High accuracy model for HBsAg loss based on longitudinal trajectories of serum qHBsAg throughout long-term antiviral therapy.

OBJECTIVE: Hepatitis B surface antigen (HBsAg) loss is the optimal outcome for patients with chronic hepatitis B (CHB) but this rarely occurs with currently approved therapies. We aimed to develop and validate a prognostic model for HBsAg loss on treatment using longitudinal data from a large, prospectively followed, nationwide cohort. DESIGN: CHB patients receiving nucleos(t)ide analogues as antiviral treatment were enrolled from 50 centres in China. Quantitative HBsAg (qHBsAg) testing was prospectively performed biannually per protocol. Longitudinal discriminant analysis algorithm was used to estimate the incidence of HBsAg loss, by integrating clinical data of each patient collected during follow-up. RESULTS: In total, 6792 CHB patients who had initiated antiviral treatment 41.3 (IQR 7.6-107.6) months before enrolment and had median qHBsAg 2.9 (IQR 2.3-3.3) log10IU/mL at entry were analysed. With a median follow-up of 65.6 (IQR 51.5-84.7) months, the 5-year cumulative incidence of HBsAg loss was 2.4%. A prediction model integrating all qHBsAg values of each patient during follow-up, designated GOLDEN model, was developed and validated. The AUCs of GOLDEN model were 0.981 (95% CI 0.974 to 0.987) and 0.979 (95% CI 0.974 to 0.983) in the training and external validation sets, respectively, and were significantly better than those of a single qHBsAg measurement. GOLDEN model identified 8.5%-10.4% of patients with a high probability of HBsAg loss (5-year cumulative incidence: 17.0%-29.1%) and was able to exclude 89.6%-91.5% of patients whose incidence of HBsAg loss is 0. Moreover, the GOLDEN model consistently showed excellent performance among various subgroups. CONCLUSION: The novel GOLDEN model, based on longitudinal qHBsAg data, accurately predicts HBsAg clearance, provides reliable estimates of functional hepatitis B virus (HBV) cure and may have the potential to stratify different subsets of patients for novel anti-HBV therapies.

Therapeutic Drug Monitoring of Voriconazole in Patients with End-Stage Liver Disease.

BACKGROUND: This study aimed to identify the factors that influence voriconazole (VCZ) plasma concentrations and optimize the doses of VCZ in patients with end-stage liver disease (ESLD). METHODS: Patients with ESLD who received a VCZ maintenance dose of 100 mg twice daily (group A, n = 57) or the VCZ maintenance dose of 50 mg twice daily (group B, n = 37), orally or intravenously, were enrolled in this study. Trough plasma concentrations (Cmin) of VCZ between 1 and 5 mg/L were considered within the therapeutic target range. RESULTS: The VCZ Cmin was determined in 94 patients with ESLD. The VCZ Cmin of patients in group A was remarkably higher than those in group B (4.85 ± 2.53 mg/L vs 2.75 ± 1.40 mg/L; P < 0.001). Compared with group A, fewer patients in group B had VCZ Cmin outside the therapeutic target (23/57 vs. 6/37, P = 0.021). Univariate and multivariate analyses suggested that both body weight and Model for End-Stage Liver Disease scores were closely associated with the VCZ Cmin in group B. CONCLUSIONS: These data indicate that dose optimization based on body weight and Model for End-Stage Liver Disease scores is required to strike an efficacy-safety balance during VCZ treatment in patients with ESLD.

Novel, high accuracy models for hepatocellular carcinoma prediction based on longitudinal data and cell-free DNA signatures.

