Weighted thyroid-stimulating hormone disturbance in prognosis of hepatitis B virus-related acute-on-chronic liver failure.

AIM: To weight the prognostic value of thyroid hormones in catastrophic acute-on-chronic liver failure (ACLF). METHODS: A retrospective cohort (n = 635) and two prospective cohorts (n = 353, and 198) were enrolled in this study. The performance of a novel developed prognostic score was assessed from aspects of reliability, discrimination, and clinical net benefit. RESULTS: Thyroid-stimulating hormone (TSH) was identified to have the most potential as a prognostic predictor for hepatitis B virus-related ACLF among thyroid hormones. The novel score (modified chronic liver failure-organ failure score [mCLIF-OFs]) was developed with weighted TSH and other scored organs in the CLIF-OFs using the retrospective cohort (n = 635). The predicted risk and observed probabilities of death were comparable across the deciles of mCLIF-OFs (Hosmer-Lemeshow χ2  = 4.28, p = 0.83; Brier scaled = 11.9). The C-index of mCLIF-OFs (0.885 [0.883-0.887]) for 30-day mortality was significantly higher than that of the CLIF-OFs, chronic liver failure-sequential organ failure assessment score (CLIF-SOFAs), CLIF-C ACLFs, Model of End-stage Liver Disease (MELD), and Child-Pugh (all p < 0.001). The absolute improvements of prediction error rates of the mCLIF-OFs compared to the above five scores were from 19.0% to 61.1%. After the analysis of probability density function, the mCLIF-OFs showed the least overlapping coefficients (27.9%) among the above prognostic scores. Additionally, the mCLIF-OFs showed greater net benefit than the above five prognostic scores over a wide range of risk threshold of death. Similar results were validated in two prospective ACLF cohorts with HBV and non-HBV etiologies. CONCLUSION: Weighted TSH portended the outcome of ACLF patients, which could be treated as a "damaged organ" of the hypothalamic-pituitary-thyroid axis. The novel mCLIF-OFs is a reliable prognostic score with better discrimination power and clinical net benefit than CLIF-OFs, CLIF-SOFAs, CLIF-C ACLFs, MELD, and Child-Pugh.

A retrospective comparative study on the diagnostic efficacy and the complications: between CassiII rotational core biopsy and core needle biopsy.

Accurate pathologic diagnosis and molecular classification of breast mass biopsy tissue is important for determining individualized therapy for (neo)adjuvant systemic therapies for invasive breast cancer. The CassiII rotational core biopsy system is a novel biopsy technique with a guide needle and a "stick-freeze" technology. The comprehensive assessments including the concordance rates of diagnosis and biomarker status between CassiII and core needle biopsy were evaluated in this study. Estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), and Ki67 were analyzed through immunohistochemistry. In total, 655 patients with breast cancer who underwent surgery after biopsy at Sir Run Run Shaw Hospital between January 2019 to December 2021 were evaluated. The concordance rates (CRs) of malignant surgical specimens with CassiII needle biopsy was significantly high compared with core needle biopsy. Moreover, CassiII needle biopsy had about 20% improvement in sensitivity and about 5% improvement in positive predictive value compared to Core needle biopsy. The characteristics including age and tumor size were identified the risk factors for pathological inconsistencies with core needle biopsies. However, CassiII needle biopsy was associated with tumor diameter only. The CRs of ER, PgR, HER2, and Ki67 using Cassi needle were 98.08% (kappa, 0.941; p<.001), 90.77% (kappa, 0.812; p<.001), 69.62% (kappa, 0.482; p<.001), and 86.92% (kappa, 0.552; p<.001), respectively. Post-biopsy complications with CassiII needle biopsy were also collected. The complications of CassiII needle biopsy including chest stuffiness, pain and subcutaneous ecchymosis are not rare. The underlying mechanism of subcutaneous congestion or hematoma after CassiII needle biopsy might be the larger needle diameter and the effect of temperature on coagulation function. In summary, CassiII needle biopsy is age-independent and has a better accuracy than CNB for distinguishing carcinoma in situ and invasive carcinoma.

Biological differences between normal and cancer-associated fibroblasts in breast cancer.

