Antisolvent fabrication of monodisperse liposomes using novel ultrasonic microreactors: Process optimization, performance comparison and intensification effect.

Liposomes as drug carriers for the delivery of therapeutic agents have triggered extensive research but it remains a grand challenge to develop a novel technology for enabling rapid and mass fabrication of monodisperse liposomes. In this work, we constructed a novel ultrasonic microfluidic technology, namely ultrasonic microreactor (USMR) with two different conjunction structure (co-flow and impinge flow, corresponding to USMR-CF and USMR-IF, respectively), to prepare uniform liposomes by antisolvent precipitation method. In this process, the monodisperse liposomes with tunable droplet sizes (DS) in 60-100 nm and a polydispersity index (PDI) less than 0.1 can easily be achieved by tuning the total flow rate, flow rate ratio, ultrasonic power, and lipid concentration within the two USMRs. Impressively, the USMR-IF is superior for reducing the PDI and tuning DS of the liposomes over the USMR-CF. More importantly, the ultrasonic can effectively reduce DS and PDI at the low TFR and support the IF-micromixer in reducing the PDI even at a high TFR. These remarkable performances are mainly due to the rapid active mixing, fouling-free property and high operation stability for USMR-IF. In addition, diverse lipid formulations can also be uniformly assembled into small liposomes with narrow distribution, such as the prepared HSPC-based liposome with DS of 59.6 nm and PDI of 0.08. The liposomes show a high stability and the yield can reach a high throughput with 108 g/h by using the USMR-IF at an initial lipid concentration of 60 mM. The results in the present work highlight a novel ultrasonic microfluidic technology in the preparation of liposomes and may pave an avenue for the rapid, fouling-free, and high throughput fabrication of different and monodisperse nanomedicines with controllable sizes and narrow distribution.

Effects of the small private online course combined with simulation-based training in a patient safety education program among nursing students: A quasi-experimental study.

Objective: This study aimed to explore the effects of the small private online course (SPOC) combined with simulation-based training in a patient safety education program among nursing students in China. Methods: A quasi-experimental design was conducted. A total of 219 nursing students from four parallel classes were selected from the nursing department of a health vocational college in Zhengzhou, China, from November 2020 to June 2021 and allocated to the intervention group (n = 113) and control group (n = 106). Based on SPOC, nursing students in the intervention group implemented simulation teaching in small groups, with three class hours each time, a total of two times, divided into three stages: pre-class preparation, teaching implementation, and after-class reflection. The control group received theoretical patient safety education through SPOC, implemented on the DingDing platform for two class hours each time, four times. All participants were invited to complete a demographic questionnaire and the Chinese version of Patient Safety Competency Self-Evaluation (PSCSE) before and after the intervention. Results: A total of 103 and 102 students from the intervention and control groups completed the study. The total scores of PSCSE in the post-test of the intervention group (176.24 ± 13.73 vs. 144.64 ± 13.75) and the control group (160.87 ± 14.88 vs. 142.57 ± 15.66) were higher than those in the pre-test (P < 0.01), and the total scores of PSCSE of the intervention group were higher than those of the control group (176.24 ± 13.73 vs. 160.87 ± 14.88, P < 0.01). After intervention, the scores of PSCSE in all dimensions were increased in the intervention group (P < 0.01); in the control group, the scores of patient safety competency in most dimensions were increased (P < 0.01), except for the dimensions of reporting and response to error and communication related to error (P＞ 0.05). Except for the dimensions of knowledge and attitude of error reporting and disclosing (P＞ 0.05), the scores of other dimensions in the intervention group were higher than those in the control group (P < 0.01). Conclusion: The patient safety education program using the SPOC combined with simulation-based training can effectively improve the patient safety competency of nursing students in terms of attitude, skills, and knowledge.

Effects of early maternal separation on the expression levels of hippocampal and prefrontal cortex genes and pathways in lactating piglets.