BACKGROUND & AIMS: Current hepatocellular carcinoma (HCC) risk scores do not reflect changes in HCC risk resulting from liver disease progression/regression over time. We aimed to develop and validate two novel prediction models using multivariate longitudinal data, with or without cell-free DNA (cfDNA) signatures. METHODS: A total of 13,728 patients from two nationwide multicenter prospective observational cohorts, the majority of whom had chronic hepatitis B, were enrolled. aMAP score, as one of the most promising HCC prediction models, was evaluated for each patient. Low-pass whole-genome sequencing was used to derive multi-modal cfDNA fragmentomics features. A longitudinal discriminant analysis algorithm was used to model longitudinal profiles of patient biomarkers and estimate the risk of HCC development. RESULTS: We developed and externally validated two novel HCC prediction models with a greater accuracy, termed aMAP-2 and aMAP-2 Plus scores. The aMAP-2 score, calculated with longitudinal data on the aMAP score and alpha-fetoprotein values during an up to 8-year follow-up, performed superbly in the training and external validation cohorts (AUC 0.83-0.84). The aMAP-2 score showed further improvement and accurately divided aMAP-defined high-risk patients into two groups with 5-year cumulative HCC incidences of 23.4% and 4.1%, respectively (p = 0.0065). The aMAP-2 Plus score, which incorporates cfDNA signatures (nucleosome, fragment and motif scores), optimized the prediction of HCC development, especially for patients with cirrhosis (AUC 0.85-0.89). Importantly, the stepwise approach (aMAP -> aMAP-2 -> aMAP-2 Plus) stratified patients with cirrhosis into two groups, comprising 90% and 10% of the cohort, with an annual HCC incidence of 0.8% and 12.5%, respectively (p <0.0001). CONCLUSIONS: aMAP-2 and aMAP-2 Plus scores are highly accurate in predicting HCC. The stepwise application of aMAP scores provides an improved enrichment strategy, identifying patients at a high risk of HCC, which could effectively guide individualized HCC surveillance. IMPACT AND IMPLICATIONS: In this multicenter nationwide cohort study, we developed and externally validated two novel hepatocellular carcinoma (HCC) risk prediction models (called aMAP-2 and aMAP-2 Plus scores), using longitudinal discriminant analysis algorithm and longitudinal data (i.e., aMAP and alpha-fetoprotein) with or without the addition of cell-free DNA signatures, based on 13,728 patients from 61 centers across mainland China. Our findings demonstrated that the performance of aMAP-2 and aMAP-2 Plus scores was markedly better than the original aMAP score, and any other existing HCC risk scores across all subsets, especially for patients with cirrhosis. More importantly, the stepwise application of aMAP scores (aMAP -> aMAP-2 -> aMAP-2 Plus) provides an improved enrichment strategy, identifying patients at high risk of HCC, which could effectively guide individualized HCC surveillance.

Endoplasmic reticulum stress promotes HBV production by enhancing use of the autophagosome/multivesicular body axis.

BACKGROUND AND AIMS: HBV infection has been reported to trigger endoplasmic reticulum (ER) stress and initiate autophagy. However, how ER stress and autophagy influence HBV production remains elusive. Here, we studied the effect of tunicamycin (TM), an N-glycosylation inhibitor and ER stress inducer, on HBV replication and secretion and examined the underlying mechanisms. APPROACH AND RESULTS: Protein disulfide isomerase (an ER marker), microtubule-associated protein 1 light chain 3 beta (an autophagosome [AP] marker), and sequestosome-1 (a typical cargo for autophagic degradation) expression were tested in liver tissues of patients with chronic HBV infection and hepatoma cell lines. The role of TM treatment in HBV production and trafficking was examined in hepatoma cell lines. TM treatment that mimics HBV infection triggered ER stress and increased AP formation, resulting in enhanced HBV replication and secretion of subviral particles (SVPs) and naked capsids. Additionally, TM reduced the number of early endosomes and HBsAg localization in this compartment, causing HBsAg/SVPs to accumulate in the ER. Thus, TM-induced AP formation serves as an alternative pathway for HBsAg/SVP trafficking. Importantly, TM inhibited AP-lysosome fusion, accompanied by enhanced AP/late endosome (LE)/multivesicular body fusion, to release HBsAg/SVPs through, or along with, exosome release. Notably, TM treatment inhibited HBsAg glycosylation, resulting in impairment of HBV virions' envelopment and secretion, but it was not critical for HBsAg/SVP trafficking in our cell systems. CONCLUSIONS: TM-induced ER stress and autophagic flux promoted HBV replication and the release of SVPs and naked capsids through the AP-LE/MVB axis.

Competitive evolved sub-clonal BCR::ABL1 and novel MSI2::PC fusion genes in myelodysplastic syndrome with isolated del(5q).

Myelodysplastic syndrome (MDS) represents a group of neoplasms with extensive heterogeneity. Recurrent mutations in dozens of driver genes have been identified in over 90% of MDS cases, although fusion genes are rarely seen. We first report the competitive evolved sub-clonal breakpoint cluster region (BCR)::ABL1 and novel MSI2::PC fusion gene in MDS with del(5q) in initial diagnosis that underwent dismal progression. However, the BCR::ABL1 clone vanished while the MSI2::PC clone rose to the major one with disease progression. A novel MSI2::PC fusion transcript was identified in initial diagnosis and disease progression of the patient through transcriptome sequencing (RNA-seq) and Quantitative reverse transcription polymerase Chain Reaction (PCR) showed MSI2::PC/ABL1 expression at initial diagnosis and disease progression. In addition, mutation screening of 300 leukemia driver genes identified ARID2 c.5046del/p.F1682Lfs*19 and ZNF292 c.4565A > G/p.Q1522R mutation in bone marrow sample at initial diagnosis and disease progression. In conclusion, the dynamic process of the two fusion and phenotype manifestations may help to understand further the molecular significance of the anomalies of BCR::ABL1, MSI2, and PC in oncogenesis.

hMSCs-derived exosome circCDK13 inhibits liver fibrosis by regulating the expression of MFGE8 through miR-17-5p/KAT2B.

