Single-cell transcriptome landscape of zebrafish liver reveals hepatocytes and immune cell interactions in understanding nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease in the world. Immunity is the major contributing factor in NAFLD; however, the interaction of immune cells and hepatocytes in disease progression has not been fully elucidated. As a popular species for studying NAFLD, zebrafish, whose liver is a complex immune system mediated by immune cells and non-immune cells in maintaining immune tolerance and homeostasis. Understanding the cellular composition and immune environment of zebrafish liver is of great significance for its application in NAFLD. Here, we established a liver atlas that consists of 10 cell types using single-cell RNA sequencing (scRNA-seq). By examining the heterogeneity of hepatocytes and analyzing the expression of NAFLD-associated genes in the specific cluster, we provide a potential target cell model to study NAFLD. Additionally, our analysis identified two subtypes of distinct resident macrophages with inflammatory and non-inflammatory functions and characterized the successive stepwise development of T cell subclusters in the liver. Importantly, we uncovered the possible regulation of macrophages and T cells on target cells of fatty liver by analyzing the cellular interaction between hepatocytes and immune cells. Our data provide valuable information for an in-depth study of immune cells targeting hepatocytes to regulate the immune balance in NAFLD.

Single-cell-resolution transcriptome map revealed novel genes involved in testicular germ cell progression and somatic cells specification in Chinese tongue sole with sex reversal.

Female-to-male sex reversals (pseudomales) are common in lower vertebrates and have been found in natural populations, which is a concern under rapid changes in environmental conditions. Pseudomales can exhibit altered spermatogenesis. However, the regulatory mechanisms underlying pseudomale spermatogenesis remain unclear. Here, we characterized spermatogenesis in Chinese tongue sole (Cynoglossus semilaevis), a species with genetic and environmental sex determination, based on a high-resolution single-cell RNA-seq atlas of cells derived from the testes of genotypic males and pseudomales. We identified five germ cell types and six somatic cell types and obtained a single-cell atlas of dynamic changes in gene expression during spermatogenesis in Chinese tongue sole, including alterations in pseudomales. We detected decreased levels of Ca2+ signaling pathway-related genes in spermatogonia, insufficient meiotic initiation in spermatocytes, and a malfunction of somatic niche cells in pseudomales. However, a cluster of CaSR genes and MAPK signaling factors were upregulated in undifferentiated spermatogonia of pseudomales. Additionally, we revealed that Z chromosome-specific genes, such as piwil2, dhx37, and ehmt1, were important for spermatogenesis. These results improve our understanding of reproduction after female-to-male sex-reversal and provide new insights into the adaptability of reproductive strategies in lower vertebrates.

Echinacoside exerts its protective effects in a type 2 diabetes mellitus injury model via the AKT pathway.

Echinacoside (ECH) is the main compound of Cistanche deserticola, which possesses antioxidant, antitumor, antifatigue, and anti-inflammatory properties. The present study investigated the protective effects of echinacoside on type 2 diabetes mellitus (T2DM)-induced injury in T2DM injury db/db mice model and insulin-resistant LO2 cell model. The results demonstrated that ECH probably alleviated T2DM-induced injury by mediating the AKT pathway, which provided a new direction for the treatment of T2DM-induced injury.

Single-Cell Atlas of the Chinese Tongue Sole (Cynoglossus semilaevis) Ovary Reveals Transcriptional Programs of Oogenesis in Fish.

Oogenesis is a highly orchestrated process that depends on regulation by autocrine/paracrine hormones and growth factors. However, many details of the molecular mechanisms that regulate fish oogenesis remain elusive. Here, we performed a single-cell RNA sequencing (scRNA-seq) analysis of the molecular signatures of distinct ovarian cell categories in adult Chinese tongue sole (Cynoglossus semilaevis). We characterized the successive stepwise development of three germ cell subtypes. Notably, we identified the cellular composition of fish follicle walls, including four granulosa cell types and one theca cell type, and we proposed important transcription factors (TFs) showing high activity in the regulation of cell identity. Moreover, we found that the extensive niche-germline bidirectional communications regulate fish oogenesis, whereas ovulation in fish is accompanied by the coordination of simultaneous and tightly sequential processes across different granulosa cells. Additionally, a systems biology analysis of the homologous genes shared by Chinese tongue sole and macaques revealed remarkably conserved biological processes in germ cells and granulosa cells across vertebrates. Our results provide key insights into the cell-type-specific mechanisms underlying fish oogenesis at a single-cell resolution, which offers important clues for exploring fish breeding mechanisms and the evolution of vertebrate reproductive systems.

