Risk of hepatocellular carcinoma in antiviral treatment-naïve chronic hepatitis B patients treated with entecavir or tenofovir disoproxil fumarate: a network meta-analysis.

BACKGROUND: Long-term antiviral treatments are associated with a significantly lower hepatocellular carcinoma (HCC) incidence in chronic hepatitis B (CHB) patients by reducing HBV DNA concentrations. However, it is still controversial whether antiviral strategies affect HCC development in antiviral treatment-naïve CHB patients. This study aimed to estimate the incidence of HCC in antiviral treatment-naïve CHB patients who were treated with Entecavir (ETV) and Tenofovir Disoproxil Fumarate (TDF) and compare the efficacy of two treatment regimens in HCC reduction. METHODS: The PubMed, Embase, China National Knowledge Infrastructure, and Wanfang databases were systematically searched until June 24, 2021. The pooled incidence and 95% confidence interval of HCC were calculated by the Freeman-Tukey double arcsine transformation method. The efficacies of ETV and TDF treatments in HCC reduction were compared through a network meta-analysis. RESULTS: A total of 27 studies were identified as eligible for this systematic review. The incidence densities in the ETV and TDF treatment groups were 2.78 (95% CI: 2.21-3.40) and 2.59 (95% CI: 1.51-3.96) per 100 persons-year among patients with preexisting cirrhosis and 0.49 (95% CI: 0.32-0.68) and 0.30 (95% CI: 0.06-0.70) per 100 persons-year among patients without preexisting cirrhosis. As the proportion of CHB patients with preexisting cirrhosis increased, the incidence density of HCC also increased gradually. Compared with other Nucleos(t)ide analogs (NAs) treatments, ETV and TDF treatments significantly lowered the risk of HCC, with hazard ratios (HRs) of 0.60 (95% CI: 0.40-0.90) and 0.56 (95% CI: 0.35-0.89), respectively. However, there was no difference in the incidence density of HCC between ETV and TDF treatments (HR = 0.92, 95% CI: 0.71-1.20) regardless of preexisting cirrhosis. CONCLUSION: ETV and TDF treatments were associated with significantly lower risks of HCC than other NAs treatments. However, no difference was observed between ETV and TDF treatments in the risk of HCC development regardless of preexisting cirrhosis among treatment-naïve CHB patients.

A unique B cell epitope-based particulate vaccine shows effective suppression of hepatitis B surface antigen in mice.

OBJECTIVE: This study aimed to develop a novel therapeutic vaccine based on a unique B cell epitope and investigate its therapeutic potential against chronic hepatitis B (CHB) in animal models. METHODS: A series of peptides and carrier proteins were evaluated in HBV-tolerant mice to obtain an optimised therapeutic molecule. The immunogenicity, therapeutic efficacy and mechanism of the candidate were investigated systematically. RESULTS: Among the HBsAg-aa119-125-containing peptides evaluated in this study, HBsAg-aa113-135 (SEQ13) exhibited the most striking therapeutic effects. A novel immunoenhanced virus-like particle carrier (CR-T3) derived from the roundleaf bat HBV core antigen (RBHBcAg) was created and used to display SEQ13, forming candidate molecule CR-T3-SEQ13. Multiple copies of SEQ13 displayed on the surface of this particulate antigen promote the induction of a potent anti-HBs antibody response in mice, rabbits and cynomolgus monkeys. Sera and purified polyclonal IgG from the immunised animals neutralised HBV infection in vitro and mediated efficient HBV/hepatitis B virus surface antigen (HBsAg) clearance in the mice. CR-T3-SEQ13-based vaccination induced long-term suppression of HBsAg and HBV DNA in HBV transgenic mice and eradicated the virus completely in hydrodynamic-based HBV carrier mice. The suppressive effects on HBsAg were strongly correlated with the anti-HBs level after vaccination, suggesting that the main mechanism of CR-T3-SEQ13 vaccination therapy was the induction of a SEQ13-specific antibody response that mediated HBV/HBsAg clearance. CONCLUSIONS: The novel particulate protein CR-T3-SEQ13 suppressed HBsAg effectively through induction of a humoural immune response in HBV-tolerant mice. This B cell epitope-based therapeutic vaccine may provide a novel immunotherapeutic agent against chronic HBV infection in humans.

The Role of Psychological Stress on Heart Autophagy in Mice With Heart Failure.

