Carbene Footprinting Directs Design of Genetically Encoded Proximity-Reactive Protein Binders.

Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI-IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRASG12D/RAF1, which lacks crystal structure, and generated a covalent binder to KRASG12D. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.

C1GALT1 in health and disease.

O-linked glycosylation (O-glycosylation) and N-linked glycosylation (N-glycosylation) are the two most important forms of protein glycosylation, which is an important post-translational modification. The regulation of protein function involves numerous mechanisms, among which protein glycosylation is one of the most important. Core 1 synthase glycoprotein-N-acetylgalactosamine 3-ß-galactosyltransferase 1 (C1GALT1) serves an important role in the regulation of O-glycosylation and is an essential enzyme for synthesizing the core 1 structure of mucin-type O-glycans. Furthermore, C1GALT1 serves a vital role in a number of biological functions, such as angiogenesis, platelet production and kidney development. Impaired C1GALT1 expression activity has been associated with different types of human diseases, including inflammatory or immune-mediated diseases, and cancer. O-glycosylation exists in normal tissues, as well as in tumor tissues. Previous studies have revealed that changes in the level of glycosyltransferase in different types of cancer may be used as potential therapeutic targets. Currently, numerous studies have reported the dual role of C1GALT1 in tumors (carcinogenesis and cancer suppression). The present review reports the role of C1GALT1 in normal development and human diseases. Since the mechanism and regulation of C1GALT1 and O-glycosylation remain elusive, further studies are required to elucidate their effects on development and disease.

Elevated soluble 4-1BB is associated with serum markers of hepatitis B virus in patients with chronic hepatitis B.

BACKGROUND: Previous studies have suggested that the costimulatory molecule 4-1BB plays pivotal roles in regulating immunity during chronic viral infection. However, up to now, there are few studies about 4-1BB in chronic hepatitis B (CHB). AIM: To clarify this issue, we report our comprehensive study results on the expression levels of 4-1BB in patients with CHB. METHODS: From September 2018 to June 2019, a total of 64 patients with CHB were recruited from the Department of Hepatology, The First Hospital of Jilin University. Peripheral blood samples were collected from 52 treatment-naïve and 12 entecavir-treated patients with CHB as well as 37 healthy donors (including 24 healthy adults and 13 healthy children). The levels of soluble 4-1BB (s4-1BB) in plasma were measured by ELISA. 4-1BB mRNA expression in peripheral blood mononuclear cells was detected by real-time quantitative PCR. RESULTS: The s4-1BB levels in the plasma of patients with CHB were significantly higher than those in healthy adults (94.390 ± 7.393 ng/mL vs 8.875 ± 0.914 ng/mL, P < 0.001). In addition, the s4-1BB level in plasma was significantly increased in patients with a higher viral load and a disease flare up. However, there were no significant differences between treatment-naïve and entecavir-treated patients. Interestingly, among treatment-naïve patients with CHB, the levels of s4-1BB in plasma had a significant positive correlation with hepatitis B surface antigen, hepatitis B virus DNA, hepatitis B e antigen, and triglyceride levels (r = 0.748, P < 0.001; r = 0.406, P = 0.004; r = 0.356, P = 0.019 and r = -0.469, P = 0.007, respectively). The 4-1BB mRNA expression was higher in the peripheral blood mononuclear cells of patients with CHB than in the peripheral blood mononuclear cells of healthy adults, but the difference was not statistically significant. CONCLUSION: These results suggest that the levels of s4-1BB may be associated with pathogenesis of hepatitis B virus and therefore may be a promising biomarker for disease progression.

Src homolog and collagen homolog1 isoforms in acute and chronic liver injuries.

Src homolog and collagen homolog (SHC) proteins are adaptor proteins bound to cell surface receptors that play an important role in signal transduction and related diseases. As an important member of the SHC protein family, SHC1 regulates cell proliferation and apoptosis, reactive oxygen species (ROS) production, and oxidative stress. Three isomeric proteins namely, p46shc, p52shc, and p66shc, are produced from the same SHC1 gene locus. All the three proteins are found in the liver, and are widely expressed in various hepatic cells. SHC1 has been proven to be associated with acute and chronic liver injuries of different etiologies, and plays important roles in liver fibrosis and hepatocellular carcinoma (HCC). Therefore, this review summarizes recent studies that discuss and explore the role of SHC1 in the occurrence and progression of liver diseases. We also provide a theoretical basis for future studies.

Timing of DAA Initiation After Curative Treatment and Its Relationship with the Recurrence of HCV-Related HCC.