Identification of 2-substituted pyrrolo[1,2-b]pyridazine derivatives as new PARP-1 inhibitors.

A library of new 2-substituted pyrrolo[1,2-b]pyridazine derivatives were rapidly assembled and identified as PARP inhibitors. Structure-activity relationship for this class of inhibitor resulted in the discovery of most potent compounds 15a and 15b that exhibited about 29- and 5- fold selective activity against PARP-1 over PARP-2 respectively. The antiproliferative activity of the as-prepared compounds were demonstrated by further celluar assay in BRCA2-deficient V-C8 and BRCA1-deficient MDA-MB-436 cell lines, displaying that compound 15b could robustly reduce the corresponding cell proliferation and growth with CC50s of 340 and 106 nM respectively. The PK property of 15b was also investigated here.

Switching Lamivudine with Adefovir Dipivoxil Combination Therapy to Entecavir Monotherapy Provides Better Viral Suppression and Kidney Safety.

Introduction: Most chronic hepatitis B (CHB) patients in China are primitively treated with a combination of lamivudine (LAM) and adefovir dipivoxil (ADV). Although antiviral resistance can be avoided with this combination therapy, using it can have harmful side effects related to ADV, specifically kidney and bone injury. This study was designed to compare viral suppression and kidney safety when switching LAM and ADV combination therapy de novo to entecavir (ETV) monotherapy in patients with CHB and compensated hepatic cirrhosis. Materials and methods: In total, 360 CHB and compensated liver cirrhosis patients who received treatment of LAM and ADV combination therapy for more than 1 year were included in this study. One hundred and eighty patients continued combination therapy to serve as a control group and the other 180 patients were switched to ETV monotherapy to serve as the experimental group. The total course of therapy was 3 years. Laboratory studies were done every 3 months to measure liver and kidney function. Studies included glomerular filtration rate (eGFR), HBV-DNA, urine ß2-microglobulin (ß2-M) and retinol binding protein (RBP). Results: In the experimental group, an HBV-DNA level below 20 IU/ml was found in 77.65%, 85.88%, and 94.77% in years 1, 2, and 3, respectively. In the control group, HBV-DNA levels were below 20 IU/ml in 69.66%, 75.42%, and 85.80% in years 1, 2, and 3, respectively. Low HBV-DNA levels in the experimental group were significantly less common than in the control group on the second and third year; P values were 0.009 and 0.006 for years 2 and 3, respectively. The cumulative genetic mutation rate was 3.49% in the experimental group and 8.88% in the control group (P=0.044). Decreases in eGFR more than 30% from baseline were found in 0%, 0.56%, and 1.74% of patients in the experimental group and 4.49%, 9.14% and 14.79% in patients in the control group in the first, second, and third year, respectively. Serum creatinine more than 50 µmol/L above baseline was found in 0%, 0% and 1.74% of patients in the experimental group and 1.12%, 4.00% and 5.32% of patients in the control group in years 1, 2, and 3, respectively. The urine ß2-M and RBP levels were abnormal more often in the experimental group than in the control group. Conclusion: Switching to ETV monotherapy can decrease HBV-DNA levels, reduce the genetic mutation rate, and prevent renal damage caused by LAM and ADV combination therapy in patients with CHB and compensated liver cirrhosis. Patients receiving LAM and ADV combination therapy de novo should be switched to ETV monotherapy immediately.

The ribosome biogenesis protein Esf1 is essential for pharyngeal cartilage formation in zebrafish.

