Impact of streptozotocin-induced diabetes on functional responses of dendritic cells and macrophages towards Burkholderia pseudomallei

Diabetes mellitus is a documented risk factor for melioidosis, a tropical infection caused by Burkholderia pseudomallei. The increased susceptibility of diabetic individuals to infections with other pathogens has been associated with immune dysregulation. However, the impact of diabetes on the functional responses of dendritic cells (DC) and macrophages during B. pseudomallei infection has not been investigated. This study compared the responses of macrophages and DC towards B. pseudomallei using bone marrow-derived DC (BMDC) and peritoneal elicited macrophages (PEM) isolated from streptozotocin-induced diabetic C57BL/6 mice exhibiting hyperglycaemia for 9 days (acute) or 70 days (chronic) and age-matched nondiabetic C57BL/6 mice. Following coincubation of BMDC and PEM with a highly virulent B. pseudomallei isolate, maturation, bacterial internalization plus intracellular survival and cytokine gene expression profiles were assessed. No significant differences in functional responses of BMDC or PEM isolated from acute diabetic and nondiabetic mice were observed. However, significant differences in BMDC and PEM function were observed when chronic diabetic and nondiabetic mice were compared. This study demonstrates that diabetic mice with extended periods of uncontrolled hyperglycaemia have impaired DC and macrophage function towards B. pseudomallei, which may contribute to the high susceptibility observed in clinical practice.     

Myeloid Cell Sirtuin-1 Expression Does Not Alter Host Immune Responses to Gram-Negative Endotoxemia or Gram-Positive Bacterial Infection

The role of sirtuin-1 (SIRT1) in innate immunity, and in particular the influence of SIRT1 on antimicrobial defense against infection, has yet to be reported but is important to define since SIRT1 inhibitors are being investigated as therapeutic agents in the treatment of cancer, Huntington’s disease, and autoimmune diseases. Given the therapeutic potential of SIRT1 suppression, we sought to characterize the role of SIRT1 in host defense. Utilizing both pharmacologic methods and a genetic knockout, we demonstrate that SIRT1 expression has little influence on macrophage and neutrophil antimicrobial functions. Myeloid SIRT1 expression does not change mortality in gram-negative toxin-induced shock or gram-positive bacteremia, suggesting that therapeutic suppression of SIRT1 may be done safely without suppression of myeloid cell-specific immune responses to severe bacterial infections.     

Unidirectional mating-type switching confers self-fertility to Thielaviopsis cerberus,the only homothallic species in the genus

Sexual reproduction is ubiquitous in nature, and nowhere is this more so than in the fungi. Heterothallic behaviour is observed when there is a strict requirement of contact between two individuals of opposite mating type for sexual reproduction to occur. In contrast, a homothallic species can complete the entire sexual cycle in isolation, although several genetic mechanisms underpin this self-fertility. These can be inferred by characterising the structure and gene-content of the mating-type locus, which contains genes that are involved in the regulation of sexual reproduction. In this study, the genetic basis of homothallism in Thielaviopsis cerberus was investigated, the only known self-fertile species within this genus. Using genome sequencing and conventional molecular techniques, two versions of the mating-type locus were identified in this species. This is typical of species that have a unidirectional mating-type switching reproductive strategy. The first version was a self-fertile locus that contained four known mating-type genes, while the second was a self-sterile version with a single mating-type gene. The conversion from a self-fertile to a self-sterile locus is likely mediated by a homologous recombination event at two direct repeats present in the self-fertile locus, resulting in the deletion of three mating-type genes and one of the repeats. Both locus versions were present in isolates that were self-fertile, while self-sterility was caused by the presence of only a switched locus. This study provides a clear example of the architectural fluidity in the mating-type loci that is common among even closely related fungal species.     

Chemistry and biology of the ramoplanin family of peptide antibiotics

The peptide antibiotic ramoplanin factor A2 is a promising clinical candidate for treatment of Gram-positive bacterial infections that are resistant to antibiotics such as glycopeptides, macrolides, and penicillins. Since its discovery in 1984, no clinical or laboratory-generated resistance to this antibiotic has been reported. The mechanism of action of ramoplanin involves sequestration of peptidoglycan biosynthesis Lipid intermediates, thus physically occluding these substrates from proper utilization by the late-stage peptidoglycan biosynthesis enzymes MurG and the transglycosylases (TGases). Ramoplanin is structurally related to two cell wall active lipodepsipeptide antibiotics, janiemycin, and enduracidin, and is functionally related to members of the lantibiotic class of antimicrobial peptides (mersacidin, actagardine, nisin, and epidermin) and glycopeptide antibiotics (vancomycin and teicoplanin). Peptidomimetic chemotherapeutics derived from the ramoplanin sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and related pathogens. Here we review the chemistry and biology of the ramoplanins including its discovery, structure elucidation, biosynthesis, antimicrobial activity, mechanism of action, and total synthesis.     

Expression of chalcone synthase and chalcone isomerase proteins in Arabidopsis seedlings

Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. The recombinant proteins were then used to immunize chickens and the resulting IgY fraction was purified from egg yolks. Immunoblots of crude protein extracts from Arabidopsis seedlings carrying wild-type and null alleles for CHS and CHI showed that the resulting antibody preparations provide useful tools for characterizing expression of the flavonoid pathway at the protein level. An initial analysis of expression patterns in seedlings shows that CHS and CHI proteins are present at high levels during a brief period of early seedling germination that just precedes the transient accumulation of flavonoid end-products.     

Wnt/beta-catenin signaling directs multiple stages of tooth morphogenesis

Wnt/beta-catenin signaling plays key roles in tooth development, but how this pathway intersects with the complex interplay of signaling factors regulating dental morphogenesis has been unclear. We demonstrate that Wnt/beta-catenin signaling is active at multiple stages of tooth development. Mutation of beta-catenin to a constitutively active form in oral epithelium causes formation of large, misshapen tooth buds and ectopic teeth, and expanded expression of signaling molecules important for tooth development. Conversely, expression of key morphogenetic regulators including Bmp4, Msx1, and Msx2 is downregulated in embryos expressing the secreted Wnt inhibitor Dkk1 which blocks signaling in epithelial and underlying mesenchymal cells. Similar phenotypes are observed in embryos lacking epithelial beta-catenin, demonstrating a requirement for Wnt signaling within the epithelium. Inducible Dkk1 expression after the bud stage causes formation of blunted molar cusps, downregulation of the enamel knot marker p21, and loss of restricted ectodin expression, revealing requirements for Wnt activity in maintaining secondary enamel knots. These data place Wnt/beta-catenin signaling upstream of key morphogenetic signaling pathways at multiple stages of tooth development and indicate that tight regulation of this pathway is essential both for patterning tooth development in the dental lamina, and for controlling the shape of individual teeth.