OBJECTIVE: To investigate the effects of human bone marrow mesenchymal stem cells (hMSCs)-derived exosome circCDK13 on liver fibrosis and its mechanism. METHODS: Exosomes derived from hMSCs were extracted and identified by flow cytometry and osteogenic and adipogenic induction, and the expressions of marker proteins on the surface of exosomes were detected by western blot. Cell proliferation was measured by CCK8 assay, the expression of active markers of HSCs by immunofluorescence, and the expressions of fibrosis-related factors by western blot. A mouse model of liver fibrosis was established by intraperitoneal injection of thioacetamide (TAA). Fibrosis was detected by HE staining, Masson staining, and Sirius red staining. Western blot was utilized to test the expressions of PI3K/AKT and NF-κB pathway related proteins, dual-luciferase reporter assay and RIP assay to validate the binding between circCDK13 and miR-17-5p as well as between miR-17-5p and KAT2B, and ChIP to validate the effect of KAT2B on H3 acetylation and MFGE8 transcription. RESULTS: hMSCs-derived exosomes inhibited liver fibrosis mainly through circCDK13. Dual-luciferase reporter assay and RIP assay demonstrated the binding between circCDK13 and miR-17-5p as well as between miR-17-5p and KAT2B. Further experimental results indicated that circCDK13 mediated liver fibrosis by regulating the miR-17-5p/KAT2B axis, and KAT2B promoted MFGE8 transcription by H3 acetylation. Exo-circCDK13 inhibited PI3K/AKT and NF-κB signaling pathways activation through regulating the miR-17-5p/KAT2B axis. CONCLUSION: hMSCs-derived exosome circCDK13 inhibited liver fibrosis by regulating the expression of MFGE8 through miR-17-5p/KAT2B axis.

Cell-free DNA methylation markers for differential diagnosis of hepatocellular carcinoma.

BACKGROUND: Aberrant DNA methylation may offer opportunities in revolutionizing cancer screening and diagnosis. We sought to identify a non-invasive DNA methylation-based screening approach using cell-free DNA (cfDNA) for early detection of hepatocellular carcinoma (HCC). METHODS: Differentially, DNA methylation blocks were determined by comparing methylation profiles of biopsy-proven HCC, liver cirrhosis, and normal tissue samples with high throughput DNA bisulfite sequencing. A multi-layer HCC screening model was subsequently constructed based on tissue-derived differentially methylated blocks (DMBs). This model was tested in a cohort consisting of 120 HCC, 92 liver cirrhotic, and 290 healthy plasma samples including 65 hepatitis B surface antigen-seropositive (HBsAg+) samples, independently validated in a cohort consisting of 67 HCC, 111 liver cirrhotic, and 242 healthy plasma samples including 56 HBsAg+ samples. RESULTS: Based on methylation profiling of tissue samples, 2321 DMBs were identified, which were subsequently used to construct a cfDNA-based HCC screening model, achieved a sensitivity of 86% and specificity of 98% in the training cohort and a sensitivity of 84% and specificity of 96% in the independent validation cohort. This model obtained a sensitivity of 76% in 37 early-stage HCC (Barcelona clinical liver cancer [BCLC] stage 0-A) patients. The screening model can effectively discriminate HCC patients from non-HCC controls, including liver cirrhotic patients, asymptomatic HBsAg+ and healthy individuals, achieving an AUC of 0.957(95% CI 0.939-0.975), whereas serum α-fetoprotein (AFP) only achieved an AUC of 0.803 (95% CI 0.758-0.847). Besides detecting patients with early-stage HCC from non-HCC controls, this model showed high capacity for distinguishing early-stage HCC from a high risk population (AUC=0.934; 95% CI 0.905-0.963), also significantly outperforming AFP. Furthermore, our model also showed superior performance in distinguishing HCC with normal AFP (< 20ng ml-1) from high risk population (AUC=0.93; 95% CI 0.892-0.969). CONCLUSIONS: We have developed a sensitive blood-based non-invasive HCC screening model which can effectively distinguish early-stage HCC patients from high risk population and demonstrated its performance through an independent validation cohort. TRIAL REGISTRATION: The study was approved by the ethic committee of The Second Xiangya Hospital of Central South University (KYLL2018072) and Chongqing University Cancer Hospital (2019167). The study is registered at ClinicalTrials.gov(# NCT04383353 ).

