Establishment and methodological evaluation of a chemiluminescence assay for detection of anti-envelope protein (E1, E2) antibodies in the serum of hepatitis C virus-infected patients.

BACKGROUND: To establish a chemiluminescence method for detecting anti-E1 and anti-E2 antibodies in the serum of patients with hepatitis C virus (HCV) infection. METHODS: The microplate was coated with recombinant envelope proteins E1 and E2 by indirect method, respectively, and the kits for detecting anti-E1 and anti-E2 antibodies were prepared. The methodological indexes were evaluated. RESULTS: The methodological indexes of the kits were as follows: precision test (the variation coefficient of anti-E1 antibody 6.71%-8.95% for within run and 9.91%-12.16% for between run, the variation coefficient of anti-E2 antibody 6.06%-8.44% for within run and 10.77%-13.98% for between run, respectively). The blank limit and detection limit were 1.18 RLIR and 3.16 RLIR for the anti-E1 antibody, and 1.26 RLIR and 3.32 RLIR for the anti-E2 antibody, respectively. The correlation coefficients (r) of anti-E1 and anti-E2 were 0.9963 and 0.9828, the analysis and measurement ranges (AMR) were 1.66-41.28 RLIR and 1.55-19.46 RLIR, and the average recovery was 96.4% and 93.7%, respectively. The rheumatoid factor and other positive serum samples had no interference or cross-reaction to the test, and the kits were stable within 15 months. The positive rates of anti-E1 and anti-E2 antibodies in 45 patients with HCV infection were 35.6% (16/45) and 44.4% (20/45), respectively. CONCLUSIONS: The kits for detecting anti-E1 and anti-E2 meet the requirements of methodology, and can be used in screening diagnosis, disease monitoring, prognosis evaluation, disease mechanism, and epidemiological studies of HCV infection. The HCV envelope proteins E1 and E2 have an immune response in HCV-infected patients.

M2ixKG: Mixing for harder negative samples in knowledge graph.

Knowledge graph embedding (KGE) involves mapping entities and relations to low-dimensional dense embeddings, enabling a wide range of real-world applications. The mapping is achieved via distinguishing the positive and negative triplets in knowledge graphs. Therefore, how to design high-quality negative triplets is critical in the effectiveness of KEG models. Existing KGE models face challenges in generating high-quality negative triplets. Some models employ simple static distributions, i.e. uniform or Bernoulli distribution, and it is difficult for these methods to be trained distinguishably because of the sampled uninformative negative triplets. Furthermore, current methods are confined to constructing negative triplets from existing entities within the knowledge graph, limiting their ability to explore harder negatives. We introduce a novel mixing strategy in knowledge graphs called M2ixKG. M2ixKG adopts mixing operation in generating harder negative samples from two aspects: one is mixing among the heads and tails in triplets with the same relation to strengthen the robustness and generalization of the entity embeddings; the other is mixing the negatives with high scores to generate harder negatives. Our experiments, utilizing three datasets and four classical score functions, highlight the exceptional performance of M2ixKG in comparison to previous negative sampling algorithms.

Adaptive pseudo-Siamese policy network for temporal knowledge prediction.

Temporal knowledge prediction is a crucial task for early event warning, which has gained increasing attention recently. It aims to predict future facts based on relevant historical facts using temporal knowledge graphs. There are two main difficulties associated with the prediction task: from the perspective of historical facts, modeling the evolutionary patterns of facts to accurately predict the query and from the query perspective, handling the two cases where the query contains seen and unseen entities in a unified framework. Driven by these two problems, we propose a novel adaptive pseudo-Siamese policy network for temporal knowledge prediction based on reinforcement learning. Specifically, we design the policy network in our model as a pseudo-Siamese network consisting of two sub-policy networks. In the sub-policy network I, the agent searches for the answer to the query along the entity-relation paths to capture static evolutionary patterns. In sub-policy network II, the agent searches for the answer to the query along relation-time paths to deal with unseen entities. Moreover, we develop a temporal relation encoder to capture the temporal evolutionary patterns. Finally, we design a gating mechanism to adaptively integrate the results of the two sub-policy networks to help the agent focus on the destination answer. To assess the performance of our model, we conduct link prediction on four benchmark datasets, and extensive experimental results demonstrate that our method achieves considerable performance compared with existing methods.

Establishment and Evaluation of Recombinant Expression of HCV Transmembrane Protein (p7) and Detection of Anti-p7 Antibody in Serum of HCV-Infected Patients by Chemiluminescence.

OBJECTIVE: Our aim was to establish a chemiluminescence method for detecting anti-transmembrane protein (p7) antibody in the serum of patients with hepatitis C virus (HCV) infection. METHODS: The p7 gene was amplified by polymerase chain reaction using the plasmid PUC-p7 containing the p7 nucleic acid sequence of the HCV 1b genotype as the template, and recombinant plasmid pGEX-KG-p7 was constructed. After p7 fusion, the protein was induced and expressed in the prokaryote, extracted, and purified; the anti-p7 antibody detection kit was prepared, and its efficacy was evaluated. RESULTS: The plasmid pGEX-KG-p7 was constructed correctly, and p7 fusion protein was obtained. The methodological indexes of the kit, the precision test, blank limit and detection limit, etc, met the requirements. The positive rate of serum anti-p7 antibody in 45 patients with HCV infection was 20%. CONCLUSIONS: The kit can be used in screening diagnosis, condition monitoring, prognosis, and disease mechanism and epidemiological study of HCV infection. The p7 protein has immune response in HCV-infected patients.

Development and application of a universal extraction-free reagent based on an algal glycolipid.

