Essential oil of Ligusticum chuanxiong Hort. Regulated P-gp protein and tight junction protein to change pharmacokinetic parameters of temozolomide in blood, brain and tumor.

ETHNOPHARMACOLOGICAL RELEVANCE: The existence of the blood-brain barrier/blood tumor barrier (BBB/BTB) severely restricts the effectiveness of anti-tumor drugs, thus glioma is still an incurable disease with a high fatality rate. Chuanxiong (Ligusticum chuanxiong Hort., Umbelliferae) was used as a messenger drug to increase the distribution of drugs in brain tissue, and its application in Chinese herbal formula for treating glioma was also the highest. AIM OF THE STUDY: Our previous researches showed that essential oil (EO) of chuanxiong could promote temozolomide (TMZ) entry into glioma cells in vitro and enhance TMZ-induced anticancer efficiency in vivo, and therefore, the aim of this study was to investigate whether EO could increase the concentration accumulation of TMZ in brain or tumor of C6 glioma rats and the related mechanisms. MATERIALS AND METHODS: The pharmacokinetics were conducted in C6 glioma rats by administering either TMZ alone or combined with EO through oral routes. TMZ concentration in blood, brain and tumor was detected using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and then pharmacokinetic parameters were calculated. The changed expressions of P-gp protein, tight junction occludin, claudin-5 and zonula occludens-1 (ZO-1) in brain of glioma rats were studied by Western blot to clarify the mechanism. Finally, the chemical composition of EO was analyzed by gas chromatography-massspectrometry (GC-MS). RESULTS: The results showed that EO significantly affected the pharmacokinetic parameters such as Tmax, Cmax and CL (p < 0.01), but did not significantly change the AUC(0→∞) of TMZ in blood (p > 0.05). However, EO markedly improved the AUC(0→∞)of TMZ in brain and tumor (p < 0.01). The calculate drug targeting index was greater than 1, indicating that EO could promote the distribution of TMZ to the brain and tumor. Western blot analysis showed that EO significantly inhibited the expression of P-gp, tight junction protein claudin-5, occludin and ZO-1. And meanwhile, the expressions of P-gp, claudin-5 and occludin also markedly down-regulated in EO-TMZ co-administration treatment. GC-MS analysis of the TIC component of EO was (E)-Ligustilide (36.93%), Terpinolene (7.245%), gamma-terpinene (7.225%) etc. CONCLUSION: EO could promote the distribution of TMZ in the brain and tumor of C6 glioma rats, which may attribute to down-regulate the expression of P-gp, claudin-5 and occludin.

LINC00470 accelerates the proliferation and metastasis of melanoma through promoting APEX1 expression.

Recently studies found that APEX1 was abnormally expressed in melanoma, indicating that it might be involved in the development of melanoma. However, the underlying mechanism and the interaction between APEX1 and LINC00470 in melanoma are not clear. Therefore, we aimed to investigate the role of LINC00470 in the development of melanoma in this work. We discovered that LINC00470 was overexpressed in melanoma tissues and cells compared with the adjacent normal tissues and cells by qPCR. The overexpression of LINC00470 promoted the proliferation and migration of melanoma cells. The functional investigation demonstrated that LINC00470 activated the transcription factor, ZNF131, to regulate the APEX1 expression, which finally promoted cell proliferation and migration. In contrast, knockdown of LINC00470 could significantly inhibit the melanoma cell proliferation and migration, and suppress the growth of tumor in vivo. Overexpression of APEX1 could reverse the impact of the silence of LINC00470 in melanoma cells. In summary, our studies revealed that LINC00470 promoted melanoma proliferation and migration by enhancing the expression of APEX1, which indicated that LINC00470 might be a therapeutic target for the treatment of melanoma.

Epidemiological and spatiotemporal analyses of HIV/AIDS prevalence among older adults in Sichuan, China between 2008 and 2019: A population-based study.