Efficacy and safety of alfosbuvir plus daclatasvir in Chinese patients with hepatitis C virus genotypes 1, 2, 3, and 6 infection: An open-label, phase 2 study.

Alfosbuvir is a novel potent HCV NS5B polymerase inhibitor in development for the treatment of chronic HCV infection. Our previous studies indicated that alfosbuvir monotherapy was well-tolerated and druggable in healthy subjects and HCV-infected patients. Here, we evaluate the efficacy and safety of alfosbuvir in combination with daclatasvir in Chinese patients with HCV genotype 1, 2, 3 or 6. In this open-label study, patients with chronic HCV infection were randomly assigned with a 1:1:1 ratio to receive 12 weeks of daclatasvir 60 mg plus alfosbuvir at a dose of 400, 600 or 800 mg (Cohort A, B or C) daily. Randomization was stratified by HCV genotype and the presence or absence of cirrhosis at screening. The primary endpoint was a sustained virologic response 12 weeks after the end of treatment (SVR12). A total of 124 patients were enrolled in the study, all of whom were available for post-treatment week 12 assessments. SVR12 was achieved in 92.7% (38/41), 95.2% (40/42) and 100% (41/41) of patients in Cohort A, B and C respectively. The most common adverse events were hepatic steatosis, upper respiratory tract infection, hypercholesterolaemia, hypertriglyceridaemia, blood bilirubin increased, and total bile acids increased. There were no discontinuations due to adverse events, and no treatment-related serious adverse events were reported. Once-daily oral administration of alfosbuvir plus daclatasvir were highly effective and safe in Chinese patients infected with HCV genotype 1, 2, 3 or 6, suggesting this regimen could be a promising drug candidate for HCV treatment irrespective of genotype. (ClinicalTrials.gov number, NCT04070235).

A Gene Signature Comprising Seven Pyroptosis-Related Genes Predicts Prognosis in Pediatric Patients with Acute Myeloid Leukemia.

INTRODUCTION: The aim of the study was to construct a pyroptosis-related risk score (RS) model for the prognosis of acute myeloid leukemia (AML). METHODS: The TARGET (training) and E-MTAB-1216 (validation) datasets were downloaded. Pyroptosis-related genes with differences in expression were identified between the recurrent and nonrecurrent samples of the training dataset. An RS prognostic model comprising seven pyroptosis-related genes was constructed using LASSO regression coefficients. The samples were classified into the high- and low-risk groups using the RS model; the differentially expressed genes (DEGs) between these groups were identified, followed by DEG functional analysis and the immunological evaluation of these groups. RESULTS: Forty-nine pyroptosis-related genes, including 22 DEGs, were screened. WT1, NPM, FLT3/ITD, and CEBPA mutations were found in most pediatric AML samples. An RS prognostic model was constructed using 7 pyroptosis-related genes. The two risk groups and prognostic data were significantly related. FLT3/ITD mutations, CEBPA mutations, and RS model status were identified as independent prognostic factors, using the clinical information. The DEGs between the two groups were correlated with immune-related pathways. Moreover, the immune cell distribution and the occurrence of immune-related pathways were notably decreased in the high-risk group. DISCUSSION/CONCLUSION: Seven pyroptosis-related genes, CHMP2A, PRKACA, CASP9, IRF2, CHMP3, HMGB1, and AIM2, can predict the prognosis and recurrence of childhood AML.

microRNA-122 inhibits hepatic stellate cell proliferation and activation in vitro and represses carbon tetrachloride-induced liver cirrhosis in mice.

