iProPhos: A Web-Based Interactive Platform for Integrated Proteome and Phosphoproteome Analysis.

Large-scale omics studies have generated a wealth of mass spectrometry-based proteomics data, which provide additional insights into disease biology spanning genomic boundaries. However, there is a notable lack of web-based analysis and visualization tools that facilitate the reutilization of these data. Given this challenge, we present iProPhos, a user-friendly web server to deliver interactive and customizable functionalities. iProPhos incorporates a large number of samples, including 1444 tumor samples and 746 normal samples across 12 cancer types, sourced from the Clinical Proteomic Tumor Analysis Consortium. Additionally, users can also upload their own proteomics/phosphoproteomics data for analysis and visualization. In iProPhos, users can perform profiling plotting and differential expression, patient survival, clinical feature-related, and correlation analyses, including protein-protein, mRNA-protein, and kinase-substrate correlations. Furthermore, functional enrichment, protein-protein interaction network, and kinase-substrate enrichment analyses are accessible. iProPhos displays the analytical results in interactive figures and tables with various selectable parameters. It is freely accessible at http://longlab-zju.cn/iProPhos without login requirement. We present two case studies to demonstrate that iProPhos can identify potential drug targets and upstream kinases contributing to site-specific phosphorylation. Ultimately, iProPhos allows end-users to leverage the value of big data in cancer proteomics more effectively and accelerates the discovery of novel therapeutic targets.

Upregulation of CYR61 by TGF-ß and YAP signaling exerts a counter-suppression of hepatocellular carcinoma.

Transforming growth factor-ß (TGF-ß) and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-ß target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-ß in liver cancer cells. In addition, TGF-ß-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-ß- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-ß- or YAP-induced malignant transformation of HCC cells in vitro and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-ß and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

Stabilization of SETD3 by deubiquitinase USP27 enhances cell proliferation and hepatocellular carcinoma progression.

The histone methyltransferase SETD3 plays critical roles in various biological events, and its dysregulation is often associated with human diseases including cancer. However, the underlying regulatory mechanism remains elusive. Here, we reported that ubiquitin-specific peptidase 27 (USP27) promotes tumor cell growth by specifically interacting with SETD3, negatively regulating its ubiquitination, and enhancing its stability. Inhibition of USP27 expression led to the downregulation of SETD3 protein level, the blockade of the cell proliferation and tumorigenesis of hepatocellular carcinoma (HCC) cells. In addition, we found that USP27 and SETD3 expression is positively correlated in HCC tissues. Notably, higher expression of USP27 and SETD3 predicts a worse survival in HCC patients. Collectively, these data elucidated that a USP27-dependent mechanism controls SETD3 protein levels and facilitates its oncogenic role in liver tumorigenesis.

Biological and Genetic Characterization of Physostegia Chlorotic Mottle Virus in Europe Based on Host Range, Location, and Time.

Application of high throughput sequencing (HTS) technologies enabled the first identification of Physostegia chlorotic mottle virus (PhCMoV) in 2018 in Austria. Subsequently, PhCMoV was detected in Germany and Serbia on tomatoes showing severe fruit mottling and ripening anomalies. We report here how prepublication data-sharing resulted in an international collaboration across eight laboratories in five countries, enabling an in-depth characterization of PhCMoV. The independent studies converged toward its recent identification in eight additional European countries and confirmed its presence in samples collected 20 years ago (2002). The natural plant host range was expanded from two to nine species across seven families, and we confirmed the association of PhCMoV presence with severe fruit symptoms on economically important crops such as tomato, eggplant, and cucumber. Mechanical inoculations of selected isolates in the greenhouse established the causality of the symptoms on a new indexing host range. In addition, phylogenetic analysis showed a low genomic variation across the 29 near-complete genome sequences available. Furthermore, a strong selection pressure within a specific ecosystem was suggested by nearly identical sequences recovered from different host plants through time. Overall, this study describes the European distribution of PhCMoV on multiple plant hosts, including economically important crops on which the virus can cause severe fruit symptoms. This work demonstrates how to efficiently improve knowledge on an emergent pathogen by sharing HTS data and provides a solid knowledge foundation for further studies on plant rhabdoviruses.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

AMPK inhibits Smad3-mediated autoinduction of TGF-ß1 in gastric cancer cells.