Craniofacial malformations are common congenital birth defects and usually caused by abnormal development of the cranial neural crest cells. Some nucleolar ribosome biogenesis factors are implicated in neural crest disorders also known as neurocristopathies. However, the underlying mechanisms linking ribosome biogenesis and neural crest cell (NCC) development remain to be elucidated. Here we report a novel zebrafish model with a CRISPR/Cas9-generated esf1 mutation, which exhibits severe NCC-derived pharyngeal cartilage loss and defects in the eyes, brain, and heart. The expression of several typical NCC markers, including sox10, dlx2a, nrp2b, crestin, vgll2a, and sox9a, was reduced in the head of the esf1 mutants, which indicates that esf1 plays a role in the development of zebrafish NCCs. We demonstrate that, similar to the yeast, loss of esf1 in zebrafish leads to defects in 18S rRNA biogenesis and ribosome biogenesis. We also show strong upregulation of p53 signaling as well as apoptosis, and poor proliferation in mutants. Inactivation of p53 rescues the early tissue defects and pharyngeal cartilage loss observed in esf1 mutants, indicating that increased cell death and pharyngeal cartilage defects observed in esf1 mutants are mediated via upregulated p53 signaling pathways. Based on transplantation analysis, we found esf1 functions in NCC in a cell autonomous fashion. Together, our results suggest that esf1 is required for NCC development and pharyngeal cartilage formation. These studies provide a potential model for investigating the relationship between ribosome biogenesis defects and craniofacial neurocristopathies.

Angiopoietin-like protein 4 improves glucose tolerance and insulin resistance but induces liver steatosis in high-fat-diet mice.

Angiopoietin-like protein 4 (Angptl4) is a secreted protein predominantly expressed in liver and adipose tissues, and has been identified as an adipokine. Angptl4 is the target gene of peroxisome proliferator­activated receptors, which are widely used as lipid­lowering and anti­diabetic drugs, and previous studies have demonstrated that Angptl4 is able to directly stimulate adipocyte lipolysis. The current study focused on how Angptl4 was involved in regulating lipid and glucose metabolism in high­fat­diet (HFD) C57 mice. In the present study, mice were divided into three groups, with standard chow mice as a normal control, adenovirus (adv)­injected HFD mice as a model control and adv­Angptl4­injected HFD mice as the Angptl4+ group. Firstly, compared with the normal control group, mice in the model control group gained more body weight with severe liver steatosis and increased serum levels of triglyceride, total cholesterol, free fatty acids, alanine aminotransferase and aspartate aminotransferase. In the Angptl4+ group, Angptl4 reduced the weight growth rate, aggravated hepatic steatosis and further increased all the aforementioned serum indexes. Secondly, compared with the normal control, the model control group had a reduced glucose tolerance and developed insulin resistance. Angptl4 expression and the phosphorylation levels of several insulin signaling pathway­associated genes, insulin receptor substrate 1, protein kinase B, janus kinase 2, signal transducer and activator of transcription 3 were downregulated in the liver samples. Adv­Angptl4 injection was observed to improve glucose tolerance and insulin resistance. The genes measured were identified to be upregulated close to normal levels. All the results suggested that Angptl4 served an important role in lipid and glucose metabolism in HFD­induced obese mice, and this may have a great significance for treatment of hyperlipidemia, diabetes, metabolic syndrome and other diseases.

Early kidney injury during long-term adefovir dipivoxil therapy for chronic hepatitis B.

AIM: To evaluate urine ß2-microglobulin (ß2-M), retinol-binding protein (RBP) excretion, and renal impairment with adefovir dipivoxil (ADV) for chronic hepatitis B. METHODS: We enrolled 165 patients with chronic hepatitis B infection who were treated with ADV monotherapy (n = 90) or ADV plus lamivudine combination therapy (n = 75). An additional 165 chronic hepatitis B patients treated with entecavir were recruited as controls. We detected serum creatinine, urine ß2-M, and RBP levels, and estimated the glomerular filtration rate (eGFR) at the initiation of antiviral therapy and every 6 mo for a period of five years. RESULTS: Urine ß2-M abnormalities were observed in patients during the first (n = 3), second (n = 7), third (n = 11), fourth (n = 16), and fifth (n = 21) year of ADV treatment. Urinary RBP abnormalities were observed in patients during the first (n = 2), second (n = 8), third (n = 12), fourth (n = 15), and fifth (n = 22) year of ADV treatment. eGFR decreased 20%-30% from baseline in 20 patients, 30%-50% in 12 patients, and > 50% in 3 patients during the five years of treatment. Further analysis indicated that decreases in eGFR of ≥ 30% relative to the baseline level correlated significantly with urine RBP and ß2-M abnormalities. In contrast, both serum creatinine and eGFR remained stable in patients treated with entecavir, and only one of these patients developed a urine ß2-M abnormality, and two developed urine RBP abnormalities during the five years of treatment. CONCLUSION: Urine RBP and ß2-M are biomarkers of renal injury during long-term ADV treatment for chronic hepatitis B, and indicate when treatment should be switched to entecavir.

