Inducing and monitoring mitochondrial pH changes with an iridium(III) complex via two-photon lifetime imaging.

Real-time monitoring of mitochondrial dynamic changes plays a key role in the development of mitochondria-targeted anticancer theranostic agents. In this work, a pH-responsive and mitochondria-targeted cyclometalated iridium(III) complex MitoIr-NH has been explored as a novel anticancer agent. MitoIr-NH displayed pH-responsive phosphorescence intensity and lifetime, accumulated in mitochondria, showed higher antiproliferative activity and induced a series of mitochondria-related events. Moreover, MitoIr-NH could simultaneously induce mitophagy and quantitatively monitor mitochondrial pH changes through two-photon phosphorescence lifetime imaging microscopy (TPPLIM) in a real-time manner.

A pan-cancer analysis identifies SOAT1 as an immunological and prognostic biomarker.

Sterol o-acyltransferase1 (SOAT1) is an enzyme that regulates lipid metabolism. Nevertheless, the predictive value of SOAT1 regarding immune responses in cancer is not fully understood. Herein, we aimed to expound the predictive value and the potential biological functions of SOAT1 in pan-cancer. Raw data related to SOAT1 expression in 33 different types of cancer were acquired from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. SOAT1 expression was significantly increased in most cancers and showed a distinct correlation with prognosis. This enhanced expression of the SOAT1 gene was confirmed by evaluating SOAT1 protein expression using tissue microarrays. In addition, we found significant positive associations between SOAT1 expression levels and infiltrating immune cells, including T cells, neutrophils, and macrophages. Moreover, the co-expression analysis between SOAT1 and immune genes showed that many immune-related genes were increased with enhanced SOAT1 expression. A gene set enrichment analysis (GSEA) revealed that the expression of SOAT1 correlated with the tumor microenvironment, adaptive immune response, interferon signaling, and cytokine signaling. These findings indicate that SOAT1 is a potential candidate marker for predicting prognosis and a promising target for tumor immunotherapy in cancers.

A novel approach for the non-invasive diagnosis of pulmonary nodules using low-depth whole-genome sequencing of cell-free DNA.

Background: Differentiating between benign and malignant pulmonary nodules is a diagnostic challenge, and inaccurate detection can result in unnecessary invasive procedures. Cell-free DNA (cfDNA) has been successfully utilized to detect various solid tumors. In this study, we developed a genome-wide approach to explore the characteristics of cfDNA sequencing reads obtained by low-depth whole-genome sequencing (LD-WGS) to diagnose pulmonary nodules. Methods: LD-WGS was performed on cfDNA extracted from 420 plasma samples from individuals with pulmonary nodules that were no more than 30 mm in diameter, as determined by computed tomography (CT). The sequencing read distribution patterns of cfDNA were analyzed and used to establish a model for distinguishing benign from malignant pulmonary nodules. Results: We proposed the concept of weighted reads distribution difference (WRDD) based on the copy number alterations (CNAs) of cfDNA to construct a benign and malignant diagnostic (BEMAD) algorithm model. In a training cohort of 360 plasma samples, the model achieved an average area under the receiver operating characteristic (ROC) curve (AUC) value of 0.84 in 10-fold cross-validation. The model was validated in an independent cohort of 60 plasma samples, obtaining an AUC value of 0.87. The BEMAD model could distinguish benign from malignant nodules at a sensitivity of 74% and a specificity of 86%. Furthermore, analysis of the critical features of the cfDNA using the BEMAD model identified repeat regions that were associated with microsatellite instability, which is an important indicator of tumorigenesis. Conclusions: This study provides a novel non-invasive diagnostic approach to discriminate between benign and malignant pulmonary nodules to avoid unnecessary invasive procedures.

Capabilities of hepatitis B surface antigen are divergent from hepatitis B virus DNA in delimiting natural history phases of chronic hepatitis B virus infection.