Background: Cancer-associated fibroblasts (CAFs) constitute the primary constituents of the tumor microenvironment (TME) and exert significant influences on cancer progression. However, adequate comprehension of CAF profiles in breast cancer, as well as the precise mechanisms underlying their promotion of cancer, remains lacking. Objectives: To discerns the biological differences between normal fibroblasts (NFs) and CAFs in breast cancer and explore the underlying mechanism. Methods: Three pairs of CAFs and NFs were isolated from breast cancer patients of diverse subtypes who had not undergone prior radiotherapy or chemotherapy. Morphological characteristics of CAFs and NFs were assessed through optical and electron microscopy, their biological attributes were examined using cell counting kits and transwell assays, and their impact on breast cancer cells was simulated using a coculture system. Furthermore, the miRNA profiles of CAFs and NFs were sequenced via an Illumina HiSeq 2500 platform. Results: CAFs exhibited higher growth rate and motility than NFs and a stronger potential to promote the malignancy of breast cancer cells. RNA sequencing of both NFs and CAFs revealed differentially expressed miRNAs with notable variability among distinct patients within their NFs and CAFs, while the enrichment of the target genes of differentially expressed miRNAs within both GO terms and KEGG pathways demonstrated significant similarity across patients with different profiles. Conclusion: CAFs have greater malignancy and higher potential to influence the growth, migration, invasion and chemoresistance of cocultured breast cancer cells than NFs. In addition, the miRNAs that are differentially expressed in CAFs when compared to NFs display substantial variability across patients with distinct breast cancer subtypes, while the enrichment of target genes regulated by these miRNAs, within GO terms and KEGG pathways, remains remarkably consistent among patients with varying profiles.

Human menstrual blood-derived stem cells alleviate autoimmune hepatitis via JNK/MAPK signaling pathway in vivo and in vitro.

Autoimmune hepatitis (AIH) is a severe globally distributed liver disease that could occur at any age. Human menstrual blood-derived stem cells (MenSCs) have shown therapeutic effect in acute lung injury and liver failure. However, their role in the curative effect of AIH remains unclear. Here, a classic AIH mouse model was constructed through intravenous injection with concanavalin A (Con A). MenSCs were intravenously injected while Con A injection in the treatment groups. The results showed that the mortality by Con A injection was significantly decreased by MenSCs treatment and liver function tests and histological analysis were also ameliorated. The results of phosphoproteomic analysis and RNA-seq revealed that MenSCs improved AIH, mainly by apoptosis and c-Jun N-terminal kinase/mitogen-activated protein signaling pathways. Apoptosis analysis demonstrated that the protein expression of cleaved caspase 3 was increased by Con A injection and reduced by MenSCs transplantation, consistent with the TUNEL staining results. An AML12 co-culture system and JNK inhibitor (SP600125) were used to verify the JNK/MAPK and apoptosis signaling pathways. These findings suggested that MenSCs could be a promising strategy for AIH.

Modification of PLAC8 by UFM1 affects tumorous proliferation and immune response by impacting PD-L1 levels in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer is characterized by a poor prognosis and lack of targeted treatments, and thus, new targeting markers and therapeutic strategies are urgently needed. We previously indicated that PLAC8 promotes tumorigenesis and exerts multidrug resistance in breast cancer. Therefore, we aimed to characterize the PLAC8-regulated network in triple-negative breast cancer. METHODS: We measured the levels of PLAC8 in breast cancer cell lines and found that PLAC8 is post-translationally modified by ubiquitin-fold modifier 1 (UFM1). Then, we revealed a new regulatory system of PD-L1 by PLAC8 in triple-negative breast cancer. We also tested the molecular functions of PLAC8 in triple-negative breast cancer cell lines and measured the expression of PLAC8 and PD-L1 in breast cancer tissues. RESULTS: PLAC8 was generally highly expressed in triple-negative breast cancer and could be modified by UFM1, which maintains PLAC8 protein stability. Moreover, PLAC8 could promote cancer cell proliferation and affect the immune response by regulating the level of PD-L1 ubiquitination. Additionally, among patients with breast cancer, the expression of PLAC8 was higher in triple-negative breast cancer than in non-triple-negative breast cancer and positively correlated with the level of PD-L1. CONCLUSIONS: Our current study discoveries a new PLAC8-regulated network in triple-negative breast cancer and provides corresponding guidance for the clinical diagnosis and immunotherapy of triple-negative breast cancer.

