A tractable method for simultaneous modifications to the head and tail of bacteriophage lambda and its application to enhancing phage-mediated gene delivery

There is considerable interest in the use of bacteriophage vectors for mammalian cell gene transfer applications, due to their stability, excellent safety profile and inexpensive mass production. However, to date, phage vectors have been plagued by mediocre performance as gene transfer agents. This may reflect the complexity of the viral infection process in mammalian cells and the need to refine each step of this process in order to arrive at an optimal, phage-based gene transfer system. Therefore, a flexible system was designed that alowed for the introduction of multiple modifications on the surface of bacteriophage lambda. Using this novel method, multiple peptides were displayed simultaneously from both the phage head and tail. Surface head display of an ubiquitinylation motif greatly increased the efficiency of phage-mediated gene transfer in a murine macrophage cell line. Gene transfer was further increased when this peptide was displayed in combination with a tail-displayed CD40-binding motif. Overall, this work provides a novel system that can be used to rationally improve bacteriophage gene transfer vectors and shows it may be possible to enhance the efficiency of phage-mediated gene transfer by targeting and optimizing multiple steps within the viral infection pathway.     

Interactions of the Cytoplasmic Domains of Human and Simian Retroviral Transmembrane Proteins with Components of the Clathrin Adaptor Complexes Modulate Intracellular and Cell Surface Expression of Envelope Glycoproteins

The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXØ, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXØ motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXØ motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXØ motifs interact with the μ1 and μ2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the β2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXØ-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved.     

The structural and functional organization of H-NS-like proteins is evolutionarily conserved in Gram-negative bacteria

The structural gene of the H-NS protein, a global regulator of bacterial metabolism, has been identified in the group of enterobacteria as well as in closely related bacteria, such as Erwinia chrysanthemi and Haemophilus influenzae . Isolated outside these groups, the BpH3 protein of Bordetella pertussis exhibits a low amino acid conservation with H-NS, particularly in the N-terminal domain. To obtain information on the structure, function and/or evolution of H-NS, we searched for other H-NS-related proteins in the latest databases. We found that HvrA, a trans -activator protein in Rhodobacter capsulatus , has a low but significant similarity with H-NS and H-NS-like proteins. This Gram-negative bacterium is phylogenetically distant from Escherichia coli . Using theoretical analysis (e.g. secondary structure prediction and DNA binding domain modelling) of the amino acid sequence of H-NS, StpA (an H-NS-like protein in E. coli ), BpH3 and HvrA and by in vivo and in vitro experiments (e.g. complementation of various H-NS-related phenotypes and competitive gel shift assay), we present evidence that these proteins belong to the same class of DNA binding proteins. In silico analysis suggests that this family also includes SPB in R. sphaeroides , XrvA in Xanthomonas oryzae and VicH in Vibrio cholerae . These results demonstrate that proteins structurally and functionally related to H-NS are widespread in Gram-negative bacteria.     

The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats

Background  

In Archeae and Bacteria, the repeated elements called CRISPRs for "clustered regularly interspaced short palindromic repeats" are believed to participate in the defence against viruses. Short sequences called spacers are stored in-between repeated elements. In the current model, motifs comprising spacers and repeats may target an invading DNA and lead to its degradation through a proposed mechanism similar to RNA interference. Analysis of intra-species polymorphism shows that new motifs (one spacer and one repeated element) are added in a polarised fashion. Although their principal characteristics have been described, a lot remains to be discovered on the way CRISPRs are created and evolve. As new genome sequences become available it appears necessary to develop automated scanning tools to make available CRISPRs related information and to facilitate additional investigations.     

Load-dependent effects of duodenal glucose on glycemia, gastrointestinal hormones, antropyloroduodenal motility, and energy intake in healthy men

Gastric emptying is a major determinant of glycemia, gastrointestinal hormone release, and appetite. We determined the effects of different intraduodenal glucose loads on glycemia, insulinemia, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and cholecystokinin (CCK), antropyloroduodenal motility, and energy intake in healthy subjects. Blood glucose, plasma hormone, and antropyloroduodenal motor responses to 120-min intraduodenal infusions of glucose at 1) 1 ("G1"), 2) 2 ("G2"), and 3) 4 ("G4") kcal/min or of 4) saline ("control") were measured in 10 healthy males in double-blind, randomized fashion. Immediately after each infusion, energy intake at a buffet meal was quantified. Blood glucose rose in response to all glucose infusions (P < 0.05 vs. control), with the effect of G4 and G2 being greater than that of G1 (P < 0.05) but with no difference between G2 and G4. The rises in insulin, GLP-1, GIP, and CCK were related to the glucose load (r > 0.82, P < 0.05). All glucose infusions suppressed antral (P < 0.05), but only G4 decreased duodenal, pressure waves (P < 0.01), resulted in a sustained stimulation of basal pyloric pressure (P < 0.01), and decreased energy intake (P < 0.05). In conclusion, variations in duodenal glucose loads have differential effects on blood glucose, plasma insulin, GLP-1, GIP and CCK, antropyloroduodenal motility, and energy intake in healthy subjects. These observations have implications for strategies to minimize postprandial glycemic excursions in type 2 diabetes.     