OBJECTIVES: To describe epidemiological trends and spatial distributions of HIV/AIDS among older adults (aged ≥50) in Sichuan Province, China during 2008-19, and provide scientific reference for HIV/AIDS prevention, intervention and treatment. METHODS: Data on HIV/AIDS cases reported in 2008-19 was extracted from the Case Report System. The Cochran-Armitage trend test was used to determine epidemic trends. Spatial autocorrelation and space-time analysis were conducted with ArcGIS10.6 and ArcGIS Pro2.4, respectively. RESULTS: A total of 77854 HIV/AIDS cases among older adults were included in the study. Newly reported cases increased from 320 in 2008 to 22189 in 2019, and the reported incidence rate (number of new reported cases/older adult population) rose from 0.001% to 0.077%. Infections through heterosexual transmission increased from 65.3% to 98.2% of total cases in older adults in this period. Spatial analysis at the county-level showed significant clustering throughout Sichuan, with the main hot spots concentrated in the southeast. Spatiotemporal analysis indicated that most of the southeastern counties/districts were Consecutive Hot Spots. CONCLUSIONS: Older adults have become a key population in the HIV/AIDS epidemic in Sichuan; comprehensive prevention and intervention measures targeted to older adults are urgently needed to control the spread of HIV/AIDS.

[Effects of weeding and fertilization on soil nitrogen mineralization and leaching in the mixed forest of Dalbergia odorifera and Santalum album]. / é“²è‰å’Œæ–½è‚¥å¯¹é™é¦™é»„æª€ä¸Žå°åº¦æª€é¦™æ··äº¤æž—åœŸå£¤æ°®ç´ çŸ¿åŒ–æ·‹æº¶çš„å½±å“.

The mixed plantation of precious tree species Dalbergia odorifera and Santalum album is an important model for the development of plantations in South China. Based on an experiment with four treatments, i.e., CK (without any treatment), weeding, fertilization, weeding+fertilization, we investigated the effects of weeding and fertilization on soil net mineralization rate, net nitrification rate, net ammonification rate, and nitrogen leaching rate. Among the four treatments, the maximum net nitrogen mineralization rate in the 0-10 cm soil layer was 18.92 and 18.13 mg·kg-1·month-1 in spring and autumn, respectively; the maximum nitrification rate was 20.35 and 18.85 mg·kg-1·month-1in spring and autumn, respectively; the maximum ammonium mineralization rate was 0.22 and 0.26 mg·kg-1·month-1 in summer and winter, respectively; the maximum nitrogen leaching was 15.98 mg·kg-1·month-1 in autumn and 86.69 mg·kg-1·year-1 in the whole year. The treatments of weeding, fertilization, weeding+fertilization reduced net mineralization rate and net nitrification rate. The annual nitrogen mineralization decreased by 26.2%, 16.1% and 6.3%, respectively, and the annual nitrogen nitrification decreased by 17.1%, 16.6%, and 1.4%, respectively. Moreover, the treatments of weeding, fertilization, weeding+fertilization inhibited soil ammonium accumulation and reduced annual nitrogen leaching by 25.2%, 8.6%, and 6.1%, respectively. Compared with weeding, fertilization and weeding+fertilization, seasonal factors had more significant effects on soil nitrogen mineralization and leaching. Weeding, fertilization and weeding+fertilization decreased soil nitrogen nitrification and ammonification to a certain extent, reduced soil nitrogen mineralization and leaching loss, which contribute to the preservation of soil fertility and nitrogen accumulation.

Genetic Diversity and Population Structure Analysis of DalbergiaOdorifera Germplasm and Development of a Core Collection Using Microsatellite Markers.

