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.

Pleurospiroketals A-E, perhydrobenzannulated 5,5-spiroketal sesquiterpenes from the edible mushroom Pleurotus cornucopiae.

Five novel perhydrobenzannulated 5,5-spiroketal sesquiterpenes, namely, pleurospiroketals A-E (1-5), were isolated from the culture of the edible mushroom Pleurotus cornucopiae. Pleurospiroketals D (4) and E (5) were obtained as an isomeric mixture with a ratio of 5:4. Their structures were established by NMR, X-ray single-crystal diffraction, and CD data analysis. Pleurospiroketals A-E (1-5) are sesquiterpenoids with a unique benzannulated 5,5-spiroketal skeleton. Compounds 1-3 showed inhibitory activity against nitric oxide production in lipopolysaccharide-activated macrophages with IC(50) values of 6.8, 12.6, and 20.8 µM, respectively.

MiR-187 regulates the proliferation, migration and invasion of human trophoblast cells by repressing BCL6-mediated activation of PI3K/AKT signaling.

INTRODUCTION: Recurrent miscarriage (RM), refers to two or more consecutive spontaneous miscarriage in a pregnant woman. RM is caused by many factors, and microRNAs play an important role in the development and pathology of RM. In the present study, we investigated the function of miR-187 in the pathogenesis of RM and its effects on human trophoblast cells. METHODS: The localization of miR-187 in the human placenta in early pregnancy was determined by in situ hybridization. QRT-PCR was used to detect the expression of miR-187 in villi of normal early pregnancy induced abortion group and recurrent spontaneous miscarriage group. Then, HTR8/SVneo cells were used to investigated the effect of miR-187 on BCL6 expression and biological activity of trophoblasts. RESULTS: We found that the expression of miR-187 in villi of RM group was higher than that of normal abortion group and miR-187 inhibited the proliferation, migration, and invasion of HTR8 cells. We also found that miR-187 promoted apoptosis, inhibited EMT, and inhibited the PI3K/AKT pathway in HTR8 cells. In addition, we also found that BCL6 is a direct target of miR-187 and is negatively regulated by miR-187. In addition, BCL6 reversed the inhibitory effects of miR-187 on HTR8/SVneo cells. These data demonstrate that miR-187-induced repression of PI3K/AKT signaling is mediated by BCL6 in HTR8 cells. DISSCUSSION: MiR-187 inhibits the proliferation, migration, and invasion of trophoblasts through a mechanism that involves regulation of BCL6.

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.].

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.

Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells.

The global pandemic of coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable neutralization assay is very important for the development of vaccines and novel drugs. In this study, a G protein-deficient vesicular stomatitis virus (VSVdG) bearing a truncated spike protein (S with C-terminal 18 amino acid truncation) was compared to that bearing the full-length spike protein of SARS-CoV-2 and showed much higher efficiency. A neutralization assay was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and hACE2-overexpressing BHK21 cells (BHK21-hACE2 cells). The experimental results can be obtained by automatically counting the number of EGFP-positive cells at 12 h after infection, making the assay convenient and high-throughput. The serum neutralizing titer measured by the VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with that measured by the wild type SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 S protein were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

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

Rationale: Monoclonal antibodies (mAbs) mostly targeting extracellular or cell surface molecules have been widely used in the treatment of various diseases. However, mAbs cannot pass through the cell membrane as efficiently as small compounds, thus limiting their use against intracellular targets. Methods to shuttle antibodies into living cells may largely expand research and application in areas based on mAbs. Hepatitis B virus X protein (HBx) is an important intracellular multi-functional viral protein in the life cycle of hepatitis B virus (HBV). HBx plays essential roles in virus infection and replication and is strongly associated with HBV-related carcinogenesis. Methods: In this study, we developed a cell-penetrating whole molecule antibody targeting HBx (9D11-Tat) by the fusion of a cell penetrating peptide (CPP) on the C-terminus of the heavy chain of a potent mAb specific to HBx (9D11). The anti-HBV effect and mechanism of 9D11-Tat were investigated in cell and mouse models mimicking chronic HBV infection. Results: Our results demonstrated that the recombinant 9D11-Tat antibody could efficiently internalize into living cells and significantly suppress viral transcription, replication, and protein production both in vitro and in vivo. Further analyses suggested the internalized 9D11-Tat antibody could greatly reduce intracellular HBx via Fc binding receptor TRIM21-mediated protein degradation. This process simultaneously stimulated the activations of NF-κB, AP-1, and IFN-ß, which promoted an antiviral state of the host cell. Conclusion: In summary, our study offers a new approach to target intracellular pathogenesis-related protein by engineered cell-penetrating mAb expanding their potential for therapeutic applications. Moreover, the 9D11-Tat antibody may provide a novel therapeutic agent against human chronic HBV infection.