In this study, we independently developed a universal nasopharyngeal swab extraction-free reagent based on a trehalose lipid for the rapid detection of pathogen nucleic acids in respiratory infectious diseases. By comparing the isothermal amplification results of a 2019-nCoV pseudovirus solution treated with different components of the extraction-free reagent, we determined the optimal composition of the extraction-free reagent to be a mixed solution of 10 mmol L-1 tris-HCl containing 0.05 mmol L-1 EDTA (TE solution), 5% glycine betaine, 0.5% Triton X-100, and 1.5% trehalose lipid. The results showed that the extraction-free reagent could cleave DNA viruses, RNA viruses, and bacteria to release nucleic acids and did not affect the subsequent nucleic acid amplification. Its efficiency was consistent with that of magnetic bead extraction. Real-time fluorescence quantitative PCR was used to analyze the stability and repeatability of the detection results of the samples treated with the extraction-free reagent and the sensitivity of the extraction-free reagent. The results showed that the extraction-free kit could stably store the pathogen nucleic acid for at least 24 hours, the detection repeatability was satisfactory, and there was no incompatibility with the detection limits of various manufacturers' nucleic acid detection reagents. In conclusion, the established nucleic acid extraction-free method can effectively lyse respiratory infectious disease pathogens to release nucleic acids (DNA and RNA) at room temperature and can directly amplify nucleic acids without extraction steps. This method takes a short time and has high efficiency. The released nucleic acid met the requirements of molecular biological detection methods such as real-time fluorescence quantitative PCR (qPCR), reverse transcription-polymerase chain reaction (RT-PCR), and isothermal nucleic acid amplification (INAA).

Clinical characteristics and association analysis of persistent low-level HBsAg expression in a physical examination population with HBV infection.

Certain patients with hepatitis B virus (HBV) infection present with persistently low levels of serum hepatitis B surface antigen (HBsAg) and have been indicated to have low rates of HBV nucleic acid replication. To explore the serological and molecular epidemiological characteristics of HBV population with low-level HBsAg in the present study, associated serum markers and virologic genotype detection were performed accordingly. Determination of HBV markers was performed using a chemiluminescence immunoassay from which 2,544 out of 45,256 adults who underwent routine health examination were tested positive for HBsAg. HBV DNA was detected by real-time fluorescent quantitative PCR. The patients were divided into low-level and high-level groups, according to their HBsAg levels (cut-off value, 10 IU/ml). The prevalence and levels of HBsAg positivity and HBV DNA in patients with HBV infection were analyzed by age, sex, serological pattern and clinical type. The fibrosis status of patients with low-level HBsAg was assessed by determining the aspartate aminotransferase-to-platelet ratio (APRI), and sequencing was employed to determine serotypes and genotypes. HBV-infected patients with low-level HBsAg (<10 IU/ml) accounted for 15.41% of the 2,544 HBsAg-positive patients, and the prevalence of HBsAg positivity exhibited a tendency to increase with age. The male-to-female ratio was ~1.9:1, and the average age was 54.98±16.28 years among HBV-infected patients with low-level HBsAg. The major serological pattern and clinical types were HBsAg/antibody against hepatitis Be antigen (anti-HBe)/antibody against hepatitis B core antigen (anti-HBc)-positive (94.90%) and chronic asymptomatic (ASC) (97.95%), respectively. HBV DNA exhibited a low-level of replication and the prevalence of HBV DNA positivity assessed by the routine method and by the enrichment method was 27.74% (97/392) and 45.92% (180/392), respectively. No significant differences among the age groups were identified in the different HBsAg level groups (P>0.05). The prevalence of HBV DNA positivity was associated with HBsAg only in patients with serological pattern HBV-M2 (HBsAg/anti-HBe/anti-HBc-positive) in the low-level HBsAg group (odds ratio: 1.30; 95% CI: 1.15-1.47; P<0.05). The APRI had no association with age, HBsAg, HBV DNA level or liver function index in ASC patients in the low-level HBsAg group (P>0.05). The prevalence of the serotype adw and genotype B was 85.53 and 89.47%, respectively. Further improvement in the systematic study of populations with low-level HBsAg has important clinical and epidemiological significance for improving the detection of HBV serological markers, elucidating the mechanisms leading to low-level HBsAg, overcoming immune tolerance to eliminate HBV infection and preventing HBV transmission.

[In vitro culture of tachyzoites of Toxoplasma gondii RH strain in HeLa cells].

OBJECTIVE: To establish a stable and efficient in vitro culture model for tachyzoites of Toxoplasma gondii RH strain. METHODS: Tachyzoites were inoculated into HeLa cells to establish an in vitro culture system. The proliferation of tachyzoites was observed under microscope by the method of Giemsa stain. At the same time, the longterm tachyzoites maintenance in HeLa cells was established, and the effect of different temperature and time on the yield and motility of tachyzoites were observed. RESULTS: The RH strain tachyzoites were cultured and maintained in HeLa cells. Most HeLa cells were destroyed 96 h after inoculation. In the long-term culture system, the proliferation of tachyzoites was stable and its virulence to mouse showed no decrease. Furthermore, tachyzoites in this system proliferated by 5-20 times and (1-5) x 10(7) tachyzoites were harvested. When cultured in HeLa cells at 37 degrees C for 72h then at 25 degrees C for another 120 h, the tachyzoites proliferated by more than 40 times with a motility rate of over 90%. However, rare HeLa cells left in the medium were found. CONCLUSION: Tachyzoites of T. gondii RH strain can be subcultured in HeLa cells for a long time, and high proliferation rate of tachyzoites can be obtained from this in vitro culture system.