CpG 684: an effective adjuvant for the inactivated COVID-19 vaccine in mice.

Aim: This study used CpG 684 as adjuvant of inactivated COVID-19 vaccine to detect a humoral and cellular immune response in mice. Materials & methods: We used 10 and 20 µg CpG 684 as adjuvants of an inactivated COVID-19 vaccine to immunize mice. IgG, IgG1, IgG2a, IgG2b and IgM binding antibodies were detected in serum by ELISA. The IFN-γ cytokine was detected by ELISPOT. Results: CpG 684 improved spike-specific IgG and IgM subtype binding antibodies and increased the neutralizing antibody titer against prototype, Delta and Beta strains. CpG 684 also improved cellular immune response. Conclusion: CpG 684 is an effective adjuvant for inactivated COVID-19 vaccine.

Liver macrophages and sinusoidal endothelial cells execute vaccine-elicited capture of invasive bacteria.

Vaccination has substantially reduced the morbidity and mortality of bacterial diseases, but mechanisms of vaccine-elicited pathogen clearance remain largely undefined. We report that vaccine-elicited immunity against invasive bacteria mainly operates in the liver. In contrast to the current paradigm that migrating phagocytes execute vaccine-elicited immunity against blood-borne pathogens, we found that invasive bacteria are captured and killed in the liver of vaccinated host via various immune mechanisms that depend on the protective potency of the vaccine. Vaccines with relatively lower degrees of protection only activated liver-resident macrophage Kupffer cells (KCs) by inducing pathogen-binding immunoglobulin M (IgM) or low amounts of IgG. IgG-coated pathogens were directly captured by KCs via multiple IgG receptors FcγRs, whereas IgM-opsonized bacteria were indirectly bound to KCs via complement receptors of immunoglobulin superfamily (CRIg) and complement receptor 3 (CR3) after complement C3 activation at the bacterial surface. Conversely, the more potent vaccines engaged both KCs and liver sinusoidal endothelial cells by inducing higher titers of functional IgG antibodies. Endothelial cells (ECs) captured densely IgG-opsonized pathogens by the low-affinity IgG receptor FcγRIIB in a "zipper-like" manner and achieved bacterial killing predominantly in the extracellular milieu via an undefined mechanism. KC- and endothelial cell-based capture of antibody-opsonized bacteria also occurred in FcγR-humanized mice. These vaccine protection mechanisms in the liver not only provide a comprehensive explanation for vaccine-/antibody-boosted immunity against invasive bacteria but also may serve as in vivo functional readouts of vaccine efficacy.

Immunogenicity and Safety of a SARS-CoV-2 Inactivated Vaccine KCONVAC in Chinese Children: Randomized, Double-blind, Placebo-controlled Phase 1 and 2 Trials.

BACKGROUND: It is important to extend the indication of severe acute respiratory syndrome coronavirus 2 vaccine to children to improve the vaccine intake rate and reduce infection in this population. METHODS: In 2 phase 1 and phase 2 randomized, double-blind and placebo-controlled trials, 84 and 480 Chinese healthy children 3 to 17 years old were enrolled, respectively, and randomized in 3:1 ratio to receive 2 doses of a severe acute respiratory syndrome coronavirus 2 inactivated vaccine, KCONVAC or placebo. The 2 doses were given 28 days apart. Adverse events (AEs) were recorded through Day 28 after each dosing. Live virus neutralizing antibody and receptor binding domain antibody (RBD-IgG) were tested before vaccination and after the second dose. RESULTS: Two doses of the vaccine, KCONVAC, elicited geometric mean titers of 142-150 for neutralizing antibody and 4154-4253 for RBD-IgG 28 days after the second dose. Seroconversion rates were 100% after 2 doses for both antibodies in both trials. The predominant AEs were injection-site pain, cough and fever. Most AEs were grade 1 or 2 in intensity. Five participants reported 6 vaccination-unrelated serious AEs in the phase 2 trial. CONCLUSIONS: Two doses of this study vaccine, KCONVAC, were well tolerated and immunogenic in children 3 to 17 years of age.

Cytotoxicity of alkaline serine protease (ASPNJ) on Jurkat cells and its correlation with changes in the expression of membrane-associated proteins.

