Genotyping of Human Brucella melitensis Biovar 3 Isolated from Shanxi Province in China by MLVA16 and HOOF

Background

Brucellosis presents a significant economic burden for China because it causes reproductive failure in host species and chronic health problems in humans. These problems can involve multiple organs. Brucellosis is highly endemic in Shanxi Province China. Molecular typing would be very useful to epidemiological surveillance. The purpose of this study was to assess the diversity of Brucella melitensis strains for epidemiological surveillance. Historical monitoring data suggest that Brucella melitensis biovar 3 is the predominant strain associated with the epidemic of brucellosis in Shanxi Province.

Methods/Principal Findings

Multiple-locus variable-number repeat analysis (MLVA-16) and hypervariable octameric oligonucleotide fingerprinting (HOOF-print) were used to type a human-hosted Brucella melitensis population (81 strains). Sixty-two MLVA genotypes (discriminatory index: 0.99) were detected, and they had a genetic similarity coefficient ranging from 84.9% to 100%. Eighty strains of the population belonged to the eastern Mediterranean group with panel 1 genotypes 42 (79 strains) and 43 (1 strain). A new panel 1 genotype was found in this study. It was named 114 MLVAorsay genotype and it showed similarity to the two isolates from Guangdong in a previous study. Brucella melitensis is distributed throughout Shanxi Province, and like samples from Inner Mongolia, the eastern Mediterranean genotype 42 was the main epidemic strain (97%). The HOOF-printing showed a higher diversity than MLVA-16 with a genetic similarity coefficient ranging from 56.8% to 100%.

Conclusions

According to the MLVA-16 and HOOF-printing results, both methods could be used for the epidemiological surveillance of brucellosis. A new genotype was found in both Shanxi and Guangdong Provinces. In areas with brucellosis, the MLVA-16 scheme is very important for tracing cases back to their origins during outbreak investigations. It may facilitate the expansion and eradication of the disease.     

Effects of germination time on seed morph ratio in a seed-dimorphic species and possible ecological significance

Background and Aims Diaspores of heteromorphic species may germinate at different times due to distinct dormancy-breaking and germination requirements, and this difference can influence life history traits. The primary aim of this study was to determine the effect of germination time of the two seed morphs of Suaeda corniculata subsp. mongolica on life history traits of the offspring.Methods Germinated brown and black seeds were sown on the 20th of each month from April to September in a simulated but near-natural habitat of the species. Phenological and vegetative traits of the maternal plants, and number, size and germination percentage of the offspring were determined.Key Results Germinated seeds sown late in the year produced smaller plants that had a higher proportion of non-dormant brown than dormant black seeds, and these brown seeds were larger than those produced by germinated seeds sown early in the year. The length of the seedling stage for brown seeds was shorter than that for black seeds, and the root/shoot ratio and reproductive allocation of plants from brown seeds were more variable than they were for plants from black seeds. Late-germinating brown seeds produced larger plants than late-germinating black seeds.Conclusions Altering the proportion of the two seed types in response to germination timing can help alleviate the adverse effects of delayed germination. The flexible strategy of a species, such as S. corniculata, that produces different proportions of dimorphic seeds in response to variation in germination timing may favour the maintenance and regeneration of the population in its unpredictable environment.     

Targeting Hedgehog signaling pathway and autophagy overcomes drug resistance of BCR-ABL-positive chronic myeloid leukemia

The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. The Hedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drug resistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or -resistant BCR-ABL⁺ CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway could markedly induce autophagy in BCR-ABL⁺ CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing could significantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our study demonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability and induce apoptosis of imatinib-sensitive or -resistant BCR-ABL⁺ cells. Moreover, this combination had little cytotoxicity in human peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARP cleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicated that simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR-ABL⁺ cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.     

Large-scale production of foreign proteins via the novel plant transient expression system in <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

Transient expression of foreign genes by Agrobacterium infiltration is a versatile technique that can be used as a rapid tool for functional protein production in plants. A reproducible protocol of large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L. was established in our study. Non-detached plants from soil-independent culture were used as the target organ, and vacuum infiltrating mediated by Agrobacterium tumefaciens harboring green fluorescent protein (GFP) gene was performed. Step-by-step optimization was performed and showed that the quality of plant material as well as agro-infiltration conditions were the major factors influencing the gene expression. Monitoring the transient GFP expression daily, the highest expression level was achieved on the 8th day post-infiltration. Evidence of anti-acidic fibroblast growth factor-single chain variable fragment (anti-aFGF-scFv) gene expression in pea seedling was also achieved using agro-mediated vacuum infiltration system. Our work proves that the system is suitable for the largescale production of pharmaceutical proteins. The in planta infiltration system described here provides a powerful tool to explore easily gene expression in Pisum sativum L. avoiding tissue culture steps and the labor-intensive generation of transgenic plants.     

Cloning and characterization of an mRNA encoding a novel G protein alpha-subunit abundant in mantle and gill of pearl oyster Pinctada fucata

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G(s)alpha (pfG(s)alpha) from the pearl oyster was isolated. The G(s)alpha cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) G(s)alpha. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG(s)alpha has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G(s)alpha mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG(s)alpha in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG(s)alpha that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G(s)alpha in MC3T3-E1 cells. These results indicated that G(s)alpha may be involved in regulation of physiological function, particularly in biological biomineralization.     

Effects of wild-type p53 expression on the quantity and activity of topoisomerase IIalpha and beta in various human cancer cell lines.

The p53 null HL-60 cell line was transfected with plasmids coding for either the wild-type p53 or mutant p53 gene. The stable expression of wild-type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR-529. HL-60 cells expressing wild-type p53 demonstrated 8- to 10-fold more VP-16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild-type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild-type p53 in the K562 cell line resulted in a 3-fold increase in sensitivity to VP-16. The quantity of topoisomerase IIalpha was not altered by the transfection as determined by immunoblotting, while the amount of the beta isoform was increased 2.5-fold in HL-60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIalpha was substantially diminished in both stable and inducible wild-type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP-16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIalpha. No changes in topoisomerase IIbeta immunostaining were observed. These results suggest that an epitope for topoisomerase IIalpha is concealed in cells expressing wild-type p53 and that a complex between topoisomerase IIalpha and p53 may be disrupted by the addition of antitumor drugs.     

Two New Triterpenoids from the Roots of Sanguisorba officinalis L.

Two new triterpenoids, octanordammar-1,11,13(17)-trien-17-ol-3,16-dione (1) and lup-12-en-15α,19β-diol-3,11-dioxo-28-oic acid (4), as well as 13 known compounds were isolated from the roots of Sanguisorba officinalis L. (Rosaceae). Their structures were determined using spectroscopic methods.