Objective: Quantitative hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA in the natural history of chronic HBV infection have not been rationally evaluated. This study aimed to re-characterize quantitative HBsAg and HBV DNA in the natural history phases. Methods: A total of 595 and 651 hepatitis B e antigen (HBeAg)-positive patients and 485 and 705 HBeAg-negative patients were assigned to the early and late cohorts, respectively. Based on the 'S-shape' receiver operating characteristic (ROC) curves, the HBeAg-positive sub-cohorts with possibly high HBV replication (PHVR) and possibly low HBV replication (PLVR) and the HBeAg-negative sub-cohorts with possibly high HBsAg expression (PHSE) and possibly low HBsAg expression (PLSE) were designated. Results: The areas under the ROC curve (AUCs) of HBsAg and HBV DNA in predicting HBeAg-positive significant hepatitis activity (SHA) in the early cohort, sub-cohort with PHVR, and sub-cohort with PLVR were 0.655 and 0.541, 0.720 and 0.606, and 0.553 and 0.725, respectively; those in the late cohort, sub-cohort with PHVR, and sub-cohort with PLVR were 0.646 and 0.501, 0.798 and 0.622, and 0.603 and 0.674, respectively. The AUCs of HBsAg and HBV DNA in predicting HBeAg-negative SHA in the early cohort, sub-cohort with PHSE, and sub-cohort with PLSE were 0.508 and 0.745, 0.573 and 0.780, and 0.577 and 0.729, respectively; those in the late cohort, sub-cohort with PHSE, and sub-cohort with PLSE were 0.503 and 0.761, 0.560 and 0.814, and 0.544 and 0.722, respectively. The sensitivity and specificity of HBsAg ≤4.602 log10 IU/ml in predicting HBeAg-positive SHA in the early cohort were 82.6% and 45.8%, respectively; those in the late cohort were 87.0% and 44.1%, respectively. The sensitivity and specificity of HBV DNA >3.301 log10 IU/ml in predicting HBeAg-negative SHA in the early cohort were 73.4% and 60.8%, respectively; those in the late cohort were 73.6% and 64.1%, respectively. Conclusion: Quantitative HBsAg and HBV DNA are valuable, but their capabilities are divergent in delimiting the natural history phases.

A Novel Non-invasive Model Based on GPR for the Prediction of Liver Fibrosis in Patients With Chronic Hepatitis B.

Background: Some controversy remains regarding conventional serum indices for the evaluation of liver fibrosis. Therefore, we aimed to combine the existing index with other serum parameters to discriminate liver fibrosis stages in patients with chronic hepatitis B (CHB). Methods: A total of 1,622 treatment-naïve CHB patients were divided into training (n = 1,211) and validation (n = 451) cohorts. Liver histology was assessed according to the Scheuer scoring scheme. All common demographic and clinical parameters were analyzed. Results: By utilizing the results of the logistic regression analysis, we developed a novel index, the product of GPR, international normalized ratio (INR), and type IV collagen (GIVPR), to discriminate liver fibrosis. In the training group, the areas under the ROCs (AUROCs) of GIVPR, APRI, FIB-4, and GPR for significant fibrosis were 0.81, 0.75, 0.72, and 0.77, respectively; the AUROCs of GIVPR, APRI, FIB-4, and GPR for advanced fibrosis were 0.82, 0.74, 0.74, and 0.78, respectively; and the AUROCs of GIVPR, APRI, FIB-4, and GPR for cirrhosis were 0.87, 0.78, 0.78, and 0.83, respectively. Similar results were also obtained in the validation group. Furthermore, the decision curve analysis suggested that GIVPR represented superior clinical benefits in both independent cohorts. Conclusion: The GIVPR constructed on GPR represents a superior predictive model for discriminating liver fibrosis in CHB patients.

Predictive performance of eLIFT for liver inflammation and fibrosis in chronic liver diseases.