Detection of circulating tumor cells: opportunities and challenges.

Circulating tumor cells (CTCs) are cells that shed from a primary tumor and travel through the bloodstream. Studying the functional and molecular characteristics of CTCs may provide in-depth knowledge regarding highly lethal tumor diseases. Researchers are working to design devices and develop analytical methods that can capture and detect CTCs in whole blood from cancer patients with improved sensitivity and specificity. Techniques using whole blood samples utilize physical prosperity, immunoaffinity or a combination of the above methods and positive and negative enrichment during separation. Further analysis of CTCs is helpful in cancer monitoring, efficacy evaluation and designing of targeted cancer treatment methods. Although many advances have been achieved in the detection and molecular characterization of CTCs, several challenges still exist that limit the current use of this burgeoning diagnostic approach. In this review, a brief summary of the biological characterization of CTCs is presented. We focus on the current existing CTC detection methods and the potential clinical implications and challenges of CTCs. We also put forward our own views regarding the future development direction of CTCs.

Metabolome and Transcriptome Profiling Unveil the Mechanisms of Polyphenol Synthesis in the Developing Endopleura of Walnut (Juglans regia L.).

Walnut (Juglans regia L.) is an important woody nut tree species, and its endopleura (the inner coating of a seed) is rich in many polyphenols. Thus far, the pathways and essential genes involved in polyphenol biosynthesis in developing walnut endopleura remain largely unclear. We compared metabolite differences between endopleura and embryo in mature walnuts, and analyzed the changes of metabolites in endopleura at 35, 63, 91, 119, and 147 days after pollination (DAP). A total of 760 metabolites were detected in the metabolome, and the polyphenol contents in endopleura were higher than those in embryos. A total of 15 types of procyanidins, 10 types of kaempferol glycosides, and 21 types of quercetin glycosides that accumulated during endopleura development were identified. The analysis of the phenylpropane metabolic pathway showed that phenylalanine was gradually transformed into proanthocyanidins and other secondary metabolites with the development of endopleura. A total of 49 unigenes related to polyphenol synthesis were identified by transcriptome analysis of endopleura. The expression patterns of PAL, C4H, 4CL, CHS, CHI, F3H, LDOX, and ANR were similar, and their expression levels were highest in endopleura at maturity. Transcriptome and metabolome analysis showed that endopleura rapidly synthesized and accumulated polyphenols during maturation. Moreover, the transcription factor MYB111 played an important role in synthesizing polyphenols in endopleura, and its expression pattern was positively correlated with the accumulation pattern of quercetin, kaempferol, and proanthocyanidins. MYB111 was co-expressed with NAP, NAC, ATR1, and other genes related to cell senescence and abiotic stress response. Our study analyzed the composition and molecular synthesis mechanism of polyphenols in walnut endopleura, and provided new perspectives and insights regarding the nutritional research of walnut nuts.

Interactome and Ubiquitinome Analyses Identify Functional Targets of Herpes Simplex Virus 1 Infected Cell Protein 0.