OBJECTIVE: This study aimed to determine the roles of microRNA (miR)-122 in the activation of hepatic stellate cells (HSCs) and liver cirrhosis. METHODS: Rat primary HSCs were incubated with transforming growth factor-beta (TGF-ß), during which miR-122 and EphB2 expression was measured. miR-122 mimic and/or pcDNA3.1 EphB2 was transfected into TGF-ß-induced HSCs. A mouse model of liver cirrhosis was established via an intraperitoneal injection of carbon tetrachloride (CCl4), followed by the injection of miR-122 agomir. Levels of serum alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured. Fibronectin (FN), alpha smooth muscle actin (α-SMA), Collagen I, miR-122, and EphB2 expression was evaluated in liver tissues and HSCs. Cell proliferation was measured using CCK-8 assay. Interactions between miR-122 and EphB2 were assessed using dual luciferase reporter assay. RESULTS: miR-122 (0.15-fold) was downregulated and EphB2 (mRNA: 5.06-fold; protein: 2.35-fold) was upregulated after TGF-ß induction of HSCs. Overexpressed miR-122 decreased proliferation and EphB2 (mRNA: 0.46-fold; protein: 0.62-fold), FN (mRNA: 0.45-fold; protein: 0.64-fold), α-SMA (mRNA: 0.48-fold; protein: 0.51-fold), and Collagen I (mRNA: 0.44-fold; protein: 0.51-fold) expression in HSCs, which was abrogated by EphB2 upregulation. miR-122 expression was reduced by 0.21-fold and serum ALT and AST levels were enhanced in mice following 8-week CCl4 induction along with increased expression of FN, α-SMA, and Collagen I in liver tissues, which was blocked by miR-122 overexpression. Moreover, EphB2 was a target gene of miR-122. CONCLUSION: miR-122 curtails HSC proliferation and activation by targeting EphB2 and suppresses liver cirrhosis in mice.

Mn-SOD alleviates methotrexate-related hepatocellular injury via GSK-3ß affecting anti-oxidative stress of HO-1 and Drp1. / Mn-SOD通过GSK-3ß影响HO-1和Drp1的抗氧化应激减轻MTXç›¸å ³è‚ç»†èƒžæŸä¼¤.

OBJECTIVES: Methotrexate (MTX) is the most common therapeutic agent that may have the risk of drug-induced liver injury. Its pathogenic mechanism is related to oxidative stress caused by mitochondrial dysfunction. Superoxide dismutase (SOD), including manganese-containing SOD (Mn-SOD), can exert its effect of anti-oxidative stress by scavenging superoxide free radicals. Accordingly, this study is performed to explore the underlying molecular mechanism via observing whether Mn-SOD could affect the damage of MTX to hepatocytes. METHODS: Human hepatocyte cell line L-02 was cultured in vitro and divided into 4 groups, including a blank group with the addition of the same volume of serum-free medium, a MTX group (40 µg/well MTX drug-treatment), a MTX+NC group (40 µg/well MTX drug-treatment+blank plasmid), and a MTX+SOD group (40 µg/well MTX drug-treatment+Mn-SOD plasmid). The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and microRNA-122 (miR-122) in the supernatant of cell culture were respectively detected by automatic biochemical analytical instrument and real-time RT-PCR to evaluate the degree of hepatocyte damage in each group. MitoSOX fluorescent probe was used to label intracellular superoxide in each group, and cell apoptosis was detected by flow cytometry. Meanwhile, the contents of glycogen synthase kinase-3 beta (GSK-3ß), hemeoxygenase-1 (HO-1), mitochondrial fission-mediated protein of dynamin-related protein 1 (Drp1), and Mn-SOD were detected by Western blotting. RESULTS: Compared with the blank group, the levels of ALT, AST, and miR-122 in the supernatant of hepatocyte culture of the MTX group and MTX+NC group were significantly elevated (all P <0.05), and that in the MTX+SOD group were significantly decreased ( P <0.05) and equivalent to that in the blank group. MitoSOX staining revealed that the MTX group and MTX+NC had the most abundant superoxide; and the amount was significantly reduced in the MTX+SOD group, without a significant difference when compared with the blank group. Furthermore, the results of flow cytometry indicated that compared with the blank group, the MTX group and MTX+NC group showed significantly increased cell apoptosis ( P <0.05); while there was obviously reduced cell apoptosis in the MTX+SOD group than that in the MTX group and MTX+NC group ( P <0.05). According to the results of Western blotting, the blank group and MTX+SOD group had higher expressions of Mn-SOD, p-GSK-3ß, and HO-1; while the MTX group and MTX+NC group exhibited remarkably lower levels of Mn-SOD, p-GSK-3ß, and HO-1 than those in the blank group ( P <0.05). Besides, a completely opposite trend was found in the expression of Drp1, which was highly expressed in the MTX group and MTX+NC group, but lowly expressed in the blank group and the MTX+SOD group. CONCLUSIONS: MTX may induce hepatocyte damage, and one of the mechanisms may be due to the decrease of intracellular Mn-SOD level, which can cause the accumulation of superoxide, affect the levels of HO-1 and Drp1 through GSK-3ß leading to mitochondrial damage and cell apoptosis. High expression of Mn-SOD intracellularly through exogenous introduction can scavenge drug-produced superoxide, affect HO-1 and Drp1 levels through GSK-3ß, activate mitochondria, protect cells against damage from oxidative stress, and inhibit hepatocyte apoptosis eventually. So exogenous introduction of SOD may be a potential therapeutic approach to block or reverse MTX-related hepatocyte injury.

