[Post-transcriptional regulation mechanism and antiviral strategy of hepatitis B virus RNA].

Chronic hepatitis B virus (HBV) infection is one of the major public health issues of ongoing global concern. Due to inadequate understanding of the HBV life cycle, there is a lack of effective drugs to cure chronic hepatitis B. During HBV replication, covalently closed circular DNA (cccDNA) serves as the template for viral replication and can be transcribed to produce five viral RNAs of 3.5, 2.4, 2.1 kb and 0.7 kb in length, which are translated to produce HBeAg, core protein, polymerase (P) protein, HBsAg and HBx proteins, respectively. Among them, the 3.5 kb pregenomic RNA (pgRNA) is also the template for viral reverse transcription. Polymerase protein recognizes and binds to the capsid assembly signal on the pgRNA to initiate capsid assembly and reverse transcription. Recent studies have revealed that the processes of splicing, nuclear export, stability, translation, and pgRNA encapsidation of HBV RNAs are regulated by a post-transcriptional regulatory network within the host cell and depend on unique post-transcriptional regulatory elements in the HBV RNA structure. The aim of this review is to overview the post-transcriptional regulatory mechanisms of HBV RNA and their applications in the study of HBV antiviral therapeutics, with the aim of providing new ideas for the development of new drugs targeting HBV RNA.

[Association between HBV viral load and severity of liver inflammation in patients with chronic hepatitis B virus infection].

Objective: To explore the relationship and dynamic changes between virological markers and hepatic pathological damage due to host anti-hepatitis B virus (HBV) immunity in the natural course of disease in chronic HBV infected patients. Methods: Two hundred and thirty-eight adult chronic HBV-infected patients who underwent liver biopsy from January 2016 to June 2022 in Taizhou Hospital, Zhejiang Province, were retrospectively selected. General clinical data such as age, gender, platelets, ALT, AST, albumin, HBV DNA, qHBsAg, HBeAg, and liver pathology diagnostic indexes such as the grade of liver necroinflammation and liver fibrotic stages of the patients were collected. The patients were grouped according to HBeAg status, and subgrouped according to different grades of liver necroinflammation and different HBV DNA loads. Statistical analyses were performed to compare the differences in HBV virologic marker levels between the groups, and the correlation between them and the indicators of hepatic inflammatory injury, such as ALT,AST, and the grade of liver necroinflammation in the patients. Results: The levels of HBV virological markers in HBeAg-positive patients with moderate or higher liver necroinflammatory grade (G≥2) were significantly lower than those with mild (no) liver necroinflammatory grade (G < 2) (P < 0.01); whereas the opposite trend was observed in HBeAg-negative patients, with the levels of HBV DNA, and qHBsAg in the G≥2 subgroup being significantly higher than those in the G < 2 subgroup (P < 0.01). Correspondingly, HBV DNA level and qHBsAg showed weak to moderately strong negative correlation with liver necroinflammatory grade and AST which was an indicator of hepatic inflammatory injury in HBeAg-positive patients (P < 0.05); whereas in HBeAg-negative patients, they showed weak to moderately strong positive correlation with hepatic inflammatory activity and ALT, AST (P < 0.001), in which qHBsAg showed only a weak positive correlation with patients' liver necroinflammatory grade (P = 0.003). Further subgroup analyses of HBeAg-positive patients according to whether the HBV DNA level was > 2×10(6) IU/ml showed weak to moderate negative correlations between HBV virological markers and liver necroinflammatory grade as well as ALT and AST in the subgroup of patients with HBV DNA > 2×10(6) IU/ml (P < 0.05); however, the negative correlation disappeared in patients who were still HBeAg positive and had HBV DNA ≤ 2×10(6) IU/ml. Moreover, HBV DNA and ALT, HBeAg and AST showed moderate positive correlation (P < 0.05). Conclusion: We speculate that the activation of host anti-HBV immunity can efficiently inhibit HBV replication by targeting the infected hepatocytes, but only in the early phase of disease progression in HBeAg positive patients with HBV DNA high (> 2×10(6) IU/ml).

[A preliminary discussion on carnosine dipeptidase 1 as a potential novel biomarker for the diagnostic and prognostic evaluation of hepatocellular carcinoma].