Introduction: In actual production, due to increased litter size when raising pigs, the management of piglets by split-suckling leads to intermittent neonatal maternal separation (MS). Early lactation is a critical period for the cognitive development of the brain of newborn piglets, and we hypothesized that intermittent MS may affect piglets' neurodevelopment and cognitive ability. Methods: To determine the effects of the MS, we selected hippocampal and prefrontal cortex (PFC) tissues from piglets for the detection of neurodevelopmental or cognitive related indicators, the control group (Con group, n = 6) was established with no MS and an experimental group (MS group, n = 6) was established with MS for 6 h/day. Piglets in the MS group were milk-supplemented during the separation period and all piglets in both treatment groups were weaned at postnatal day (PND) 35. On PND 35, three male piglets from each group were sacrificed for hippocampus and PFC samples used for reference transcriptome sequencing. Following bioinformatics analysis, Gene ontology (GO) enrichment, Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, and candidate gene screening and pathway were performed for differentially expressed genes. Results: The results showed that a total of 1,632 differential genes were identified in the hippocampus of the MS group, including 1,077 up-regulated differential genes, 555 down-regulated differential genes, and 655 significant GO entries. Analysis of the PFC of the MS group revealed 349 up-regulated genes, 151 down-regulated differential genes, and 584 significant GO entries. Genes associated with neurodevelopment were screened for large fold differences in the hippocampus, and genes associated with cognition were screened for large fold differences in the PFC. Quantitative real-time PCR (qRT-PCR) was used to verify the sequencing data. Western blot (WB) experiments revealed that MS inhibited the neurodevelopment-related WNT signaling pathway in the hippocampus and the cognitive-related PI3K-AKT signaling pathway in the PFC. Discussion: Taken together, these findings suggest that intermittent MS may affect some cognitive functions in piglets by damaging hippocampal and PFC genes or pathways.

Testis cell pyroptosis mediated by CASP1 and CASP4: possible sertoli cell-only syndrome pathogenesis.

BACKGROUND: Sertoli cell-only syndrome (SCOS) is the most serious pathological type of non-obstructive azoospermia. Recently, several genes related to SCOS have been identified, including FANCM, TEX14, NR5A1, NANOS2, PLK4, WNK3, and FANCA, but they cannot fully explain the pathogenesis of SCOS. This study attempted to explain spermatogenesis dysfunction in SCOS through testicular tissue RNA sequencing and to provide new targets for SCOS diagnosis and therapy. METHODS: We analyzed differentially expressed genes (DEGs) based on RNA sequencing of nine patients with SCOS and three patients with obstructive azoospermia and normal spermatogenesis. We further explored the identified genes using ELISA and immunohistochemistry. RESULTS: In total, 9406 DEGs were expressed (Log2|FC|≥ 1; adjusted P value < 0.05) in SCOS samples, and 21 hub genes were identified. Three upregulated core genes were found, including CASP4, CASP1, and PLA2G4A. Thus, we hypothesized that testis cell pyroptosis mediated by CASP1 and CASP4 might be involved in SCOS occurrence and development. ELISA verified that CASP1 and CASP4 activities in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenesis. Immunohistochemical results showed that CASP1 and CASP4 in the normal spermatogenesis group were mainly expressed in the nuclei of spermatogenic, Sertoli, and interstitial cells. CASP1 and CASP4 in the SCOS group were mainly expressed in the nuclei of Sertoli and interstitial cells because of the loss of spermatogonia and spermatocytes. CASP1 and CASP4 expression levels in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenisis. Furthermore, the pyroptosis-related proteins GSDMD and GSDME in the testes of patients with SCOS were also significantly higher than those in control patients. ELISA also showed that inflammatory factors (IL-1 ß, IL-18, LDH, and ROS) were significantly increased in the SCOS group. CONCLUSIONS: For the first time, we found that cell pyroptosis-related genes and key markers were significantly increased in the testes of patients with SCOS. We also observed many inflammatory and oxidative stress reactions in SCOS. Thus, we propose that testis cell pyroptosis mediated by CASP1 and CASP4 could participate in SCOS occurrence and development.

Screening and validation of potential markers associated with uterine corpus endometrial carcinoma and polycystic ovary syndrome based on bioinformatics methods.

