Serum M2BPGi level is a novel predictive biomarker for the responses to pegylated interferon-α treatment in HBeAg-positive chronic hepatitis B patients.

Serum Mac-2-binding protein glycosylation isomer (M2BPGi) level was found to be a useful prognostic marker for hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients treated with nucleoside/nucleotide analogs (NUCs) therapy, and the aim of our study is to evaluate the clinical implementation of M2BPGi level in the prediction of antiviral responses to pegylated-interferon-α (PEG-IFN-α) treatment in HBeAg-positive CHB patients. Ninety-six CHB patients who received PEG-IFN-α treatment for at least 48 weeks were recruited. The serum M2BPGi, alanine aminotransferase (ALT), hepatitis B surface antigen (HBsAg), HBeAg, and HBV DNA levels at baseline, weeks 4, 12, and 24 after PEG-IFN-α treatment were determined and their associations with antiviral responses were evaluated and the virological response (VR) rate and serological response (SR) rate after 48 weeks of treatment were 65.6% and 35.4%, respectively. Baseline serum M2BPGi level was significantly different between VR and non-VR (P = 0.002) or SR and non-SR groups (P = 0.012). Multivariate analyses suggested that baseline serum M2BPGi level was independently associated with VR and SR of PEG-IFN-α treatment at week 48. The area under the ROC curve (AUC) of baseline M2BPGi was 0.682 in predicting VR, which was superior to HBsAg (AUC = 0.566) or HBV DNA (AUC = 0.567). The AUC of baseline M2BPGi in predicting SR was 0.655, which was also higher than that of HBsAg (AUC = 0.548) or HBV DNA (AUC = 0.583). These results suggested that baseline serum M2BPGi level was a novel predictor of VR and SR for PEG-IFN-α treatment in HBeAg-positive CHB patients.

Serum WFA+ -M2BP levels for evaluation of early stages of liver fibrosis in patients with chronic hepatitis B virus infection.

BACKGROUND & AIMS: Accurate evaluation of liver fibrosis is crucial for predicting progression of chronic hepatitis B virus (HBV) infection. We assessed the utility of a novel fibrosis glycobiomarker Wisteria floribunda agglutinin-positive Mac-2-binding protein (WFA+ -M2BP) for evaluating liver fibrosis and disease progression in patients with chronic HBV infection. METHODS: We enrolled 774 patients with chronic HBV infection, with or without fibrosis, diagnosed by liver biopsy/FibroScan. Patients who underwent liver biopsy (n = 297) were divided into training (n = 221) and validation (n = 76) groups. Serum WFA+ -M2BP values were measured and compared with FIB-4 index, aspartate aminotransferase (AST)-to-platelet ratio (APRI) and AST-to-alanine aminotransferase ratio (AAR) using receiver-operating characteristic (ROC) analysis. RESULTS: Serum WFA+ -M2BP levels increased significantly with fibrosis progression (P < 0.0001). Area under the ROC curve of WFA+ -M2BP for diagnosing significant fibrosis was higher than that of FIB-4 (P = 0.198), APRI (P = 0.017) and AAR (P < 0.001), with sensitivity and specificity in the training set of 60.5% and 79.8% and validation set of 59.5% and 82.1%, respectively. Serum WFA+ -M2BP levels were significantly correlated with FibroScan values (P < 0.0001) and improved the accuracy of FibroScan in assessing significant fibrosis. Changes in WFA+ -M2BP levels were parallel with those in FibroScan values during nucleot(s)ide analogues therapy in patients with chronic HBV infection. CONCLUSIONS: WFA+ -M2BP is an accurate serum indicator for assessing early stages of liver fibrosis and may monitor regression of fibrosis during the treatment of chronic HBV infection. WFA+ -M2BP provides a simple and reliable alternative or complementary method to liver biopsy and FibroScan.

[Laparoscopic percutaneous extraperitoneal closure does not affect vas deferens orientation or testicular volume and perfusion].

OBJECTIVE: To investigate the influence of single-port laparoscopic percutaneous extraperitoneal closure (LPEC) on the orientation of the vas deferens and the volume and perfusion of the testis in pediatric patients undergoing inguinal hernia repair. METHODS: A total of 92 consecutively enrolled boys diagnosed with unilateral inguinal hernia underwent single-port LPEC between June 2013 and June 2014. The orientation of the vas deferens and the testicular volume and perfusion of the patients were ultrasonographically assessed preoperatively and at 1 and 6 months after surgery. RESULTS: All the surgical procedures were performed successfully without conversion or serious perioperative complications. Ultrasonography showed no angulation or distortion of the vas deferens on the surgical side during a six-month follow-up period. Similarly, no obvious changes were observed in the testicular volume or perfusion. CONCLUSIONS: Single-port LPEC is safe and effective in the treatment of pediatric inguinal hernia and does not affect the orientation of the vas deferens or testicular volume and perfusion.

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection.

Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T- and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated.

[Functional analysis of hepatitis B virus immune escape mutants with insertion mutations in the surface antigen].

