Primers with 5' flaps improve real-time PCR

Primers that contain portions noncomplementary to the target region are usually used to add to the PCR product a utility sequence such as a restriction site or a universal probe binding site. We have demonstrated that primers with short 5'AT-rich overhangs increase real-time PCR fluorescent signal. The improvement is particularly significant for difficult to amplify templates, such as highly variable viral sequences or bisulfite-treated DNA.     

<Emphasis Type="Italic">Lactococcus lactis</Emphasis> M4, a potential host for the expression of heterologous proteins

Background  

Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins.     

Three sorting nexins drive the degradation of apoptotic cells in response to PtdIns(3)P signaling

Apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Phagosome maturation requires phosphatidylinositol 3-phosphate [PtdIns(3)P], yet how PtdIns(3)P triggers phagosome maturation remains largely unknown. Through a genomewide PtdIns(3)P effector screen in the nematode Caenorhabditis elegans , we identified LST-4/SNX9, SNX-1, and SNX-6, three BAR domain-containing sorting nexins, that act in two parallel pathways to drive PtdIns(3)P-mediated degradation of apoptotic cells. We found that these proteins were enriched on phagosomal surfaces through association with PtdIns(3)P and through specific protein-protein interaction, and they promoted the fusion of early endosomes and lysosomes to phagosomes, events essential for phagosome maturation. Specifically, LST-4 interacts with DYN-1 (dynamin), an essential phagosome maturation initiator, to strengthen DYN-1's association to phagosomal surfaces, and facilitates the maintenance of the RAB-7 GTPase on phagosomal surfaces. Furthermore, both LST-4 and SNX-1 promote the extension of phagosomal tubules to facilitate the docking and fusion of intracellular vesicles. Our findings identify the critical and differential functions of two groups of sorting nexins in phagosome maturation and reveal a signaling cascade initiated by phagocytic receptor CED-1, mediated by PtdIns(3)P, and executed through these sorting nexins to degrade apoptotic cells.     

Sequential rather than interactive effects of multiple stressors as drivers of phytoplankton community change in a large lake

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Discrimination between Francisella tularensis and Francisella-like endosymbionts when screening ticks by PCR

The presence of Francisella-like endosymbionts in tick species known to transmit tularemia poses a potential diagnostic problem for laboratories that screen tick samples by PCR for Francisella tularensis. Tick samples initially considered positive for F. tularensis based on standard 16S rRNA gene PCR were found to be positive only for Francisella-like endosymbionts using a multitarget F. tularensis TaqMan assay (ISFtu2, tul4, and iglC) and 16S rRNA gene sequencing. Specificity of PCR-based diagnostics for F. tularensis should be carefully evaluated with appropriate specimen types prior to diagnostic use.     

Towards a structure for a TSG-6.hyaluronan complex by modeling and NMR spectroscopy: insights into other members of the link module superfamily

The Link module from human TSG-6, a hyaladherin with roles in ovulation and inflammation, has a hyaluronan (HA)-binding groove containing two adjacent tyrosine residues that are likely to form CH-pi stacking interactions with sequential rings in the sugar. We have used this observation to construct a model of a protein.HA complex, which was then tested against existing experimental information and by acquisition of new NMR data sets of [(13)C, (15)N]HA (8-mer) complexed with unlabeled protein. A major finding of this analysis was that acetamido side chains of two GlcNAc rings fit into hydrophobic pockets on either side of the adjacent tyrosines, providing a selectivity mechanism of HA over other polysaccharides. Furthermore, two basic residues have a separation that matches that of glucuronic acids in the sugar, consistent with the formation of salt bridges; NMR experiments at a range of pH values identified protein groups that titrate due to their proximity to a free carboxylate in HA. Sequence alignment and construction of homology models for all human Link modules in their HA-bound states revealed that many of these features are conserved across the superfamily, thus allowing the prediction of functionally important residues. In the case of cartilage link protein, its two Link modules were docked together (using bound HA as a guide), identifying hydrophobic residues likely to form an intra-Link module interface as well as amino acids that could be involved in supporting intermolecular interactions between link proteins and chondroitin sulfate proteoglycans. Here, we propose a mechanism for ternary complex formation that generates higher order helical structures, as may exist in cartilage aggregates.     

Modulation of macrophage phenotype by soluble product(s) released from neutrophils

The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-alpha, 168% more IL-6, and 61% less TGF-beta1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-alpha staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-alpha and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-alpha and IL-6 release from LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-alpha release from LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-alpha and superoxide release from murine peritoneal macrophages. The TNF-alpha inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.