Background: Endometrial cancer (UCEC) is a commonly occurring tumor in females, and polycystic ovary syndrome (PCOS) is closely related to UCEC, but the molecular mechanisms remain unclear. This article aims to explore potential molecular mechanisms in UCEC and PCOS, as well as identify prognostic genes for UCEC. Methods: Bioinformatics methods were employed to screen for DEGs in UCEC and PCOS. The shared DEGs were analyzed by constructing a protein-protein interaction (PPI) network using the String database and Cytoscape software. The enrichment analysis was performed using Metascape. The shared DEGs associated with the prognosis of UCEC were identified through univariate and lasso Cox regression methods. A multivariate Cox regression model was constructed and internally validated. The expression and test efficiency of the key prognostic genes were verified using external datasets for UCEC and PCOS. Furthermore, the Gepia database was utilized to analyze the expression of key prognostic genes and their correlation with the disease-free survival (RFS) of UCEC. Tumor mutation burden (TMB), immune infiltration, and the correlation of immune cells were assessed for the prognostic genes of UCEC. Results: There were 151 shared DEGs identified between UCEC and PCOS through bioinformatics screening. These shared DEGs were primarily enriched in leukocyte activation. Following model construction and verification, nine genes were determined to be prognostic for UCEC from the shared DEGs. Among them, TSPYL5, KCNJ15, RTN1, HMOX1, DCAF12L1, VNN2, and ANXA1 were confirmed as prognostic genes in UCEC through external validation. Additionally, RTN1 was identified as a key gene in both UCEC and PCOS. Gepia analysis revealed that higher expression of RTN1 was associated with RFS in UCEC. Immune infiltration analysis of the shared DEGs demonstrated significant differences in the expression of various immune cells between UCEC high and low TMB groups. The seven key prognostic genes in UCEC exhibited regulatory relationships with immune cells. Conclusion: This study identified TSPYL5, KCNJ15, RTN1, HMOX1, DCAF12L1, VNN2, and ANXA1 as the key prognostic DEGs of UCEC. These genes are associated with UCEC survival, TMB, immune cell infiltration, and immune cell regulation. Among them, RTN1 may serve as a potential biomarker for both UCEC and PCOS.

Amplicon-based sequencing and co-occurence network analysis reveals notable differences of microbial community structure in healthy and dandruff scalps.

BACKGROUND: Dandruff is a chronic, recurring, and common scalp problem that is caused by several etiopathogeneses with complex mechanisms. Management of this condition is typically achieved via antifungal therapies. However, the precise roles played by microbiota in the development of the condition have not been elucidated. Despite their omnipresence on human scalp little is known about the co-occurrence/co-exclusion network of cutaneous microbiota. RESULTS: We characterized the scalp and hair surface bacterial and fungal communities of 95 dandruff-afflicted and healthy individuals residing in China. The degree distributions of co-occurrence/co-exclusion network in fungi-bacteria and bacteria-bacteria were higher in the healthy group (P < 0.0001), whereas the betweenness values are higher in the dandruff group (P < 0.01). Meanwhile, the co-occurrence/co-exclusion network among fungi-fungi and fungi-bacteria showed that compared to the healthy group, the dandruff group had more positive links (P < 0.0001). In addition, we observed that Malassezia slooffiae, Malassezia japonica and Malassezia furfur, were more abundant in the dandruff group than in the healthy group. These microbiota were co-exclusion by either multiple bacterial genera or Malassezia sp. in healthy group. The lactic acid bacteria on the scalp and hair surface, especially the genera Lactobacillus and Lactococcus, exhibit a negative correlation with multiple bacterial genera on the scalp and hair surface. Lactobacillus plantarum and Pediococcus lactis isolated on the healthy human scalp can inhibit the growth of Staphylococcus epidermidis in vitro. CONCLUSIONS: We showed that microbial networks on scalp and hair surface with dandruff were less integrated than their healthy counterparts, with lower node degree and more positive and stronger links which were deemed to be unstable and may be more susceptible to environmental fluctuations. Lactobacillus bacteria have extensive interactions with other bacteria or fungi in the scalp and hair surface micro-ecological network and can be used as targets for improving scalp health.

