Simultaneous determination of flavonoids and triterpenoids in Cyclocarya paliurus leaves using high-performance liquid chromatography

Background: Cyclocarya paliurus is an endangered plant and endemic to China. The leaves of C. paliurus have been used in drug formulations and as ingredients in functional foods in China. The aim of this study was to develop an effective method to extract most of the compounds and to establish a simplified HPLC analytical method to determine the contents of major bioactive compounds simultaneously.Materials and methods: High-performance liquid chromatography (HPLC) coupled with a photodiode array detector (PDA) was used for the simultaneous determination of the major flavonoids and triterpenoids in C. paliurus leaves.Results: Ultrasonic extraction in 100% methanol for 30 min was adopted as the optimal extraction method for C. paliurus leaves. The separation conditions were optimized using a Phenomenex C18 ODS column (250 mm × 4.6 mm, 5 μm) with a mobile phase of acetonitrile and 0.02% formic acid and a detection wavelength of 205 nm. The validation data indicated that this new HPLC analytical method successfully quantified the provenance and seasonal variations of seven major compounds (three flavonoids and four triterpenoids) in C. paliurus leaves.Conclusion: The study provided a novel and simplified approach to simultaneously determine the quantity of major flavonoids and triterpenoids in C. paliurus leaves. The results could promote the optimization of silvicultural systems for quality control of C. paliurus.Key words: Cyclocarya paliurus; HPLC; flavonoids; triterpenoid

Molecular characters and recombinant expression of the carboxylesterase gene of the meadow moth Loxostege sticticalis L. (Lepidoptera: Pyralidae)

Insect carboxylesterases are enzymes that catalyze the hydrolysis of ester and amide moieties, which play important roles in insecticide resistance, specifically allelochemical tolerance and developmental regulation. We obtained the cDNA encoding carboxylesterase gene of Loxostege sticticalis (LstiCarE) by a cDNA library screen. The full cDNA of LstiCarE is 1,980 bp in length, containing an open reading frame (ORF) of 1,875 bp, which encodes a preprotein of 625 amino acid residues. The LstiCarE contains the catalytic triad (Ser-His-Glu), the pentapeptide GxSxG motif and GxxHxxD/E motif, which are typical characteristic of esterases. The GxSxG and GxxHxxD/E motifs of LstiCarE are modified as GCSAG and GxxHxxQ, respectively. The 3-D model structure of LstiCarE showed that Ser197, His440 and Glu321 are aggregated together, which form the catalytic triad. The recombinant LstiCarE were successfully expressed in BL21 cells using recombinant plasmid DNA, and showed high carboxylesterase activity. However, the biochemical and physiological functions of carboxylesterase gene in L. sticticalis requires further investigation.Key words: Carboxylesterase gene, Loxostege sticticalis, recombinant expression

A transient homotypic interaction model for the influenza A virus NS1 protein effector domain

Influenza A virus NS1 protein is a multifunctional virulence factor consisting of an RNA binding domain (RBD), a short linker, an effector domain (ED), and a C-terminal 'tail'. Although poorly understood, NS1 multimerization may autoregulate its actions. While RBD dimerization seems functionally conserved, two possible apo ED dimers have been proposed (helix-helix and strand-strand). Here, we analyze all available RBD, ED, and full-length NS1 structures, including four novel crystal structures obtained using EDs from divergent human and avian viruses, as well as two forms of a monomeric ED mutant. The data reveal the helix-helix interface as the only strictly conserved ED homodimeric contact. Furthermore, a mutant NS1 unable to form the helix-helix dimer is compromised in its ability to bind dsRNA efficiently, implying that ED multimerization influences RBD activity. Our bioinformatical work also suggests that the helix-helix interface is variable and transient, thereby allowing two ED monomers to twist relative to one another and possibly separate. In this regard, we found a mAb that recognizes NS1 via a residue completely buried within the ED helix-helix interface, and which may help highlight potential different conformational populations of NS1 (putatively termed 'helix-closed' and 'helix-open') in virus-infected cells. 'Helix-closed' conformations appear to enhance dsRNA binding, and 'helix-open' conformations allow otherwise inaccessible interactions with host factors. Our data support a new model of NS1 regulation in which the RBD remains dimeric throughout infection, while the ED switches between several quaternary states in order to expand its functional space. Such a concept may be applicable to other small multifunctional proteins

Conformational adaptation of Asian macaque TRIMCyp directs lineage specific antiviral activity

