Partial Protective Effect of Intranasal Immunization with Recombinant Toxoplasma gondii Rhoptry Protein 17 against Toxoplasmosis in Mice

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects a variety of mammals, including humans. An effective vaccine for this parasite is therefore needed. In this study, RH strain T. gondii rhoptry protein 17 was expressed in bacteria as a fusion with glutathione S-transferase (GST) and the recombinant proteins (rTgROP17) were purified via GST-affinity chromatography. BALB/c mice were nasally immunised with rTgROP17, and induction of immune responses and protection against chronic and lethal T. gondii infections were investigated. The results revealed that mice immunised with rTgROP17 produced high levels of specific anti-rTgROP17 IgGs and a mixed IgG1/IgG2a response of IgG2a predominance. The systemic immune response was associated with increased production of Th1 (IFN-γand IL-2) and Th2 (IL-4) cytokines, and enhanced lymphoproliferation (stimulation index, SI) in the mice immunised with rTgROP17. Strong mucosal immune responses with increased secretion of TgROP17-specific secretory IgA (SIgA) in nasal, vaginal and intestinal washes were also observed in these mice. The vaccinated mice displayed apparent protection against chronic RH strain infection as evidenced by their lower liver and brain parasite burdens (59.17% and 49.08%, respectively) than those of the controls. The vaccinated mice also exhibited significant protection against lethal infection of the virulent RH strain (survival increased by 50%) compared to the controls. Our data demonstrate that rTgROP17 can trigger strong systemic and mucosal immune responses against T. gondii and that ROP17 is a promising candidate vaccine for toxoplasmosis.     

Structural modulation of gut microbiota during alleviation of type 2 diabetes with a Chinese herbal formula

The gut microbiota is hypothesized to have a critical role in metabolic diseases, including type 2 diabetes (T2D). A traditional Chinese herbal formula, Gegen Qinlian Decoction (GQD), can alleviate T2D. To find out whether GQD modulates the composition of the gut microbiota during T2D treatment, 187 T2D patients were randomly allocated to receive high (HD, n=44), moderate (MD, n=52), low dose GQD (LD, n=50) or the placebo (n=41) for 12 weeks in a double-blinded trial. Patients who received the HD or MD demonstrated significant reductions in adjusted mean changes from baseline of fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) compared with the placebo and LD groups. Pyrosequencing of the V3 regions of 16S rRNA genes revealed a dose-dependent deviation of gut microbiota in response to GQD treatment. This deviation occurred before significant improvement of T2D symptoms was observed. Redundancy analysis identified 47 GQD-enriched species level phylotypes, 17 of which were negatively correlated with FBG and 9 with HbA1c. Real-time quantitative PCR confirmed that GQD significantly enriched Faecalibacterium prausnitzii, which was negatively correlated with FBG, HbA1c and 2-h postprandial blood glucose levels and positively correlated with homeostasis model assessment of β-cell function. Therefore, these data indicate that structural changes of gut microbiota are induced by Chinese herbal formula GQD. Specifically, GQD treatment may enrich the amounts of beneficial bacteria, such as Faecalibacterium spp. In conclusion, changes in the gut microbiota are associated with the anti-diabetic effects of GQD.     

Mixed Polyethylene Glycol-Modified Breviscapine-Loaded Solid Lipid Nanoparticles for Improved Brain Bioavailability: Preparation,Characterization, and In Vivo Cerebral Microdialysis Evaluation in Adult Sprague Dawley Rats