Hepatitis B surface antigen levels during natural history of chronic hepatitis B: a Chinese perspective study.

AIM: To determine the baseline hepatitis B surface antigen (HBsAg) levels during the different phases of chronic hepatitis B (CHB) patients in China. METHODS: Six hundred and twenty-three hepatitis B virus or un-infected patients not receiving antiviral therapy were analyzed in a cross-sectional study. The CHB patients were classified into five phases: immune-tolerant (IT, n = 108), immune-clearance (IC, n = 161), hepatitis B e antigen negative hepatitis (ENH, n = 149), low-replicative (LR, n = 135), and liver cirrhosis (LC, n = 70). HBsAg was quantified (Abbott ARCHITECT assay) and correlated with hepatitis B virus (HBV) DNA, and serum alanine aminotransferase/aspartate aminotransferase (ALT/AST) in each phase of CHB was also determined. RESULTS: Median HBsAg titers were different in each phase of CHB (P < 0.001): IT (4.85 log10 IU/mL), IC (4.36 log10 IU/mL), ENH (2.95 log10 IU/mL), LR (3.18 log10 IU/mL) and LC (2.69 log10 IU/mL). HBsAg titers were highest in the IT phase and lowest in the LC phase. Serum HBsAg titers showed a strong correlation with HBV viral load in the IC phase (r = 0.683, P < 0.001). No correlation between serum HBsAg level and ALT/AST was observed. CONCLUSION: The mean baseline HBsAg levels differ significantly during the five phases of CHB, providing evidence on the natural history of HBV infection. HBsAg quantification may predict the effects of immune-modulator or oral nucleos(t)ide analogue therapy.

De novo combined lamivudine and adefovir dipivoxil therapy vs entecavir monotherapy for hepatitis B virus-related decompensated cirrhosis.

AIM: To compare efficacy of combined lamivudine (LAM) and adefovir dipivoxil (ADV) therapy with that of entecavir (ETV) monotherapy for hepatitis B virus (HBV)-related decompensated liver cirrhosis. METHODS: A total of 120 naïve patients with HBV-related decompensated cirrhosis participated in this study. Sixty patients were treated with combined LAM and ADV therapy (LAM + ADV group), while the other 60 were treated with ETV monotherapy (ETV group) for two years. Tests for liver and kidney function, alpha-fetoprotein, HBV serum markers, HBV DNA load, prothrombin time (PT), and ultrasonography or computed tomography scan of the liver were performed every 1 to 3 mo. Repeated measure ANOVA and the χ(2) test were performed to compare the efficacy, side effects, and the cumulative survival rates at 48 and 96 wk. RESULTS: Forty-five patients in each group were observed for 96 wk. No significant differences in HBV DNA negative rates and alanine aminotransferase (ALT) normalization rates at weeks 48 (χ(2) = 2.12 and 2.88) and 96 (χ(2) = 3.21 and 3.24) between the two groups were observed. Hepatitis B e antigen seroconversion rate in the LAM + ADV group at week 96 was significantly higher in the ETV group (43.5% vs 36.4%, χ(2) = 4.09, P < 0.05). Viral breakthrough occurred in 2 cases (4.4%) by week 48 and in 3 cases (6.7%) by week 96 in the LAM + ADV group, and no viral mutation was detected. In the ETV group, viral breakthrough occurred in 1 case (2.2%) at the end of week 96. An increase in albumin (F = 18.9 and 17.3), decrease in total bilirubin and in ALT (F = 16.5, 17.1 and 23.7, 24.8), reduced PT (F = 22.7 and 24.5), and improved Child-Turcotte-Pugh and the model for end-stage liver disease scores (F = 18.5, 17.8, and 24.2, 23.8) were observed in both groups. The cumulative rates of mortality and liver transplantation were 16.7% (10/60) and 18.3% (11/60) in the LAM + ADV and ETV groups, respectively. CONCLUSION: Both LAM + ADV combination therapy and ETV monotherapy can effectively inhibit HBV replication, improve liver function, and decrease mortality.