Dalbergia odorifera T. Chen (Fabaceae) is a woody tree species indigenous to Hainan Island in China. Due to its high medicinal and commercial value, this tree species has been planted over 3500 ha² in southern China. There is an urgent need for improvement of the D. odorifera germplasm, however, limited information on germplasm collection, conservation, and assessment of genetic resources is available. Therefore, we have built a database of 251 individuals collected across the whole of southern China, which included 42 wild trees and 210 cultivated trees, with the following objectives. (1) Evaluate genetic diversity and population structure of the database using 19 microsatellite markers and (2) develop a core collection for improvement and breeding programs. Totally, the 19 microsatellite markers harbored 77 alleles across the database with the polymorphic information content (PIC) ranging from 0.03 to 0.66. Medium genetic diversity level was inferred by Nei's gene diversity (0.38), Shannon's information index (0.65), and observed (0.33) and expected heterozygosity (0.38). Structure analysis showed that four was the optimum cluster size using the model-based Bayesian procedure, and the 251 D. odorifera individuals were grouped into five populations including four pure ones (RP1-4) and one mixed one (MIX) based on their maximum membership coefficients. Among these populations, the expected heterozygosity varied from 0.30 (RP3) to 0.38 (RP4). Analysis of molecular variance (AMOVA) showed 11% genetic variation existed among populations, and moderate population differentiation was inferred by the matrix of pairwise Fst (genetic differentiation among populations), which was in the range of 0.031 to 0.095. Moreover, a core collection of 31 D. odorifera individuals including six wild and 25 cultivated trees was developed, which was only 12.4% of the database but conserved the whole genetic diversity. The results of this study provided additional insight into the genetic structure of the large D. odorifera germplasm, and the core collection will be useful for the efficient and sustainable utilization of genetic resources, as well as efficient improvement in breeding programs.

Characterization of the chloroplast genome of Cassia siamea Lain, a rosewood species from southeast China.

Cassia siamea is a rosewood species in Southwest China with high wood and medicinal value. To clarify genetic background of C. siamea, we sequenced chloroplast genome by Illumina Hiseq and PacBio Sequel. The whole genome was 148,437 bp in length, containing a large single copy region (77,723 bp), a small single copy region (18,462 bp) and a pair of inverted repeats regions (26,126 bp). The cp genome contained 102 genes (71 protein-coding genes, 27 tRNAs and 4 rRNAs). The phylogenetic analysis indicated that C. siamea is close to Senna tora within Cassiinae/Caesalpiniaceae. The complete chloroplast genome of C. siamea will provide useful resources for the development and utilization of this species and the phylogenetic study of Fabaceae.

[Prevalence of Hepatitis B Surface Antigens and Hepatitis B Surface Antibodies in 1-12 Years-old Children in Mianyang,Sichuan].

OBJECTIVE: To determine the prevalence of hepatitis B surface antigens (HBsAg) and hepatitis B surface antibodies (anti-HBs) in 1-12 years-old children in Mianyang city,Sichuan Province. METHODS: Children born after the implementation of Hepatitis B immunization policy were selected using a stratified random cluster sampling strategy from January to December 2015. A total of 72 623 eligible children participated in the study,which included a questionnaire survey and blood tests (0.3 mL vein blood) for HBsAg and anti-HBs with ELISA method. Repeated tests were performed on the blood samples with a HBsAg positive result. RESULTS: About 0.24% of the children were HBsAg positive; 64.50% were anti-HBs positive; 35.26% were both HBsAg and anti-HBs negative. The standardized rates based on the 2010 population census were: 0.24% HBsAg positive,64.05% anti-HBs positive,and 35.71% both HBsAg and anti-HBs negative. HBsAg positive rates increased with age, ranging from 0% to 0.65% (P<0.001). Rural children had a higher HBsAg positive rate (0.32%) than their urban counterparts (0.16%,P<0.001). Those with a family history of Hepatitis B had a higher HBsAg positive rate (1.53%) than those without a family history (0.22%,P<0.001). Anti-HBs positive rates decreased withage,ranging from 47.85% to 71.43% (P<0.001). Rural children had a lower anti-HBs positive rate (62.06%)than their urban counterparts (66.81%,P<0.001). The prevalence of both HBsAg and anti-HBs negative cases increased with age,ranging from 28.57% to 51.98% (P<0.001). Rural children had a higher rate of both HBsAg and anti-HBs negative (37.62%) than their urban counterparts (33.03%,P<0.001). About 35.37% of the children who had negative HBsAg and anti-HBs had not received Hepatitis B immunization. CONCLUSION: Hepatitis B vaccinations are highly effective in Mianyang. However,there are disparities in anti-HBs positive rates between the children with different characteristics. A certain proportion of children are still susceptible to hepatitis B infection. It is necessary to attach importance to neonatal hepatitis B vaccination,surveillance on anti-HBs,and strengthened immunization for the children who are lack of antibody protection.