We aim to study the inhibitory effect of alkaline serine protease (ASPNJ) on lymphocytic leukemia Jurkat cells and its related mechanism through examining the expression of membrane proteins or membrane-associated proteins. MTT assay and trypan blue staining were used to detect the inhibitory effect of ASPNJ on the proliferation and growth of Jurkat cells. Wright-Giemsa staining was used to observe the effect of ASPNJ on the morphology of Jurkat cells. The effect of ASPNJ on Jurkat cell apoptosis was detected by flow cytometry. Two-dimensional electrophoresis-mass spectrometry (2-DE-MS) was used to detect and identify the differentially expressed proteins of Jurkat cells treated with ASPNJ (4 µg/mL, 3 h), of which three were selected and verified by Western blot. ASPNJ significantly inhibited the proliferation of leukemia cells (Raji, U937, and Jurkat), caused obvious morphological changes, and induced apoptosis of Jurkat cells. ASPNJ also increased the sensitivity of Jurkat cells to vincristine (VCR). Seven differentially expressed proteins were obtained through 2DE-MS, of which Peroxiredoxin-6 (PRDX6), Calcium-binding protein (CHP1), and 40S ribosomal protein SA (RPSA) were validated. ASPNJ can cause significant toxic effects on Jurkat cells and enhance the effects of VCR. The mechanism of action of ASPNJ on Jurkat cells may be related to differentially expressed proteins such as PRDX6. This study provides a new experimental basis and direction for antileukemia research.

Comparison of the immunogenicity of nasal-spray rVSV vector, adenovirus vector, and inactivated COVID-19-based vaccines in rodent models.

Intranasal (i.n.) vaccines can induce mucosal and systemic immunity against respiratory pathogens. Previously, we demonstrated that the recombinant vesicular stomatitis virus (rVSV)-based COVID-19 vaccine rVSV-SARS-CoV-2, with poor immunogenicity via the intramuscular route (i.m.), is more suitable for i.n. administration in mice and nonhuman primates. Here, we found that the rVSV-SARS-CoV-2 Beta variant was more immunogenic than the wild-type strain and other variants of concern (VOCs) in golden Syrian hamsters. Furthermore, the immune responses elicited by rVSV-based vaccine candidates via the i.n. route were significantly higher than those of two licensed vaccines: the inactivated vaccine KCONVAC delivered via the i.m. route and the adenovirus-based Vaxzevria delivered i.n. or i.m. We next assessed the booster efficacy of rVSV following two i.m. doses of KCONVAC. Twenty-eight days after receiving two i.m. doses of KCONVAC, hamsters were boosted with a third dose of KCONVAC (i.m.), Vaxzevria (i.m. or i.n.), or rVSVs (i.n.). Consistent with other heterologous booster studies, Vaxzevria and rVSV elicited significantly higher humoral immunity than the homogenous KCONVAC. In summary, our results confirmed that two i.n. doses of rVSV-Beta elicited significantly higher humoral immune responses than commercial inactivated and adeno-based COVID vaccines in hamsters. As a heterologous booster dose, rVSV-Beta induced potent, persistent, and broad-spectrum humoral and mucosal neutralizing responses against all VOCs, highlighting its potential to be developed into a nasal-spray vaccine.

Emerging trends and research foci of epithelial-mesenchymal transition in gliomas: A scientometric analysis and review.

Background: Epithelial-mesenchymal transition (EMT) is a key factor in the invasion and migration of glioma cells, and the study of EMT in gliomas has become a hot topic over the past decade. Scientometric analysis is gaining more attention since it can obtain hot topics and emerging trends in a research field. This article analyzed the research related to EMT in gliomas for the first time, including descriptions of research situations, evaluations of research foci, and predictions of emerging trends. Methods: We searched the topic-related original articles from January 2012 to December 2021 in the Web of Science Core Collection (WoSCC) by using a specific strategy, and a total of 1,217 publications were obtained. The WoS platform, VOS viewer, and CiteSpace were used to analyze the annual distribution of publications and citations, authors and density of keywords, and other analyses including countries, institutions, references, clustering, burst analysis, and the timeline view of keywords. Results: Scientometric analysis identified that the study of EMT in gliomas has developed fast and received continuous attention in the last decade. Based on the results of data analysis, most publications on the topic came from China, and the United States had the highest betweenness centrality. The top 10 co-cited references revealed the landmark documents that had greatly promoted the development of this field. The major focus is on the cellular and molecular mechanisms of EMT in gliomas, and the therapy related to EMT target and non-coding RNAs has been developing fast in recent years. Conclusions: This study revealed the intimate connections between EMT and gliomas, and the complex mechanisms regulating EMT in gliomas had been studied widely in the last decade. Exploring the deep mechanisms of EMT in gliomas is the foundation of the targeted inhibitions, which can promote the development of therapies for gliomas.