Objective: To explore carnosine dipeptidase 1 (CNDP1) potential value as a diagnostic and prognostic evaluator of hepatocellular carcinoma (HCC). Methods: A gene chip and GO analysis were used to screen the candidate marker molecule CNDP1 for HCC diagnosis. 125 cases of HCC cancer tissues, 85 cases of paracancerous tissues, 125 cases of liver cirrhosis tissues, 32 cases of relatively normal liver tissue at the extreme end of hepatic hemangioma, 66 cases from serum samples of HCC, and 82 cases of non-HCC were collected. Real-time fluorescent quantitative PCR, immunohistochemistry, western blot, and enzyme-linked immunosorbent assay were used to detect the differences in mRNA and protein expression levels of CNDP1 in HCC tissue and serum. Receiver operating characteristic (ROC) curves and Kaplan-Meier survival were used to analyze and evaluate the value of CNDP1 in the diagnosis and prognosis of HCC patients. Results: The expression level of CNDP1 was significantly reduced in HCC cancer tissues. The levels of CNDP1 were significantly lower in the cancer tissues and serum of HCC patients than those in liver cirrhosis patients and normal controls. ROC curve analysis showed that the area under the curve of serum CNDP1 in the diagnosis of HCC patients was 0.753 2 (95% CI 0.676-0.830 5), and the sensitivity and specificity were 78.79% and 62.5%, respectively. The combined detection of serum CNDP1 and serum alpha-fetoprotein (AFP) significantly improved the diagnostic accuracy (AUC = 0.820 6, 95% CI 0.753 5-0.887 8). The diagnostic sensitivity and specificity of serum CNDP1 for AFP-negative HCC patients were 73.68% and 68.75% (AUC = 0.793 1, 95% CI 0.708 8-0.877 4), respectively. In addition, the level of serum CNDP1 distinguished small liver cancer (tumor diameter < 3 cm) (AUC = 0.757 1, 95% CI 0.637 4-0.876 8). Kaplan-Meier survival analysis showed that CNDP1 was associated with a poor prognosis in HCC patients. Conclusion: CNDP1 may be a potential biomarker for the diagnostic and prognostic evaluation of HCC, and it has certain complementarity with serum AFP.

[Antisense oligodeoxynucleotides: the certain but limited efficacy and the uncovering mechanisms for the cure of chronic hepatitis B].

Recently, several phase I and phase II clinical trials of antisense oligodeoxynucleotides (ASOs) targeting to the commonly shared conserved sequences of HBV transcripts brought us some promising results. Particularly in the report of phase IIb clinical trial of Bepirovirsen (GSK3228836), approximately 9-10% patients with low baseline serum HBsAg (> 100 IU/ml & < 3 000 IU/ml) achieved functional cure after 24 weeks' of Bepirovirsen treatment. After reviewing the results of other clinical trials, one would be impressed to know that ALG-020572 (Aligos), RO7062931 (Roche) and GSK3389404 (GSK) all failed to sufficiently suppress serum HBsAg expression though the hepatocyte-targeted delivery of these ASOs were enhanced via N-acetyl galactosamine conjugation. Bepirovirsen enabled some patients to achieve sustained disappearance of serum HBsAg. The analysis of its distribution in different tissues of patients after drug administration showed that only a few fractions of ASOs entered liver tissues and far fewer eventually entered hepatocytes. Taking into consideration that only a few hepatocytes could be expected positive for HBsAg staining among these participants with low serum HBsAg level. We suspect that the mechanistic contribution of ASOs declining the serum HBsAg is not only via directly acting on the HBV transcripts in hepatocytes, but also via entering non-parenchymal cells such as Kupffer cells and resulting in stimulation and activation of innate immunity. Eventually the serum HBsAg declines in most participants and even disappears in a small fraction of patients with low baseline HBsAg level, via attack the infected hepatocytes evidenced by the aberrant elevation of ALT. Nevertheless, the functional cure of CHB remains a challenging issue and more efforts are needed.

IL-17A as a new circulating bioindicator for non-small cell lung cancer diagnosis and prognosis.