[Awareness of Acquired Immune Deficiency Syndrome and Its Influencial Factors among College Students in Universities Implementing "Red Ribbon in Colleges" Event across China].

Objective To investigate the college students'awareness of acquired immune deficiency syndrome (AIDS) and its influencial factors in universities implementing "Red Ribbon in Colleges" event.Methods A multistage random sampling method was used to select 2057 students from 8 universities. Among them,1977 students were surveyed by quantitative questionnaires and 80 students were interviewed qualitatively. SPSS 24.0 software was used for data processing and analysis.Results A total of 1977 students was investigated,among whom the overall awareness rate of AIDS-related knowledge was 88.21%. The awareness rate of the three major routes of AIDS transmission (i.e. sexual transmission,blood transmission,and mother-to-child transmission) were more than 90%,and the awareness rate of "Can mosquito bites spread AIDS" was lowest (72.99%). The awareness rate of AIDS-related knowledge was significantly higher among students who had attended the "Red Ribbon in Colleges" event (95.36%,P<0.001),followed by those who had received AIDS prevention education as a freshman (92.22%,P<0.001),had filled in AIDS prevention questionnaires (91.42%,P<0.001),and had attended other AIDS-related health education events in schools or colleges (91.36%,P<0.001),and the differences were statistically significant when compared with students who had not attended any of these events. Multivariate unconditional Logistic regression analysis showed that attending the "Red Ribbon in Colleges" event (OR=2.973,95%CI=1.684-5.249,P<0.001),receiving AIDS prevention education as a freshman (OR=2.944,95%CI=2.073-4.181,P<0.001),attending other AIDS-related health education in schools or colleges (OR=1.590,95%CI=1.141-2.217,P=0.006),majoring in engineering disciplines (OR=2.756,95%CI=1.846-4.114,P<0.001),junior students (OR=1.916,95%CI=1.216-3.020,P=0.005),living in central China (OR=1.868,95%CI=1.085-3.218,P=0.024),and living in northern China (OR=1.774,95%CI=1.159-2.716,P=0.008) were favourable factors for high awareness rate. Qualitative analysis indicated that students rated highly of the "Red Ribbon in Colleges" event.Conclusion The "Red Ribbon in Colleges" event and other AIDS education programs help to increase the college students'awareness of AIDS-related knowledge.

[Prevalence of Overweight and Obesity in Adult Populations in Mianyang,Sichuan Province].