Determination of ribose and phosphorus contents in Haemophilus influenzae type b capsular polysaccharide by a quantitative NMR method using a single internal standard.

The ribose and phosphorus contents in Haemophilus influenzae type b (Hib) capsular polysaccharide (CPS) are two important chemical indexes for the development and quality control of Hib conjugate vaccine. A quantitative 1H- and 31P-NMR method using a single internal standard was developed for simultaneous determination of ribose and phosphorus contents in Hib CPS. Hexamethylphosphoramide (HMPA) was successfully utilized as an internal standard in quantitative 1H-NMR method for ribose content determination. The ribose and phosphorus contents were found to be affected by the concentration of polysaccharide solution. Thus, 15-20 mg·L-1 was the optimal concentration range of Hib CPS in D2O solution for determination of ribose and phosphorus contents by this method. The ribose and phosphorus contents obtained by the quantitative NMR were consistent with those obtained by traditional chemical methods. In conclusion, this quantitative 1H- and 31P-NMR method using a single internal standard shows good specificity, accuracy and precision, providing a valuable approach for the quality control of Hib glycoconjugate vaccines.

Rapid Degradation of SARS-CoV-2 Spike S Protein by A Specific Serine Protease.

The S protein of SARS-CoV-2 is a crucial structural and functional component for virus entry. Due to the constant mutation of the virus, there are very limited ways to prevent and control COVID-19. This experiment used a macroscopic SDS-PAGE method and proved that the S protein of wild-type SARS-CoV-2 virus, especially the S1 subunit, is very sensitive to alkaline serine protease with acidic pI (ASPNJ), NJ represents Neanthes japonica (Izuka) from which ASP is purified). ASPNJ cleaves proteins when the carbonyl group of the peptide bond is contributed by arginine or lysine. ASPNJ can degrade the S protein very quickly and effectively in vitro with relative selectivity. It can be inferred that the S, S1 and RBD of SARS-CoV-2 variants can also be easily degraded by ASPNJ. This rapid and strong degradation of the S protein by ASPNJ may become a potential new treatment strategy.

Immunogenicity and Safety of a 3-Dose Regimen of a SARS-CoV-2 Inactivated Vaccine in Adults: A Randomized, Double-Blind, Placebo-Controlled Phase 2 Trial.

BACKGROUND: Control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic needs effective vaccines. METHODS: In a phase 2 randomized, double-blind, placebo-controlled trial, 500 adults aged 18-59 years or ≥60 years were randomized in 2:2:1 ratio to receive 3 doses of 5 µg or 10 µg of a SARS-CoV-2 inactivated vaccine, or placebo separated by 28 days. Adverse events (AEs) were recorded through day 28 after each dosing. Live virus or pseudovirus neutralizing antibodies, and receptor binding domain immunoglobulin G (RBD-IgG) antibody were tested after the second and third doses. RESULTS: Two doses of the vaccine elicited geometric mean titers (GMTs) of 102-119, 170-176, and 1449-1617 for the 3 antibodies in younger adults. Pseudovirus neutralizing and RBD-IgG GMTs were similar between older and younger adults. The third dose slightly (<1.5 fold) increased GMTs. Seroconversion percentages were 94% or more after 2 doses, which were generally similar after 3 doses. The predominant AEs were injection-site pain. All the AEs were grade 1 or 2 in intensity. No serious AE was deemed related to study vaccination. CONCLUSIONS: Two doses of this vaccine induced robust immune response and had good safety profile. A third dose given 28 days after the second dose elicited limited boosting antibody response.

The Alterations in Methylene Blue/Light-Treated Frozen Plasma Proteins Revealed by Proteomics.