Objective: The easy liver fibrosis test (eLIFT) is a novel predictor of liver fibrosis in chronic liver disease (CLD). This study aimed to evaluate the predictive value of the eLIFT for liver inflammation and fibrosis in CLD patients. Methods: We enrolled 1125 patients with CLD who underwent liver biopsy. The predictive accuracy for liver inflammation and fibrosis of the eLIFT was assessed and compared to that of the aspartate aminotransferase-to-platelet ratio index (APRI), fibrosis-4 score (FIB-4), and gamma-glutamyl transpeptidase-to-platelet ratio (GPR) by ROC (Receiver Operating Characteristic) analysis and decision curve analysis (DCA). Results: The areas under the ROC curves (AUROCs) of the eLIFT for assessing liver inflammation G ≥ 2 and G ≥ 3 were 0.77 (0.75-0.80) and 0.81 (0.79-0.84), with cut-offs of 8.0 and 11.0, respectively. The AUROCs of the eLIFT for predicting fibrosis stages S ≥ 2 and S4 were 0.72 (0.70-0.76) and 0.76 (0.72-0.80), with cut-offs of 9.0 and 10.0, respectively. In discriminating G≥2 inflammation, the AUROC of the eLIFT was better than that of the FIB-4, with no difference compared with the GPR, but lower than that of the APRI. When discriminating G≥3 inflammation, the AUROC of the eLIFT was comparable to that of the APRI and GPR but superior to that of the FIB-4. There were no significant differences between the four indexes for predicting S≥2 and S4. Conclusion: The eLIFT is a potentially useful noninvasive predictor of liver inflammation and fibrosis in patients with CLD.

Predictive Performances of Blood Parameter Ratios for Liver Inflammation and Advanced Liver Fibrosis in Chronic Hepatitis B Infection.

OBJECTIVE: Blood parameter ratios, including neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), and monocyte to lymphocyte ratio (MLR), have been reported that they are correlated to the progression of liver disease. This study is aimed at evaluating the predictive value of PLR, NLR, and MLR for liver inflammation and fibrosis in patients with chronic hepatitis B (CHB). METHODS: We recruited 457 patients with CHB who underwent a liver biopsy and routine laboratory tests. Liver histology was assessed according to the Scheuer scoring system. The predictive accuracy for liver inflammation and fibrosis was assessed by receiver operating characteristics (ROC) analysis. RESULTS: PLR and NLR presented significantly reverse correlation to liver inflammation and fibrosis. However, these correlations were not observed for MLR and liver histology. The AUROCs of PLR for assessing G2-3 and G3 were 0.676 and 0.705 with cutoffs 74.27 and 68.75, respectively. The AUROCs of NLR in predicting inflammatory scores G2-3 and G3 were 0.616 and 0.569 with cutoffs 1.36 and 1.85, respectively. The AUROCs of PLR for evaluating fibrosis stages S3-4 and S4 were 0.723 and 0.757 with cutoffs 79.67 and 74.27, respectively. The AUROCs of NLR for evaluating fibrosis stages S3-4 and S4 were 0.590 with cutoff 1.14. CONCLUSION: Although PLR has similar predictive power of progressive liver fibrosis compared with APRI, FIB-4, and GPR in CHB patients, it has the advantage of less cost and easy application with the potential to be widely used in clinical practice.

DZNep, an inhibitor of the histone methyltransferase EZH2, suppresses hepatic fibrosis through regulating miR-199a-5p/SOCS7 pathway.

BACKGROUND: Hepatic fibrosis is a common response to chronic liver injury. Recently, the role of DZNep (a histone methyltransferase EZH2 inhibitor) in repressing pulmonary and renal fibrosis was verified. However, the potential effect of DZNep on hepatic fibrosis has not been elucidated. METHODS: The hepatic fibrosis model was established in rats treated with CCl4 and in hepatic stellate cells (HSCs) treated with TGF-ß1. The liver tissues were stained with H&E and Masson's trichrome. The expression of EZH2, SOCS7, collagen I, αSMA mRNA and miR-199-5p was assessed using qPCR, immunohistochemical or western blot analysis. A dual-luciferase reporter assay was carried out to validate the regulatory relationship of miR-199a-5p with SOCS7. RESULTS: The EZH2 level was increased in CCl4-treated rats and in TGF-ß1-treated HSCs, whereas DZNep treatment significantly inhibited EZH2 expression. DZNep repressed hepatic fibrosis in vivo and in vitro, as evidenced by the decrease of hepatic fibrosis markers (α-SMA and Collagen I). Moreover, miR-199a-5p expression was repressed by DZNep in TGF-ß1-activated HSCs. Notably, downregulation of miR-199a-5p decreased TGF-ß1-induced expression of fibrosis markers. SOCS7 was identified as a direct target of miR-199a-5p. The expression of SOCS7 was decreased in TGF-ß1-activated HSCs, but DZNep treatment restore d SOCS7 expression. More importantly, SOCS7 knockdown decreased the effect of DZNep on collagen I and α SMA expression in TGF-ß1-activated HSCs. CONCLUSIONS: DZNep suppresses hepatic fibrosis through regulating miR-199a-5p/SOCS7 axis, suggesting that DZNep may represent a novel treatment for fibrosis.