We have previously shown that adenine monophosphate-activated protein kinase (AMPK) regulates transforming growth factor ß (TGF-ß)-triggered Smad3 phosphorylation. Here we report that AMPK inhibits TGF-ß1 production. First, metformin reduced mRNA levels of TGF-ß1 in gastric cancer cells, in parallel to the decrease of its protein abundance. The effects were more prominent in the cells containing LKB1, an upstream kinase of AMPK. Second, knockdown of Smad3 by siRNA abrogated the expression of TGF-ß1. Third, metformin suppressed firefly luciferase activity whose transcription was driven by TGF-ß1 promoter. In accordance, deletion of the putative binding site of Smad3 in the TGF-ß1 promoter region severely impaired the promoter activity and response to metformin. Fourth, in support of our in vitro study, clinical treatment of type 2 diabetes with metformin significantly reduced the plasma level of TGF-ß1. Finally, immunohistochemical studies revealed that TGF-ß1 was highly expressed in human gastric cancer tissues as compared with adjacent normal tissues. In contrast, p-AMPK exhibited opposite changes. Furthermore, the survival rate of gastric cancer patients was positively correlated with p-AMPK and negative with TGF-ß1. Therefore, our present studies depict a mechanism underlying AMPK suppression of TGF-ß1 autoinduction, which is mediated through inhibition of Smad3 phosphorylation and activation. Collectively, our study sheds a light on the potential usage of AMPK activators in the treatment of TGF-ß1-mediated gastric cancer progression.

Metformin attenuates trauma-induced heterotopic ossification via inhibition of Bone Morphogenetic Protein signalling.

AMP-activated protein kinase (AMPK) is an intracellular sensor of energy homoeostasis that is activated under energy stress and suppressed in energy surplus. AMPK activation leads to inhibition of anabolic processes that consume ATP. Osteogenic differentiation is a process that highly demands ATP during which AMPK is inhibited. The bone morphogenetic proteins (BMPs) signalling pathway plays an essential role in osteogenic differentiation. The present study examines the inhibitory effect of metformin on BMP signalling, osteogenic differentiation and trauma-induced heterotopic ossification. Our results showed that metformin inhibited Smad1/5 phosphorylation induced by BMP6 in osteoblast MC3T3-E1 cells, concurrent with up-regulation of Smad6, and this effect was attenuated by knockdown of Smad6. Furthermore, we found that metformin suppressed ALP activity and mineralization of the cells, an event that was attenuated by the dominant negative mutant of AMPK and mimicked by its constitutively active mutant. Finally, administration of metformin prevented the trauma-induced heterotopic ossification in mice. In conjuncture, AMPK activity and Smad6 and Smurf1 expression were enhanced by metformin treatment in the muscle of injured area, concurrently with the reduction of ALK2. Collectively, our study suggests that metformin prevents heterotopic ossification via activation of AMPK and subsequent up-regulation of Smad6. Therefore, metformin could be a potential therapeutic drug for heterotopic ossification induced by traumatic injury.

KLF2 inhibits TGF-ß-mediated cancer cell motility in hepatocellular carcinoma.

Feedback regulation plays a pivotal role in determining the intensity and duration of TGF-ß signaling and subsequently affecting the pathophysiological roles of TGF-ß, including those in liver malignancy. KLF2, a member of the Krüppel-like factor (KLF) family transcription factors, has been implicated in impeding hepatocellular carcinoma (HCC) development. However, the underlying molecular mechanisms are not fully understood. In the present study, we found that TGF-ß stimulates the expression of KLF2 gene in several HCC cell lines. KLF2 protein is able to inhibit TGF-ß/Smad signaling in HCC cells as assessed by luciferase reporter assay. Further studies indicated that KLF2 inhibits the transcriptional activity of Smad2/3 and Smad4 and ameliorates TGF-ß-induced target gene expression, therefore creating a novel negative feedback loop in TGF-ß signaling. Functionally, stably expression of KLF2 in HCCLM3 cells attenuated TGF-ß-induced cancer cell motility in wound-healing and transwell assays by interfering with TGF-ß-mediated upregulation of MMP2. Together, our results revealed that KLF2 protein has a tumor-suppressive function in HCC through a negative feedback loop over TGF-ß signaling.