INTRODUCTION: The aim of this study was to investigate the modified proteins in methylene blue/light-treated frozen plasma (MB-FP) compared with fresh frozen plasma (FFP) in order to gain a better application of MB/light-treated plasma in clinic transfusion. METHODS: MB-FP and FFP were collected from Changchun central blood station, and a trichloroacetic acid/acetone precipitation method was used to remove albumin for the enrichment of lower abundance proteins. The plasma protein in MB-FP and FFP were separated using two-dimensional gel electrophoresis (2-DE) and the differentially expressed protein spots were analyzed using mass spectrometry. Finally, the differentially expressed proteins were tested using Western blot and enzyme-linked immunosorbent assay (ELISA). RESULTS: Approximately 14 differentially expressed protein spots were detected in the MB-FP, and FFP was chosen as the control. After 2-DE comparison analysis and mass spectrometry, 8 significantly differentially expressed protein spots were identified, corresponding to 6 different proteins, including complement C1r subcomponent (C1R), inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4), keratin, type II cytoskeletal 1 (KRT1), hemopexin (HPX), fibrinogen gamma chain (FGG), and transthyretin (TTR). Western blot showed no significant difference in the expression level of KRT1 between MB-FP and FFP (p > 0.05). Both Western blot and ELISA indicated that the level of HPX was significantly higher in FFP than in MB-FP (p < 0.05). CONCLUSION: This comparative proteomics study revealed that some significantly modified proteins occur in MB-FP, such as C1R, ITI-H4, KRT1, HPX, FGG, and TTR. Our findings provide more theoretical data for using MB-FP in transfusion medicine. However, the relevance of the data for the transfusion of methylene blue/light-treated plasma remains unclear. The exact modification of these proteins and the effects of these modified proteins on their functions and their effects in clinical plasma infusion need to be further studied.

Enhanced protective immunity against SARS-CoV-2 elicited by a VSV vector expressing a chimeric spike protein.

SARS-CoV-2 and SARS-CoV are genetically related coronavirus and share the same cellular receptor ACE2. By replacing the VSV glycoprotein with the spikes (S) of SARS-CoV-2 and SARS-CoV, we generated two replication-competent recombinant viruses, rVSV-SARS-CoV-2 and rVSV-SARS-CoV. Using wild-type and human ACE2 (hACE2) knock-in mouse models, we found a single dose of rVSV-SARS-CoV could elicit strong humoral immune response via both intranasal (i.n.) and intramuscular (i.m.) routes. Despite the high genetic similarity between SARS-CoV-2 and SARS-CoV, no obvious cross-neutralizing activity was observed in the immunized mice sera. In macaques, neutralizing antibody (NAb) titers induced by one i.n. dose of rVSV-SARS-CoV-2 were eight-fold higher than those by a single i.m. dose. Thus, our data indicates that rVSV-SARS-CoV-2 might be suitable for i.n. administration instead of the traditional i.m. immunization in human. Because rVSV-SARS-CoV elicited significantly stronger NAb responses than rVSV-SARS-CoV-2 in a route-independent manner, we generated a chimeric antigen by replacing the receptor binding domain (RBD) of SARS-CoV S with that from the SARS-CoV-2. rVSV expressing the chimera (rVSV-SARS-CoV/2-RBD) induced significantly increased NAbs against SARS-CoV-2 in mice and macaques than rVSV-SARS-CoV-2, with a safe Th1-biased response. Serum immunized with rVSV-SARS-CoV/2-RBD showed no cross-reactivity with SARS-CoV. hACE2 mice receiving a single i.m. dose of either rVSV-SARS-CoV-2 or rVSV-SARS-CoV/2-RBD were fully protected against SARS-CoV-2 challenge without obvious lesions in the lungs. Our results suggest that transplantation of SARS-CoV-2 RBD into the S protein of SARS-CoV might be a promising antigen design for COVID-19 vaccines.

Immunogenicity and safety of a severe acute respiratory syndrome coronavirus 2 inactivated vaccine in healthy adults: randomized, double-blind, and placebo-controlled phase 1 and phase 2 clinical trials.