Gastrointestinal and immunological responses of senior dogs to chicory and mannan-oligosaccharides

Thirty-four senior dogs (pointers 8-11 years, beagles 9-11 years) were used to evaluate the effects of oligosaccharides on nutritional and immunological characteristics. Dogs were randomly allotted to treatments [1% chicory (CH), 1% mannan-oligosaccharide (MOS), 1% chicory + 1% MOS (CM), or no supplementation (control, CON)] in a parallel design with a 4 week baseline period followed by a 4 week treatment period. Dietary supplementation with MOS or CM tended (P = 0.07) to increase food intake due, in part, to an increase in fermentable fibre and a decrease in energy content of the diet. Although wet faecal output increased (P < 0.05) for dogs supplemented with MOS or CM, when corrected for food intake, no differences were noted. The CM treatment increased (P < 0.05) faecal score (1 = hard and dry, 5 = watery liquid), although these scores remained in a desirable range (3 to 3.5). Chicory supplementation increased (P = 0.07) fat digestibility. Chicory or MOS increased (P < or = 0.05) faecal bifidobacteria concentrations 0.4 and 0.5 log10 cfu/g DM, respectively, compared to the CON, while MOS decreased (P < 0.05) faecal E. coli concentrations. Oligosaccharides did not affect white blood cell (WBC) concentrations, but CH and CM tended to increase (P = 0.10) neutrophil concentrations compared to control dogs. Peripheral lymphocyte concentrations were decreased in dogs supplemented with MOS (P = 0.06) and CM (P < 0.05). Chicory and MOS alter faecal microbial populations and certain indices of the immune system of senior dogs.     

Nicotine inhibits cytokine production by placenta cells via NFkappaB: potential role in pregnancy-induced hypertension

Pregnancy-induced hypertension (PIH), also known as preeclampsia, is one of the major causes of maternal and fetal death. While the precise cause of PIH is not known, aberrant cytokine production and placenta participation are considered to be important factors. Gestational cigarette smoking, which is widely accepted to be harmful to both the mother and fetus, is protective against PIH. Based on the antiinflammatory activity of nicotine, the major component of cigarettes, we examined the effect of nicotine and other cholinergic agonists on placental inflammatory responses ex vivo. We observed that nicotine and other cholinergic agonists significantly suppress placenta cytokine production following stimulation. Placenta cells express the alpha7 nicotinic acetylcholine receptor (alpha7nAChR), and using cholinergic antagonists, we demonstrated that the antiinflammatory effect of nicotine and other cholinergic agonists is, in part, mediated through the nAChR pathway. By contrast, cholinergic stimulation had no effect on the expression of soluble fms-like tyrosine kinase (sFlt), an antiangiogenic substance implicated in maternal vascular dysfunction during PIH. Mechanistic studies reveal that cholinergic agonists exert their antiinflammatory effects through the NFkappaB pathway. Taken together, our results suggest that cholinergic agonists, including nicotine, may reduce cytokine production by placenta cells via NFkappaB to protect against PIH.     

PHENOPSIS, an automated platform for reproducible phenotyping of plant responses to soil water deficit in Arabidopsis thaliana permitted the identification of an accession with low sensitivity to soil water deficit

The high-throughput phenotypic analysis of Arabidopsis thaliana collections requires methodological progress and automation. Methods to impose stable and reproducible soil water deficits are presented and were used to analyse plant responses to water stress. Several potential complications and methodological difficulties were identified, including the spatial and temporal variability of micrometeorological conditions within a growth chamber, the difference in soil water depletion rates between accessions and the differences in developmental stage of accessions the same time after sowing. Solutions were found. Nine accessions were grown in four experiments in a rigorously controlled growth-chamber equipped with an automated system to control soil water content and take pictures of individual plants. One accession, An1, was unaffected by water deficit in terms of leaf number, leaf area, root growth and transpiration rate per unit leaf area. Methods developed here will help identify quantitative trait loci and genes involved in plant tolerance to water deficit.