OBJECTIVE: Lung cancer (LC) is the highest contributor to cancer-associated mortality worldwide. Approximately 85% of all LC incidences involve non-small cell LC (NSCLC). Unfortunately, owing to a significant lack of sensitive and robust bioindicators, most patient diagnoses occur at advanced stages of the disease, thereby resulting in extremely poor patient outcomes. Herein, we elucidated the role of interleukin-17A (IL-17A) among NSCLC patients. MATERIALS AND METHODS: Circulating IL-17A content was measured using enzyme-linked immunosorbent assay (ELISA), and its diagnostic and prognostic abilities were assessed using the receiver operating characteristic (ROC) curve and Kaplan-Meier analysis, respectively. RESULTS: Our analysis revealed that circulating IL-17A levels were significantly augmented among NSCLC vs. control samples. Moreover, based on our area under the curve (AUC) analysis, circulating IL-17A levels fared considerably better than the standard bioindicator carcinoembryonic antigen (CEA) in both testing and validation cohorts. Notably, we also revealed that the circulating IL-17A levels were accurately and reliably predicted in early-stage NSCLC patients. Besides, we demonstrated a strong correlation between elevated circulating IL-17A expression and worse prognosis among NSCLC patients. CONCLUSIONS: Herein, we demonstrated that circulating IL-17A levels can serve as reliable and potent diagnostic and prognostic bioindicators for NSCLC.

[A study of the clinical curative effect of nucleos(t)ide analogues treated to pegylated interferon-α add-on therapy in patients with chronic hepatitis B].

Objective: To investigate the hepatitis B surface antigen (HBsAg) clearance condition and its predictive factors after treatment with nucleos(t)ide analogues to pegylated interferon-α add-on therapy in patients with chronic hepatitis B. Methods: Patients with chronic hepatitis B who visited the First Affiliated Hospital of Zhengzhou University from 2018~2019 were prospectively enrolled. HBsAg≤ 1500 IU/mL, hepatitis B e antigen-negative, HBV DNA undetectable, received antiviral treatment with nucleos(t)ide analogues for at least one year, and pegylated interferon-α add-on therapy for 48 weeks were included. The primary endpoint of study was to determine the proportion of HBsAg clearance at 72 weeks. Concurrently, the predictive factors for HBsAg clearance were analyzed. Quantitative and qualitative data were analyzed using a t-test or non-parametric test and a Fisher's exact test. Results: A total of 38 cases were included in this study, of which 13 cases obtained HBsAg clearance at 48 weeks of therapy and another six cases obtained HBsAg clearance throughout the extended treatment period of 72 weeks, accounting for 50.00% of all enrolled patients. There was a significant difference in HBsAg dynamics between the HBsAg clearance group and the non-clearance group (P < 0.05). Univariate logistic regression analysis showed that patients' age, baseline, 12-and 24-week HBsAg levels, and early HBsAg reduction were predictive factors for HBsAg clearance at 72 weeks of treatment. Multivariate logistic regression analysis showed that age (OR = 1.311; P = 0.016; 95% confidence interval: 1.051~1.635) and HBsAg levels at 24 weeks of treatment (OR = 4.481; P = 0.004; 95% confidence interval: 1.634~12.290) were independent predictors for HBsAg clearance. Conclusion: Hepatitis B e antigen-negative, nucleos(t)ide analogue treated, HBsAg ≤ 1500 IU/mL, and HBV DNA undetectable, peg-IFNα add-on treatment for 48 weeks could promote HBsAg clearance in patients with chronic hepatitis B. Six of the sixteen cases (37.50%) who did not obtain HBsAg clearance at week 48 did so with the course of therapy extended to week 72. Hence, the optimal individualized treatment strategy should be customized according to the predictors rather than the fixed 48-week course. Age (≤ 38), baseline HBsAg level (≤2.86 log(10)IU/ml), HBsAg level at 24 weeks (≤ 0.92 log(10)IU/ml), and 12-week HBsAg decrease from baseline (≥ 0.67 log(10)IU/ml) indicate that patients are highly likely to obtain HBsAg clearance at the 72 weeks of combination therapy, in which the combined indicator based on HBsAg level ≤0.92 log(10)IU/ml at 24 weeks will identify 85.0% to 100.0% of patients with HBsAg clearance.

[Some HBeAg-negative chronic hepatitis B patients treated with nucleos(t)ide analogue can achieve HBsAg loss after drug withdrawal: stop-to-cure may be coming].