Clustering Cases of Chlamydia psittaci Pneumonia in Coronavirus Disease 2019 Screening Ward Staff.

We describe 4 cases of Chlamydia psittaci pneumonia among medical staff in a coronavirus disease 2019 (COVID-19) screening ward, as well as the experience of dealing with this nosocomial infection event. Atypical pneumonia, in addition to COVID-19, should be considered when clustering cases occur, even during a COVID-19 pneumonia pandemic.

Population pharmacokinetics, safety and dosing optimization of voriconazole in patients with liver dysfunction: A prospective observational study.

AIMS: Voriconazole is a broad-spectrum antifungal agent for the treatment of invasive fungal infections. There is limited information about the pharmacokinetics and appropriate dosage of voriconazole in patients with liver dysfunction. This study aimed to explore the relationship between voriconazole trough concentration (Ctrough ) and toxicity, identify the factors significantly associated with voriconazole pharmacokinetic parameters and propose an optimised voriconazole dosing regimen for patients with liver dysfunction. METHODS: The study prospectively enrolled 51 patients with 272 voriconazole concentrations. Receiver operating characteristic curves were used to explore the relationship between voriconazole Ctrough and toxicity. The pharmacokinetic data was analysed with nonlinear mixed-effects method. Dosing simulations stratified by total bilirubin (TBIL, TBIL-1: TBIL < 51 µmol/L; TBIL-2: 51 µmol/L ≤ TBIL < 171 µmol/L; TBIL-3: TBIL ≥ 171 µmol/L) were performed. RESULTS: Receiver operating characteristic curve analysis revealed that voriconazole Ctrough of ≤ 5.1 mg/L were associated with significantly lower the incidence of adverse events. A 1-compartment pharmacokinetic model with first-order absorption and elimination was used to describe the data. Population pharmacokinetic parameters of clearance, volume of distribution and oral bioavailability were 0.88 L/h, 148.8 L and 88.4%, respectively. Voriconazole clearance was significantly associated with TBIL and platelet count. The volume of distribution increased with body weight. Patients with TBIL-1 could be treated with a loading dose of 400 mg every 12 hours (q12h) for first day, followed by a maintenance dose of 100 mg q12h administered orally or intravenously. TBIL-2 and TBIL-3 patients could be treated with a loading dose of 200 mg q12h and maintenance doses of 50 mg q12h or 100 mg once daily and 50 mg once daily orally or intravenously, respectively. CONCLUSIONS: Lower doses and longer dosing intervals should be considered for patients with liver dysfunction. TBIL-based dosing regimens provide a practical strategy for achieving voriconazole therapeutic range and therefore maximizing treatment outcomes.

CAT1 silencing inhibits TGF-ß1-induced mouse hepatic stellate cell activation in vitro and hepatic fibrosis in vivo.

Hepatic fibrosis is characterized by abnormal accumulation of extracellular matrix (ECM). Hepatic stellate cells (HSCs) are the primary cells that produce ECM in response to hepatic injury, and transforming growth factor-beta (TGF-ß) has been regarded as the central stimulus responsible for HSC-mediated ECM production. In the present study, we attempted to identify a critical factor in HSC activation and the underlying mechanism. By analyzing online microarray expression profiles, we found that the expression of high-affinity cationic amino acid transporter 1 (CAT1) was upregulated in hepatic fibrosis models and activated HSCs. We isolated and identified mouse HSCs (MHSCs) and found that in these cells, CAT1 was most highly upregulated by TGF-ß1 stimulation in both time- and dose-dependent manners. In vitro, CAT1 overexpression further enhanced, while CAT1 silencing inhibited, the effect of TGF-ß1 in promoting MHSC activation. In vivo, CAT1 silencing significantly improved the hepatic fibrosis induced by both CCl4 and non-alcoholic fatty liver disease (NAFLD). In summary, CAT1 was significantly upregulated in TGF-ß1-activated MHSCs and mice with hepatic fibrosis. CAT1 silencing inhibited TGF-ß1-induced MHSC activation in vitro and fibrogenic changes in vivo. CAT1 is a promising target for hepatic fibrosis treatment that requites further investigation in human cells and clinical practice.