BACKGROUND: The significant morbidity and mortality resulted from the infection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for urgent development of effective and safe vaccines. We report the immunogenicity and safety of an inactivated SARS-CoV-2 vaccine, KCONVAC, in healthy adults. METHODS: Phase 1 and phase 2 randomized, double-blind, and placebo-controlled trials of KCONVAC were conducted in healthy Chinese adults aged 18 to 59 years. The participants in the phase 1 trial were randomized to receive two doses, one each on Days 0 and 14, of either KCONVAC (5 or 10 µg/dose) or placebo. The participants in the phase 2 trial were randomized to receive either KCONVAC (at 5 or 10 µg/dose) or placebo on Days 0 and 14 (0/14 regimen) or Days 0 and 28 (0/28 regimen). In the phase 1 trial, the primary safety endpoint was the proportion of participants experiencing adverse reactions/events within 28 days following the administration of each dose. In the phase 2 trial, the primary immunogenicity endpoints were neutralization antibody seroconversion and titer and anti-receptor-binding domain immunoglobulin G seroconversion at 28 days after the second dose. RESULTS: In the phase 1 trial, 60 participants were enrolled and received at least one dose of 5-µg vaccine (n = 24), 10-µg vaccine (n = 24), or placebo (n = 12). In the phase 2 trial, 500 participants were enrolled and received at least one dose of 5-µg vaccine (n = 100 for 0/14 or 0/28 regimens), 10-µg vaccine (n = 100 for each regimen), or placebo (n = 50 for each regimen). In the phase 1 trial, 13 (54%), 11 (46%), and seven (7/12) participants reported at least one adverse event (AE) after receiving 5-, 10-µg vaccine, or placebo, respectively. In the phase 2 trial, 16 (16%), 19 (19%), and nine (18%) 0/14-regimen participants reported at least one AE after receiving 5-, 10-µg vaccine, or placebo, respectively. Similar AE incidences were observed in the three 0/28-regimen treatment groups. No AEs with an intensity of grade 3+ were reported, expect for one vaccine-unrelated serious AE (foot fracture) reported in the phase 1 trial. KCONVAC induced significant antibody responses; 0/28 regimen showed a higher immune responses than that did 0/14 regimen after receiving two vaccine doses. CONCLUSIONS: Both doses of KCONVAC are well tolerated and able to induce robust immune responses in healthy adults. These results support testing 5-µg vaccine in the 0/28 regimen in an upcoming phase 3 efficacy trial. TRIAL REGISTRATION: http://www.chictr.org.cn/index.aspx (No. ChiCTR2000038804, http://www.chictr.org.cn/showproj.aspx?proj=62350; No. ChiCTR2000039462, http://www.chictr.org.cn/showproj.aspx?proj=63353).

An Effective Approach for Reliability-Based Sensitivity Analysis with the Principle of Maximum Entropy and Fractional Moments.

The reliability-based sensitivity analysis requires to recursively evaluate a multivariate structural model for many failure probability levels. This is in general a computationally intensive task due to irregular integrations used to define the structural failure probability. In this regard, the performance function is first approximated by using the multiplicative dimensional reduction method in this paper, and an approximation for the reliability-based sensitivity index is derived based on the principle of maximum entropy and the fractional moment. Three examples in the literature are presented to examine the performance of this entropy-based approach against the brute-force Monte-Carlo simulation method. Results have shown that the multiplicative dimensional reduction based entropy approach is rather efficient and able to provide reliability estimation results for the reliability-based sensitivity analysis of a multivariate structural model.

Screening of groundwater remedial alternatives for brownfield sites: a comprehensive method integrated MCDA with numerical simulation.

Brownfield sites pollution and remediation is an urgent environmental issue worldwide. The screening and assessment of remedial alternatives is especially complex owing to its multiple criteria that involves technique, economy, and policy. To help the decision-makers selecting the remedial alternatives efficiently, the criteria framework conducted by the U.S. EPA is improved and a comprehensive method that integrates multiple criteria decision analysis (MCDA) with numerical simulation is conducted in this paper. The criteria framework is modified and classified into three categories: qualitative, semi-quantitative, and quantitative criteria, MCDA method, AHP-PROMETHEE (analytical hierarchy process-preference ranking organization method for enrichment evaluation) is used to determine the priority ranking of the remedial alternatives and the solute transport simulation is conducted to assess the remedial efficiency. A case study was present to demonstrate the screening method in a brownfield site in Cangzhou, northern China. The results show that the systematic method provides a reliable way to quantify the priority of the remedial alternatives.

A novel acidic serine protease, ASPNJ inhibits proliferation, induces apoptosis and enhances chemo-susceptibility of acute promyelocytic leukemia cell.