CXXC5: A novel regulator and coordinator of TGF-ß, BMP and Wnt signaling.

CXXC5 is a member of the CXXC-type zinc-finger protein family. Proteins in this family play a pivotal role in epigenetic regulation by binding to unmethylated CpG islands in gene promoters through their characteristic CXXC domain. CXXC5 is a short protein (322 amino acids in length) that does not have any catalytic domain, but is able to bind to DNA and act as a transcription factor and epigenetic factor through protein-protein interactions. Intriguingly, increasing evidence indicates that expression of the CXXC5 gene is controlled by multiple signaling pathways and a variety of transcription factors, positioning CXXC5 as an important signal integrator. In addition, CXXC5 is capable of regulating various signal transduction processes, including the TGF-ß, Wnt and ATM-p53 pathways, thereby acting as a novel and crucial signaling coordinator. CXXC5 plays an important role in embryonic development and adult tissue homeostasis by regulating cell proliferation, differentiation and apoptosis. In keeping with these functions, aberrant expression or altered activity of CXXC5 has been shown to be involved in several human diseases including tumourigenesis. This review summarizes the current understanding of CXXC5 as a transcription factor and signaling regulator and coordinator.

Feedback regulation of TGF-ß signaling.

Transforming growth factor beta (TGF-ß) is a multi-functional polypeptide that plays a critical role in regulating a broad range of cellular functions and physiological processes. Signaling is initiated when TGF-ß ligands bind to two types of cell membrane receptors with intrinsic Ser/Thr kinase activity and transmitted by the intracellular Smad proteins, which act as transcription factors to regulate gene expression in the nucleus. Although it is relatively simple and straight-forward, this TGF-ß/Smad pathway is regulated by various feedback loops at different levels, including the ligand, the receptor, Smads and transcription, and is thus fine-tuned in terms of signaling robustness, duration, specificity, and plasticity. The precise control gives rise to versatile and context-dependent pathophysiological functions. In this review, we firstly give an overview of TGF-ß signaling, and then discuss how each step of TGF-ß signaling is finely controlled by distinct modes of feedback mechanisms, involving both protein regulators and miRNAs.

Smad7 Protein Interacts with Receptor-regulated Smads (R-Smads) to Inhibit Transforming Growth Factor-ß (TGF-ß)/Smad Signaling.

TGF-ß is a pleiotropic cytokine that regulates a wide range of cellular actions and pathophysiological processes. TGF-ß signaling is spatiotemporally fine-tuned. As a key negative regulator of TGF-ß signaling, Smad7 exerts its inhibitory effects by blocking receptor activity, inducing receptor degradation or interfering with Smad-DNA binding. However, the functions and the molecular mechanisms underlying the actions of Smad7 in TGF-ß signaling are still not fully understood. In this study we report a novel mechanism whereby Smad7 antagonizes TGF-ß signaling at the Smad level. Smad7 oligomerized with R-Smad proteins upon TGF-ß signaling and directly inhibited R-Smad activity, as assessed by Gal4-luciferase reporter assays. Mechanistically, Smad7 competes with Smad4 to associate with R-Smads and recruits the E3 ubiquitin ligase NEDD4L to activated R-Smads, leading to their polyubiquitination and proteasomal degradation. Similar to the R-Smad-Smad4 oligomerization, the interaction between R-Smads and Smad7 is mediated by their mad homology 2 (MH2) domains. A positive-charged basic region including the L3/ß8 loop-strand module and adjacent amino acids in the MH2 domain of Smad7 is essential for the interaction. These results shed new light on the regulation of TGF-ß signaling by Smad7.