Herpes simplex virus 1 (HSV-1) can productively infect multiple cell types and establish latent infection in neurons. Infected cell protein 0 (ICP0) is an HSV-1 E3 ubiquitin ligase crucial for productive infection and reactivation from latency. However, our knowledge about its targets especially in neuronal cells is limited. We confirmed that, like in non-neuronal cells, ICP0-null virus exhibited major replication defects in primary mouse neurons and Neuro-2a cells. We identified many ICP0-interacting proteins in Neuro-2a cells, 293T cells, and human foreskin fibroblasts by mass spectrometry-based interactome analysis. Co-immunoprecipitation assays validated ICP0 interactions with acyl-coenzyme A thioesterase 8 (ACOT8), complement C1q binding protein (C1QBP), ovarian tumour domain-containing protein 4 (OTUD4), sorting nexin 9 (SNX9), and vimentin (VIM) in both Neuro-2a and 293T cells. Overexpression and knockdown experiments showed that SNX9 restricted replication of an ICP0-null but not wild-type virus in Neuro-2a cells. Ubiquitinome analysis by immunoprecipitating the trypsin-digested ubiquitin reminant followed by mass spectrometry identified numerous candidate ubiquitination substrates of ICP0 in infected Neuro-2a cells, among which OTUD4 and VIM were novel substrates confirmed to be ubiquitinated by transfected ICP0 in Neuro-2a cells despite no evidence of their degradation by ICP0. Expression of OTUD4 was induced independently of ICP0 during HSV-1 infection. Overexpressed OTUD4 enhanced type I interferon expression during infection with the ICP0-null but not wild-type virus. In summary, by combining two proteomic approaches followed by confirmatory and functional experiments, we identified and validated multiple novel targets of ICP0 and revealed potential restrictive activities of SNX9 and OTUD4 in neuronal cells.

Human menstrual blood-derived stem cell transplantation suppresses liver injury in DDC-induced chronic cholestasis.

BACKGROUND: Cholestatic liver injury can lead to serious symptoms and prognoses in the clinic. Currently, an effective medical treatment is not available for cholestatic liver injury. Human menstrual blood-derived stem cells (MenSCs) are considered as an emerging treatment in various diseases. This study aimed to explore the treatment effect of MenSCs in cholestatic liver injury. METHODS: The treatment effect of MenSCs on chronic cholestatic liver injury was verified in 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC)-induced C57/BL6 mice. Pathological, fibrosis area in the liver tissue and serum liver enzymes were tested. Proteomics and western blot were used to explore the related targets and molecular mechanisms. Adeno-associated virus (AAV) 9-infected mice were applied for verification. RESULTS: MenSCs markedly improved the survival rate of the DDC-treated mice (60% vs. 100%), and decreased the mouse serum aspartate aminotransferase (AST) (169.4 vs. 108.0 U/L, p < 0.001), alanine aminotransferase (ALT) (279.0 vs. 228.9 U/L, p < 0.01), alkaline phosphatase (ALP) (45.6 vs. 10.6 U/L, p < 0.0001), direct bilirubin (DBIL) (108.3 vs. 14.0 µmol/L, p < 0.0001) and total bilirubin (TBIL) (179.2 vs. 43.3 µmol/L, p < 0.0001) levels as well as intrahepatic cholestasis, bile duct dilation and fibrotic areas (16.12 vs. 6.57%, p < 0.05). The results further indicated that MenSCs repaired the DDC-induced liver tight junction (TJ) pathway and bile transporter (OATP2, BSEP and NTCP1) injury, thereby inhibiting COL1A1, α-SMA and TGF-ß1 activation by upregulating liver ß-catenin expression. CONCLUSIONS: MenSC transplantation could be an effective treatment method for cholestatic liver injury in mice. MenSCs may exhibit therapeutic effects by regulating ß-catenin expression.

Host MOV10 is induced to restrict herpes simplex virus 1 lytic infection by promoting type I interferon response.

Moloney leukemia virus 10 protein (MOV10) is an interferon (IFN)-inducible RNA helicase implicated in antiviral activity against RNA viruses, yet its role in herpesvirus infection has not been investigated. After corneal inoculation of mice with herpes simplex virus 1 (HSV-1), we observed strong upregulation of both MOV10 mRNA and protein in acutely infected mouse trigeminal ganglia. MOV10 suppressed HSV-1 replication in both neuronal and non-neuronal cells, and this suppression required the N-terminus, but not C-terminal helicase domain of MOV10. MOV10 repressed expression of the viral gene ICP0 in transfected cells, but suppressed HSV-1 replication independently of ICP0. MOV10 increased expression of type I IFN in HSV-1 infected cells with little effect on IFN downstream signaling. Treating the cells with IFN-α or an inhibitor of the IFN receptor eliminated MOV10 suppression of HSV-1 replication. MOV10 enhanced IFN production stimulated by cytoplasmic RNA rather than DNA. IKKε co-immunoprecipitated with MOV10 and was required for MOV10 restriction of HSV-1 replication. Mass spectrometry identified ICP27 as a viral protein interacting with MOV10. Co-immunoprecipitation results suggested that this interaction depended on the RGG box of ICP27 and both termini of MOV10. Overexpressed ICP27, but not its RGG-Box deletion mutant, rendered MOV10 unable to regulate HSV-1 replication and type I IFN production. In summary, MOV10 is induced to restrict HSV-1 lytic infection by promoting the type I IFN response through an IKKε-mediated RNA sensing pathway, and its activity is potentially antagonized by ICP27 in an RGG box dependent manner.