Effect of gas disturbance on combustion characteristics of flammable refrigerants near LFLs.

Refrigerants with low global warming potential (GWP), will play an important role in the research of alternative refrigerants and the development of new refrigerants, but most of them are flammable. And the actual environment of the refrigeration system varies with different factors, especially gas disturbance. In this paper, the combustion characteristics of four refrigerants, i.e., isobutane (R600a), ethylene (R1150), propane (R290), and 2,3,3,3-Tetrafluoroprop-1-ene (R1234yf) under the conventional static and the gas disturbance were studied. Firstly, the flame characteristics and lower flammability limits (LFLs) of the refrigerants were investigated. Then the special combustion phenomena were observed and analyzed in the presence of the gas disturbance, and an improved method for better defining the LFLs of the flammable gases under the disturbance was proposed. Finally, the experimental results show that the LFLs of the three natural refrigerants under specific low disturbance are lower than that in normal conditions, but except for R1234yf. It was found that the specific disturbance may enhance the combustion intensity by comparing the burning time of visible flame under the two conditions. The results have guiding significance for the safe application of flammable refrigerants.

[Differentially expressed long non-coding RNAs in spermatozoa with different fertilizing abilities].

OBJECTIVE: To search for conception capacity-related long non-coding RNAs (lncRNA) and explore their possible roles in fertilization. METHODS: We obtained 10 semen samples, 5 of high and the other 5 of low fertilizing ability, extracted large RNAs, established a cDNA library, and performed RNA sequencing with the HiSeq 2000 sequencing system. Using the bioinformatics method, we assembled and predicted lncRNAs, screened differentially expressed genes between the two groups by NOIseq, analyzed the lncRNAs with the box plot and volcano plot, and determined their expression patterns by hierarchical cluster analysis. We examined the functional classification of differentially expressed lncRNAs by pathway and gene ontology (GO) enrichment and predicted those of some lncRNAs by lncRNA-mRNA interaction analysis and intersection analysis with up- and down-stream cis-acting elements. RESULTS: A total of 147 1615 lncRNAs were identified in all the semen samples, including 463 596 novel ones and 8 019 known ones, with 4 052 differentially expressed lncRNAs, 985 upregulated and the other 3 067 downregulated. Box plot and volcano plot filtering analyses showed statistically significant differences in the expressions of the lncRNAs between the two groups, and so did hierarchical cluster analysis. GO functional annotations manifested the involvement of the differentially expressed lncRNAs in the metabolic process, biological regulation, membrane and organelle formation, and protein-nucleotide binding. Pathway analysis showed that the differentially expressed lncRNAs were related to transport and catabolism, cell motility, signaling molecular interactions, signaling transduction, and signaling pathways in the development and immune systems. The functions of the 5 lncRNAs predicted were shown to be associated with sperm motility, acrosomal reaction and signal transduction during fertilization. CONCLUSIONS: Differentially expressed lncRNAs may play an important role in fertilization and become biomarkers for the assessment of sperm quality.

Laboratory and Molecular Characterization of Dengue Viruses in a 2014 Outbreak in Guangfo Region, Southern China.

Non-specific symptoms and low viremia levels make early diagnosis of dengue virus (DENV) infection challenging. This study aimed to i) identify laboratory markers that can be used to predict a DENV-positive diagnosis and ii) perform a molecular characterization of DENVs from the 2014 Guangdong epidemic. This retrospective study analyzed 1,044 patients from the Guangdong epidemic who were clinically suspected cases of dengue. Viral RNA was detected by real-time RT-PCR, and viral-specific NS1 antigen was detected using enzyme-linked immuno sorbent assay. A molecular phylogenetic analysis was performed for the with the DENV C-prM gene junction. Patients with dengue infection had leukopenia (2.8 × 109/L), thrombocytopenia (109.0 × 109/L), elevated aspartate aminotransferase (56.0 IU/L) and alanine aminotransferase (43.5 IU/L), and prolonged activated partial thromboplastin time (APTT, 33.5 s) (all P ＜ 0.001) compared to patients without dengue. The positive predictive value of leukopenia and thrombocytopenia for DENV infection were 96.9% and 93.0%, respectively. Leukopenia, thrombocytopenia, elevated aminotransferases, and prolonged APTT were useful predictive markers for an early diagnosis of DENV infection. Phylogenetic analysis indicated that the DENVs from the 2014 epidemic were closely related to a 2010 New Delhi strain and a 2013 Guangzhou strain. The 2014 epidemic consisted of co-circulating DENV-1 genotypes I and V from multiple origins. Efficient dengue surveillance can facilitate rapid response to future outbreaks.