Hepatitis C virus infection is a major cause of chronic hepatitis, leading to cirrhosis and hepatocellular carcinoma (HCC). Many studies agree that interferon (IFN)-based antiviral therapy can reduce the risk of HCC recurrence in patients with chronic hepatitis C who have achieved a sustained virological response (SVR). The recent introduction of direct-acting antivirals (DAA) has resulted in excitingly high SVR rates. However, as an IFN-free regimen, DAAs only exert antiviral activity without an immune response. The benefit of DAA-based regimens for HCC recurrence in patients with cirrhosis and following successful curative treatment remains controversial. Additionally, the time span between curative-intent therapy and the DAA regimen is an independent risk factor for HCC recurrence, irrespective of the DAA response. HCC patients who are eligible for potentially curative therapy by liver resection or ablation should defer DAA therapy; however, the accurate timing remains unclear. In this study, we reviewed the timing of DAA initiation after curative treatment and its effect on the recurrence of related HCC.

Validation of the GALAD Model and Establishment of GAAP Model for Diagnosis of Hepatocellular Carcinoma in Chinese Patients.

PURPOSE: GALAD is a statistical model for estimating the likelihood of having hepatocellular carcinoma (HCC) based on gender, age, AFP, AFP-L3, and PIVKA-II. We aimed to assess its performance and build new models in China, where hepatitis B virus (HBV) is the leading etiology of HCC. PATIENTS AND METHODS: We built the GALAD-C model with the same five variables in GALAD, and the GAAP model with gender, age, AFP, and PIVKA-II, using logistic regression based on 242 patients with HCC and 283 patients with chronic liver disease (CLD). We also collected 50 patients with other malignant liver tumors (OMTs) and 50 healthy controls (HCs). A test dataset (169 patients with HCC and 139 with CLD) was used to test the performance of GAAP. RESULTS: The GALAD-C and GAAP models achieved comparable performance (area under the receiver operating characteristic curve [AUC], 0.922 vs 0.914), and both were superior to GALAD, PIVKA-II, AFP, and AFP-L3% (AUCs, 0.891, 0.869, 0.750, and 0.711) for discrimination of HCC from CLD for the entire dataset. The AUCs of the GALAD, GALAD-C and GAAP models were excellent for the hepatitis C virus (HCV) subgroup (0.939, 0.958 and 0.954), and for discrimination HCC from HCs (0.988, 0.982, and 0.979), but were relatively lower for the HBV subgroup (0.855, 0.904, and 0.894), and for HCC within Milan Criteria (0.810, 0.841, and 0.840). They were not superior to AFP (0.873) for discrimination of HCC from OMT (0.873, 0.809, and 0.823). GAAP achieved an AUC of 0.922 in the test dataset. CONCLUSION: GALAD was excellent for discrimination of HCC from CLD in the HCV subgroup of a cohort of Chinese patients. The GAAP and GALAD-C models achieved better performance compared with GALAD. These three models exhibited better performance in patients with an HCV etiology than those with HBV.

IL-33 Inhibits Hepatitis B Virus through Its Receptor ST2 in Hydrodynamic HBV Mouse Model.

Interleukin-33 has been demonstrated to be associated with liver damage. However, its potential value in hepatitis B virus (HBV) infection remains unknown. This study was designed to investigate the role of IL-33 in hydrodynamic HBV mouse model. Different doses of IL-33 were used to treat HBV wild-type, ST2 knockout, CD8+ T depletion, NK depletion C57BL/6 mice and C.B-17 SCID and nod SCID mouse, respectively. The concentrations of HBV DNA, HBsAg, HBeAg, and molecules related to liver function were detected in the collected serum at different time points from model mice. Intrahepatic HBcAg was visualized by immunohistochemical staining of liver tissues. In vitro, hepG2 cells were transfected with pAAV-HBV 1.2, then treated with IL-33. The results showed that IL-33 significantly reduced HBV DNA and HBsAg in a dose-dependent manner in HBV wild-type mice. However, in the IL-33 specific receptor ST2 knockout mice, their antiviral effects could not be exerted. Through immunodeficient animal models and in vivo immune cell depletion mouse model, we found that IL-33 could not play antiviral effects without NK cells. Moreover, IL-33 could reduce the levels of HBsAg and HBeAg in the supernatant of HBV-transfected hepG2 cells in vitro. Our study revealed that IL-33 could inhibit HBV through ST2 receptor in the HBV mouse model, and this effect can be impaired without NK cell. Additionally, IL-33 had the direct anti-HBV effect in vitro, indicating that IL-33 could be a potent inducer of HBV clearance and a promising drug candidate.

Evaluation of a new point-of-care oral anti-HCV test for screening of hepatitis C virus infection.

