Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6'-dibehenate)-a novel adjuvant inducing both strong CMI and antibody responses

Incorporation of the glycolipid trehalose 6,6'-dibehenate (TDB) into cationic liposomes composed of the quaternary ammonium compound dimethyldioctadecylammonium (DDA) produce an adjuvant system which induces a powerful cell-mediated immune response and a strong antibody response, desirable for a high number of disease targets. We have used differential scanning calorimetry (DSC) to investigate the effect of TDB on the gel-fluid phase transition of DDA liposomes and to demonstrate that TDB is incorporated into DDA liposome bilayers. Transmission Electron Microscopy (TEM) and cryo-TEM confirmed that liposomes were formed when a lipid film of DDA containing small amounts of TDB was hydrated in an aqueous buffer solution at physiological pH. Furthermore, time development of particle size and zeta potential of DDA liposomes incorporating TDB during storage at 4 degrees C and 25 degrees C, indicates that TDB effectively stabilizes the DDA liposomes. Immunization of mice with the mycobacterial fusion protein Ag85B-ESAT-6 in DDA-TDB liposomes induced a strong, specific Th1 type immune response characterized by substantial production of the interferon-gamma cytokine and high levels of IgG2b isotype antibodies. The lymphocyte subset releasing the interferon-gamma was identified as CD4 T cells.     

Isolation of deoxyribonucleic acid from mammalian tissues

1. DNA has been isolated from different mammalian tissues. The DNA preparations were free from RNA, protein and polysaccharides and have a similar range of sedimentation coefficients (approx. 24s). 2. Protein was removed by a two-stage extraction with a phenol-cresol mixture by using a detergent with 4-aminosalicylate in the first stage and sodium chloride in the second. 3. Polysaccharides remained in solution when DNA was precipitated with 2-butoxyethanol in the presence of 0.5m-sodium chloride and 1.5m-sodium benzoate. 4. Ribosomal RNA was removed by precipitation in the presence of 3m-sodium chloride at 0 degrees , when DNA remained soluble.     

Evaluation of a national universal coverage campaign of long-lasting insecticidal nets in a rural district in north-west Tanzania

ABSTRACT: BACKGROUND: Insecticide-treated nets (ITN) are one of the most effective measures for preventing malaria. Mass distribution campaigns are being used to rapidly increase net coverage in at-risk populations. This study had two purposes: to evaluate the impact of a universal coverage campaign (UCC) of long-lasting insecticidal nets (LLINs) on LLIN ownership and usage, and to identify factors that may be associated with inadequate coverage. METHODS: In 2011 two cross-sectional household surveys were conducted in 50 clusters in Muleba district, north-west Tanzania. Prior to the UCC 3,246 households were surveyed and 2,499 afterwards. Data on bed net ownership and usage, demographics of household members and household characteristics including factors related to socio-economic status were gathered, using an adapted version of the standard Malaria Indicator Survey. Specific questions relating to the UCC process were asked. RESULTS: The proportion of households with at least one ITN increased from 62.6% (95% Confidence Interval (CI) = 60.9-64.2) before the UCC to 90.8% (95% CI = 89.0-92.3) afterwards. ITN usage in all residents rose from 40.8% to 55.7%. After the UCC 58.4% (95% CI = 54.7-62.1) of households had sufficient ITNs to cover all their sleeping places. Households with children under five years (OR = 2.4, 95% CI = 1.9-2.9) and small households (OR = 1.9, 95% CI = 1.5-2.4) were most likely to reach universal coverage. Poverty was not associated with net coverage. Eighty percent of households surveyed received LLINs from the campaign. CONCLUSIONS: The UCC in Muleba district of Tanzania was equitable, greatly improving LLIN ownership and, more moderately, usage. However, the goal of universal coverage in terms of the adequate provision of nets was not achieved. Multiple, continuous delivery systems and education activities are required to maintain and improve bed net ownership and usage.     

Aminoimidazoles as bioisosteres of acylguanidines: novel, potent, selective and orally bioavailable inhibitors of the sodium hydrogen exchanger isoform-1

Inhibition of the sodium hydrogen exchanger isoform-1 (NHE-1) has been shown to limit damage to the myocardium under ischemic conditions in animals. While most known NHE-1 inhibitors are acylguanidines, this report describes the design and synthesis of a series of heterocyclic inhibitors of NHE-1 including aminoimidazoles with undiminished in vitro activity and oral bioavailability.     