Integrative Proteomic Analysis of Multiple Posttranslational Modifications in Inflammatory Response.

Posttranslational modifications (PTMs) of proteins, particularly acetylation, phosphorylation, and ubiquitination, play critical roles in the host innate immune response. PTMs' dynamic changes and the crosstalk among them are complicated. To build a comprehensive dynamic network of inflammation-related proteins, we integrated data from the whole-cell proteome (WCP), acetylome, phosphoproteome, and ubiquitinome of human and mouse macrophages. Our datasets of acetylation, phosphorylation, and ubiquitination sites helped identify PTM crosstalk within and across proteins involved in the inflammatory response. Stimulation of macrophages by lipopolysaccharide (LPS) resulted in both degradative and non-degradative ubiquitination. Moreover, this study contributes to the interpretation of the roles of known inflammatory molecules and the discovery of novel inflammatory proteins.

A genome variation map provides insights into the genetics of walnut adaptation and agronomic traits.

BACKGROUND: Common walnut (Juglans regia L.) is one of the top four most consumed nuts in the world due to its health benefits and pleasant taste. Despite its economic importance, the evolutionary history and genetic control of its adaptation and agronomic traits remain largely unexplored. RESULTS: We report a comprehensive walnut genomic variation map based on whole-genome resequencing of 815 walnut accessions. Evolutionary analyses suggest that Chinese J. regia diverged from J. sigillata with extensive hybridizations after the split of the two species. In contrast to annual crops, the genetic diversity and heterozygous deleterious mutations of Chinese common walnut trees have continued to increase during the improvement process. Selective sweep analyses identify 902 genes uniquely selected in the improved common walnut compared to its progenitor population. Five major-effect loci are identified to be involved in walnut adaptations to temperature, precipitation, and altitude. Genome-wide association studies reveal 27 genomic loci responsible for 18 important agronomic traits, among which JrFAD2 and JrANR are the potentially major-effect causative genes controlling linoleic acid content and color of the endopleura of the nut, respectively. CONCLUSIONS: The largest genomic resource for walnuts to date has been generated and explored in this study, unveiling their evolutionary history and cracking the genetic code for agronomic traits and environmental adaptation of this economically crucial crop tree.

Integrative proteomics reveals the role of E3 ubiquitin ligase SYVN1 in hepatocellular carcinoma metastasis.

BACKGROUND: Tumor metastasis is a major factor for poor prognosis of hepatocellular carcinoma (HCC), but the relationship between ubiquitination and metastasis need to be studied more systematically. We analyzed the ubiquitinome of HCC in this study to have a more comprehensive insight into human HCC metastasis. METHODS: The protein ubiquitination levels in 15 HCC specimens with vascular invasion and 15 without vascular invasion were detected by ubiquitinome. Proteins with significantly different ubiquitination levels between HCCs with and without vascular invasion were used to predict E3 ubiquitin ligases associated with tumor metastasis. The topological network of protein substrates and corresponding E3 ubiquitin ligases was constructed to identify the key E3 ubiquitin ligase. Besides, the growth, migration and invasion ability of LM3 and HUH7 hepatoma cell lines with and without SYVN1 expression interference were measured by cell proliferation assay, subcutaneous tumor assay, umphal vein endothelium tube formation assay, transwell migration and invasion assays. Finally, the interacting proteins of SYVN1 were screened and verified by protein interaction omics, immunofluorescence, and immunoprecipitation. Ubiquitin levels of related protein substrates in LM3 and HUH7 cells were compared in negative control, SYVN1 knockdown, and SYVN1 overexpression groups. RESULTS: In this study, our whole-cell proteomic dataset and ubiquitinomic dataset contained approximately 5600 proteins and 12,000 ubiquitinated sites. We discovered increased ubiquitinated sites with shorter ubiquitin chains during the progression of HCC metastasis. In addition, proteomic and ubiquitinomic analyses revealed that high expression of E3 ubiquitin-protein ligase SYVN1 is related with tumor metastasis. Furthermore, we found that SYVN1 interacted with heat shock protein 90 (HSP90) and impacted the ubiquitination of eukaryotic elongation factor 2 kinase (EEF2K). CONCLUSIONS: The ubiquitination profiles of HCC with and without vascular invasion were significantly different. SYVN1 was the most important E3 ubiquitin-protein ligase responsible for this phenomenon, and it was related with tumor metastasis and growth. Therefore, SYVN1 might be a potential therapeutic target for HCC.