OBJECTIVE: To evaluate the influence of insertion mutations occurring in the hydrophobic region, between amino acids 114 and 115, of the hepatitis B surface antigen (HBsAg) on viral antigenicity and replication. METHODS: Hepatitis B virus (HBV) DNA was obtained from patients with HBsAg-positive chronic hepatitis B (CHB) infection and subjected to sequence analysis and comparison to GenBank reference sequences for HBV genotype B (AB073826) and genotype C (AF286594). Insertion mutations detected in the HBsAg region were used to make recombinant expression plasmids via site-directed mutagenesis. After transfecting the recombinant HBsAg into Huh7 cells, the mutants' effects on viral antigenicity and replication were evaluated by chemiluminescence microparticle immunoassay (CMIA) and Southern blot hybridization, respectively. The viral antigenicity of each mutant was predicted by bioinformatic analysis, using the Jameson-Wolf method to predict the antigenic index, the Hopp-Woods method to predict hydrophilicity, the Emini method to predict the probability of a region lying of the protein's surface, and the Karplus-Schulz method to predict the flexibility of the protein backbone. RESULTS: Two CHB patients harbored HBV with insertion mutations in HBsAg: one with two (NT) and one with three (NTT) inserted amino acids between 114 and 115. The NTT recombinant HBsAg mutant showed no impact on viral replication and reacted weakly with anti-HBs in CMIA (P = 0.02). The antigen indices for the insertion of NTT were 1.00, -0.16, and 0.18, and insertion of the three amino acids affected the index values of five proximal amino acid sites (with an average increase of 0.13). The hydrophilic indices for the insertion of NTT were 0.2, -0.4, and -0.4, with no significant effect on the proximal amino acids. The insertion of the three amino acids changed both the surface probability (range: -0.55 to 2.97; affecting eight proximal amino acids) and the flexibility (range: -0.01 to 1.1; affecting five proximal amino acids). CONCLUSION: The NTT three amino acids insertion in the HBsAg open reading frame, between 114 and 115 of the wild-type sequence, detected in a CHB patient may play a role in HBV immune escape without influencing the viral replicative capacity.

MxA induction may predict sustained virologic responses of chronic hepatitis B patients with IFN-alpha treatment.

The objective of this study was to find potential biomarkers for predicting sustained virologic responses to interferon-alpha (IFN-alpha) treatment in chronic hepatitis B (CHB) patients. A total of 101 CHB patients were treated with pegylated IFN-alpha2a for 48 weeks and followed up for 24 weeks, including 34 IFN responders (IFN-Rs) and 67 IFN nonresponders (IFN-NRs). After peripheral blood mononuclear cells (PBMCs) and Epstein-Barr virus-transferred B (EBV-B) cell lines were treated with different concentrations of IFN-alpha in vitro, activated IFN-stimulated gene factor3 (ISGF3) and IFN-gamma-activation factor (GAF) were measured by EMSA, and MxA, OAS1, and PKR mRNA were measured by real-time PCR. Polymorphisms in the MxA promoter were genotyped to find the possible association. IFN-alpha-activated ISGF3 and GAF levels were similar between IFN-NRs and IFN-Rs. However, MxA mRNA induction in IFN-Rs was higher than that in IFN-NRs, and such discrepancy increased when highly concentrated IFN was used to stimulate. The OAS1 and PKR mRNA induction have a similar pattern between IFN-Rs and IFN-NRs. In addition, frequency of the MxA-88G/T genotype was significantly different between IFN-Rs and IFN-NRs, and this polymorphism was also functional because MxA mRNA induction in patients with GG genotype was lower than those with GT genotype. Regression analysis showed that MxA mRNA induction after 10,000 IU/mL IFN stimulation could serve as an independent factor for predicting IFN-alpha, with an area under curve (AUC) of 0.838, a positive predictive value of 68% for IFN-Rs, and a negative predictive value of 89% for IFN-NRs. MxA mRNA induced by IFN-alpha might predict sustained virologic responses to IFN-alpha treatment in CHB patients.

Logic Synthesis of Recombinase-Based Genetic Circuits.

A synthetic approach to biology is a promising technique for various applications. Recent advancements have demonstrated the feasibility of constructing synthetic two-input logic gates in Escherichia coli cells with long-term memory based on DNA inversion induced by recombinases. Moreover, recent evidences indicate that DNA inversion mediated by genome editing tools is possible. Powerful genome editing technologies, such as CRISPR-Cas9 systems, have great potential to be exploited to implement large-scale recombinase-based circuits. What remains unclear is how to construct arbitrary Boolean functions based on these emerging technologies. In this paper, we lay the theoretical foundation formalizing the connection between recombinase-based genetic circuits and Boolean functions. It enables systematic construction of any given Boolean function using recombinase-based logic gates. We further develop a methodology leveraging existing electronic design automation (EDA) tools to automate the synthesis of complex recombinase-based genetic circuits with respect to area and delay optimization. In silico experimental results demonstrate the applicability of our proposed methods as a useful tool for recombinase-based genetic circuit synthesis and optimization.