Nucleoside/Nucleotide analogues (NAs) are widely used for the antiviral treatment of chronic hepatitis B (CHB), however, it is difficult to achieve serum hepatitis B surface antigen (HBsAg) loss with NAs therapy. In recent years, several prospective trails have reported that HBsAg loss (functional cure or clinical cure) also occurs in a small number of hepatitis B e antigen (HBeAg) negative CHB patients who discontinued long-term treatment with NAs. Accordingly, the "stop-to-cure" strategy is proposed. Although the mechanism has not been fully elucidated, the known factors related to serum HBsAg loss with NAs withdrawal include HBV genotype, duration of NAs treatment, serum HBsAg and HBV RNA levels at end-of-treatment, and ethnic differences. In the review, we discuss the best time to stop NAs therapy, the potential markers for predicting relapse after cessation of NAs and the possible mechanism of "stop-to-cure" in HBeAg-negative CHB patients, and propose some suggestions on the time of retreatment.

[Natural history and disease progression of chronic hepatitis B virus infection].

OBJECTIVE: To better understand and revise the natural history and disease progression of chronic hepatitis B virus (HBV) infection through analysis of a single-center large-scale cohort of indivi-duals with chronic HBV infection. METHODS: Patients with chronic HBV infection who had undergone liver biopsy in the Fifth Medical Center of Chinese People's Liberation Army (PLA) General Hospital from January 2014 to October 2020 were retrospectively recruited. Based on patient's hepatitis B e antigen (HBeAg) states and pathologic diagnosis, they were categorized into four disease progression statuses (or phases according to the old-terminology in the updated guidelines of chronic hepatitis B (CHB), such as European Association for the Study of the Liver (EASL) 2017, Clinical Practice Guidelines on the Management of Hepatitis B Virus Infection: HBeAg-positive chronic HBV infection (immune tolerance), HBeAg-positive CHB (immune active HBeAg positive), HBeAg-negative chronic HBV infection (inactive carrier), and HBeAg-negative CHB (immune reactive HBeAg negative). Then the demographic, laboratory tests and liver histological results of the patients in different disease progression stages were compared. Age differences between the two groups were evaluated using Mann-Whitney U test. RESULTS: A total of 760 eligible patients with a median age of 29 (interquartile range: 16-39) years were enrolled. Among them, 197 were underage individuals (age < 18 years) and 563 were adults; and 456 were males and 304 females. According to the pathological diagnosis, the patients were classified, and in each of the above four natural disease phases there were 173, 329, 95, and 163 individuals, respectively. Further comparison of the ages of the patients of the four disease progression statuses revealed that patients of HBeAg-negative CHB had a median age at 37 years, which was reasonably higher than those with HBeAg-positive CHB in immune active phase (37 vs. 24 years, P < 0.001), but was relatively younger than those with HBeAg-negative chronic HBV infection (37 vs. 39 years, P= 0.240). CONCLUSION: According to this study, it could be speculated that HBeAg-negative CHB patients probably not all reactivate from individuals of HBeAg-negative chronic HBV infection. Instead, certain HBeAg-negative CHB patients may also come from HBeAg-positive CHB patients who have undergone HBeAg clearance or seroconversion and still remain in the immune active state.

[Consideration on the possible etiological mechanisms and countermeasures about severe acute hepatitis of unknown origin in children].

Since April 2022, severe acute hepatitis of unknown origin in children has spread to 35 countries and regions around the world, and more than 1 010 cases have been reported. Since the severe acute hepatitis of unknown origin involves a wide range of areas and has a high rate, it is critical to identify the etiology and establish effective preventive, diagnostic and therapeutic measures as soon as possible. This study discusses the possible mechanisms and countermeasures of the severe acute hepatitis of unknown origin in children. It speculates that the occurrence of the recent severe acute hepatitis might be related to adenovirus, adeno-associated virus infection, and the COVID-19 epidemic, while the difference in HLA polymorphism among different races might be related to the fact that reported cases were more common in Europe and the United States. Based on the currently available evidence, it can be preliminarily judged that the risk of large-scale outbreak of severe acute hepatitis of unknown origin in children would be low in China, but the persistent awareness and vigilance of the etiology is still needed.

[Mechanism and clinical significance of HBV reactivation after anti-HCV therapy].

Direct-acting antivirals (DAAs) can strongly inhibit the replication of hepatitis C virus (HCV) and effectively clear the infection, but it may cause hepatitis B virus (HBV) reactivation, leading to severe liver damage and fulminate hepatitis in patients with HCV/HBV coinfection. In this review, we summarized the different replication process of HCV and HBV in infected hepatocytes and consequent innate immune response, and then discussed the molecular mechanism and clinical significance of HBV reactivation, and put forward the clinical precaution.

