Effects of quaternary ammonium silane coatings on mixed fungal and bacterial biofilms on tracheoesophageal shunt prostheses

Two quaternary ammonium silanes (QAS) were used to coat silicone rubber tracheoesophageal shunt prostheses, yielding a positively charged surface. One QAS coating [(trimethoxysilyl)-propyldimethyloctadecylammonium chloride] was applied through chemical bonding, while the other coating, Biocidal ZF, was sprayed onto the silicone rubber surface. The sprayed coating lost its stability within an hour, while the chemically bonded coating appeared stable. Upon incubation in an artificial throat model, allowing simultaneous adhesion and growth of yeast and bacteria, all coated prostheses showed significant reductions in the numbers of viable yeast (to 12% to 16%) and bacteria (to 27% to 36%) compared with those for silicone rubber controls, as confirmed using confocal laser scanning microscopy after live/dead staining of the biofilms. In situ hybridization with fluorescently labeled oligonucleotide probes showed that yeasts expressed hyphae on the untreated and Biocidal ZF-coated prostheses but not on the QAS-coated prostheses. Whether this is a result of the positive QAS coating or is due to the reduced number of bacteria is currently unknown. In summary, this is the first report on the inhibitory effects of positively charged coatings on the viability of yeasts and bacteria in mixed biofilms. Although the study initially aimed at reducing voice prosthetic biofilms, its relevance extends to all biomedical and environmental surfaces where mixed biofilms develop and present a problem.     

Investigation of mechanism-based thrombin inhibitors: Implications of a highly conserved water molecule for the binding of coumarins within the S pocket

The synthesis of novel coumarins bearing on the lateral side chain in the 3-position an amine or a guanidine group is described. In vitro evaluation highlighted 14d which possesses a meta aniline side chain as a very potent THR inhibitor. Surprisingly, the introduction of a guanidine moiety always led to a decrease in THR inhibiting properties. We, thus, used docking experiments to rationalize the SAR in the series. This study showed the crucial role of a conserved water molecule in the specificity pocket of THR during docking simulation in order to explain the inactivity of guanidine derivatives.     

Exploring the interactions of gliadins with model membranes: Effect of confined geometry and interfaces

Mechanisms leading to the assembly of wheat storage proteins into proteins bodies within the endoplasmic reticulum (ER) of endosperm cells are unresolved today. In this work, physical chemistry parameters which could be involved in these processes were explored. To model the confined environment of proteins within the ER, the dynamic behavior of γ‐gliadins inserted inside lyotropic lamellar phases was studied using FRAP experiments. The evolution of the diffusion coefficient as a function of the lamellar periodicity enabled to propose the hypothesis of an interaction between γ‐gliadins and membranes. This interaction was further studied with the help of phospholipid Langmuir monolayers. γ‐ and ω‐gliadins were injected under DMPC and DMPG monolayers and the two‐dimensional (2D) systems were studied by Brewster angle microscopy (BAM), polarization modulation infrared reflection‐absorption spectroscopy (PM‐IRRAS), and surface tension measurements. Results showed that both gliadins adsorbed under phospholipid monolayers, considered as biological membrane models, and formed micrometer‐sized domains at equilibrium. However, their thicknesses, probed by reflectance measurements, were different: ω‐gliadins aggregates displayed a constant thickness, consistent with a monolayer, while the thickness of γ‐gliadins aggregates increased with the quantity of protein injected. These different behaviors could find some explanations in the difference of aminoacid sequence distribution: an alternate repeated ‐ unrepeated domain within γ‐gliadin sequence, while one unique repeated domain was present within ω‐gliadin sequence. All these findings enabled to propose a model of gliadins self‐assembly via a membrane interface and to highlight the predominant role of wheat prolamin repeated domain in the membrane interaction. In the biological context, these results would mean that the repeated domain could be considered as an anchor for the interaction with the ER membrane and a nucleus point for the formation and growth of protein bodies within endosperm cells. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 610–622, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Rescue of key features of the p63‐null epithelial phenotype by inactivation of Ink4a and Arf

Mice lacking p63 cannot form skin, exhibit craniofacial and skeletal defects, and die soon after birth. The p63 gene regulates a complex network of target genes, and disruption of p63 has been shown to affect the maintenance of epithelial stem cells, the differentiation of keratinocytes, and the preservation of the adhesive properties of stratified epithelium. Here, we show that inactivation of p63 in mice is accompanied by aberrantly increased expression of the Ink4a and Arf tumour suppressor genes. In turn, anomalies of the p63‐null mouse affecting the skin and skeleton are partially ameliorated in mice lacking either Ink4a or Arf. Rescue of epithelialization is accompanied by restoration of keratinocyte proliferative capacity both in vivo and in vitro and by expression of markers of squamous differentiation. Thus, in the absence of p63, abnormal upregulation of Ink4a and Arf is incompatible with skin development.     