Genome-wide identification of agronomically important genes in outcrossing crops using OutcrossSeq.

Many important crops (e.g., tuber, root, and tree crops) are cross-pollinating. For these crops, no inbred lines are available for genetic study and breeding because they are self-incompatible, clonally propagated, or have a long generation time, making the identification of agronomically important genes difficult, particularly in crops with a complex autopolyploid genome. In this study, we developed a method, OutcrossSeq, for mapping agronomically important loci in outcrossing crops based on whole-genome low-coverage resequencing of a large genetic population, and designed three computation algorithms in OutcrossSeq for different types of outcrossing populations. We applied OutcrossSeq to a tuberous root crop (sweet potato, autopolyploid), a tree crop (walnut tree, highly heterozygous diploid), and hybrid crops (double-cross populations) to generate high-density genotype maps for the outcrossing populations, which enable precise identification of genomic loci underlying important agronomic traits. Candidate causative genes at these loci were detected based on functional clues. Taken together, our results indicate that OutcrossSeq is a robust and powerful method for identifying agronomically important genes in heterozygous species, including polyploids, in a cost-efficient way. The OutcrossSeq software and its instruction manual are available for downloading at www.xhhuanglab.cn/tool/OutcrossSeq.html.

Transcriptome Analysis of Walnut (Juglans regia L.) Embryos Reveals Key Developmental Stages and Genes Involved in Lipid Biosynthesis and Polyunsaturated Fatty Acid Metabolism.

Walnut (Juglans regia L.) is a widely cultivated woody oilseed tree species, and its embryo is rich in polyunsaturated fatty acids. Thus far, the pathways and essential genes involved in oil biosynthesis in developing walnut embryos remain largely unclear. Our analyses revealed that a mature walnut embryo accumulated 69% oil, in which 71% were polyunsaturated fatty acids with 64% linoleic acid and 7% linolenic acid. RNA sequencing generated 39 384 unigenes in 24 cDNA libraries prepared from walnut embryos collected at 49, 63, 77, 91, 105, 119, 133, and 147 days after pollination (DAP). The principal components analysis (PCA) of samples and cluster analysis of differentially expressed genes (DEGs) showed that the total samples were divided into three main groups: 49 DAP, 63-119 DAP, and 133-147 DAP. We identified 108 unigenes associated with lipid biosynthesis, including 60 unigenes for fatty acid biosynthesis, 33 for triacylglycerol biosynthesis, 7 for oil bodies, and 8 for transcription factors. The expression levels of the genes encoding WRI1, ACCase, ACP, KASII, SAD, FAD2, FAD3, and PDAT were upregulated at 63-119 DAP relative to the levels at 49 DAP. Additionally, the lipid biosynthesis in walnut embryos began to increase while oil contents increased from 15 to 69%. We identified eight SAD, three FAD2, one FAD3, one FAD5, one FAD6, and three FAD7/8 genes. In addition, SAD, FAD2, and FAD3 were highly abundantly expressed in the walnut embryo, and their FPKM values achieved were 834, 2205, and 9038, respectively. High expression levels of FAD2 and FAD3 may be the reason why walnuts are rich in polyunsaturated fatty acids. Subcellular localization confirmed that the JrFAD3 protein played a role in the endoplasmic reticulum rather than the plastid, suggesting that linolenic acid was mainly synthesized in the endoplasmic reticulum. Weighted gene coexpression network analysis (WGCNA) showed that ACP, ENO, VAMP727, and IDD14 were coexpressed with WRI1. Our study provides large-scale and comprehensive transcriptome data of walnut embryo development. These data lay the foundation for the metabolic engineering of walnuts to increase oil contents and modify fatty acid compositions.