[Clinical application of serum Golgi protein 73 in patients with chronic liver diseases].

Golgi protein 73 (GP73) is a transmembrane protein on the Golgi apparatus and can be cut and released into the blood. In recent years, an increasing number of clinical studies have shown that the elevated serum GP73 level is closely related to liver diseases. And thus GP73 is expected to be used as a new serum marker for assessing progress of chronic liver diseases. Herein, the clinical application of serum GP73 in chronic hepatitis, liver fibrosis, liver cirrhosis and hepatocellular carcinoma with different etiologies was reviewed based on available literatures; and a research outlook in this field is made.

[A short half-life of cccDNA offer or ignite hope for hepatitis B cure under nucleos(t)ide analogues treatment].

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the template for HBV replication. Currently, there is a lack of therapeutic drugs that directly target cccDNA. Therefore, blocking cccDNA supplements as fast as possible and reducing the existing cccDNA is the key to achieving a complete cure of chronic hepatitis B. Previous studies have suggested that cccDNA had a long half-life, but a recent study showed that it only took a few months to update cycle of cccDNA pool, and its number was much less than previously predicted. In the future, with the advent of new antiviral drugs that can completely inhibit HBV replication, it is expected that the cccDNA pool will be completely cleared due to its supplement complete blockade, so as to achieve virological cure of chronic hepatitis B.

[The mechanisms of the translation of polymerase from HBV pregenomic RNA].

Hepatitis B virus (HBV) is an important pathogen that causes different liver diseases such as viral hepatitis and liver cirrhosis. HBV pregenomic RNA (pgRNA) plays a crucial role in HBV life cycle, which is not only the translation template of core (C) and polymerase (P), but also the template of reverse transcription. The ratio of P protein to core protein is tightly regulated. Since P and core are both translated by pgRNA and the open reading frame (ORF) of P is located downstream of the ORF of core, how to initiate P protein translation is a key scientific question. Previous studies suggest that P can be translated through different mechanisms, such as leaky scanning and reinitiation. In this review, we summarized the proposed mechanisms relevant to the translation of polymerase from HBV pgRNA through literature review and derivation.

[The Clinical Significance and Mechanism of the Effect for Hepatitis B Virus Protein on Host Immune].

The cytotoxic effect targeting hepatitis B virus (HBV) infected hepatocytes from virus-specific cytotoxic T cells and the neutralizing antibodies secreted by virus-specific B cells play an important role in the immune control and elimination of HBV. In patients with chronic hepatitis B, the liver immune microenvironment usually presents a suppression state, and virus-specific immune cells are mostly exhausted. Studies on the interaction between HBV and host immunity during infection, especially the influence of various viral proteins on immune cell function, will contribute to understanding the mechanism of the chronicity of HBV infection, disease progression, and optimization of immunotherapy against HBV. The review summarized the suppressive effects of HBV viral proteins on the host innate immunity and adaptive immune system, to help us understanding the mechanism(s) relevant to the observation that a CHB patient with HBeAg loss and lower HBsAg level is more likley achieving functionall cure. and expect to provide new sights for accelerate virus clearance and achieve functional cure of chronic hepatitis B, by removing the HBV viral proteins and consequently, liberting host immune from suppression state.

[A possible mechanism for low-level viremia occurrence in nucleos(t)ide analog-treated chronic hepatitis B patients].

The first-line nucleos(t)ide analogs (NAs) based antiviral drugs can effectively inhibit HBV replication and slow down the progression of chronic hepatitis B. However, about 20% of patients receiving standard NAs antiviral therapy will still develop low-level viremia (LLV). Therefore, understanding the occurrence mechanism of LLV will help to optimize antiviral treatment regimens and improve the prognosis of patients with chronic hepatitis B. This article systematically summarizes the possible mechanisms of LLV occurrence, and the important factor of NAs failure. Taking into account the unique limitations of NAs competitive inhibition of virus replication, weakening host's immune response is not enough to directly eliminate infected hepatocytes. This makes it difficult to achieve a complete virological response in some patients with the active compensatory proliferation of residual infected hepatocytes and the accompanying effective removal or dilution of covalent, closed, circular DNA (cccDNA) pools. Therefore, it is speculated that activating host immunity can eliminate infected liver cells and may be more conducive to address LLV.