RAPD analysis of the interspecific somatic hybrids: Solanum bulbocastanum (+) S. tuberosum

The diploid Mexican species S. bulbocastanum (blb) was used as a source of late blight resistance in somatic hybridization with the potato (S. tuberosum, tbr) dihaploid H-8105. The produced 2x blb (+) 2x tbr H-8105 somatic hybrids did not retain the blb parent's characteristic high resistance to P. infestans. The revealed aneuploidy of blb (+) tbr H-8105 hybrids indicated a possible loss of individual blb chromosome(s) carrying the resistance genes. Four hybrid clones differing in terms of their ploidy, morphology and growth potential were analysed for the presence of all twelve blb chromosomes (linkage groups). The RAPD markers assigned to particular chromosomes were selected on the basis of the linkage map of S. bulbocastanum constructed by Naess et al., Mol. Gen. Genom. 265 (2001) 694-704. Of the 86 markers analysed, twelve (14%) were common for blb and H-8105, while 34 (40%) and 40 (46%) markers were specific for the blb and H-8105 genome, respectively; this confirms the differences between the nuclear genomes of the two species. Seventeen markers (20%) present in one or the other parent were absent in the hybrids, and only one new marker was found in the hybrids. The poorly growing, aneuploid and chimeric hybrids had the same band profiles as the well growing, morphologically normal hybrids, except for two bands that were present only in normal hybrids. The presence of eleven blb linkage groups in the blb (+) tbr H-8105 hybrids was confirmed. The markers specific for the second linkage group (chromosome 2) of blb were not present in the RAPD patterns of the somatic hybrids analysed, suggesting a loss or rearrangement of this chromosome in the combined nuclear genome of the hybrids.     

Regulation of ploidy and senescence by the AMPK‐related kinase NUAK1

Senescence is an irreversible cell‐cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss‐of‐function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK‐related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant‐negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.     

Cost-effectiveness of pooled nucleic acid amplification testing for acute HIV infection after third-generation HIV antibody screening and rapid testing in the United States: a comparison of three public health settings

Background

Detection of acute HIV infection (AHI) with pooled nucleic acid amplification testing (NAAT) following HIV testing is feasible. However, cost-effectiveness analyses to guide policy around AHI screening are lacking; particularly after more sensitive third-generation antibody screening and rapid testing.

Methods and Findings

We conducted a cost-effectiveness analysis of pooled NAAT screening that assessed the prevention benefits of identification and notification of persons with AHI and cases averted compared with repeat antibody testing at different intervals. Effectiveness data were derived from a Centers for Disease Control and Prevention AHI study conducted in three settings: municipal sexually transmitted disease (STD) clinics, a community clinic serving a population of men who have sex with men, and HIV counseling and testing sites. Our analysis included a micro-costing study of NAAT and a mathematical model of HIV transmission. Cost-effectiveness ratios are reported as costs per quality-adjusted life year (QALY) gained in US dollars from the societal perspective. Sensitivity analyses were conducted on key variables, including AHI positivity rates, antibody testing frequency, symptomatic detection of AHI, and costs. Pooled NAAT for AHI screening following annual antibody testing had cost-effectiveness ratios exceeding US$200,000 per QALY gained for the municipal STD clinics and HIV counseling and testing sites and was cost saving for the community clinic. Cost-effectiveness ratios increased substantially if the antibody testing interval decreased to every 6 months and decreased to cost-saving if the testing interval increased to every 5 years. NAAT was cost saving in the community clinic in all situations. Results were particularly sensitive to AHI screening yield.

Conclusions

Pooled NAAT screening for AHI following negative third-generation antibody or rapid tests is not cost-effective at recommended antibody testing intervals for high-risk persons except in very high-incidence settings. Please see later in the article for the Editors'' Summary