Cefotiam Treatment in Children: Evidence of Subtherapeutic Levels.

BACKGROUND: Cefotiam, a second-generation cephalosporin, is a broad-spectrum antibiotic with good antibacterial action against both gram-negative and gram-positive bacteria. It is used widely in clinical practice, although bacterial drug resistance makes its clinical use problematic. The authors hypothesized that subtherapeutic concentrations of cefotiam leads to bacterial resistance. The present study was conducted to evaluate whether the standard cefotiam dosing regimen resulted in a subtherapeutic concentrations in children. METHOD: Data were prospectively collected from pediatric patients with suspected or confirmed community-acquired pneumonia who were receiving cefotiam at the standard dosing regimen (40-80 mg/kg, 2 or 3 times daily). A blood sample was collected after 70%-100% of the dosing interval, and plasma concentrations were determined by high-performance liquid chromatography using an ultraviolet detector. RESULTS: The data from 88 patients (age, 3.0 ± 2.8 years; weight, 15.4 ± 8.3 kg) were used for analysis. The average of cefotiam concentrations was 0.06 mcg/mL (range: <0.05-0.79 mcg/mL). Most patients (n = 72, 81.8%) had concentrations below 0.1 mcg/mL; only 2 patients had concentrations higher than 0.4 mcg/mL. CONCLUSIONS: The standard dosing regimen for cefotiam resulted in extremely low plasma concentrations in children; such low concentrations may lead to antimicrobial drug resistance. Thus, an increase in cefotiam dosage in children to 80 mg/kg 4 times daily is recommended (maximum dose on the label).

Coexistence of Pure Red Cell Aplasia and Autoimmune Haemolytic Anaemia Associated with Thymoma.

Thymoma is an uncommon neoplasia derived from the epithelial cells of the thymus, which leads to immune dysregulation and is associated with a series of autoimmune diseases. However, the concurrence of these disease entities is rare, and the exact mechanisms of these diseases are still unclear. We have admitted several cases who were diagnosed with thymoma, autoimmune haemolytic anaemia, and pure red cell aplasia. These cases were the first to report the concurrence of these three disorders. After thymectomy, anaemia improved, haemolytic cells decreased, and haemoglobin was normalized.

Psychological Impact and Coping Strategies of Frontline Medical Staff in Hunan Between January and March 2020 During the Outbreak of Coronavirus Disease 2019 (COVID­19) in Hubei, China.

BACKGROUND Throughout China, during the recent epidemic in Hubei province, frontline medical staff have been responsible for tracing contacts of patients infected with coronavirus disease 2019 (COVID­19). This study aimed to investigate the psychological impact and coping strategies of frontline medical staff in Hunan province, adjacent to Hubei province, during the COVID­19 outbreak between January and March 2020. MATERIAL AND METHODS A cross-sectional observational study included doctors, nurses, and other hospital staff throughout Hunan province between January and March 2020. The study questionnaire included five sections and 67 questions (scores, 0-3). The chi-squared χ² test was used to compare the responses between professional groups, age-groups, and gender. RESULTS Study questionnaires were completed by 534 frontline medical staff. The responses showed that they believed they had a social and professional obligation to continue working long hours. Medical staff were anxious regarding their safety and the safety of their families and reported psychological effects from reports of mortality from COVID­19 infection. The availability of strict infection control guidelines, specialized equipment, recognition of their efforts by hospital management and the government, and reduction in reported cases of COVID­19 provided psychological benefit. CONCLUSIONS The COVID­19 outbreak in Hubei resulted in increased stress for medical staff in adjacent Hunan province. Continued acknowledgment of the medical staff by hospital management and the government, provision of infection control guidelines, specialized equipment and facilities for the management of COVID­19 infection should be recognized as factors that may encourage medical staff to work during future epidemics.

Developing and Preliminary Validating an Automatic Cell Classification System for Bone Marrow Smears: a Pilot Study.