OBJECTIVE: To determine the prevalence of overweight and obesity in adult populations in Mianyang,Sichuan province. METHODS: A questionnaire survey involving physical examinations were conducted in 262 434 adults (≥18 yr.) selected through multi-stage random sampling from November 2014 to September 2015 in Mianyang city. RESULTS: Among the people surveyed,79 981 (30.48%) were overweight,and 23 010 (8.77%) had obesity. The male participants had a higher percentage (32.28%) of overweight than females (29.10%, P<0.01),but lower obesity rate (8.53%) than females (8.95%, P<0.01). Those aged 50-59 yr. had the highest prevalence of overweight (39.26%) and obesity (12.07%). The participants with up to junior middle school education were most likely to be overweight (35.02%) and obese (10.57%). Overweight and obesity were most prevalent in the unemployed: 36.87% and 12.65%,respectively. Fucheng had higher prevalence of overweight (30.01%) and obesity (10.14%) than Jiangyou (29.97% and 7.46% respectively,all P<0.01). The prevalence of overweight and obesity was higher in the smokers (32.88% and 9.46%) than in the non-smokers (30.02% and 8.64%,all P<0.01). Those who drank alcohols had higher prevalence of overweight and obesity (33.43% and 9.72%) than those who did not drink alcohols (29.78% and 8.54% respectively,all P<0.01). CONCLUSION: High prevalence of overweight and obesity in adult populations in Mianyang is evident,especially in those who are 50-59 years old,unemployed,drinking alcohols,smoking,and have low educational level.

DNA methylation-mediated silencing of matricellular protein dermatopontin promotes hepatocellular carcinoma metastasis by α3ß1 integrin-Rho GTPase signaling.

Dermatopontin (DPT), a tyrosine-rich, acidic matricellular protein, has been implicated in several human cancers. However, its biological functions and molecular mechanisms in cancer progression, particular hepatocellular carcinoma (HCC), remain unknown. We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis. The overexpression of DPT dramatically suppressed HCC cell migration in vitro and intrahepatic metastasis in vivo. We further revealed that the down-regulation of DPT in HCC was due to epigenetic silencing by promoter DNA methylation. And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling. Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3ß1 integrin. Our study provides new evidence for epigenetic control of tumor microenvironment, and suggests matricellular protein DPT may serve as a novel prognostic marker and act as a HCC metastasis suppressor.

Monoamine oxidase A suppresses hepatocellular carcinoma metastasis by inhibiting the adrenergic system and its transactivation of EGFR signaling.

BACKGROUND & AIMS: Monoamine oxidase A (MAOA), a catecholamine neurotransmitter degrading enzyme, is closely associated with neurological and psychiatric disorders. However, its role in cancer progression remains unknown. METHODS: Hepatocellular carcinoma (HCC) tissue arrays (n=254) were used to investigate the correlation between MAOA expression and clinicopathological findings. In vitro invasion and anoikis assays, and in vivo intrahepatic and lung metastasis models were used to determine the role of MAOA in HCC metastasis. Quantitative real-time PCR, western blotting, immunohistochemical staining and HPLC analysis were performed to uncover the mechanism of MAOA in HCC. RESULTS: We found that MAOA expression was significantly downregulated in 254 clinical HCC samples and was closely correlated with cancer vasoinvasion, metastasis, and poor prognoses. We then demonstrated that MAOA suppressed norepinephrine/epinephrine (NE/E)-induced HCC invasion and anoikis inhibition, and uncovered that the effects of NE/E on HCC behaviors were primarily mediated through alpha 1A (ADRA1A) and beta 2 adrenergic receptors (ADRB2). In addition to the canonical signaling pathway, which is mediated via adrenergic receptors (ADRs), we found that ADR-mediated EGFR transactivation was also involved in NE-induced HCC invasion and anoikis inhibition. Notably, we found that MAOA could synergize with EGFR inhibitors or ADR antagonists to abrogate NE-induced HCC behaviors. CONCLUSIONS: Taken together, the results of our study may provide insights into the application of MAOA as a novel predictor of clinical outcomes and indicate that increasing MAOA expression or enzyme activity may be a new approach that can be used for HCC treatment.

Lumican Accelerates Wound Healing by Enhancing α2ß1 Integrin-Mediated Fibroblast Contractility.