Nomogram for predicting advanced liver fibrosis and cirrhosis in patients with chronic liver disease.

BACKGROUND: We aimed to formulate a novel predictive nomogram to discriminate liver fibrosis stage in patients with chronic liver disease. METHODS: Nomograms were established based on the results of multivariate analysis. The predictive accuracy of the nomograms was assessed by ROC analysis and calibration. Decision curve analysis (DCA) was used to determine the clinical benefit of the nomograms. RESULTS: INR, platelets, and N-terminal propeptide type III collagen (PIIINP) were independent predictors for advanced liver fibrosis (≥ S3) and cirrhosis (S4) in patients with chronic liver disease in the training cohort. In the training set, the areas under the ROCs (AUROCs) of nomogram S3S4, APRI, FIB-4, and GPR for stage ≥ S3 were 0.83, 0.71, 0.68, and 0.74, respectively; the AUROCs of nomogram S4, APRI, FIB-4, and GPR for stage S4 were 0.88, 0.74, 0.78, and 0.79, respectively. The calibrations showed optimal agreement between the prediction by the established nomograms and actual observation. In the validation set, the AUROCs of nomogram S3S4, APRI, FIB-4, and GPR for stage ≥ S3 were 0.86, 0.79, 0.78, and 0.81, respectively; the AUROCs of nomogram S4, APRI, FIB-4, and GPR for stage S4 were 0.88, 0.77, 0.81, and 0.83, respectively. Furthermore, the decision curve analysis suggested that the nomograms represent better clinical benefits in both independent cohorts than APRI, FIB-4, and GPR. CONCLUSION: The constructed nomograms could be a superior tool for discriminating advanced fibrosis and cirrhosis in chronic liver disease.

INR-to-platelet ratio (INPR) as a novel noninvasive index for predicting liver fibrosis in chronic hepatitis B.

Objective: We aimed to investigate whether a novel noninvasive index, i.e., the international normalized ratio-to-platelet ratio (INPR), was a variable in determining liver fibrosis stage in patients with chronic hepatitis B (CHB). Methods: A total of 543 treatment-naïve CHB patients were retrospectively enrolled. Liver histology was assessed according to the Metavir scoring scheme. All common demographic and clinical parameters were analyzed. Results: Based on routine clinical parameters (age, sex, HBeAg status, HBV DNA, hematological parameters, coagulation index, and liver biochemical indicators), a novel index, i.e., the INR-to-platelet ratio (INPR), was developed to magnify the unfavorable effects of liver fibrosis on INR and platelets. The AUCs of INPR for predicting significant fibrosis, advanced fibrosis, and cirrhosis were 0.74, 0.76 and 0.86, respectively. Compared with APRI, FIB-4, and GPR, the INPR had comparable predictive efficacy for significant fibrosis and better predictive performance for advanced fibrosis and cirrhosis. Conclusion: INPR could be an accurate, easily calculated and inexpensive index to assess liver fibrosis in patients with CHB. Further studies are needed to verify this indicator and compare it with other noninvasive methods for predicting liver fibrosis in CHB patients.

Identification of parameters in routine blood and coagulation tests related to the severity of COVID-19.

Objective: This study aimed to identify the predictive value of simple markers in routine blood and coagulation tests for the severity of coronavirus disease 2019 (COVID-19). Methods: A total of 311 consecutive COVID-19 patients, including 281 patients with mild/moderate COVID-19 and 30 patients with severe/life-threatening COVID-19, were retrospectively enrolled. Logistic modeling and ROC curve analyses were used to assess the indexes for identifying disease severity. Results: Lymphocyte and eosinophil counts of COVID-19 patients in the severe/life-threatening group were significantly lower than those of patients in the mild/moderate group (P < 0.001). Coagulation parameters, high-sensitivity C-reactive protein (hsCRP) levels and procalcitonin levels were higher in the severe/life-threatening group compared with the mild/moderate group (all P < 0.05). Univariate and multivariate logistic models revealed that hsCRP and fibrinogen degradation products (FDPs) were predictors of severe COVID-19 (OR = 1.072, P = 0.036; and OR = 1.831, P = 0.036, respectively). The AUROCs of hsCRP and FDP for predicting severe/life-threatening COVID-19 were 0.850 and 0.766, respectively. The optimal cutoffs of hsCRP and FDP for the severe/life-threatening type of COVID-19 were 22.41 mg/L and 0.95 µg/ml, respectively. Conclusion: Serum CRP and FDP levels are positively related to the severity of COVID-19. This finding indicates that CRP and FDP levels may potentially be used as early predictors for severe illness and help physicians triage numerous patients in a short time.