TRIMCyps are anti-retroviral proteins that have arisen independently in New World and Old World primates. All TRIMCyps comprise a CypA domain fused to the tripartite domains of TRIM5α but they have distinct lentiviral specificities, conferring HIV-1 restriction in New World owl monkeys and HIV-2 restriction in Old World rhesus macaques. Here we provide evidence that Asian macaque TRIMCyps have acquired changes that switch restriction specificity between different lentiviral lineages, resulting in species-specific alleles that target different viruses. Structural, thermodynamic and viral restriction analysis suggests that a single mutation in the Cyp domain, R69H, occurred early in macaque TRIMCyp evolution, expanding restriction specificity to the lentiviral lineages found in African green monkeys, sooty mangabeys and chimpanzees. Subsequent mutations have enhanced restriction to particular viruses but at the cost of broad specificity. We reveal how specificity is altered by a scaffold mutation, E143K, that modifies surface electrostatics and propagates conformational changes into the active site. Our results suggest that lentiviruses may have been important pathogens in Asian macaques despite the fact that there are no reported lentiviral infections in current macaque populations

A Non-Specific Effect Associated with Conditional Transgene Expression Based on Cre-loxP Strategy in Mice

Transgenes flanked by loxP sites have been widely used to generate transgenic mice where the transgene expression can be controlled spatially and temporally by Cre recombinase. Data from this approach has led to important conclusions in cancer, neurodevelopment and neurodegeneration. Using this approach to conditionally express micro RNAs (miRNAs) in mice, we found that Cre-mediated recombination in neural progenitor cells caused microcephaly in five of our ten independent transgenic lines. This effect was not associated with the types or the quantity of miRNAs being expressed, nor was it associated with specific target knockdown. Rather, it was correlated with the presence of multiple tandem transgene copies and inverted (head-to-head or tail-to-tail) transgene repeats. The presence of these inverted repeats caused a high level of cell death in the ventricular zone of the embryonic brain, where Cre was expressed. Therefore, results from this Cre-loxP approach to generate inducible transgenic alleles must be interpreted with caution and conclusions drawn in previous reports may need reexamination

Functional Analysis of General Odorant Binding Protein 2 from the Meadow Moth, Loxostege sticticalis L. (Lepidoptera: Pyralidae)

Odorant binding proteins play a crucial role in transporting semiochemicals across the sensillum lymph to olfactory receptors within the insect antennal sensilla. In this study, the general odorant binding protein 2 gene was cloned from the antennae of Loxostege sticticalis, using reverse transcription PCR and rapid amplification of cDNA ends. Recombinant LstiGOBP2 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR assays indicated that LstiGOBP2 mRNA is expressed mainly in adult antennae, with expression levels differing with developmental age. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the LstiGOBP2 protein has binding affinity to a broad range of odorants. Most importantly, trans-11-tetradecen-1-yl acetate, the pheromone component of Loxostege sticticalis, and trans-2-hexenal and cis-3-hexen-1-ol, the most abundant plant volatiles in essential oils extracted from host plants, had high binding affinities to LstiGOBP2 and elicited strong electrophysiological responses from the antennae of adults

Disparities and risks of sexually transmissible infections among men who have sex with men in China: a meta-analysis and data synthesis.

BACKGROUND: Sexually transmitted infections (STIs), including Hepatitis B and C virus, are emerging public health risks in China, especially among men who have sex with men (MSM). This study aims to assess the magnitude and risks of STIs among Chinese MSM. METHODS: Chinese and English peer-reviewed articles were searched in five electronic databases from January 2000 to February 2013. Pooled prevalence estimates for each STI infection were calculated using meta-analysis. Infection risks of STIs in MSM, HIV-positive MSM and male sex workers (MSW) were obtained. This review followed the PRISMA guidelines and was registered in PROSPERO. RESULTS: Eighty-eight articles (11 in English and 77 in Chinese) investigating 35,203 MSM in 28 provinces were included in this review. The prevalence levels of STIs among MSM were 6.3% (95% CI: 3.5-11.0%) for chlamydia, 1.5% (0.7-2.9%) for genital wart, 1.9% (1.3-2.7%) for gonorrhoea, 8.9% (7.8-10.2%) for hepatitis B (HBV), 1.2% (1.0-1.6%) for hepatitis C (HCV), 66.3% (57.4-74.1%) for human papillomavirus (HPV), 10.6% (6.2-17.6%) for herpes simplex virus (HSV-2) and 4.3% (3.2-5.8%) for Ureaplasma urealyticum. HIV-positive MSM have consistently higher odds of all these infections than the broader MSM population. As a subgroup of MSM, MSW were 2.5 (1.4-4.7), 5.7 (2.7-12.3), and 2.2 (1.4-3.7) times more likely to be infected with chlamydia, gonorrhoea and HCV than the broader MSM population, respectively. CONCLUSION: Prevalence levels of STIs among MSW were significantly higher than the broader MSM population. Co-infection of HIV and STIs were prevalent among Chinese MSM. Integration of HIV and STIs healthcare and surveillance systems is essential in providing effective HIV/STIs preventive measures and treatments. TRIAL REGISTRATION: PROSPERO NO: CRD42013003721