BACKGROUND: Hepatitis C virus (HCV) infection is a public health issue for which an effective universal screening method is urgently needed. An oral anti-HCV test could provide a noninvasive and rapid screening strategy for HCV infection. This study evaluated the performance of a new point-of-care oral assay developed by Well for the detection of HCV antibody. METHODS: Individuals from three centers with and without HCV infection were enrolled. All participants were tested for oral HCV antibody using the Well assay and for serum HCV antibody using established tests (ARCHITECT i2000 anti-HCV assay and InTec serum anti-HCV assay). For participants who obtained positive results, HCV RNA was tested for verification. Some patients underwent the OraQuick HCV test at the same time, and some self-tested with the Well assay during the same period. RESULTS: A total of 1179 participants, including 486 patients with chronic HCV infection, 108 patients with other liver diseases, and 585 individuals who underwent physical examination, were enrolled. The Well anti-HCV test had a sensitivity of 91.88% (95% confidence interval [CI]: 88.97-94.09%) and a specificity of 98.00% (96.58-98.86%) for oral HCV antibody detection. The consistency between the Well and InTec assays was 97.02% (1138/1179). The consistency between the Well and OraQuick assays was 98.50% (197/200). Furthermore, the results of self-testing were highly consistent with those of researcher-administered tests (Kappa = 0.979). In addition, the HCV RNA results also showed that HCV RNA could only be detected on 1 of the 39 false-negative samples, and for 172 positive HCV RNA results, 171 could be detected by the Well oral anti-HCV assay. CONCLUSIONS: The Well oral anti-HCV test offers high sensitivity and specificity and performed comparably to both the OraQuick assay and InTec assay for HCV diagnosis. Thus, the Well test represents a new tool for universal HCV screening to identify infected patients, particularly in regions with limited medical resources.

The Potential Effects of Diabetes Mellitus on Liver Fibrosis in Patients with Primary Biliary Cholangitis.

BACKGROUND The impact of diabetes mellitus (DM) on the natural progression of primary biliary cholangitis (PBC) has not yet been determined. The objective of this study was to determine whether DM is associated with increased liver damage in PBC. MATERIAL AND METHODS There were 168 treatment-naïve PBC patients, including 37 patients with DM, enrolled in this study between 2012 and 2018. Patient demographics, clinical features, and biochemical and histopathological parameters were collected. Disease severity was assessed by pathological data, Child Pugh grade, and noninvasive indicators. Relevant risks for PBC-related cirrhosis were assessed by univariate and multivariate analyses. RESULTS The noninvasive scores predicting fibrosis were all significantly higher in PBC-DM versus PBC-only patients (fibrosis-4 score: 4.08 versus 3.21, P=0.029; aminotransferase-to-platelet ratio index: 1.46 versus 1.09, P=0.036; red blood cell distribution width to platelet ratio: 0.12 versus 0.08, P=0.016; Mayo Risk Score: 1.52 versus 0.19, P=0.011; the Newcastle model: 2.85 versus 2.07, P=0.009; albumin-bilirubin score: -1.92 versus -2.10, P=0.023). Cirrhosis occurred at a higher rate (62.2% versus 42.0%, P=0.030) in PBC-DM patients, but Child Pugh grade and pathological differences could not be accurately determined. A multivariate analysis revealed DM increased the risk of PBC-related cirrhosis, with a resulting adjusted odds ratio of 2.351 (95% confidence interval, 1.022-5.409). CONCLUSIONS The results of this retrospective, single-center study suggest that DM is associated with more severe liver fibrosis in PBC. Consequently, improved management of DM might alter the prognosis of PBC patients.

Isolated elevated aspartate aminotransferase in an asymptomatic woman due to macro-aspartate aminotransferase: A case report.

BACKGROUND: Macro-aspartate aminotransferase (AST), a macroenzyme, is a high-molecular mass complex formed by self-polymerization or association with other serum components that are difficult for the kidney to clear, leading to the isolated elevation of serum AST activity. Cases of macro-AST formation are rare, with only 3 published in the English language literature up to September 2019 in China. In this paper, we present a case in which an asymptomatic woman with persistent isolated elevated AST was confirmed as having macro-AST by the polyethylene glycol precipitation method. CASE SUMMARY: A 34-year-old woman was referred to our clinic for elevated AST levels with normal levels of other liver-associated enzymes on November 12, 2018. Her AST level of liver function test had been abnormal for 7 mo before she came to the clinic. The patient was asymptomatic with a normal physical examination. There was no relevant family history and no alcohol consumption or smoking. She had a several-month history of traditional Chinese medical taking and had stopped it 1 year prior. The laboratory tests in our clinic showed only the elevation of AST (89.5 U/L) with no other significant abnormalities. We performed the precipitation technique with polyethylene glycol to confirm the presence of macro-AST. Then for almost a year, her AST level still fluctuated in the abnormal range. CONCLUSION: This case highlights that clinical physicians should be familiar with this rare condition of persistent isolated AST elevation due to the presence of macro-AST to avoid unnecessary investigation and patient anxiety.