OBJECTIVE: Psychological stress in chronic heart failure (CHF) is associated with systemic neurohormonal and immune system responses and increased mortality. Autophagy refers to the biological process of degradation and recycling of dysfunctional cellular components. We investigated the role of psychological stress on autophagy function in CHF mice. METHODS: C57BL/6 mice underwent transverse aortic constriction, with or without combined acoustic and restraint stress, and cardiac function was assessed by echocardiography analysis. Serum corticosterone and angiotensin II (Ang II) were determined using enzyme-linked immunosorbent assay (ELISA). Autophagy and oxidative stress were measured with immunohistochemistry and quantitative polymerase chain reaction, and chloroquine and rapamycin were used to detect autophagy flux. In vivo, cardiomyocytes were cultured with or without Ang II or N-acetylcysteine, and autophagy and oxidative stress were also detected. RESULTS: A 1-week stress exposure significantly increased serum levels of corticosterone and Ang II (p = .000), increased levels of oxidative stress, induced overt heart failure, and increased mortality (p = .002). Furthermore, stress exposure unregulated messenger RNA expression of Bcl-2-interacting coiled-coil protein 1 (10.891 [3.029] versus 4.754 [1.713], p = .001), cysteine-rich domain containing beclin-1 interacting (6.403 [1.813] versus 3.653 [0.441], p = .006), and autophagy 7 (111.696 [4.049] versus 6.189 [1.931], p = .017), increased expression of autophagosomal, and decreased clearance of autophagosomes. In vitro, Ang II significantly increased autophagy flux in cultured cardiomyocytes, which could be partly inhibited by N-acetylcysteine. CONCLUSIONS: Psychological stress may contribute to the development of CHF by enhancing heart oxidative stress and impairing autophagy flux.

Rapid fluorescent lateral-flow immunoassay for hepatitis B virus genotyping.

Hepatitis B virus (HBV) genotyping plays an important role in the clinical management of chronic hepatitis B (CHB) patients. However, the current nucleic acid based techniques are expensive, time-consuming, and inconvenient. Here, we developed a novel DNA-independent HBV genotyping tool based on a one-step fluorescent lateral flow immunoassay (LFIA). Epitope-targeting immunization and screening techniques were used to develop HBV genotype specific monoclonal antibodies (mAbs). These mAbs were used to develop a multitest LFIA with a matched scanning luminoscope for HBV genotyping (named the GT-LFIA). The performance of this novel assay was carefully evaluated in well-characterized clinical cohorts. The GT-LFIA, which can specifically differentiate HBV genotypes A, B, C, and D in a pretreatment-free single test, was successfully developed using four genotype specific mAbs. The detection limits of the GT-LFIA for HBV genotypes A, B, C, and D were 2.5-10.0 IU HBV surface antigen/mL, respectively. Among the sera from 456 CHB patients, 439 (96.3%; 95% confidence interval (CI), 94.1-97.8%) were genotype-differentiable by the GT-LFIA and 437 (99.5%; 95% CI, 98.4-99.9%) were consistent with viral genome sequencing. In the 21 patients receiving nucleos(t)ide analogue therapy, for end-of-treatment specimens that were HBV DNA undetectable and were not applicable for DNA-dependent genotyping, the GT-LFIA presented genotyping results that were consistent with those obtained in pretreatment specimens by viral genome sequencing and the GT-LFIA. In conclusion, the novel GT-LFIA is a convenient, fast, and reliable tool for differential HBV genotyping, especially in patients with low or undetectable HBV DNA levels.

Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice.

Autophagic dysfunction is observed in diabetes mellitus. Resveratrol has a beneficial effect on diabetic cardiomyopathy. Whether the resveratrol-induced improvement in cardiac function in diabetes is via regulating autophagy remains unclear. We investigated the mechanisms underlying resveratrol-mediated protection against heart failure in diabetic mice, with a focus on the role of sirtuin 1 (SIRT1) in regulating autophagic flux. Diabetic cardiomyopathy in mice was induced by streptozotocin (STZ). Long-term resveratrol treatment improved cardiac function, ameliorated oxidative injury and reduced apoptosis in the diabetic mouse heart. Western blot analysis revealed that resveratrol decreased p62 protein expression and promoted SIRT1 activity and Rab7 expression. Inhibiting autophagic flux with bafilomycin A1 increased diabetic mouse mortality and attenuated resveratrol-induced down-regulation of p62, but not SIRT1 activity or Rab7 expression in diabetic mouse hearts. In cultured H9C2 cells, redundant or overactive H2O2 increased p62 and cleaved caspase 3 expression as well as acetylated forkhead box protein O1 (FOXO1) and inhibited SIRT1 expression. Sirtinol, SIRT1 and Rab7 siRNA impaired the resveratrol amelioration of dysfunctional autophagic flux and reduced apoptosis under oxidative conditions. Furthermore, resveratrol enhanced FOXO1 DNA binding at the Rab7 promoter region through a SIRT1-dependent pathway. These results highlight the role of the SIRT1/FOXO1/Rab7 axis in the effect of resveratrol on autophagic flux in vivo and in vitro, which suggests a therapeutic strategy for diabetic cardiomyopathy.