[Application of the new serum markers in the research and development of innovative hepatitis B drugs].

Chronic hepatitis B (CHB) poses a serious threat to the health of the Chinese people. Viral markers are of great significance during antiviral therapy and research and development of innovative drugs. However, traditional serovirological markers such as HBV DNA and hepatitis B surface antigen (HBsAg) have certain limitations in assessing the efficacy of antiviral treatment and accurately reflecting the transcriptional activity of covalently closed circular DNA (cccDNA). In recent years, the role of new viral markers such as HBV RNA, hepatitis B core-related antigen (HBcrAg) and quantitative anti-hepatitis B core protein (qAnti-HBc) have attracted more and more attention because of accurately reflecting the transcription activity of cccDNA, determining the therapeutic effect and predicting the risk of recurrence after drug discontinuation. This article briefly introduces the application of new and old markers in various stages of antiviral therapy in CHB patients, and combines the application of new viral markers in the evaluation of drug efficacy, and expounds its possible role in the research and development of new antiviral therapy.

[Diagnostic value of liver stiffness measurement for the evaluation of liver fibrosis in nonalcoholic fatty liver].

Objective: To investigate the clinical and diagnostic value of liver stiffness measurement (LSM) for the evaluation and comparison of aspartate aminotransferas/platelet ratio index (APRI), fibrosis 4 indexes (FIB-4) and NAFLD fibrosis score (NFS) with liver fibrosis staging in relation to nonalcoholic fatty liver disease (NAFLD). Methods: 103 cases with NAFLD who met the inclusion criteria confirmed by liver biopsy were selected for retrospective analysis. The results of serological tests and LSM were recorded. The APRI, FIB-4 and NFS were calculated. The accuracy and applicability of four liver fibrosis models in the diagnosis of liver fibrosis in NAFLD patients were compared with the receiver operating characteristic curve (ROC), and the diagnostic cut-off value of LSM was established. Results: Varying degrees of LSM, APRI, FIB-4 and NFS had shown positive correlations with the increasing degree of liver fibrosis. Among them, LSM was positively correlated with the degree of liver fibrosis, and the correlation coefficient was r = 0.727, P < 0.0001. Consistent with this, the area under the receiver operating characteristic curve, sensitivity, and specificity of LSM diagnosis of liver fibrosis in different stages was significantly higher than APRI, FIB-4 and NFS. Area under receiver operating characteristic curve of LSM was 0.862 and 0.928 for significant liver fibrosis (f ≥ 2), and advanced liver fibrosis (f ≥ 3). Conclusion: LSM has a good diagnostic exclusion value for NAFLD-induced fibrosis, and its sensitivity and specificity are better than APRI, FIB-4 and NFS.

[Exploring the mechanism of liver enzyme abnormalities in patients with novel coronavirus-infected pneumonia].

Objective: To explore and analyze the possible mechanism of liver injury in patients with coronavirus disease 2019 (novel coronavirus pneumonia, NCP). Methods: The correlation between ALT, AST and other liver enzyme changes condition and NCP patients' disease status reported in the literature was comprehensively analyzed. ACE2 expression in liver tissue for novel coronavirus was analyzed based on single cell sequencing (GSE115469) data. RNA-Seq method was used to analyze Ace2 expression and transcription factors related to its expression in liver tissues at various time-points after hepatectomy in mouse model of acute liver injury with partial hepatectomy. t-test or Spearman rank correlation analysis was used for statistical analysis. Results: ALT and AST were abnormally elevated in some patients with novel coronavirus infection, and the rate and extent of ALT and AST elevation in severe NCP patients were higher than those in non-severe patients. Liver tissue results of single cell sequencing and immunohistochemistry showed that ACE2 was only expressed in bile duct epithelial cells of normal liver tissues, and very low in hepatocytes. In a mouse model of acute liver injury with partial hepatectomy, Ace2 expression was down-regulated on the first day, but it was elevated up to twice of the normal level on the third day, and returned to normal level on seventh day when the liver recovered and hepatocyte proliferation stopped. Whether this phenomenon suggests that the bile duct epithelial cells with positive expression of Ace2 participate in the process of liver regeneration after partial hepatectomy deserves further study. In RNA-Seq data, 77 transcription factors were positively correlated with the expression of Ace2 (r > 0.2, FDR < 0.05), which were mainly enriched in the development, differentiation, morphogenesis and cell proliferation of glandular epithelial cells. Conclusion: We assumed that in addition to the over activated inflammatory response in patients with NCP, the up-regulation of ACE2 expression in liver tissue caused by compensatory proliferation of hepatocytes derived from bile duct epithelial cells may also be the possible mechanism of liver tissue injury caused by 2019 novel coronavirus infection.