[Effect of TSP-2 antibody against a single epitope of mouse Toll-like receptor 2 extracellular domain on zymosan A-induced peritonitis in mice].

OBJECTIVE: To observe the effect of the antibody TSP-2 against a single epitope of mouse Toll-like receptor 2 extracellular domain (mTLR2ECD) on the inflammation in mice with zymosan A-induced peritonitis. METHODS: In mice with peritonitis induced by intraperitoneal injection of zymosan A, pretreatments with PBS, normal rabbit IgG and TSP-2 antibody at two different doses (2.5 and 5.0 mg/kg) were administered via the tail vein. Six hours after intraperitoneal injection of zymosan A, Evans blue was injected through the tail vein, and the frequency of writhing of the mice within 20 min were recorded. The mice were then sacrificed for peritoneal lavage, and the lavage fluid was collected to assess the exudation of Evans blue in the supernatant. The peritoneal leukocyte count, mast cell degranulation and release of such inflammatory mediators as platelet activating factor (PAF) and tumor necrosis factor-alpha (TNFalpha) in the lavage fluid were observed by cell counting, specific cell staining, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with PBS or rabbit IgG groups, TSP-2 treatment resulted in significantly reduced writhing response of the mice and lowered Evans blue exudation and leukocyte count in the peritoneal lavage, with also decreased degranulation of the mast cells induced by C48/80. CONCLUSION: TSP-2 antibody against a single epitope of mTLR2ECD inhibits the inflammatory response in mice with zymosan A-induced peritonitis.

Genomic analysis of a Chinese isolate of Getah-like virus and its phylogenetic relationship with other Alphaviruses.

An alphavirus, M-1 strain, was isolated from a pool of culicine mosquitoes collected in Hainan island of China during an arbovirus survey in 1964. In the present study, we determined the complete nucleotide sequence of the M-1 strain using RT-PCR and RACE techniques. The M-1 genome is 11,690 nucleotides (nt) in length and contains two open reading frames (ORFs) encoding four nonstructural proteins and five structural proteins, respectively. Searches using Blast and comparison analyses suggested that M-1 is closely linked to Sagiyama virus (SAGV, AB032553) with 98% identity and Getah viruse (GETV, AY702913) with 97.8% identity in the full-length nucleotide sequence. However, compared with SAGV, there is 1 deletion (3 nucleotides in length) in the Capsid region, a deletion in the 3' untranslated region (10 nucleotides in length) and 2 insertions in the 3' untranslated region involving a total of 5 nucleotides. Interestingly, from the 5' UTR to the end of coding region, M-1 share the highest identity with GETV, even though the identity of 3' UTR drops dramatically to 76.2%. Furthermore, phylogenetic analysis based on the complete genomic sequences and sequences for structural or non-structural proteins of M-1 and 15 alphaviruses showed that M-1 grouped with GETV first and then grouped together with SAGV. Based on the comparison analysis and phylogenetic analysis, we conclude that M-1 strain can be considered as a strain that is a Chinese isolate of Getah-like virus.

[Cloning and expression of murine Toll-like receptor-2 N terminal and preparation of its antibody].

OBJECTIVE: To prepare the recombinant murine Toll-like receptor-2 N-terminal (mTLR-2N) fusion protein and obtain anti-mTLR-2N polyclonal antibody. METHODS: The gene encoding 153 amino acids of mTLR-2N was amplified by PCR and cloned into pET32A vector with sequence verification. The recombinant fusion protein was expressed in E. coli and purified by Probond resin column. Rabbits were immunized with fusion protein to obtain the polyclonal anti-sera, and the antibodies were identified by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. RESULTS: The recombinant fusion protein was efficiently expressed and purified. The polyclonal antibodies could bind to the fusion protein expressed in different vectors as the antigens in ELISA, and also bind with RAW264.7 cells expressing mTLR-2 and CHO cells transfected with full-length mTLR-2 gene. CONCLUSION: The recombinant mTLR-2N fusion protein is obtained and the anti-mTLR-2N polyclonal antibody can recognize natural mTLR-2 on the cell surface.