Performance evaluation of HBsAg by Lumipulse HBsAg-HQ: The agreement with HBsAg by Architect HBsAg-QT and the effectiveness in predicting liver tissue pathological states of chronic hepatitis B patients.

BACKGROUND: A novel high-sensitivity HBsAg quantification assay, Lumipulse HBsAg-HQ, was developed. However, its performance in practical application has not yet been adequately investigated. OBJECTIVES: The aim of the study was to evaluate the agreement of serum HBsAg by Lumipulse HBsAg-HQ (HBsAg-HQ) with HBsAg by Architect HBsAg-QT (HBsAg-QT) and comparatively investigate the efficacy of serum HBsAg-HQ and HBsAg-QT in predicting the liver tissue pathological states of chronic hepatitis B (CHB) patients. MATERIAL AND METHODS: A total of 147 HBeAg-positive and 128 HBeAg-negative patients were enrolled. HBsAg-HQ and HBsAg-QT were measured using CLEIA Lumipulse G1200 and CMIA Abbott Architect I2000 automatic analyzer, respectively. The Scheuer standard was used for the pathological diagnosis of liver tissue samples. RESULTS: In both HBeAg-positive and HBeAg-negative patients, HBsAg-HQ was significantly positively correlated with HBsAg-QT (r = 0.913 and r = 0.959, respectively), the overall disagreement rates between HBsAg-HQ and HBsAg-QT were 2.72% (4/147) and 4.69% (6/128), respectively. In HBeAg-positive patients, the area under the ROC curve (AUC) of HBsAg-HQ and HBsAg-QT for predicting the grade ≥G3 (0.686 and 0.684, respectively) and stage ≥S4 (0.739 and 0.745, respectively) were the greatest compared with other pathological states; the optimal cutoffs of HBsAg-HQ and HBsAg-QT for predicting the grade ≥G3 were <2.244 × 107 mIU/mL and <3.589 × 107 mIU/mL, and those for predicting the stage ≥S4 were 7.328 × 106 mIU/mL and <6.194 × 106 mIU/mL, respectively. CONCLUSIONS: HBsAg-HQ is highly correlated and in agreement with HBsAg-QT in both HBeAg-positive and HBeAg-negative patients; HBsAg-HQ and HBsAg-QT are very valuable in predicting the grade ≥G3 and stage ≥S4 in HBeAg-positive patients.

Population-wide sampling of retrotransposon insertion polymorphisms using deep sequencing and efficient detection.

Active retrotransposons play important roles during evolution and continue to shape our genomes today, especially in genetic polymorphisms underlying a diverse set of diseases. However, studies of human retrotransposon insertion polymorphisms (RIPs) based on whole-genome deep sequencing at the population level have not been sufficiently undertaken, despite the obvious need for a thorough characterization of RIPs in the general population. Herein, we present a novel and efficient computational tool called Specific Insertions Detector (SID) for the detection of non-reference RIPs. We demonstrate that SID is suitable for high-depth whole-genome sequencing data using paired-end reads obtained from simulated and real datasets. We construct a comprehensive RIP database using a large population of 90 Han Chinese individuals with a mean ×68 depth per individual. In total, we identify 9342 recent RIPs, and 8433 of these RIPs are novel compared with dbRIP, including 5826 Alu, 2169 long interspersed nuclear element 1 (L1), 383 SVA, and 55 long terminal repeats. Among the 9342 RIPs, 4828 were located in gene regions and 5 were located in protein-coding regions. We demonstrate that RIPs can, in principle, be an informative resource to perform population evolution and phylogenetic analyses. Taking the demographic effects into account, we identify a weak negative selection on SVA and L1 but an approximately neutral selection for Alu elements based on the frequency spectrum of RIPs. SID is a powerful open-source program for the detection of non-reference RIPs. We built a non-reference RIP dataset that greatly enhanced the diversity of RIPs detected in the general population, and it should be invaluable to researchers interested in many aspects of human evolution, genetics, and disease. As a proof of concept, we demonstrate that the RIPs can be used as biomarkers in a similar way as single nucleotide polymorphisms.

