. Expression patterns of genes in these two clusters were validated in normal and talpid³ mutant wing buds by in situ hybridisation and demonstrated to be responsive to application of Shh. Expression of several genes in the Hoxd13 cluster was also shown to be responsive to manipulation of protein kinase A (PKA) activity, thus demonstrating regulation by Gli repression. Genes in the Hoxd13 cluster were then sub-clustered by computational comparison of 3D expression patterns in normal wing buds to produce syn-expression groups. Hoxd13 and Sall1 are syn-expressed in the posterior region of early chick wing buds together with 6 novel genes which are likely to be functionally related and represent secondary targets of Shh signalling. Other groups of syn-expressed genes were also identified, including a group of genes involved in vascularisation.

System-wide changes to SUMO modifications in response to heat shock.

Covalent conjugation of the small ubiquitin-like modifier (SUMO) proteins to target proteins regulates many important eukaryotic cellular mechanisms. Although the molecular consequences of the conjugation of SUMO proteins are relatively well understood, little is known about the cellular signals that regulate the modification of their substrates. Here, we show that SUMO-2 and SUMO-3 are required for cells to survive heat shock. Through quantitative labeling techniques, stringent purification of SUMOylated proteins, advanced mass spectrometric technology, and novel techniques of data analysis, we quantified heat shock-induced changes in the SUMOylation state of 766 putative substrates. In response to heat shock, SUMO was polymerized into polySUMO chains and redistributed among a wide range of proteins involved in cell cycle regulation; apoptosis; the trafficking, folding, and degradation of proteins; transcription; translation; and DNA replication, recombination, and repair. This comprehensive proteomic analysis of the substrates of a ubiquitin-like modifier (Ubl) identifies a pervasive role for SUMO proteins in the biologic response to hyperthermic stress.

The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation.

The chicken talpid(3) mutant, with polydactyly and defects in other embryonic regions that depend on hedgehog (Hh) signalling (e.g. the neural tube), has a mutation in KIAA0568. Similar phenotypes are seen in mice and in human syndromes with mutations in genes that encode centrosomal or intraflagella transport proteins. Such mutations lead to defects in primary cilia, sites where Hh signalling occurs. Here, we show that cells of talpid(3) mutant embryos lack primary cilia and that primary cilia can be rescued with constructs encoding Talpid3. talpid(3) mutant embryos also develop polycystic kidneys, consistent with widespread failure of ciliogenesis. Ultrastructural studies of talpid(3) mutant neural tube show that basal bodies mature but fail to dock with the apical cell membrane, are misorientated and almost completely lack ciliary axonemes. We also detected marked changes in actin organisation in talpid(3) mutant cells, which may explain misorientation of basal bodies. KIAA0586 was identified in the human centrosomal proteome and, using an antibody against chicken Talpid3, we detected Talpid3 in the centrosome of wild-type chicken cells but not in mutant cells. Cloning and bioinformatic analysis of the Talpid3 homolog from the sea anemone Nematostella vectensis identified a highly conserved region in the Talpid3 protein, including a predicted coiled-coil domain. We show that this region is required to rescue primary cilia formation and neural tube patterning in talpid(3) mutant embryos, and is sufficient for centrosomal localisation. Thus, Talpid3 is one of a growing number of centrosomal proteins that affect both ciliogenesis and Hh signalling.

mRNA translation regulation by the Gly-Ala repeat of Epstein-Barr virus nuclear antigen 1.

The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5' untranslated region (UTR) of the c-myc mRNA to the 5' UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA.

Integration of growth and specification in chick wing digit-patterning.

In the classical model of chick wing digit-patterning, the polarizing region--a group of cells at the posterior margin of the early bud--produces a morphogen gradient, now known to be based on Sonic hedgehog (Shh), that progressively specifies anteroposterior positional identities in the posterior digit-forming region. Here we add an integral growth component to this model by showing that Shh-dependent proliferation of prospective digit progenitor cells is essential for specifying the complete pattern of digits across the anteroposterior axis. Inhibiting Shh signalling in early wing buds reduced anteroposterior expansion, and posterior digits were lost because all prospective digit precursors formed anterior structures. Inhibiting proliferation also irreversibly reduced anteroposterior expansion, but instead anterior digits were lost because all prospective digit precursors formed posterior structures. When proliferation recovered in such wings, Shh transcription was maintained for longer than normal, suggesting that duration of Shh expression is controlled by a mechanism that measures proliferation. Rescue experiments confirmed that Shh-dependent proliferation controls digit number during a discrete time-window in which Shh-dependent specification normally occurs. Our findings that Shh signalling has dual functions that can be temporally uncoupled have implications for understanding congenital and evolutionary digit reductions.

The chicken talpid3 gene encodes a novel protein essential for Hedgehog signaling.