Breviscapine is used in the treatment of ischemic cerebrovascular diseases, but it has a low bioavailability in the brain due to its poor physicochemical properties and the activity of P-glycoprotein efflux pumps located at the blood–brain barrier. In the present study, breviscapine-loaded solid lipid nanoparticles (SLN) coated with polyethylene glycol (PEG) derivatives were formulated and evaluated for their ability to enhance brain bioavailability. The SLNs were either coated with polyethylene glycol (40) (PEG-40) stearate alone (Bre-GBSLN-PS) or a mixture of PEG-40 stearate and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 (DSPE-PEG₂₀₀₀) (Bre-GBSLN-PS-DSPE) and were characterized both in vitro and in vivo. The mean particle size, polydispersity index, and entrapment efficiency for Bre-GBSLN-PS and Bre-GBSLN-PS-DSPE were 21.60 ± 0.10 and 22.60 ± 0.70 nm, 0.27 ± 0.01 and 0.26 ± 0.04, and 46.89 ± 0.73% and 47.62 ± 1.86%, respectively. The brain pharmacokinetic parameters revealed that the brain bioavailability of breviscapine from the Bre-GBSLN-PS and Bre-GBSLN-PS-DSPE was significantly enhanced (p < 0.01) with the area under concentration–time curve (AUC) of 1.59 ± 0.39 and 1.42 ± 0.58 μg h/mL of breviscapine, respectively, in comparison to 0.11 ± 0.02 μg h/mL from the commercial breviscapine injection. The ratios of the brain AUC for scutellarin in comparison with the plasma scutellarin AUC for commercial breviscapine injection, Bre-GBSLN-PS, and Bre-GBSLN-PS-DSPE were 0.66%, 2.82%, and 4.51%, respectively. These results showed that though both SLN formulations increased brain uptake of breviscapine, Bre-GBSLN-PS-DSPE which was coated with a binary combination of PEG-40 stearate and DSPE-PEG₂₀₀₀ had a better brain bioavailability than Bre-GBSLN-PS. Thus, the coating of SLNs with the appropriate PEG derivative combination could improve brain bioavailability of breviscapine and can be a promising tool for brain drug delivery.KEY WORDS: breviscapine, microdialysis, mixed PEGylation, P-glycoprotein (P-gp), solid lipid nanoparticles     

拟南芥核苷糖转换相关基因研究进展

核苷糖是植物细胞壁多聚糖合成时的活化底物.综述了近年来拟南芥核苷糖转换相关基因的克隆及功能分析等方面的研究进展.使人们能够深入认识核苷糖转换酶在植物生长发育中的重要作用,并且为人工改造植物细胞壁提供了理论依据.然而,对核苷糖转换酶的转录调控和代谢调节等方面研究还有待加强.     

Comparison of the expression of Bacillus thuringiensis full-length and N-terminally truncated vip3A gene in Escherichia coli

AIMS: Studies were performed to demonstrate the function of the putative signal peptide of Vip3A proteins in Escherichia coli. METHODS AND RESULTS: The full-length vip3A-S184 gene was isolated from a soil-isolated Bacillus thuringiensis, and the vip3AdeltaN was constructed by deleting 81 nucleotides at the 5'-terminus of vip3A-S184. Both were transformed and expressed in E. coli. About 19.2% of Vip3A-S184 proteins secreted soluble proteins and others formed inclusion bodies in the periplasmic space. In contrast, the Vip3AdeltaN was insoluble and formed inclusion bodies in the cytoplasm. Bioassay indicated that Vip3A-S184 showed different toxicity against Spodoptera exigua, Helicoverpa armigera and S. litura, but Vip3AdeltaN showed no toxicity to either of them because of the deletion of the first 27 amino acids at the N-terminus. CONCLUSIONS: The results suggest that the deleted N-terminal sequences were essential for the secretion of Vip3A-S184 protein in E. coli and might be required for toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The function of the putative signal peptide of Vip3A protein in E. coli was investigated. These would be helpful to make clear the unknown secretion pathway of Vip3A protein in B. thuringiensis and determine the receptor-binding domain or toxic fragment of Vip3A-S184 protein.     

Identification of Two Species of Yeast-like Symbiotes in the Brown Planthopper, <Emphasis Type="Italic">Nilaparvata lugens</Emphasis>

To determine the species of the yeast-like symbionts (YLS) in the brown planthoppers (BPH), Nilaparvata lugens, YLS were first isolated and purified by ultracentrifugation from the fat bodies of BPH, and then 18S rDNA and internal transcribed spacer (ITS)–5.8S rDNA sequences of YLS were amplified with the different general primers for fungi. The results showed that the two different 18S and ITS–5.8S rDNA sequences of YLS were obtained. One 2291-bp DNA sequence, which contained 18S and ITS–5.8S rDNA, showed the high similarity to Cryptococcus and was named Cryp-Like symbiotes. Another 1248-bp DNA sequence, which contained a part of 18S and ITS–5.8S rDNA, showed the high similarity to Pichia guilliermondii and was named Pichia-Like symbiotes. It was further proved that Cryp- and Pichia-Like symbiotes existed in BPH through nested PCR with specific primers for two symbiotes and in situ hybridization analysis using digoxigenin-labeled probes. Our results showed that BPH harbored more than one species of eukaryotic YLS, which suggested that diversity of fungal endosymbiotes may be occurred in planthoppers, just like bacterial endosymbiotes.     

