Combinatorial materials research applied to the development of new surface coatings IV. A high-throughput bacterial biofilm retention and retraction assay for screening fouling-release performance of coatings

A high-throughput bacterial biofilm retention screening method has been augmented to facilitate the rapid analysis and down-selection of fouling-release coatings for identification of promising candidates. Coatings were cast in modified 24-well tissue culture plates and inoculated with the marine bacterium Cytophaga lytica for attachment and biofilm growth. Biofilms retained after rinsing with deionised water were dried at ambient laboratory conditions. During the drying process, retained biofilms retracted through a surface de-wetting phenomenon on the hydrophobic silicone surfaces. The retracted biofilms were stained with crystal violet, imaged, and analysed for percentage coverage. Two sets of experimental fouling-release coatings were analysed with the high-throughput biofilm retention and retraction assay (HTBRRA). The first set consisted of a series of model polysiloxane coatings that were systematically varied with respect to ratios of low and high MW silanol-terminated PDMS, level of cross-linker, and amount of silicone oil. The second set consisted of cross-linked PDMS-polyurethane coatings varied with respect to the MW of the PDMS and end group functionality. For the model polysiloxane coatings, HTBRRA results were compared to data obtained from field immersion testing at the Indian River Lagoon at the Florida Institute of Technology. The percentage coverage calculations of retracted biofilms correlated well to barnacle adhesion strength in the field (R(2)=0.82) and accurately identified the best and poorest performing coating compositions. For the cross-linked PDMS-polyurethane coatings, the HTBRRA results were compared to combinatorial pseudobarnacle pull-off adhesion data and good agreement in performance was observed. Details of the developed assay and its implications in the rapid discovery of new fouling-release coatings are discussed.     

Epithelial expression of TLR4 is modulated in COPD and by steroids,salmeterol and cigarette smoke

The toll-like receptors (TLRs) are a key component of host defense in the respiratory epithelium. Cigarette smoking is associated with increased susceptibility to infection, while COPD is characterised by bacterial colonisation and infective exacerbations. We found reduced TLR4 gene expression in the nasal epithelium of smokers compared with non-smoking controls, while TLR2 expression was unchanged. Severe COPD was associated with reduced TLR4 expression compared to less severe disease, with good correlation between nasal and tracheal expression. We went on to examine the effect of potential modulators of TLR4 expression in respiratory epithelium pertinent to airways disease. Using an airway epithelial cell line, we found a dose-dependent downregulation in TLR4 mRNA and protein expression by stimulation with cigarette smoke extracts. Treatment with the corticosteroids fluticasone and dexamethasone resulted in a dose-dependent reduction in TLR4 mRNA and protein. The functional significance of this effect was demonstrated by impaired IL-8 and HBD2 induction in response to LPS. Stimulation with salmeterol (10^-6 M) caused upregulation of TLR4 membrane protein presentation with no upregulation of mRNA, suggesting a post-translational effect. The effect of dexamethasone and salmeterol in combination was additive, with downregulation of TLR4 gene expression, and no change in membrane receptor expression. Modulation of TLR4 in respiratory epithelium may have important implications for airway inflammation and infection in response to inhaled pathogens.     

Protein Interaction between Ameloblastin and Proteasome Subunit α Type 3 Can Facilitate Redistribution of Ameloblastin Domains within Forming Enamel

Enamel is a bioceramic tissue composed of thousands of hydroxyapatite crystallites aligned in parallel within boundaries fabricated by a single ameloblast cell. Enamel is the hardest tissue in the vertebrate body; however, it starts development as a self-organizing assembly of matrix proteins that control crystallite habit. Here, we examine ameloblastin, a protein that is initially distributed uniformly across the cell boundary but redistributes to the lateral margins of the extracellular matrix following secretion thus producing cell-defined boundaries within the matrix and the mineral phase. The yeast two-hybrid assay identified that proteasome subunit α type 3 (Psma3) interacts with ameloblastin. Confocal microscopy confirmed Psma3 co-distribution with ameloblastin at the ameloblast secretory end piece. Co-immunoprecipitation assay of mouse ameloblast cell lysates with either ameloblastin or Psma3 antibody identified each reciprocal protein partner. Protein engineering demonstrated that only the ameloblastin C terminus interacts with Psma3. We show that 20S proteasome digestion of ameloblastin in vitro generates an N-terminal cleavage fragment consistent with the in vivo pattern of ameloblastin distribution. These findings suggest a novel pathway participating in control of protein distribution within the extracellular space that serves to regulate the protein-mineral interactions essential to biomineralization.     