Receptor for Activated C Kinase 1 (RACK1) Promotes Dishevelled Protein Degradation via Autophagy and Antagonizes Wnt Signaling.

Wnt signaling plays a critical role in embryonic development, tissue homeostasis, and cancer development. Dishevelled (Dvl) is an essential and central component in Wnt signaling, and its stability and activity is tightly regulated. It has been shown that Dvl can be degraded via both the proteasome and autophagy-lysosome pathways. Here we report that receptor for activated C kinase 1 (RACK1) negatively regulates Dishevelled stability and Wnt signaling. RACK1 interacts with Dvl proteins and promotes their lysosomal degradation, and this effect is enhanced by autophagy induction. RACK1 also interacts with LC3 and enhances the association of LC3 with Dvl2, thereby leading to degradation of Dvl proteins through autophagy. These findings reveal a novel regulatory function of RACK1 in Wnt signaling by modulating Dvl stability.

Activin Regulates Self-renewal and Differentiation of Trophoblast Stem Cells by Down-regulating the X Chromosome Gene Bcor.

The development of a functional placenta is largely dependent upon proper proliferation and differentiation of trophoblast stem cells (TSCs). Activin signaling has long been regarded to play important roles during this process, but the exact mechanism is largely unknown. Here, we demonstrate that the X chromosome gene BCL-6 corepressor (Bcor) is a critical downstream effector of activin to fine-tune mouse TSC fate decision. Bcor was specifically down-regulated by activin A in TSCs in a dose-dependent manner, and immediately up-regulated upon TSC differentiation. Knockdown of Bcor partially compensated for the absence of activin A in maintaining the self-renewal of TSCs together with FGF4, while promoting syncytiotrophoblast differentiation in the absence of FGF4. Moreover, the impaired trophoblast giant cell and spongiotrophoblast differentiation upon Bcor knockdown also resembled the function of activin. Reporter analysis showed that BCOR inhibited the expression of the key trophoblast regulator genes Eomes and Cebpa by binding to their promoter regions. Our findings provide us with a better understanding of placental development and placenta-related diseases.

Small C-terminal Domain Phosphatase 3 Dephosphorylates the Linker Sites of Receptor-regulated Smads (R-Smads) to Ensure Transforming Growth Factor ß (TGFß)-mediated Germ Layer Induction in Xenopus Embryos.

Germ layer induction is one of the earliest events shortly after fertilization that initiates body formation of vertebrate embryos. In Xenopus, the maternally deposited transcriptional factor VegT promotes the expression of zygotic Nodal/Activin ligands that further form a morphogen gradient along the vegetal-animal axis and trigger the induction of the three germ layers. Here we found that SCP3 (small C-terminal domain phosphatase 3) is maternally expressed and vegetally enriched in Xenopus embryos and is essential for the timely induction of germ layers. SCP3 is required for the full activation of Nodal/Activin and bone morphogenetic protein signals and functions via dephosphorylation in the linker regions of receptor-regulated Smads. Consistently, the linker regions of receptor-regulated Smads are heavily phosphorylated in fertilized eggs, and this phosphorylation is gradually removed when embryos approach the midblastula transition. Knockdown of maternal SCP3 attenuates these dephosphorylation events and the activation of Nodal/Activin and bone morphogenetic protein signals after midblastula transition. This study thus suggested that the maternal SCP3 serves as a vegetally enriched, intrinsic factor to ensure a prepared status of Smads for their activation by the upcoming ligands during germ layer induction of Xenopus embryos.

Mammalian actin-binding protein 1/HIP-55 is essential for the scission of clathrin-coated pits by regulating dynamin-actin interaction.

Actin and dynamin work cooperatively to drive the invagination and scission of clathrin-coated pits (CCPs). However, little is known about the mechanism that orchestrates the spatiotemporal recruitment of dynamin and actin. Here, we have identified the mammalian actin-binding protein 1 (mAbp1; also called HIP-55 or SH3P7), which could bind to clathrin, actin, as well as dynamin, as an adaptor that links the dynamic recruitment of dynamin and actin for the scission of CCPs. Live-cell imaging reveals that mAbp1 is specifically recruited at a late stage of the long-lived CCPs. mAbp1 knockdown impaired CCP scission by reducing dynamin recruitment at the plasma membrane. However, actin disruption remarkably eliminates mAbp1 recruitment and thus dynamin recruitment. These data suggest that by binding to both clathrin and F-actin, mAbp1 is specifically recruited at a late stage of CCP formation, which subsequently recruits dynamin to CCPs.