Right ventricular volume and function by three-dimensional echocardiography: results of the echocardiographic measurements in normal Chinese adults (EMINCA) II.

Three-dimensional (3D) echocardiography is an emerging technique for assessing right ventricular (RV) volume and function, but 3D-RV normal values from a large Chinese population are still lacking. The aim of the present study was to establish normal values of 3D-RV volume and function in healthy Chinese volunteers. A total of 1117 Han Chinese volunteers from 28 laboratories in 20 provinces of China were enrolled, and 3D-RV images of 747 volunteers with optimal image quality were ultimately analyzed by a core laboratory. Both vendor-dependent and vendor-independent software platforms were used to analyze the 3D-RV images. We found that men had larger RV volumes than women did in the whole population, even after indexing to body surface area, and older individuals had smaller RV volumes. The normal RV volume was significantly smaller than that recommended by the American Society of Echocardiography/European Association of Cardiovascular Imaging guidelines in both sexes. There were significant differences in 3D-RV measurements between the two vendor ultrasound systems and the different software platforms. The echocardiographic measurements in normal Chinese adults II study revealed normal 3D-RV volume and function in a large Chinese population, and there were significant differences between the sexes, ages, races, and vendor groups. Thus, normal 3D-RV values should be stratified by sex, age, race, and vendor.

Profile of clinical characteristics and serologic markers of sporadic hepatitis E in a community cohort study.

Hepatitis E virus (HEV) is a pathogen of global significance, but the value of HEV-related markers in the diagnosis of hepatitis E remains controversial. Previous studies on hepatitis E profiles have been mainly cross-sectional and conducted among inpatients in large hospitals, and hepatitis E cases have been primarily defined by limited partial markers. In this community-based study, 4,110 active hepatitis cases from a population of nearly 600,000 were followed over 48 months and serial serum samples were collected. Both HEV pathogen (HEV RNA and antigen) and anti-HEV antibody markers were used to determine HEV infection status and the relationship between hepatitis and HEV infection. In total, 98 hepatitis E patients were identified and all available isolates from 58 patients belonged to HEV genotype 4. The mean age of the patients was 58.14 years, with an overwhelming proportion of males (70.4%). Hepatitis E accounted for 22.86% of active hepatitis cases with alanine aminotransferase levels ≥15.0-fold the upper limit of normal, suggesting the need to include HEV in routine testing for these patients. Ninety-two hepatitis E patients were positive for at least 2 of HEV antigen, anti-HEV IgM, and HEV RNA markers at presentation, and 90.22% of them were positive for HEV antigen and anti-HEV IgM. HEV antigen, HEV RNA, and anti-HEV IgM positivity were observed in 89.80%, 82.65%, and 93.88% of hepatitis E patients at presentation, respectively. However, only 57.14% of anti-HEV IgM positivity occurred in hepatitis E patients. These findings will advance our understanding of hepatitis E and improve diagnosis.

Unpredictable chronic mild stress promotes atherosclerosis in high cholesterol-fed rabbits.