Circumventing intratumoral heterogeneity to identify potential therapeutic targets in hepatocellular carcinoma.

BACKGROUND & AIMS: Identifying target genetic mutations in hepatocellular carcinoma (HCC) for therapy is made challenging by intratumoral heterogeneity. Circulating cell-free DNAs (cfDNA) may contain a more complete mutational spectrum compared to a single tumor sample. This study aimed to identify the most efficient strategy to identify all the mutations within heterogeneous HCCs. METHODS: Whole exome sequencing (WES) and targeted deep sequencing (TDS) were carried out in 32 multi-regional tumor samples from five patients. Matched preoperative cfDNAs were sequenced accordingly. Intratumoral heterogeneity was measured using the average percentage of non-ubiquitous mutations (present in parts of tumor regions). Profiling efficiencies of single tumor specimen and cfDNA were compared. The strategy with the highest performance was used to screen for actionable mutations. RESULTS: Variable levels of heterogeneity with branched and parallel evolution patterns were observed. The heterogeneity decreased at higher sequencing depth of TDS compared to measurements by WES (28.1% vs. 34.9%, p<0.01) but remained unchanged when additional samples were analyzed. TDS of single tumor specimen identified an average of 70% of the total mutations from multi-regional tissues. Although genome profiling efficiency of cfDNA increased with sequencing depth, an average of 47.2% total mutations were identified using TDS, suggesting that tissue samples outperformed it. TDS of single tumor specimen in 66 patients and cfDNAs in four unresectable HCCs showed that 38.6% (26/66 and 1/4) of patients carried mutations that were potential therapeutic targets. CONCLUSIONS: TDS of single tumor specimen could identify actionable mutations targets for therapy in HCC. cfDNA may serve as secondary alternative in profiling HCC genome. LAY SUMMARY: Targeted deep sequencing of single tumor specimen is a more efficient method to identify mutations in hepatocellular carcinoma made from mixed subtypes compared to circulating cell-free DNA in blood. cfDNA may serve as secondary alternative in profiling HCC genome. Identifying mutations may help clinicians choose targeted therapy for better individual treatments.

Pregnane glycosides from Solanum nigrum.

Two new pregnane saponins, solanigroside A (1) and solanigroside B (2), along with two known compounds (3 and 4), were isolated from 60% ethanolic extract of the dried herb of Solanum nigrum L. The structures of 1 and 2 were elucidated as 5alpha-pregn-16-en-3beta-ol-20-one 3-O-beta-D-xylopyranosyl-(1 --> 3)-O-[alpha-L-arabinopyranosyl-(1 --> 2)]-O-beta-D-glucopyranosyl-(1 --> 4)-O-[alpha-L-rhamnopyranosyl-(1 --> 2)]-O-beta-D-galactopyranoside (1) and 5alpha-pregn-16-en-3beta-ol-20-one 3-O-beta-D-glucopyranosyl-(1 --> 2)-O-[beta-D-glucopyranosyl-(1 --> 3)]-O-beta-D-glucopyranosyl-(1 --> 4)-O-beta-D-galactopyranoside (2), respectively, on the basis of extensive spectroscopic analysis as well as comparison with reported spectroscopic data of related compounds. This paper deals with the isolation and structural characterisation of pregnane glycosides from S. nigrum L.

Steroidal saponins from Solanum nigrum.

Six new steroidal saponins, solanigrosides C-H (2-7), and one known saponin, degalactotigonin (1), were isolated from the whole plant of Solanum nigrum. Their chemical structures were elucidated using spectroscopic analysis, chemical degradation, and derivatization. All seven compounds were tested for their cytotoxicity using four human tumor cell lines (HepG2, NCI-H460, MCF-7, SF-268). Only compound 1 was cytotoxic, with IC50 values of 0.25-4.49 microM.