[Clinical application of automated digital image analysis for morphology review of peripheral blood leukocyte].

OBJECTIVE: To explore the clinical application of automated digital image analysis in leukocyte morphology examination when review criteria of hematology analyzer are triggered. METHODS: The reference range of leukocyte differentiation by automated digital image analysis was established by analyzing 304 healthy blood samples from Peking University First Hospital. Six hundred and ninty-seven blood samples from Peking University First Hospital were randomly collected from November 2013 to April 2014, complete blood cells were counted on hematology analyzer, blood smears were made and stained at the same time. Blood smears were detected by automated digital image analyzer and the results were checked (reclassification) by a staff with abundant morphology experience. The same smear was examined manually by microscope. The results by manual microscopic differentiation were used as"golden standard", and diagnostic efficiency of abnormal specimens by automated digital image analysis was calculated, including sensitivity, specificity and accuracy. The difference of abnormal leukocytes detected by two different methods was analyzed in 30 samples of hematological and infectious diseases. RESULTS: Specificity of identifying abnormalities of white blood cells by automated digital image analysis was more than 90% except monocyte. Sensitivity of neutrophil toxic abnormities (including Döhle body, toxic granulate and vacuolization) was 100%; sensitivity of blast cells, immature granulates and atypical lymphocytes were 91.7%, 60% to 81.5% and 61.5%, respectively. Sensitivity of leukocyte differential count was 91.8% for neutrophils, 88.5% for lymphocytes, 69.1% for monocytes, 78.9% for eosinophils and 36.3 for basophils. The positive rate of recognizing abnormal cells (blast, immature granulocyte and atypical lymphocyte) by manual microscopic method was 46.7%, 53.3% and 10%, respectively. The positive rate of automated digital image analysis was 43.3%, 60% and 10%, respectively. There was no statistic significance between two methods (χ(2) = 0.067, 0.271, 0.000, all P>0.05). Automated digital image analysis could be used to morphology examination with abnormal leukocytes and optimize review criteria of hematology analyzer. CONCLUSION: Sensitivity and specificity of recognizing abnormal blood leukocytes by automated digital image analysis are satisfactory, which can be used as a tool of leukocyte morphology review when review criteria of hematology analyzer are triggered.

Ultrastructural Changes of Brain Tissues Surrounding Hematomas after Intracerebral Hemorrhage.

Our knowledge about pathophysiology of intracerebral hemorrhage (ICH) mainly originates from preclinical models of ICH. In this study, cerebral ultrastructure surrounding hematoma and its correlation with clinical severity were investigated in ICH patients. Thirty patients with basal ganglia hemorrhage and 6 control subjects were enrolled. Surgical evacuation was performed for patients with a blood loss >30 ml. Stroke severity was assessed using the Glasgow Coma Scale (GCS) and the National Institute of Health Stroke Scale (NIHSS). Transmission electron microscopy (TEM) was used to evaluate the ultrastructural characteristics of tissue specimens. Neural cells surrounding the hematomas showed evidence of cell swelling and necrosis. Decreased numbers of organelles and mitochondrial cristae were accompanied by cytoplasmic vacuolization, nuclear membrane invagination and breakdown, and intranuclear chromatic agglutination. These changes resulted in disintegration together with malacia, disappearance of the nucleus and nucleolus, and karyopyknosis. More serious ultrastructural damage was seen in patients with greater NIHSS scores, lower GCS scores, and greater bleeding volumes (p < 0.001). These findings suggest that neural cells undergo unfavorable ultrastructural changes that are responsible for dysfunction after ICH.

Follistatin-like 1 (Fstl1) is a bone morphogenetic protein (BMP) 4 signaling antagonist in controlling mouse lung development.