OBJECTIVES: Chronic psychological stress is associated with an increased risk of atherosclerosis in humans. Experimental studies using various stress models have yielded controversial results. This study investigated the effects of unpredictable chronic mild stress (UCMS) on atherogenesis in New Zealand white rabbits. METHODS: Rabbits were fed with a cholesterol-enriched (1%) diet for 4 to 16 weeks, with or without concomitant UCMS treatment. Atherosclerosis was assessed in the abdominal aorta by serial sectioning and morphological analysis. Expressions of inflammatory factors were measured with immunohistochemistry and quantitative polymerase chain reaction. Serum nitrate/nitrite levels were determined with Griess assay, and corticosterone and inflammatory markers were determined using enzyme-linked immunosorbent assay. RESULTS: High-cholesterol feeding resulted in hypercholesterolemia and formation of atherosclerotic plaques in the aorta. UCMS exposure significantly increased the plaque size (p = .003) and decreased the plaque stability (decreased the contents of collagen and smooth muscle and increased the amount of macrophage and matrix metalloproteinases). The proatherogenic effects of UCMS were unrelated to changes in serum cholesterol level but accompanied by increased blood pressure (p < .001) and vascular inflammation (up-regulation of tumor necrosis factor α, C-reactive protein, and monocyte chemoattractant protein 1, all p values < .01). Serum concentrations of nitrate/nitrite were lower in UCMS-treated animals (p = .01). Vessels from UCMS-treated animals exhibited augmented phosphorylation of p38 and c-Jun N-terminal kinase and activation of nuclear factor κB. CONCLUSIONS: Chronic psychological stress may contribute to the development of atherosclerosis by enhancing vascular inflammation and decreasing endothelial nitric oxide bioavailability.

Evidence basis for using dexmedetomidine to enhance the quality of paravertebral block: A systematic review and meta-analysis of randomized controlled trials.

Background: Dexmedetomidine is considered an adjunct to local anaesthesia (LA) to prolong peripheral nerve block time. However, the results from a previous meta-analysis were not sufficient to support its use in paravertebral block (PVB). Therefore, we performed an updated meta-analysis to evaluate the efficacy of dexmedetomidine combined with LA in PVB. Methods: We performed an electronic database search from the date of establishment to April 2022. Randomized controlled trials (RCTs) investigating the combination of dexmedetomidine and LA compared with LA alone for PVB in adult patients were included. Postoperative pain scores, analgesic consumption, and adverse reactions were analyzed. Results: We identified 12 trials (701 patients) and found that the application of dexmedetomidine as a PVB adjunct reduced the postoperative pain severity of patients 12 and 24 h after surgery compared to a control group. Expressed as mean difference (MD) (95% CI), the results were -1.03 (-1.18, -0.88) (p < 0.00001, I2 = 79%) for 12 h and -1.08 (-1.24, -0.92) (p < 0.00001, I2 = 72%) for 24 h. Dexmedetomidine prolonged the duration of analgesia by at least 173.27 min (115.61, 230.93) (p < 0.00001, I2 = 81%) and reduced postoperative oral morphine consumption by 18.01 mg (-22.10, 13.92) (p < 0.00001, I2 = 19%). We also found no statistically significant differences in hemodynamic complications between the two groups. According to the GRADE system, we found that the level of evidence for postoperative pain scores at 12 and 24 h was rated as moderate. Conclusion: Our study shows that dexmedetomidine as an adjunct to LA improves the postoperative pain severity of patients after surgery and prolongs the duration of analgesia in PVB without increasing the incidence of adverse effects.

Erratum: A cell-penetrating whole molecule antibody targeting intracellular HBx suppresses hepatitis B virus via TRIM21-dependent pathway: Erratum.

[This corrects the article DOI: 10.7150/thno.20047.].

Molecular Evolution of Attachment Glycoprotein (G) and Fusion Protein (F) Genes of Respiratory Syncytial Virus ON1 and BA9 Strains in Xiamen, China.

Monitoring viral transmission and analyzing the genetic diversity of a virus are imperative to better understand its evolutionary history and the mechanism driving its evolution and spread. Especially, effective monitoring of key antigenic mutations and immune escape variants caused by these mutations has great scientific importance. Thus, to further understand the molecular evolutionary dynamics of respiratory syncytial virus (RSV) circulating in China, we analyzed nasopharyngeal swab specimens derived from hospitalized children ≤5 years old with acute respiratory tract infections (ARIs) in Xiamen during 2016 to 2019. We found that infants under 6 months of age (52.0%) were the main population with RSV infection. The prevalent pattern "BBAA" of RSV was observed during the epidemic seasons. RSV ON1 and BA9 genotypes were the dominant circulating strains in Xiamen. Interestingly, we observed four Xiamen-specific amino acid substitution combinations in the G protein and several amino acid mutations primarily occurring at antigenic sites Ø and V in the F protein. Our analyses suggest that introduction of new viruses and local evolution are shaping the diversification of RSV strains in Xiamen. This study provides new insights on the evolution and spread of the ON1 and BA9 genotypes at local and global scales. IMPORTANCE Monitoring the amino acid diversity of the RSV G and F genes helps us to find the novel genotypes, key antigenic mutations affecting antigenicity, or neutralizing antibody-resistant variants produced by natural evolution. In this study, we analyzed the molecular evolution of G and F genes from RSV strains circulating in Xiamen, China. These data provide new insights on local and global transmission and could inform the development of control measures for RSV infections.