Neuronal Development in Caenorhabditis elegans Is Regulated by Inhibition of an MLK MAP Kinase Pathway

We show that loss-of-function mutations in kinases of the MLK-1 pathway (mlk-1, mek-1, and kgb-1/jnk) function cell-autonomously in neurons to suppress defects in synapse formation and axon termination caused by rpm-1 loss of function. Our genetic analysis also suggests that the phosphatase PPM-1, like RPM-1, is a potential inhibitor of kinases in the MLK-1 pathway.     

Functional prediction of hypothetical proteins in human adenoviruses

Assigning functional information to hypothetical proteins in virus genomes is crucial for gaining insight into their proteomes. Human adenoviruses are medium sized viruses that cause a range of diseases. Their genomes possess proteins with uncharacterized function known as hypothetical proteins. Using a wide range of protein function prediction servers, functional information was obtained about these hypothetical proteins. A comparison of functional information obtained from these servers revealed that some of them produced functional information, while others provided little functional information about these human adenovirus hypothetical proteins. The PFP, ESG, PSIPRED, 3d2GO, and ProtFun servers produced the most functional information regarding these hypothetical proteins.     

Characterization of compost produced from separated pig manure and a variety of bulking agents at low initial C/N ratios

The aim of this study was to investigate the composting of separated pig manure solids with or without a variety of bulking agents at a low initial C/N ratio (12.5-23.3). Compost stability was investigated using an oxygen uptake rate (OUR) test and compost maturity was investigated using a germination index test. All treatments showed typical patterns of compost temperature. Temperatures above 60 °C were achieved by Day 2, followed by a thermophilic phase (50-60 °C), which lasted for 1 to 2 weeks followed by a cooling phase. The stability of one of treatments which did not contain any bulking agent - OUR of 25 mmol O₂ kg⁻¹ OM hour⁻¹ - was negatively affected by its initial high water content (69%). The addition of a bulking agent and initial water content below 60% were necessary to compost the separated solid fraction of pig manure at a low initial C/N ratio.     

Impact of PGC-1α on the topology and rate of superoxide production by the mitochondrial electron transport chain

Reactive oxygen species (ROS) play an important role in normal signaling events and excessive ROS are associated with many pathological conditions. The amount of ROS in cells is dependent on both the production of ROS by the mitochondrial electron transport chain and their removal by ROS-detoxifying enzymes. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of mitochondrial functions and a key regulator of the ROS-detoxifying program. However, the impact of PGC-1α on the topology and rate of superoxide production by the mitochondrial electron transport chain is not known. We report here, using mitochondria from muscle creatine kinase–PGC-1α transgenic mice, that PGC-1α does not affect the topology of ROS production, but increases the capacity of complexes I and III to generate ROS. These changes are associated with increased mitochondrial respiration and content of respiratory chain complexes. When normalizing ROS production to mitochondrial respiration, we find that PGC-1α preserves the percentage of free radical leak by the electron transport chain. Together, these data demonstrate that PGC-1α regulates the intrinsic properties of mitochondria in such a way as to preserve a tight coupling between mitochondrial respiration and ROS production.     

Individual vocal production in a sperm whale (Physeter macrocephalus) social unit

The vocal repertoires of group‐living animals may communicate individual or group identity. Female and juvenile sperm whales live in long‐term social units that can be assigned to vocal clans based on the pattern of clicks in coda vocalizations. An unusual set of circumstances allowed us to record the vocalizations of photo‐identified individuals within a single social unit over a 41 d period. Using click interpulse intervals, we were able to assign codas to individuals and investigate coda production at the individual level within a social unit for the first time. Adult females in the unit vocalized at approximately equal rates. A calf and juvenile, both male, vocalized less often than the adult females. Repertoires were indistinguishable for all unit members apart from a mother and her calf, which possessed significantly different repertoires—even from one another. We suggest that similarity among the coda repertoires of most unit members indicates a function in advertising unit identity. In contrast, the distinctive repertoires of the calf and its mother may facilitate reunions between these whales. We hypothesize that sperm whales may be able to vary their vocal repertoires as their reproductive status alters the trade‐off between the benefits of individual and group identification.