Acidic serine protease (ASPNJ) purified from Neanthes japonica, is a fibrinolytic enzyme. Earthworm fibrinolytic enzyme has been recently reported with anti-tumor activity on human hepatoma cells. To investigate if ASPNJ play therapeutic effects on emergent blood cancer, acute promyelocytic leukemia (APL), we tested the effects of ASPNJ on APL cell line NB4. Our results showed that ASPNJ inhibited the growth of NB4 cells in a dose and time dependent manner. Cell apoptosis was induced by ASPNJ with obvious morphological changes. The sensitivity of cells to cytarabine and doxorubicin were greatly increased respectively by combination with ASPNJ. In contrast to inhibitory effects on NB4 cells, ASPNJ showed much less effect on normal human neutrophils survival. There were no effects of hemolysis and agglutination observed on normal human erythrocytes following ASPNJ treatment. Conclusively, our data suggest that ASPNJ may become a new candidate for leukemia therapeutic approaches.

[Effects of purine nucleotide on the expressions of FSH and LH and the ultrastructure of endocrine cells in the pituitary gland of heroin-addicted male rats].

OBJECTIVE: To investigate the effects of purine nucleotide on the expressions of follicle-stimulating hormone (FSH) and luteotrophic hormone (LH) and the ultrastructures of the distal somatotrophic and gonadotrophic cells in the pituitary gland of heroin-addicted and -withdrawal rats. METHODS: Ninety-two male Wistar rats were randomly divided into a control group (ip saline for 14 d), a nucleotide group (ip AMP and GMP for 10 d), a heroin group (ip heroin for 10 d), a heroin + nucleotide group (ip AMP and GMP + heroin for 10 d), a 3 d withdrawal group (ip heroin for 10 d and killed at 14 d), a 9 d withdrawal group (ip heroin for 10 d and killed at 20 d), a 3 d nucleotide group (ip nucleotide for 3 d after 10 d heroin administration and killed at 14 d), and a 9 d nucleotide group (ip nucleotide for 9 d after 10 d heroin administration and killed at 20 d). Changes in the mRNA expressions of FSH and LH in the pituitary gland of the rats were analyzed by semi-quantitative RT-PCR, and alterations in the ultrastructures of the distal somatotrophic and gonadotrophic cells were observed under the microscope. RESULTS: The expression of FSH mRNA was significantly increased in the nucleotide, heroin + nucleotide, 3 d nucleotide and 9 d nucleotide groups (0.099 +/- 0.018, 0.177 +/- 0.046, 0.151 +/- 0.030 and 0.184 +/- 0.028) as compared with the control group (0.045 +/- 0.009) (P < 0.01); and so was that of LH mRNA in the heroin + nucleotide, 3 d nucleotide and 9 d nucleotide groups (0.950 +/- 0.169, 0.990 +/- 0.171 and 0.960 +/- 0.147) in comparison with the control group (0.700 +/- 0.099) (P < 0.01). In the heroin group, the nuclei of the distal somatotrophic and gonadotrophic cells exhibited morphological abnormality, unclear membrane, slightly pyknotic matrix, marginal and agglutinated heterochromatin, dilated rough endoplasmic reticula, swollen mitochondria, broken and vacuolated cristae in the cytoplasm, obviously decreased number of secretory granules, and myelin bodies in some cells. However, the heroin + nucleotide group showed no significant changes in the ultrastructures of somatotrophic and gonadotrophic cells compared with the control group. CONCLUSION: Short-term use of heroin does not obviously affect the expressions of FSH and LH mRNA in the pituitary gland of rats, while heroin + nucleotide, or nucleotide following heroin withdrawal can enhance their expressions significantly. Heroin damages the ultrastructures of the distal somatotrophic and gonadotrophic cells in the pituitary gland of male rats, and purine nucleotide can diminish or inhibit this damage.

Heroin affects purine nucleotides metabolism in rat brain.

OBJECTIVE: To explore the effects of heroin on purine nucleotides metabolism in rat brain. METHODS: Biochemical changes in association with heroin administration were compared between heroin-administered rats and non-heroin rats. HPLC method was used to detect the absolute content of purine nucleotides in brain tissues. Concentrations of uric acid (UA), blood urea nitrogen (BUN) and creatinine (Cre) in plasma were measured. Enzymatic activities of adenosine deaminase (ADA) and xanthine oxidase (XO) in brain tissue were analyzed. Real-time PCR was used to determine the relative level of transcripts of ADA, XO, adenine phosphoribosyl transferase (APRT), hypoxanthine-guaninephosphoribosyl transferase (HGPRT) and adenosine kinase (AK) in brain tissue. RESULTS: Compared with those in the saline group, the content of AMP and GTP of heroin group decreased significantly; the UA concentration in plasma, ADA and XO activities and the mRNA level of ADA and XO in brain tissues in heroin group increased significantly; the mRNA level of AK, APRT and HGPRT in brain tissues in heroin group decreased significantly (P<0.01). CONCLUSION: Heroin administration may enhance the catabolism and inhibit the anabolism of purine nucleotides in brain. There may be a deficiency of purine nucleotides, especially GTP and AMP in rat brain exposed to heroin. Our findings may provide a new potential approach to study the mechanism of heroin addiction.