[Establishment and preliminary application of serum Golgi protein 73 based noninvasive diagnostic model for compensated stage hepatitis B cirrhosis].

Objective: To establish and evaluate diagnostic efficacy and applicability of serum Golgi protein (GP) 73 based non-invasive diagnostic model with other conventional serological indicators for compensated stage hepatitis B cirrhosis. Methods: 666 cases with chronic hepatitis B (CHB) who had visited to the Fifth Medical Center of People's Liberation Army General Hospital from January 2010 to December 2017 were selected as the study subjects, and were classified according to compensated stage cirrhosis into clinical and pathological diagnosis group based on whether or not the liver histological examination was performed. A diagnostic model of compensated stage hepatitis B cirrhosis in the clinical diagnosis group was established. The current clinically used diagnostic model of liver cirrhosis, aspartate aminotransferase/platelet ratio index (APRI), fibrosis index (FIB)-4 and liver stiffness measurement (LSM) were compared. Eventually, the diagnostic model was verified step by step by pathological diagnosis group. Results: The area under the receiver operating characteristic curve (AUC) of GP73 and APRI, FIB-4, and LSM for cirrhosis patients in the clinical diagnosis group were 0.842, 0.857, 0.864, and 0.832, respectively. The diagnostic efficiency of the four indicators were of similar (P value > 0.05). A diagnostic model of compensated stage hepatitis B cirrhosis (GAPA) using logistic regression analysis was established: LogitP = 1/ [1 + exp (1.614-0.054 × GP73-0.045 × Age + 0.030 × PLT-0.015 × ALP)]. The AUC of the model was as high as 0.940 and the optimal cut-off value were 0.41. The corresponding diagnostic sensitivity and specificity were 0.92 and 0.82, respectively. The diagnostic efficiency was better than that of APRI, FIB-4, LSM and GP73 alone (P < 0.05). The AUC of GAPA was 0.877 in the pathological diagnosis group, which was similar to the diagnostic efficacy of LSM (0.891) and FIB-4 (0.847) (P > 0.1), but still superior to that of APRI (0.811) and GP73 alone (0.780) (P < 0.001). Conclusion: GAPA, a diagnostic model for compensated stage hepatitis B cirrhosis established in this study, has a good diagnostic efficacy in both the clinical and pathological diagnosis group, and has certain auxiliary diagnostic value in the areas where resources are relatively scarce or where LSM has not been developed.

[Exosome-mediated CRISPR/Cas9 system targets to cut the intercellular transmission function of hepatitis B virus genome].

Objective: To determine whether single-stranded guided RNA (gRNA) and Cas9 protein exist in the exosome secreted by cells transfected with CRISPR/Cas9 expression plasmid. Furthermore, to explore whether CRISPR/Cas9 system can edit target genes of peripheral cells through the intercellular transmission of exosomes. Methods: (1) The CRISPR/Cas9 expression plasmid was transfected into HuH7 cells. The supernatant was collected and the exosomes were concentrated and purified by differential centrifugation. The morphology and particle size of exosomes were determined by electronic microscopy and Malvern laser scatter granulometry. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the levels of gRNA and Cas9 protein. (2) HuH7 cells transfected with pBB4.5 1.2×HBV and HBV specific CRISPR/Cas9 expression plasmids were co-cultured. After 2 days, HBV DNA was extracted and sequenced by PCR. Results: There were not only full-length gRNA and Cas9 protein in the exosomes of Huh7 cells transfected with CRISPR / Cas9 expression plasmid. In addition, gene-editing functions were delivered between the cells to achieve the destruction of HBV genome of surrounding cells. Conclusion: The CRISPR-Cas9 gene-editing system has the potential to deliver exosomes between cells via carrying functional gRNA and Cas9 proteins. This phenomenon hints that we are in the process of using the CRISPR/Cas9 system for gene therapy. Therefore, it is necessary to consider the potential effects of exosomes-carried gRNA and Cas9 proteins on the surrounding cells of the distal tubules.