An Optimized FI-RSV Vaccine Effectively Protects Cotton Rats and BALB/c Mice without Causing Enhanced Respiratory Disease.

BACKGROUND: Despite considerable efforts toward vaccine development in past decades, no effective vaccines against respiratory syncytial virus (RSV) are available. Recently, we showed that an optimized formalin concentration can preserve prefusion protein (pre-F) on RSV-infected cells and protect mice against RSV infection without causing enhanced respiratory disease (ERD). Here, we sought to further stabilize pre-F on RSV virions by optimizing the production of FI-RSV. METHODS: Freshly produced RSV virions were treated with formalin under different concentrations to obtained an opti-FI-RSV vaccine with high pre-F level. Immunogenicity and safety of opti-FI-RSV were evaluated in Balb/c mice and cotton rats. RESULTS: Using 0.0156-0.1778% formalin, we successfully preserved pre-F on virions. This opti-FI-RSV exhibited improved immunogenicity and efficacy without causing ERD. Surprisingly, opti-FI-RSV, with a pre-F-dominant immunogen, still caused ERD after immunization with a suboptimal dose or when the neutralizing antibody titers declined. ERD was avoided by coadministering opti-FI-RSV with CpG + MPLA adjuvant, which subsequently induced a Th1-biasing immune response and, more importantly, significantly improved antibody avidity. CONCLUSIONS: Our study provides a new method to obtain a novel FI-RSV vaccine with a high pre-F level and may provide a reference for developing other inactivated vaccines. Our findings also emphasize that appropriate adjuvants are critical for nonreplicating vaccines.

Specific determination of hepatitis B e antigen by antibodies targeting precore unique epitope facilitates clinical diagnosis and drug evaluation against hepatitis B virus infection.

Hepatitis B e antigen (HBeAg) is a widely used marker both for chronic hepatitis B (CHB) clinical management and HBV-related basic research. However, due to its high amino acid sequence homology to hepatitis B core antigen (HBcAg), most of available anti-HBe antibodies are cross-reactive with HBcAg resulting in high interference against accurate measurement of the status and level of HBeAg. In the study, we generated several monoclonal antibodies (mAbs) targeting various epitopes on HBeAg and HBcAg. Among these mAbs, a novel mAb 16D9, which recognizes the SKLCLG (aa -10 to -5) motif on the N-terminal residues of HBeAg that is absent on HBcAg, exhibited excellent detection sensitivity and specificity in pairing with another 14A7 mAb targeting the HBeAg C-terminus (STLPETTVVRRRGR, aa141 to 154). Based on these two mAbs, we developed a novel chemiluminescent HBeAg immunoassay (NTR-HBeAg) which could detect HBeAg derived from various HBV genotypes. In contrast to widely used commercial assays, the NTR-HBeAg completely eliminated the cross-reactivity with secreted HBcAg from precore mutant (G1896A) virus in either cell culture or patient sera. The improved specificity of the NTR-HBeAg assay enables its applicability in cccDNA-targeting drug screening in cell culture systems and also provides an accurate tool for clinical HBeAg detection.

RNA Interference inhibits hepatitis B virus of different genotypes in vitro and in vivo.

BACKGROUND: Hepatitis B virus (HBV) infection increases the risk of liver disease and hepatocellular carcinoma. Small interfering RNA (siRNA) can be a potential new tool for HBV therapy. Given the high heterogeneity of HBV strains and the sensitivity towards sequences changes of siRNA, finding a potent siRNA inhibitor against the conservative site on the HBV genome is essential to ensure a therapeutic application. RESULTS: Forty short hairpin RNA (shRNA) expression plasmids were constructed to target conserved regions among nine HBV genotypes. HBV 1.3-fold genome plasmids carrying various genotypes were co-transfected with shRNA plasmids into either Huh7 cells or mice. The levels of various viral markers were examined to assess the anti-HBV efficacy of siRNA. Four (B245, B376, B1581 and B1789) were found with the ability to potently inhibit HBV RNA, DNA, surface antigen (HBsAg), e antigen (HBeAg) and core antigen (HBcAg) expression in HBV genotypes A, B, C, D and I (a newly identified genotype) in Huh7 cells and in mice. No unusual cytotoxicity or off-target effects were noted. CONCLUSIONS: Such siRNA suggests an alternate way of inhibiting various HBV genotypes in vitro and in vivo, promising advances in the treatment of HBV.