Blocking hedgehog signaling after ablation of the dorsal neural tube allows regeneration of the cardiac neural crest and rescue of outflow tract septation

Cardiac neural crest cells (CNCC) migrate into the caudal pharynx and arterial pole of the heart to form the outflow septum. Ablation of the CNCC results in arterial pole malalignment and failure of outflow septation, resulting in a common trunk overriding the right ventricle. Unlike preotic cranial crest, the postotic CNCC do not normally regenerate. We applied the hedgehog signaling inhibitor, cyclopamine (Cyc), to chick embryos after CNCC ablation and found normal heart development at day 9 suggesting that the CNCC population was reconstituted. We ablated the CNCC, and labeled the remaining neural tube with DiI/CSRE and applied cyclopamine. Cells migrated from the neural tube in the CNCC-ablated, cyclopamine-treated embryos but not in untreated CNCC-ablated embryos. The newly generated cells followed the CNCC migration pathways, expressed neural crest markers and supported normal heart development. Finally, we tested whether reducing hedgehog signaling caused redeployment of the dorsal–ventral axis of the injured neural tube, allowing generation of new neural crest-like cells. The dorsal neural tube marker, Pax7, was maintained 12 h after CNCC ablation with Cyc treatment but not in the CNCC-ablated alone. This disruption of dorsal–ventral neural patterning permits a new wave of migratory cardiac neural crest-like cells.     

Identification of contact residues and definition of the CAR-binding site of adenovirus type 5 fiber protein

The binding of adenovirus (Ad) fiber knob to its cellular receptor, the coxsackievirus and Ad receptor (CAR), promotes virus attachment to cells and is a major determinant of Ad tropism. Analysis of the kinetics of binding of Ad type 5 (Ad5) fiber knob to the soluble extracellular domains of CAR together (sCAR) and each immunoglobulin (Ig) domain (IgV and IgC2) independently by surface plasmon resonance demonstrated that the IgV domain is necessary and sufficient for binding, and no additional membrane components are required to confer high-affinity binding to Ad5 fiber knob. Four Ad5 fiber knob mutations, Ser408Glu and Pro409Lys in the AB loop, Tyr477Ala in the DG loop, and Leu485Lys in beta strand F, effectively abolished high-affinity binding to CAR, while Ala406Lys and Arg412Asp in the AB loop and Arg481Glu in beta strand E significantly reduced the level of binding. Circular dichroism spectroscopy showed that these mutations do not disorder the secondary structure of the protein, implicating Ser408, Pro409, Tyr477, and Leu485 as contact residues, with Ala406, Arg412, and Arg481 being peripherally or indirectly involved in CAR binding. The critical residues have exposed side chains that form a patch on the surface, which thus defines the high-affinity interface for CAR. Additional site-directed mutagenesis of Ad5 fiber knob suggests that the binding site does not extend to the adjacent subunit or toward the edge of the R sheet. These findings have implications for our understanding of the biology of Ad infection, the development of novel Ad vectors for targeted gene therapy, and the construction of peptide inhibitors of Ad infection.     

Decolourisation of synthetic textile dyes by Phlebia tremellosa

Phlebia tremellosa decolourised eight synthetic textile dyes (200 mg l(-1)) by greater than 96% within 14 days under stationary incubation conditions. High performance liquid chromatography analysis of culture supernatants indicated that Remazol Black B was degraded by the fungus, however, complete mineralisation did not occur as a colourless organic breakdown product accumulated. Laccase activity was detectable in culture supernatants after 5 days when the fungus was grown in the presence of an artificial textile effluent, with activity reaching a maximum of 15 U l(-1) on day 14.     

CheY-dependent methylation of the asparagine receptor, McpB, during chemotaxis in Bacillus subtilis

For the Gram-positive organism Bacillus subtilis, chemotaxis to the attractant asparagine is mediated by the chemoreceptor McpB. In this study, we show that rapid net demethylation of B. subtilis McpB results in the immediate production of methanol, presumably due to the action of CheB. We also show that net demethylation of McpB occurs upon both addition and removal of asparagine. After each demethylation event, McpB is remethylated to nearly prestimulus levels. Both remethylation events are attributable to CheR using S-adenosylmethionine as a substrate. Therefore, no methyl transfer to an intermediate carrier need be postulated to occur during chemotaxis in B. subtilis as was previously suggested. Furthermore, we show that the remethylation of asparagine-bound McpB requires the response regulator, CheY-P, suggesting that CheY-P acts in a feedback mechanism to facilitate adaptation to positive stimuli during chemotaxis in B. subtilis. This hypothesis is supported by two observations: a cheRBCD mutant is capable of transient excitation and subsequent oscillations that bring the flagellar rotational bias below the prestimulus value in the tethered cell assay, and the cheRBCD mutant is capable of swarming in a Tryptone swarm plate.     

The human HLA-A*0201 allele, expressed in hamster cells, is not a high-affinity receptor for adenovirus type 5 fiber

The coxsackie B virus and adenovirus receptor (CAR) and the major histocompatibility complex (MHC) class I alpha2 domain have been identified as high-affinity cell receptors for adenovirus type 5 (Ad5) fiber. In this study we show that CAR but not MHC class I allele HLA-A*0201 binds to Ad5 with high affinity when expressed on hamster cells. When both receptors are coexpressed on the cell surface of hamster cells, Ad5 fiber bind to a single high-affinity receptor, which is CAR.