Diabetes with hypertension as risk factors for adult dengue hemorrhagic fever in a predominantly dengue serotype 2 epidemic: a case control study

Background

Dengue hemorrhagic fever (DHF) is a severe form of dengue, characterized by bleeding and plasma leakage. A number of DHF risk factors had been suggested. However, these risk factors may not be generalized to all populations and epidemics for screening and clinical management of patients at risk of developing DHF. This study explored demographic and comorbidity risk factors for DHF in adult dengue epidemics in Singapore in year 2006 (predominantly serotype 1) and in year 2007–2008 (predominantly serotype 2).

Methods

A retrospective case-control study was conducted with 149 DHF and 326 dengue fever (DF) patients from year 2006, and 669 DHF and 1,141 DF patients from year 2007–2008. Demographic and reported comorbidity data were collected from patients previously. We performed multivariate logistic regression to assess the association between DHF and demographic and co-morbidities for year 2006 and year 2007–2008, respectively.

Results

Only Chinese (adjusted odds ratio [AOR] = 1.90; 95% confidence interval [CI]: 1.01–3.56) was independently associated with DHF in year 2006. In contrast, age groups of 30–39 years (AOR = 1.41; 95% CI:1.09–1.81), 40–49 years (AOR = 1.34; 95% CI:1.09–1.81), female (AOR = 1.57; 95% CI:1.28–1.94), Chinese (AOR = 1.67; 95% CI:1.24–2.24), diabetes (AOR = 1.78; 95% CI:1.06–2.97), and diabetes with hypertension (AOR = 2.16; 95%CI:1.18–3.96) were independently associated with DHF in year 2007–2008. Hypertension was proposed to have effect modification on the risk of DHF outcome in dengue patients with diabetes. Chinese who had diabetes with hypertension had 2.1 (95% CI:1.07–4.12) times higher risk of DHF compared with Chinese who had no diabetes and no hypertension.

Conclusions

Adult dengue patients in Singapore who were 30–49 years, Chinese, female, had diabetes or diabetes with hypertension were at greater risk of developing DHF during epidemic of predominantly serotype 2. These risk factors can be used to guide triaging of patients who require closer clinical monitoring and early hospitalization in Singapore, when confirmed in more studies.     

Cloning and expression analysis of a water stress-induced gene from Brassica oleracea.

Plant growth and productivity are greatly affected by water stress, such as drought and salinity. Here we report on the cloning and expression analysis of a water stress-induced gene from Brassica oleracea (designated as BoWS, GenBank accession number AY571333) by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoWS consisted of 594 bp and contained a 285 bp open reading frame (ORF) encoding a 95-amino-acid protein. The deduced protein had a calculated molecular mass of 10.53 kDa and an isoelectric point of 6.93. The sequence similarity and comparative analysis showed that BoWS was 84% identical to Arabidopsis thaliana putative water stress-induced protein (GenBank accession number AAM67282). Southern blot analysis indicated that BoWS was a low-copy gene. Northern blot analysis revealed that the expression of BoWS was upregulated by abscisic acid (ABA), mannitol, NaCl, drought, salicylic acid (SA) and hydrogen peroxide (H(2)O(2)). Our results indicate that BoWS is extremely related to the water-deficit stress in B. oleracea.     

Direct electrochemistry and electrocatalysis of heme-proteins entrapped in agarose hydrogel films

Three heme-proteins, including myoglobin (Mb), hemoglobin (Hb) and horseradish peroxidase (HRP), were immobilized on edge-plane pyrolytic graphite (EPG) electrodes by agarose hydrogel. The proteins entrapped in the agarose film undergo fast direct electron transfer reactions, corresponding to FeIII = e- --> FeII. The formal potential (E degrees'), the apparent coverage (Gamma), the electron transfer coefficient (alpha) and the apparent electron transfer rate constant (ks) were calculated by integrating cyclic voltammograms or performing nonlinear regression analysis of square wave voltammetric (SWV) experimental data. The E degrees's are linearly dependent on solution pH (redox Bohr effect), indicating that the electron transfer was proton-coupled. Ultraviolet visible (UV-Vis) and reflection-absorption infrared (RAIR) spectra suggest that the conformation of proteins in the agarose film are little different from that proteins alone, and the conformation changes reversibly in the range of pH 3.0-10.0. Atomic force microscopy (AFM) images of the agarose film indicate a stable and crystal-like structure formed possibly due to the synergistic interaction of hydrogen bonding between N,N-dimethylformamide (DMF), agarose hydrogel and heme-proteins. This suggests a strong interaction between the heme-proteins and the agarose hydrogel. DMF plays an important role in immobilizing proteins and enhancing electron transfer between proteins and electrodes. The mechanisms for catalytic reduction of hydrogen peroxide and nitric oxide (NO) by proteins entrapped in agarose hydrogel were also explored.