Quantitative evaluation of protective antibody response induced by hepatitis E vaccine in humans.

Efficacy evaluation through human trials is crucial for advancing a vaccine candidate to clinics. Next-generation sequencing (NGS) can be used to quantify B cell repertoire response and trace antibody lineages during vaccination. Here, we demonstrate this application with a case study of Hecolin®, the licensed vaccine for hepatitis E virus (HEV). Four subjects are administered the vaccine following a standard three-dose schedule. Vaccine-induced antibodies exhibit a high degree of clonal diversity, recognize five conformational antigenic sites of the genotype 1 HEV p239 antigen, and cross-react with other genotypes. Unbiased repertoire sequencing is performed for seven time points over six months of vaccination, with maturation pathways characterize for a set of vaccine-induced antibodies. In addition to dynamic repertoire profiles, NGS analysis reveals differential patterns of HEV-specific antibody lineages and highlights the necessity of the long vaccine boost. Together, our study presents a quantitative strategy for vaccine evaluation in small-scale human studies.

Upregulation of lncRNA RMRP promotes the activation of cardiac fibroblasts by regulating miR­613.

Long non­coding RNAs (lncRNAs) have been reported to serve a key role in a variety of cardiovascular diseases, including in cardiac fibrosis. The present study aimed to investigate the biological role and underlying mechanisms of the induction of cardiac fibroblasts by the lncRNA, RNA component of mitochondrial RNA processing endoribonuclease (RMRP). The results demonstrated that RMRP expression was upregulated in the presence of cardiac fibrosis in an abdominal aortic banding­treated rat model. Treatment with angiotensin II increased RMRP expression in cardiac fibroblasts, while the knockdown of RMRP by small­interfering RNA inhibited cardiac fibroblast proliferation, differentiation and collagen accumulation. To further investigate the underlying mechanisms of this interaction, microRNA (miR)­613 was predicted to be a target miR of RMRP and sequence alignment, luciferase activity and MS2 RNA immunoprecipitation were performed to detect the interaction between RMRP and miR­613. The results suggested that RMRP negatively regulated miR­613 in cardiac fibroblasts. Furthermore, miR­613 was indicated to mediate the promoting effect of RMRP on cardiac fibroblast activation. The current study suggested that RMRP promoted cardiac fibroblast activation by acting as a competing endogenous RNA for miR­613. Therefore, RMRP may be a novel target for the prevention or treatment of cardiac fibrosis.

The Optimal Concentration of Formaldehyde is Key to Stabilizing the Pre-Fusion Conformation of Respiratory Syncytial Virus Fusion Protein.

BACKGROUND: To date, there is no licensed vaccine available to prevent respiratory syncytial virus (RSV) infection. The valuable pre-fusion conformation of the fusion protein (pre-F) is prone to lose high neutralizing antigenic sites. The goals of this study were to stabilize pre-F protein by fixatives and try to find the possibility of developing an inactivated RSV vaccine. METHODS: The screen of the optimal fixative condition was performed with flow cytometry. BALB/c mice were immunized intramuscularly with different immunogens. The serum neutralizing antibody titers of immunized mice were determined by neutralization assay. The protection and safety of these immunogens were assessed. RESULTS: Fixation in an optimal concentration of formaldehyde (0.0244%-0.0977%) or paraformaldehyde (0.0625%-1%) was able to stabilize pre-F. Additionally, BALB/c mice inoculated with optimally stabilized pre-F protein (opti-fixed) induced a higher anti-RSV neutralization (9.7 log2, mean value of dilution rate) than those inoculated with unstable (unfixed, 8.91 log2, p < 0.01) or excessively fixed (exce-fixed, 7.28 log2, p < 0.01) pre-F protein. Furthermore, the opti-fixed immunogen did not induce enhanced RSV disease. CONCLUSIONS: Only the proper concentration of fixatives could stabilize pre-F and the optimal formaldehyde condition provides a potential reference for development of an inactivated RSV vaccine.

Robust in vitro assay for analyzing the neutralization activity of serum specimens against hepatitis B virus.