Combined Direct and Indirect CT Venography (Combined CTV) in Detecting Lower Extremity Deep Vein Thrombosis.

This study aimed to evaluate the diagnostic accuracy of combined direct and indirect CT venography (combined CTV) in the detection of lower extremity deep vein thrombosis (LEDVT). The institutional review board approved the study protocol, and patients or qualifying family members provided informed consent. A total of 96 consecutive patients undergoing combined CTV were prospectively enrolled. A combined examination with digital subtraction angiography (DSA) plus duplex ultrasonography (US) was used as the criterion standard. Three observers were blinded to clinical, DSA, and US results, and they independently analyzed all combined CTV datasets. Interobserver agreement was expressed in terms of the Cohen k value for categorical variables. Accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of combined CTV in the detection of LEDVT were determined by using patient- and location-based evaluations. Of the 96 patients, DSA plus US revealed LEDVT in 125 segmental veins in 63 patients. Patient-based evaluation with combined CTV yielded an accuracy of 96.9% to 97.9%, a sensitivity of 95.2% to 96.8%, a specificity of 100% to 100%, a PPV of 100% to 100%, and an NPV of 91.7% to 94.3% in the detection of LEDVT. Location-based evaluation yielded similar results. Through combined direct and indirect CTV, patients obtained a combined CT angiogram on the diseased limb and an indirect CT angiogram on the opposite side. The image quality of combined CTV was superior to an indirect venogram. Combined CTV shows promising diagnostic accuracy in the detection of LEDVT with 3-dimensional modeling of the lower limb venous system.

Analysis of closed abdominal injury in pregnant women.

OBJECTIVE: To explore the characteristics of closed abdominal injury in pregnancy women and its treatment. METHODS: The clinical data of 37 pregnancy patients with closed abdominal injury treated in our hospital from June 1993 to June 2003 were collected and analyzed. RESULTS: All the 37 patients were treated with operation. Among them 2 early pregnancy patients with intestinal rupture and 1 patient with retroperitoneal hematoma were treated under laparoscope; in other 34 pregnancy patients laparotomy was performed. Of the 34 patients 8 used cesarean section because premature separation of placenta and enlarged womb interrupted the management of intra-abdominal organ injury. In the 37 patients 33 (89.1%) were cured, 4 (10.8%) die, postoperative complication rate was 16.2% (6/37). Two patients (5.4%) suffered from abdominal cavity infection, 3 (8.1%) from pulmonary infection, and 1 (2.7%) had multi-organ failure. CONCLUSIONS: For pregnancy patients with closed abdominal injury, besides obsteric diseases intra-abdominal injury should be given much attention. Accurate diagnosis and timely treatment can gain the time to save the life of both mother and fetus.

Eryngiolide A, a cytotoxic macrocyclic diterpenoid with an unusual cyclododecane core skeleton produced by the edible mushroom Pleurotus eryngii.

Eryngiolide A (1), the first member of C20 diterpenoids with the skeleton deriving from a cyclododecane core fused with two γ-lactone units, was isolated from the solid culture of the edible mushroom Pleurotus eryngii. The structure of 1 was elucidated by extensive analysis of NMR spectra. Compound 1 exhibited moderate cytotoxicity against two human cancer lines in vitro.

Studies on the fluorescence fiber-optic DNA biosensor using p-hydroxyphenylimidazo[f]1,10-phenanthroline ferrum(III) as indicator.

A complex Fe(phen)(2).PHPIP.3ClO(4).2H(2)O, where phen=1,10-phenanthroline and PHPIP=p-hydroxyphenylimidazo[f]1,10-phenanthroline, was synthesized and acted as a good fluorescence indicator based on its interaction with double-duplex DNA. Then a fiber-optic DNA biosensor of fluorimetric detection was developed based on the recognition of target DNA in DNA hybridization assays. A probe ssDNA was covalently immobilized onto the surface of quartz optical fibers and then the probe ssDNA hybridized with complementary ssDNA introduced into the local environment of the sensor. The hybridization with complementary strands was monitored in real time by fluorimetric detection. Several factors affecting the probe immobilization, target DNA hybridization, and indicator binding reactions were optimized to maximize the sensitivity and shorten the assay time. Using this method, a sequence of the 16-mer oligonucleotides could be quantified over the range from 4.98 x 10(-7) to 4.88 x 10(-6) M and a detection limit of 1.08 x 10(-7) M. And the designed optic-fiber biosensor could be conveniently regenerated by thermal denature. The utility of the novel hybridization indicator could provide a simple, rapid, low toxicity and reusable detection.