[Expression of human Toll-like receptor-9 in E.coli and the preparation of antibody].

AIM: To express N terminal fragment of human Toll-like receptor-9 (hTLR-9) in E.coli and prepare its antiserum. METHODS: The thioredoxin fusion expression plasmid of terminal fragment of hTLR-9 gene (707-1 167 bp) was constructed and then transformed into E.coli BL21 (DE3). The fusion protein was expressed in E.coli BL21 (DE3) under induction of 0.5 mmol/L IPTG. Inclusion body dissolved in urea was used as immunogen to prepare mouse anti-hTLR-9 antibody. RESULTS: The fusion protein was expressed in the form of inclusion body with molecular mass of about 31kD. The purity of fusion protein reached to 90%; The result of immunohistochemical staining showed that the antisera against recombinant fusion protein could react specifically to HEK293 cells transfected with hTLR-9. CONCLUSION: The fusion protein of hTLR-9 N-terminal fragment was expressed in E.coli BL21 (DE3). The prepared anti-hTLR-9 antibody can react specifically to HEK293 cells transfected with full length hTLR-9.

Efficient RNAi-based gene family knockdown via set cover optimization.

OBJECTIVE: We address the problem of selecting an efficient set of initiator molecules (siRNAs) for RNA interference (RNAi)-based gene family knockdown experiments. Our goal is to select a minimal set of siRNAs that (a) cover a targeted gene family or a specified subset of it, (b) do not cover any untargeted genes, and (c) are individually highly effective at inducing knockdown. METHODS AND MATERIAL: We give two formalizations of the gene family knockdown problem. First, we show that the problem, minimizing the number of siRNAs required to knock down a family of genes, is NP-Hard via a reduction to the set cover problem. Second, we generalize the basic problem to incorporate additional biological constraints and optimality criteria. To solve the resulting set-cover variants, we modify the classical branch-and-bound algorithm to include some of these biological criteria. We find that in many typical cases these constraints reduce the search space enough that we are able to compute exact minimal siRNA covers within reasonable time. For larger cases, we propose a probabilistic greedy algorithm for finding minimal siRNA covers efficiently. We apply our methods to two different gene families, the FREP genes from Biomphalaria glabrata and the olfactory genes from Caenorhabditis elegans. RESULTS AND CONCLUSION: Our computational results on real biological data show that the probabilistic greedy algorithm produces siRNA covers as good as the branch-and-bound algorithm in most cases. Both algorithms return minimal siRNA covers with high predicted probability that the selected siRNAs will be effective at inducing knockdown. We also examine the role of "off-target" interactions: the constraint of avoiding covering untargeted genes can, in some cases, substantially increase the complexity of the resulting solution. Overall, we find that in many common cases our approach significantly reduces the number of siRNAs required in gene family knockdown experiments, as compared to knocking down genes independently.

[Inhibition of fibrosarcoma growth in mice by antibody against B cell epitope on mouse Toll-like receptor-2 extracellular domain].