Anti-HBs is a well-known marker of protective capability against HBV. However, little is known about the association between the qAnti-HBs determined by immunoassays and the neutralization activity (NAT) derived from functional assays. We developed an in vitro assay for direct measurement of the NAT of human sera. The new assay was highly sensitive, with an analytical sensitivity of 9.6 ± 1.3 mIU/mL for the HBIG standard. For serum detection, the maximum fold dilution required to produce ≥50% inhibition (MDF50) of HBV infection was used as the quantitative index. In vitro NAT evaluations were conducted for a cohort of 164 HBV-free healthy individuals. The results demonstrated that the NAT positively correlated with the qAnti-HBs (R2 = 0.473, p < 0.001). ROC analysis indicated that the optimal cutoff value of the qAnti-HBs to discriminate significant NAT (MDF50 ≥ 8) was 62.9 mIU/mL, with an AUROC of 0.920. Additionally, we found that the qAnti-HBc was another independent parameter positively associated with the NAT (R2 = 0.300, p < 0.001), which suggested that antibodies against other HBV proteins generated by previous HBV exposure possibly also contribute to the NAT. In summary, the new cell-based assay provides a robust tool to analyse the anti-HBV NAT. Abbreviations: HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; Anti-HBs: Hepatitis B surface antibody; HBeAg: Hepatitis B e antigen; Anti-HBc: Hepatitis B core antibody; qAnti-HBs: quantitative hepatitis B surface antibody; qAnti-HBc: quantitative hepatitis B core antibody; qHBeAg: quantitative hepatitis B e antigen; NAT: neutralization activity; HBIG: hepatitis B immune globulin; NTCP: Na+-taurocholate cotransporting polypeptide; IRES: internal ribosome entry site; ccHBV: cell culture derived hepatitis B virus; GE/cell: genome equivalent per cell; MOI: multiplicity of infection; Dpi: day post infection; HepG2-TetOn: a HepG2-derived cell line that expresses the doxycycline-regulated transactivator; ROC: receiver operating characteristic curve; AUROC: area under receiver operating characteristic curve; LLOQ: the lower limits of quantification; MDF50: the maximum fold dilution required to produce ≥50% inhibition; IC50: half maximal inhibitory concentration.

BACE1 gene silencing alleviates isoflurane anesthesia­induced postoperative cognitive dysfunction in immature rats by activating the PI3K/Akt signaling pathway.

Postoperative cognitive dysfunction (POCD) is a severe complication characterized by cognitive dysfunction following anesthesia and surgery. The aim of the present study was to investigate the effects of ß­site amyloid precursor protein cleavage enzyme 1 (BACE1) gene silencing on isoflurane anesthesia­induced POCD in immature rats via the phosphatidylinositol­3­kinase (PI3K)/protein kinase B (Akt) signaling pathway. Rat models were established and then transfected with BACE1 small interfering RNA and wortmannin (an inhibitor of PI3K). Blood gas analysis was performed, and a series of behavioral experiments were conducted to evaluate the cognitive function, learning ability and locomotor activity of rats. Reverse transcription quantitative polymerase chain reaction and western blot analysis were employed to determine the mRNA and protein expression of the associated genes. An ELISA was used to detect the inflammatory indicators and the content of amyloid precursor protein (APP) and amyloid­ß (Aß). Apoptosis of the hippocampal CA1 region was observed by terminal deoxynucleotidyl transferase dUTP nick­end labeling staining. Initially, it was revealed that the percentage of stagnation time in rats was increased by BACE1 gene silencing; the escape latency and swimming distance were markedly reduced from the 4th to the 6th day, the time the rats spent in first passing the target area was shortened, and the times of passing the target area were increased by BACE1 gene silencing, demonstrating that BACE1 gene silencing enhanced the spatial memory ability of rats. Additionally, it was determined that silencing BACE1 improved the pathological state induced by isoflurane anesthesia in immature rats, and attenuated the inflammatory response and the levels of APP and Aß in hippocampal tissues. Furthermore, it was suggested that silencing BACE1 may have promoted the activation of the PI3K/Akt signaling pathway, thereby inhibiting the apoptosis of the hippocampal CA1 region. Taken together, these results indicated that BACE1 gene silencing may improve isoflurane anesthesia­induced POCD in immature rats by activating the PI3K/Akt signaling pathway and inhibiting the Aß generated by APP.