Lumican is a dermatan sulfate proteoglycan highly expressed in connective tissue and has the ability to regulate collagen fibril assembly. Previous studies have shown that lumican is involved in wound healing, but the precise effects of lumican on reepithelialization and wound contraction, the two pivotal aspects of skin wound healing, have not been investigated. Here we explored the roles of lumican in fibroblast contractility, a main aspect of skin wound healing, by adopting mice skin wound healing model and the corresponding in vitro cellular experiments. Our results showed that lumican can promote skin wound healing by facilitating wound fibroblast activation and contraction but not by promoting keratinocyte proliferation and migration. Silencing of integrin α2 completely abolished the pro-contractility of lumican, indicating lumican enhances fibroblast contractility via integrin α2. Our study for the first time demonstrated that lumican can affect fibroblast's mechanical property, which is pivotal for many important pathological processes, such as wound healing, fibrosis, and tumor development, suggesting that lumican might have a potential to be used to modulate these processes.

Xiamenmycin attenuates hypertrophic scars by suppressing local inflammation and the effects of mechanical stress.

Hypertrophic scarring is a common disease affecting millions of people around the world, but there are currently no satisfactory drugs to treat the disease. Exaggerated inflammation and mechanical stress have been shown to be two main mechanisms of excessive fibrotic diseases. Here we found that a benzopyran natural product, xiamenmycin, could significantly attenuate hypertrophic scar formation in a mechanical stretch-induced mouse model. The compound suppressed local inflammation by reducing CD4+ lymphocyte and monocyte/macrophage retention in fibrotic foci and blocked fibroblast adhesion with monocytes. Both in vivo and in vitro studies found that the compound inhibited the mechanical stress-induced profibrotic effects by suppressing proliferation, activation, fibroblast contraction, and inactivating FAK, p38, and Rho guanosine triphosphatase signaling. Taken together, the compound could simultaneously suppress both the inflammatory and mechanical stress responses, which are the two pivotal pathological processes in hypertrophic scar formation, thus suggesting that xiamenmycin can serve as a potential agent for treating hypertrophic scar formation and other excessive fibrotic diseases.

[Correlation analysis of major agronomic characters and the polysaccharide contents in Dendrobium officinale].

OBJECTIVE: In order to provide theoretical and technological basis for the germplasm innovation and variety breeding in Dendrobium officinale, a study of the correlation between polysaccharide content and agronomic characters was conducted. METHODS: Based on the polysaccharide content determination and the agronomic characters investigation of 30 copies (110 individual plants) of Dendrobium officinale germplasm resources, the correlation between polysaccharide content and agronomic characters was analyzed via path and correlation analysis. RESULTS: Correlation analysis results showed that there was a significant negative correlation between average spacing and polysaccharide content, the correlation coefficient was -0.695. And the blade thickness was positively correlated with the polysaccharide content, but the correlation was not significant. The path analysis results showed that the stem length was the maximum influence factor to the polysaccharide, and it was positive effect, the direct path coefficient was 1.568. CONCLUSION: According to thess results, the polysaccharide content can be easily and intuitively estimated by the agronomic characters investigating data in the germpalsm resources screening and variety breeding. Therefore, it is a visual and practical technology guidance in quality variety breeding of Dendrobium officinale.

Identification and characterization of an anti-fibrotic benzopyran compound isolated from mangrove-derived Streptomyces xiamenensis.

An anti-fibrotic compound produced by Streptomycesn xiamenensis, found in mangrove sediments, was investigated for possible therapeutic effects against fibrosis. The compound, N-[[3,4-dihydro-3S-hydroxy-2S-methyl-2-(4'R-methyl-3'S-pentenyl)-2H-1-benzopyran-6-yl]carbonyl]-threonine (1), was isolated from crude extracts and its structure, including the absolute configuration was determined by extensive spectroscopic data analyses, Mosher's method, Marfey's reagent and quantum mechanical calculations. In terms of biological effects, this compound inhibits the proliferation of human lung fibroblasts (WI26), blocks adhesion of human acute monocytic leukemia cells (THP-1) to a monolayer of WI26 cells, and reduces the contractile capacity of WI26 cells in three-dimensional free-floating collagen gels. Altogether, these data indicate that we have identified a bioactive alkaloid (1) with multiple inhibitory biological effects on lung excessive fibrotic characteristics, that are likely involved in fibrosis, suggesting that this molecule might indeed have therapeutic potential against fibrosis.