Mass Spectrometry Analysis of Newly Emerging Coronavirus HCoV-19 Spike Protein and Human ACE2 Reveals Camouflaging Glycans and Unique Post-Translational Modifications.

The coronavirus disease 2019 (COVID-19) pandemic has led to worldwide efforts to understand the biological traits of the newly identified human coronavirus (HCoV-19) virus. In this mass spectrometry (MS)-based study, we reveal that out of 21 possible glycosites in the HCoV-19 spike protein (S protein), 20 are completely occupied by N-glycans, predominantly of the oligomannose type. All seven glycosylation sites in human angiotensin I converting enzyme 2 (hACE2) were found to be completely occupied, mainly by complex N-glycans. However, glycosylation did not directly contribute to the binding affinity between HCoV-19 S protein and hACE2. Additional post-translational modification (PTM) was identified, including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hACE2. Refined structural models of HCoV-19 S protein and hACE2 were built by adding N-glycan and PTMs to recently published cryogenic electron microscopy structures. The PTM and glycan maps of HCoV-19 S protein and hACE2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of HCoV-19, as well as knowledge for developing desperately needed remedies and vaccines.

Mass Spectrometry Analysis of SARS-CoV-2 Nucleocapsid Protein Reveals Camouflaging Glycans and Unique Post-Translational Modifications.

The devastating coronavirus disease 2019 (COVID-19) pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its viral components. Compared to the Spike protein, which is the primary target for currently available vaccines or antibodies, knowledge about other virion structural components is incomplete. Using high-resolution mass spectrometry, we report a comprehensive post-translational modification (PTM) analysis of nucleocapsid phosphoprotein (NCP), the most abundant structural component of the SARS-CoV-2 virion. In addition to phosphoryl groups, we show that the SARS-CoV-2 NCP is decorated with a variety of PTMs, including N-glycans and ubiquitin. Based on newly identified PTMs, refined protein structural models of SARS-CoV-2 NCP were proposed and potential immune recognition epitopes of NCP were aligned with PTMs. These data can facilitate the design of novel vaccines or therapeutics targeting NCP, as valuable alternatives to the current vaccination and treatment paradigm that is under threat of the ever-mutating SARS-CoV-2 Spike protein.

Proteomic Analysis of Cerebrospinal Fluid in Children with Acute Enterovirus-Associated Meningoencephalitis Identifies Dysregulated Host Processes and Potential Biomarkers.

Enteroviruses (EVs) are major causes of viral meningoencephalitis in children. To better understand the pathogenesis and identify potential biomarkers, cerebrospinal fluid proteome in children (n = 52) suffering from EV meningoencephalitis was compared to that in EV-negative control subjects (n = 53) using the BoxCar acquisition technique. Among 1697 proteins identified, 1193 with robust assay readouts were used for quantitative analyses. Differential expression analyses identified 154 upregulated and 227 downregulated proteins in the EV-positive group. Functional analyses showed that the upregulated proteins are mainly related to activities of lymphocytes and cytokines, inflammation, and responses to stress and viral invasion, while the downregulated proteins are mainly related to neuronal integrity and activity as well as neurogenesis. According to receiver operating characteristic analysis results, Rho-GDP-dissociation inhibitor 2 exhibited the highest sensitivity (96.2%) and specificity (100%) for discriminating EV-positive from EV-negative patients. The chemokine CXCL10 was most upregulated (>300-fold) with also high sensitivity (92.3%) and specificity (94.3%) for indicating EV positivity. Thus, this study uncovered perturbations of multiple host processes due to EV meningoencephalitis, especially the general trend of enhanced immune responses but impaired neuronal functions. The identified dysregulated proteins may also prompt biomarker development.