A novel alkaline serine protease with fibrinolytic activity from the polychaete, Neanthes japonica.

A new protease named NJP with fibrinolytic activity was isolated from Neanthes japonica (Izuka), by a combination of ammonium sulfate fractionation, hydrophobic chromatography, ion-exchange chromatography and gel filtration. The molecular mass of NJP was approximately 28.6-33.5kDa as estimated by MALDI-TOF mass spectrometry and SDS-PAGE, which revealed a monomeric form of the protease. The isoelectric point of NJP determined by 2-DE was 9.2. NJP was stable in the range of pH 7.0-11.0 with a maximum enzymatic activity at 40°C and pH 9.0. The hydrolyzing activity of NJP on fibrinogen started from the Aα-chain, followed by the Bß-chain, and the γ-chain at last. NJP had also a higher specificity for the chromogenic substrate S-2238 for thrombin. NJP activity was completely inhibited by PMSF. Analysis of partial amino acid sequences showed that NJP had very low homology with other known fibrinolytic enzymes. These results indicate that NJP is a novel alkaline thrombin-like serine protease. Thus NJP may have potential applications in the prevention and treatment of thrombosis.

Identification of active loci of a human endogenous retrovirus in neurons of patients with amyotrophic lateral sclerosis.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of motor neurons, of unknown etiology. Previous studies showed reverse transcriptase in serum of ALS patients at levels comparable to human immunodeficiency virus-infected patients; however, the source and significance of the retroviral elements is uncertain. METHODS: Expression of a human endogenous retrovirus (HERV-K) was determined in autopsy brain tissue of patients with ALS and compared to control populations by real-time polymerase chain reaction followed by sequencing of the amplified genes and confirmed by immunostaining. RESULTS: HERV-K pol transcripts were increased in patients with ALS compared to those with chronic systemic illness, but could not be detected in Parkinson disease or in the accidental death controls. Sequencing revealed several actively transcribed loci in the HML-2 and 3 subfamilies of HERV-K, with a specific pattern of expression including intact open reading frames and the transcription of a unique locus in ALS. The frequency of intact pol transcripts was highest in the motor cortex, and the reverse transcriptase protein was localized to cortical neurons of ALS patients. HERV-K expression strongly correlated with TDP-43, a multifunctional protein known to be dysregulated in ALS. INTERPRETATION: We have identified a specific pattern of HERV-K expression in ALS, which may potentially define the pathophysiology of ALS. Targeting of activated genome-encoded retroviral elements may open new prospects for the treatment of ALS.

Neanthes japonica (Iznka) fibrinolytic enzyme reduced cerebral infarction, cerebral edema and increased antioxidation in rat models of focal cerebral ischemia.

Thrombolytic agent is increasingly being used in treating acute ischemic stroke. A novel protease with strong thrombolytic activity, Neanthes japonica (Iznka) fibrinolytic enzyme (NJF) discovered in our laboratory has been reported with characteristics of direct hydrolyzing fibrin and fibrinogen. The neuroprotective effect of NJF and urokinase (UK) was tested in rat models of middle cerebral artery occlusion (MCAO). The model was successfully produced by introducing an intraluminal suture into the left middle cerebral artery (MCA). NJF (0.25, 0.5, 1mg/kg) was injected intravenously 1h after the onset of reperfusion. Compared with vehicle group, MCAO animals treated with NJF showed dose dependent reduction in cerebral infarction with improved neurological outcome. Meanwhile, ischemia induced cerebral edema was reduced in a dose dependent manner. Treatment with NJF at 0.5mg/kg was almost equivalent to UK at 15,000U/kg dosage in the reduction of cerebral infarction and cerebral edema. Biomedical assay showed that NJF treatment suppressed lipid peroxidation and restored superoxide dismutase (SOD) activities in brain tissue. These results suggest that NJF posses neuroprotective potential in rat MCAO and reperfusion model. Neuroprotection shown by NJF may be attributed to inhibition of lipid peroxidation, increase in endogenous antioxidant defense enzymes.