Bone marrow smear examination is an indispensable diagnostic tool in the evaluation of hematological diseases, but the process of manual differential count is labor extensive. In this study, we developed an automatic system with integrated scanning hardware and machine learning-based software to perform differential cell count on bone marrow smears to assist diagnosis. The initial development of the artificial neural network was based on 3000 marrow smear samples retrospectively archived from Sir Run Run Shaw Hospital affiliated to Zhejiang University School of Medicine between June 2016 and December 2018. The preliminary field validating test of the system was based on 124 marrow smears newly collected from the Second Affiliated Hospital of Harbin Medical University between April 2019 and November 2019. The study was performed in parallel of machine automatic recognition with conventional manual differential count by pathologists using the microscope. We selected representative 600,000 marrow cell images as training set of the algorithm, followed by random captured 30,867 cell images for validation. In validation, the overall accuracy of automatic cell classification was 90.1% (95% CI, 89.8-90.5%). In a preliminary field validating test, the reliability coefficient (ICC) of cell series proportion between the two analysis methods were high (ICC ≥ 0.883, P < 0.0001) and the results by the two analysis methods were consistent for granulocytes and erythrocytes. The system was effective in cell classification and differential cell count on marrow smears. It provides a useful digital tool in the screening and evaluation of various hematological disorders.

HBV down-regulates PTEN expression via Nrf2/GSK3ß signaling pathway. / HBV通过Nrf2/GSK3ß信号通路下调PTEN的表达.

OBJECTIVES: To investigate the effect of HBV infection on PTEN expression, and to explore the possible molecular mechanisms. METHODS: HepG2 cells and HepG2.2.15 cells were cultured under suitable conditions for 48 hours, and the expressions of PTEN, Nrf2 and pGSK3ß in HepG2 and HepG2.2.15 cells were detected by Western blotting. After the blank plasmid (EV) and the plasmid pWXL-Nrf2 were transiently transfected into HepG2 and HepG2.2.15 cells, respectively, the HepG2 and HepG2.2.15 cells were treated with the selective inhibitor of GSK3ß (25 nmol/L LiCl). After 48 h, the expressions of Nrf2, pGSK3ß and PTEN in HepG2 and HepG2.2.15 cells were examined by Western blotting. RESULTS: Expression of PTEN was reduced and the levels of Nrf2 and pGSK3ß were increased in HepG2.2.15 cells compared with those in the HepG2 cells (all P<0.05). After transfection with pWXL-Nrf2, the protein expression of Nrf2 and pGSK3ß in cells were significantly increased while the protein expression of PTEN was decreased (all P<0.05). Furthermore, LiCl treatment up-regulated the protein expression of Nrf2 and pGSK3ß, and eventually suppressed the production of PTEN (all P<0.05). CONCLUSIONS: HBV may down-regulate PTEN expression via Nrf2/GSK3ß signaling pathway, which may provide new ideas for the targeting therapy of hepatocellular carcinoma.

Interaction of lncRNA-MALAT1 and miR-124 regulates HBx-induced cancer stem cell properties in HepG2 through PI3K/Akt signaling.

BACKGROUND: Hepatitis B virus X protein (HBx) plays a crucial role in initiating and promoting HBV-induced hepatocellular carcinoma (HCC) development. Reports indicated that HBx promotes cancer stem cell (CSC) generation, which may be associated with HBV-related HCC. Noncoding RNA miR-124 and long noncoding RNA (lncRNA)-metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) were considered to be involved deeply in the progress of HBx-related HCC. Hence, the underlying mechanism of miR-124 and lncRNA-MALAT1 in regulating HBx-promoted CSC needs to be studied. MATERIALS AND METHODS: In present study, HepG2-X cell line was induced by transfect HBx into HepG2 cells. Overexpressing of miR-124 or silencing of lncRNA-MALAT1 was completed by transfecting miR-124 mimic or shMALAT1 into HepG2-X cells. HBx-induced CSC properties and tumorigenic potential of HepG2 cells were determined by detecting CSC marker expression, colony formation assay, and xenograft tumorigenesis. The mechanism of HBx-induced CSC properties was explored by PI3K/Akt inhibitor. Interaction of miR-124 and lncRNA-MALAT1 was detected by luciferase reporter assay. RESULTS: HBx promoted CSC properties through upregulating stemness markers and reprogramming proteins, and contributed to tumorigenicity of HepG2-X cells both in vivo and in vitro. Inhibition of Akt activation blocked the HBx-stimulated reprogramming proteins and stemness markers. HBx upregulated lncRNA-MALAT1 expression while downregulating miR-124 expression in HepG2-X cells. miR-124 interacts with lncRNA-MALAT1 by direct targeting. Overexpression of miR-124 or silencing of lncRNA-MALAT1 both blocked HBx-induced CSC generation, stemness-related factor activation and tumorigenicity via PI3K/Akt signaling. CONCLUSION: Our results demonstrated that miR-124 interact with lncRNA-MALAT1 and involve in regulating HBx-induced CSC properties via PI3K/Akt signaling.