P22077 inhibits LPS-induced inflammatory response by promoting K48-linked ubiquitination and degradation of TRAF6.

Inflammation is a biological process associated with multiple human disorders such as autoimmune diseases and metabolic diseases. Therefore, alleviation of inflammation is important for disease prevention or treatment. Recently, deubiquitinating enzymes (DUBs), especially ubiquitin specific protease-7 (USP7) attracts increasing attention as a potential drug target for inflammation. As an inhibitor of USP7, P22077 has been used to study the roles of USP7 in inflammatory response and neuroblastoma growth. However, the role and precise mechanism of P22077 in anti-inflammatory is still indistinct. In this study, we demonstrated that P22077 could attenuate the release of pro-inflammatory factors including TNF-α, IL-1ß, IL-6 and NO, suppress mRNA expression of COX-2 and iNOS, and inhibit activation of NF-κB and MAPKs signaling pathways in Raw264.7 cells and mouse peritoneal macrophages after LPS stimulation. In vivo study showed that P22077 could relieve inflammatory response and reduce the lung injury in C57BL/6 mice with LPS-induced endotoxemia. Mechanically, P22077 might play an anti-inflammatory role by promoting tumor necrosis factor receptor-associated factor 6 (TRAF6) degradation via K48-linked polyubiquitination. These findings provide a rationale for the role of the P22077 in anti-inflammatory pathway and the promising clinical application of P22077 to treat inflammatory diseases.

Plasminogen as a prognostic biomarker for HBV-related acute-on-chronic liver failure.

BACKGROUNDHBV-related acute-on-chronic liver failure (HBV-ACLF) is hallmarked by high short-term mortality rates, calling for accurate prognostic biomarkers for initial risk stratification.METHODSThree tandem mass tag-labeled (TMT-labeled) quantitative proteomic studies were performed on 10 patients with HBV-related acute hepatic decompensation and on 20 patients with HBV-ACLF. Candidate biomarkers were preliminarily verified in a cross-sectional cohort (n = 144) and further confirmed in 2 prospective cohorts (n = 207 and n = 148).RESULTSPlasminogen, a potential prognostic biomarker for HBV-ACLF, was identified by TMT quantitative proteomics and preliminarily verified in the cross-sectional cohort. Further validation with a prospective cohort (n = 207) showed that plasminogen levels at admission were significantly lower (P < 0.001) in HBV-ACLF nonsurvivors than in survivors. The cumulative survival duration of patients with high plasminogen levels was significantly longer (P < 0.001) than that of patients with low plasminogen levels. During hospitalization, plasminogen levels significantly decreased (P = 0.008) in the deterioration group but significantly increased (P < 0.001) in the improvement group. Additionally, plasminogen levels gradually increased in survivors but gradually decreased in nonsurvivors. The P5 score, a prognostic panel incorporating plasminogen levels, hepatic encephalopathy occurrence, age, international normalized ratio (INR), and total bilirubin, was significantly superior to the Child-Pugh, Model for End-stage Liver Disease (MELD), Chronic Liver Failure Consortium ACLF (CLIF-C ACLF), Chinese Group on the Study of Severe Hepatitis B (COSSH), and HINT (a prognostic score based on hepatic encephalopathy occurrence, INR, neutrophil count, and thyroid-stimulating hormone) scores (all P < 0.05). The performances of the plasminogen level and P5 score were validated in a second multicenter, prospective cohort (n = 148).CONCLUSIONSPlasminogen is a promising prognostic biomarker for HBV-ACLF, and sequential plasminogen measurements could profile the clinical course of HBV-ACLF. P5 is a high-performance prognostic score for HBV-ACLF.FUNDINGThe National Key Research and Development Program (2017YFC1200204); the National Natural Science Foundation of China (81400589, 81600497); the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (81121002); the Chinese High-Tech Research and Development Programs (2012AA020204); the National S&T Major Project (2012ZX10002004); and the Zhejiang Provincial Medicine and Health Science and Technology Project (2016147735).