Talpid3 is a classical chicken mutant with abnormal limb patterning and malformations in other regions of the embryo known to depend on Hedgehog signaling. We combined the ease of manipulating chicken embryos with emerging knowledge of the chicken genome to reveal directly the basis of defective Hedgehog signal transduction in talpid3 embryos and to identify the talpid3 gene. We show in several regions of the embryo that the talpid3 phenotype is completely ligand independent and demonstrate for the first time that talpid3 is absolutely required for the function of both Gli repressor and activator in the intracellular Hedgehog pathway. We map the talpid3 locus to chromosome 5 and find a frameshift mutation in a KIAA0586 ortholog (ENSGALG00000012025), a gene not previously attributed with any known function. We show a direct causal link between KIAA0586 and the mutant phenotype by rescue experiments. KIAA0586 encodes a novel protein, apparently specific to vertebrates, that localizes to the cytoplasm. We show that Gli3 processing is abnormal in talpid3 mutant cells but that Gli3 can still translocate to the nucleus. These results suggest that the talpid3 protein operates in the cytoplasm to regulate the activity of both Gli repressor and activator proteins.

Self-inhibition of synthesis and antigen presentation by Epstein-Barr virus-encoded EBNA1.

The glycine-alanine repeat domain (GAr) of Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) prevents major histocompatibility complex (MHC) class I-restricted presentation of EBNA1 epitopes to cytotoxic T cells. This effect has previously been attributed to the ability of GAr to inhibit its own proteasomal degradation. Here we show, both in vitro and in vivo, that GAr also inhibits messenger RNA translation of EBNA1 in cis and that this effect can be distinguished from its effect on proteasomal degradation. Hence, inhibition of messenger RNA translation, but not protein degradation, is essential to prevent antigen presentation on MHC class I molecules. Thus, by minimizing translation of the EBNA1 transcript, cells expressing EBNA1 avoid cytotoxic T cell recognition. At the same time, blocking degradation maintains the EBNA1 expression level.

Peptidase allergen Der p 1 initiates apoptosis of epithelial cells independently of tight junction proteolysis.

Loss of epithelial cell polarity, which can arise following disruption of tight junctions (TJs), is a precursor to the care-fully orchestrated removal of moribund cells from epithelia in apoptosis. Ordinarily, this cycle of events has minimally disruptive effects on the function of the epithelial barrier, but some agents have been identified that induce apoptosis and promote epithelial leakiness. The allergen Der p 1 is a cysteine peptidase that cleaves TJ adhesion proteins and induces apoptosis in epithelial cells. This suggests the possibility that, at least for some inducers of apoptosis, these events might be causally linked. We report here that Der p 1 induces epithelial apoptosis before outright cell detachment and that apoptosis occurs within the same time span as increased paracellular permeability in polarized epithelial monolayers. Whilst TJ-deficient BEAS-2B cells were resistant to Der p 1-induced apoptosis, the cell line 1HAEo-, which was also TJ deficient, was sensitive to Der p 1, providing evidence against TJ proteolysis as a cause of apoptosis. To provide direct evidence, we propagated cells that normally express TJs in low calcium medium that prevented intercellular junction assembly. These cells retained full susceptibility to Der p 1, indicating that Der p 1-induced apoptosis is independent from TJ proteolysis.

Physiological electric fields control the G1/S phase cell cycle checkpoint to inhibit endothelial cell proliferation.

Vascular endothelial cell (VEC) proliferation is a key event in angiogenesis and is tightly regulated. Electric potential differences exist around the vascular endothelium and give rise to endogenous electric fields (EFs), whether these EFs influence VEC proliferation is unclear. We exposed cultured VECs to applied EFs of physiological strengths for up to 72 h. EF at 50 or 100 mV/mm did not influence cell proliferation, but at 200 mV/mm, cell density, cell growth rate, and mitosis index decreased significantly. EF-induced reduction in VEC proliferation was not due to increased apoptosis, because caspase apoptosis inhibitor Z-VAD-FMK (20 microM), had no effect on this response. Rather, EF responses were mediated via decreased entry of cells into S phase from G1 phase, as shown by flow cytometry. Western blot showed that EFs decreased G1-specific cyclin E expression and increased cyclin/cyclin-dependent kinase complex inhibitor p27kipl expression. Thus EFs controlled VEC proliferation through induction of cell cycle arrest at G1 by down-regulation of cyclin E expression and up-regulation of p27kipl expression, rather than by promoting apoptosis. If control of the cell cycle by endogenous EFs extends beyond VECs, this would be of widespread biological significance in vivo.

p53 Stability and activity is regulated by Mdm2-mediated induction of alternative p53 translation products.

Activation of the p53 tumour suppressor protein can lead to cell-cycle arrest or apoptosis. p53 function is controlled by the mdm2 oncogene product, which targets p53 for proteasomal degradation. In this report we demonstrate that Mdm2 induces translation of the p53 mRNA from two alternative initiation sites, giving full-length p53 and another protein with a relative molecular mass (M(r)) of approximately 47K; we designate this protein as p53/47. This translation induction requires Mdm2 to interact directly with the nascent p53 polypeptide. The alternatively translated p53/47 does not contain the Mdm2-binding site and it lacks the most amino-terminal transcriptional-activation domain of p53. Increased expression of p53/47 stabilizes p53 in the presence of Mdm2, and alters the expression levels of p53-induced gene products. These results show how the interaction of Mdm2 with p53 leads to a change in the ratio of full-length p53 to p53/47 by inducing translation of both p53 proteins and the subsequent selective degradation of full-length p53. Thus, Mdm2 controls the expression levels of p53 through a dual mechanism that involves induction of synthesis and targeting for degradation.