Hypoxia stabilizes microtubule networks and decreases tumor cell chemosensitivity to anticancer drugs through Egr-1.

The hypoxic environment of solid tumor causes the tumor cells survive and which could protect them from death by facilitating resistance to therapy. Here, we provide evidence that hypoxia can increase tumor cell viability and proliferation through an Egr-1-dependant pathway. Hypoxia protected the microtubules from disassembly, and Egr-1 was colocalized with microtubules in different cell cycle stages. Knockdown of Egr-1 with its siRNA overcame the protection effect of hypoxia and increased the sensitivity of tumor cells to vinblastine under hypoxic conditions. Our results suggest a novel approach for increasing the sensitivity of tumor cells to chemotherapeutics that target microtubule assembly.

Development of the diagnostic immunoassay to detect anti-PreS1(21-47aa) antibody--a marker suggesting the health improvement of hepatitis B patients.

BACKGROUND: A new immunoassay has been developed for the detection of the anti-PreS1(21-47aa) antibody in sera of hepatitis B virus (HBV)-infected patients. Anti-PreS1(21-47aa) antibody involves virus neutralization and is a new marker for diagnosing acute and chronic B hepatitis. METHODS: The expression plasmids pGEXS I and pGEXS II, which expressed glutathione S-transferase (GST) fusion proteins containing a copy of PreS1(21-47aa) peptide and two orderly joined copies of PreS1(21-47aa) peptide, were constructed. The soluble expression products were purified by affinity chromatography. RESULTS: The two PreS1(21-47aa) fusion proteins were both successfully applied in the immunoassay based on biotin-protein A and streptavidin-HRP, and could detect the anti-PreS1(21-47aa) antibody with high sensitivity in sera from hepatitis B patients. The anti-PreS1(21-27aa) antibody was detected during the recovery phase of acute hepatitis B patients, but it was found only in few of the chronic carriers by the established conventional system. CONCLUSIONS: The follow-up study suggested that the presence of the anti-PreS1(21-27aa) antibody correlated well with the recovery of patients from hepatitis and the improvement in health.

Expression, Purification and Preliminary Clinical Use of Recombinant HBsAg GST-PreS1(21--47 aa) Fusion Proteins.

Expression plasmids pGEXSI and pGEXSII containing one copy and two orderly joined copies of PreS1(21--47 aa) DNA fragment, respectively, were constructed. GST-PreS1(21--47 aa) and GST-2xPreS1(21--47 aa) fusion proteins were highly expressed in E.Coli TG1, induced by IPTG. The expression level of GST-PreS1(21--47 aa) was about 30% of total soluble proteins in the lysate of expression bacteria, and GST-2xPreS1(21--47 aa) was about 15% of total soluble proteins, asestimated by SDS-PAGE. 50 mg GST-PreS1(21--47 aa) or 20 mg GST-2xPreS1(21--47 aa) with purity over 90% was obtained, respectively, from 1 L culture by using affinity chromatography of glutathione-Sepharose 4B. Direct ELISA results showed that antigenicity of GST-2xPreS1(21--47 aa) was better than GST-PreS1(21--47 aa) and synthetic peptide. Using GST-2xPreS1(21--47 aa) as coated antigen, a sensitive indirect ELISA for detection of anti-PreS1(21--47 aa) antibody, based on protein A-biotin and streptavidin-HRP, was established. The results from 99 sera samples of hepatitis B patients showed that anti-PreS1(21--47 aa) antibody was detected in nearly half of acute hepatitis B patients during recovery, but it was detected only in a few chronic hepatitis patients. Clinical follow-up study suggested that appearance of anti-PreS1(21--47 aa) was related to the course of the disease and recovery of patients. Detection system established in the study is promising for clinical application.