OBJECTIVE: To observe the effect of the antibody to a B cell epitope on mouse Toll-like receptor-2 (mTLR-2) extracellular domain on the growth of murine fibrosarcoma. METHODS: An immunogen was prepared by conjugating a 20-mer peptide, synthesized on the basis of prediction for B cell dominant epitope of mTLR-2, to a carrier protein. Rabbit polyclonal antibodies against B cell epitope of mTLR-2 were prepared and purified, and characterized by Western blotting, immunohistochemistry and flow cytometry. Murine fibrosarcoma S180 cells were inoculated into the left hindlimbs of Swiss mice, and in the treatment group, the injected cells were mixed with 100 microg anti-mTLR-2 antibody TSP-1, with the same dose of irrelevant rabbit IgG in the IgG control group and with buffer solution only in the blank control group. The mice in each group were anesthetized and sacrificed on days 10, 14, and 18 following the injections, respectively, and the weight of the tumors was measured and the inhibition rate calculated. RESULTS: A distinct band for the antibody TSP-1 was shown at the relative molecular mass of 95 000 by Western blotting. The antibody TSP-1 could also react with native mTLR-2 expressed on J774A.1 cells, as identified by immunohistochemistry and flow cytometry. The growth of the fibrosarcoma S180 was obviously inhibited by injections with the antibody TSP-1 mixture, and the weight of tumor in mice treated with TSP-1 was significantly lower than that in the two control groups (P<0.05), with an inhibition rate of 77% on day 10, 51% on day 14 and 53% on day 18. CONCLUSION: Local application of the antibody against B cell epitope on mTLR-2 extracellular domain can inhibit the growth of murine fibrosarcoma, especially in the early stage of tumor proliferation.

[Preparation and characterization of anti-morphine antiserum].

OBJECTIVE: To prepare the holoantigens of morphine and morphine-6-succinyl, and to evaluate the efficacy of both of the haptens in eliciting immune responses and their effects on morphine withdrawal symptoms in male mice. METHODS: Morphine-6-succinyl was prepared using acid anhydride mixture, and conjugated, along with morphine, with Blue carrier in the presence of carbodi imide. Immunization of mice with both the holoantigens and Freund adjuvant was performed, followed by determination of the antibody titer in the mouse serum with competitive ELISA and morphine dependence evaluation. RESULTS: The titers of the antibodies exceeded 1:8 000 in the serum of mice immunized with both holoantigens, and morphine- 6-succinyl induced a higher titer that was maintained for a longer period. The inhibition rates of the antisera to morphine and morphine-6-auccinyl had both reached 50%, and increased in a dose-dependent manner. CONCLUSION: Both morphine carrier conjugates can elicit high-titer antibody response in mice and relieve morphine addiction.

[Cloning of mouse TLR-2 gene and its expression in Pichia pastroris].

AIM: To clone the mouse TLR-2 gene and to express it in Pichia pastroris. METHODS: Full-length gene encoding mouse Toll-like receptor 2 (mTLR-2) was amplified by RT-PCR, cloned into pUCm-T vector, and confirmed by sequencing. The target gene was then inserted into Pichia pastroris expression vector pPICZalphaC, which was transformed into Pichia pastroris. The recombinant Pichia pastroris was confirmed by PCR and RT-PCR. Expressed protein was identified by SDS-PAGE and Western blot. RESULTS: The full-length mTLR-2 gene(GenBank accession No.AY179346) was cloned. The homology of the cloned gene to published mTLR-2 gene reached 99.84%. The recombinant expression plasmid pPICZ- mTLR-2 was constructed successfully. SDS-PAGE analysis showed that the relative molecular mass(M(r)) of recombinant protein was about 97 000. Western blot analysis showed expressed product can react to rabbit anti- mTLR-2 antibody. CONCLUSION: The full-length mTLR-2 gene is cloned and the recombinant protein can be expressed in Pichia pastroris correctly.

NS2a-NS2b sequence analysis of DEN-4 virus strains.

To study the genetic variability of DEN-4 Chinese isolates, and to trace the origin of DV4 Chinese isolates, we cloned and sequenced the NS2a-NS2b junction of 5 isolates and prototype DV4 (H-241). Our results show that isolates from the 1990 Guangdong epidemic, which were isolated in the early, middle, and late periods of the epidemic, share the same sequence in the NS2a-NS2b junction. The sequence similarity between isolates from the Guangdong epidemic in 1990 and DV4 H-241 is 96%; these isolates can be grouped into genotype I. The sequence similarity between the isolate from the Guangdong epidemic in 1987 and Dominica strain 814669 is 96%; this isolate can be grouped into genotype II. For the first time, our results show that there are also 2 DV4 genotypes in the Guangdong area of southern China, and these isolates perhaps were introduced from other epidemic areas outside of China.