Reconfigurable neuromorphic computation in biochemical systems.

Implementing application-specific computation and control tasks within a biochemical system has been an important pursuit in synthetic biology. Most synthetic designs to date have focused on realizing systems of fixed functions using specifically engineered components, thus lacking flexibility to adapt to uncertain and dynamically-changing environments. To remedy this limitation, an analog and modularized approach to realize reconfigurable neuromorphic computation with biochemical reactions is presented. We propose a biochemical neural network consisting of neuronal modules and interconnects that are both reconfigurable through external or internal control over the concentrations of certain molecular species. Case studies on classification and machine learning applications using the DNA strain displacement technology demonstrate the effectiveness of our design in both reconfiguration and autonomous adaptation.

Synthesizing Configurable Biochemical Implementation of Linear Systems from Their Transfer Function Specifications.

The ability to engineer synthetic systems in the biochemical context is constantly being improved and has a profound societal impact. Linear system design is one of the most pervasive methods applied in control tasks, and its biochemical realization has been proposed by Oishi and Klavins and advanced further in recent years. However, several technical issues remain unsolved. Specifically, the design process is not fully automated from specification at the transfer function level, systems once designed often lack dynamic adaptivity to environmental changes, matching rate constants of reactions is not always possible, and implementation may be approximative and greatly deviate from the specifications. Building upon the work of Oishi and Klavins, this paper overcomes these issues by introducing a design flow that transforms a transfer-function specification of a linear system into a set of chemical reactions, whose input-output response precisely conforms to the specification. This system is implementable using the DNA strand displacement technique. The underlying configurability is embedded into primitive components and template modules, and thus the entire system is adaptive. Simulation of DNA strand displacement implementation confirmed the feasibility and superiority of the proposed synthesis flow.

Serum HBeAg sero-conversion correlated with decrease of HBsAg and HBV DNA in chronic hepatitis B patients treated with a therapeutic vaccine.

Currently, there are various approaches for developing therapeutic vaccines for chronic hepatitis B patients. Previously, an antigen-antibody-based therapeutic vaccine (YIC) has been conducted in a double-blind placebo controlled phase IIb clinical trial in 242 chronic hepatitis B patients. At the end of follow-up for 24 weeks, HBeAg sero-conversion rate was 21.6% in the 60 µg immunized group, compared to 9% in the alum immunized control group (p=0.03). To analyze the correlation between HBeAg-seroconversion, and decrease of serum HBsAg and HBV DNA, serum samples were back quantified for serum HBsAg and HBV DNA collected at baseline, end of treatment, and end of follow-up from patients who were treated either with 60 µg of YIC, or with placebo. Patients were dichotomized to HBeAg sero-converted and non-converted groups in comparison with patients in the placebo group. The correlations between HBeAg seroconversion and the decrease of HBsAg, HBV DNA and ALT levels during study period were analyzed using a logistic regression model. Results showed marked and sustained reduction of HBsAg, HBV DNA and ALT level in HBeAg sero-converted patients compared to those in patients of HBeAg non-converted and placebo groups. Reduction of HBV DNA and elevation of ALT was markedly associated with HBeAg seroconversion with an adjusted OR of 0.09 (95%CI: 0.01-0.62) and 0.08 (95%CI: 0.02-0.37) respectively after adjusted by age and sex, while reduction of HBsAg level was close to of significance (p=0.054). Analysis indicated that HBeAg sero-conversion was a reasonable endpoint for therapeutic vaccination.

Polymorphisms of microsomal triglyceride transfer protein in different hepatitis B virus-infected patients.

AIM: To identify the two polymorphisms of microsomal triglyceride transfer protein (MTP) gene in the Chinese population and to explore their correlation with both hepatitis B virus (HBV) self-limited infection and persistent infection. METHODS: A total of 316 subjects with self-limited HBV infection and 316 patients with persistent HBV infection (195 subjects without familial history), matched with age and sex, from the Chinese Han population were enrolled in this study. Polymorphisms of MTP at the promoter region -493 and at H297Q were determined by the allele specific polymerase chain reaction (PCR). RESULTS: The ratio of males to females was 2.13:1 for each group and the average age in the self-limited and chronic infection groups was 38.36 and 38.28 years, respectively. None of the allelic distributions deviated significantly from that predicted by the Hardy-Weinberg equilibrium. There was a linkage disequilibrium between H297Q and -493G/T (D' = 0.77). As the c2 test was used, the genotype distribution of MTP-493G/T demonstrated a significant difference between the self-limited infection group and the entire chronic group or the chronic patients with no family history (c2 = 8.543, P = 0.015 and c2 = 7.199, P = 0.019). The allele distribution at the MTP-493 position also demonstrated a significant difference between the study groups without family history (c2 = 6.212, P = 0.013). The T allele emerged as a possible protective factor which may influence the outcomes of HBV infection (OR: 0.59; 95% CI: 0.389-0.897). CONCLUSION: The polymorphism of the MTP gene, T allele at -493, may be involved in determining the HBV infection outcomes, of which the mechanism needs to be further investigated.