Lung morphogenesis is a well orchestrated, tightly regulated process through several molecular pathways, including TGF-ß/bone morphogenetic protein (BMP) signaling. Alteration of these signaling pathways leads to lung malformation. We investigated the role of Follistatin-like 1 (Fstl1), a secreted follistatin-module-containing glycoprotein, in lung development. Deletion of Fstl1 in mice led to postnatal lethality as a result of respiratory failure. Analysis of the mutant phenotype showed that Fstl1 is essential for tracheal cartilage formation and alveolar maturation. Deletion of the Fstl1 gene resulted in malformed tracheal rings manifested as discontinued rings and reduced ring number. Fstl1-deficient mice displayed septal hypercellularity and end-expiratory atelectasis, which were associated with impaired differentiation of distal alveolar epithelial cells and insufficient production of mature surfactant proteins. Mechanistically, Fstl1 interacted directly with BMP4, negatively regulated BMP4/Smad1/5/8 signaling, and inhibited BMP4-induced surfactant gene expression. Reducing BMP signaling activity by Noggin rescued pulmonary atelectasis of Fstl1-deficient mice. Therefore, we provide in vivo and in vitro evidence to demonstrate that Fstl1 modulates lung development and alveolar maturation, in part, through BMP4 signaling.

Clinical analysis of 102 cases of Epstein-Barr virus infections in Chinese children.

The purpose of this study is to investigate the clinical manifestations and disease severity, to evaluate the recent trend of clinical manifestations and differences in the clinical and laboratory findings of EBV-associated IM (infectious mononucleosis) according to the age of children. We retrospectively collected cases on hospitalized patients a majority of 7 years old with characteristic symptoms of IM and serologically diagnosed EBV-associated IM at Shaanxi Provincial Peoples University Hospital in Xi'an from Apr, 2012 to Oct, 2013. All their medical records were reviewed and analyzed. For each patient, clinical, laboratory data and outcome were collected retrospectively and compared to previous studies to evaluate the differences between the clinical and laboratory findings of patients of different ages. The clinical manifestations in children with EB virus infection varied. There were 60 (58.8%) cases of children with infectious mononucleosis, 26 (25.49%) cases of Epstein-barr virus infection,16 cases of the atypical EB virus infection, accounting for 15.67%. 78% children were under 7 years of age, 12% were 7 to 14 years of age. There are differences in the symptoms and signs among the different age groups. The clinical manifestations in children with EB virus infection involved multiple systems and produced harm is heavier and should be paid attention to during the treatment. The disease continues to occur mostly in children under 10 years of age. When compared to previous Chinese studies about 15 years ago, the age distribution was similar and the incidence of hepatosplenomegaly was lower in our study.

Improving the Comprehension of Pathogenicity and Phylogeny in 'Candidatus Phytoplasma meliae' through Genome Characterization.

'Candidatus Phytoplasma meliae' is a pathogen associated with chinaberry yellowing disease, which has become a major phytosanitary problem for chinaberry forestry production in Argentina. Despite its economic impact, no genome information of this phytoplasma has been published, which has hindered its characterization at the genomic level. In this study, we used a metagenomics approach to analyze the draft genome of the 'Ca. P. meliae' strain ChTYXIII. The draft assembly consisted of twenty-one contigs with a total length of 751.949 bp, and annotation revealed 669 CDSs, 34 tRNAs, and 1 set of rRNA operons. The metabolic pathways analysis showed that ChTYXIII contains the complete core genes for glycolysis and a functional Sec system for protein translocation. Our phylogenomic analysis based on 133 single-copy genes and genome-to-genome metrics supports the classification as unique 'Ca. P. species' within the MPV clade. We also identified 31 putative effectors, including a homolog to SAP11 and others that have only been described in this pathogen. Our ortholog analysis revealed 37 PMU core genes in the genome of 'Ca. P. meliae' ChTYXIII, leading to the identification of 2 intact PMUs. Our work provides important genomic information for 'Ca. P. meliae' and others phytoplasmas for the 16SrXIII (MPV) group.