Engineered antifouling microtopographies - effect of feature size, geometry, and roughness on settlement of zoospores of the green alga Ulva

The effect of feature size, geometry, and roughness on the settlement of zoospores of the ship fouling alga Ulva was evaluated using engineered microtopographies in polydimethylsiloxane elastomer. The topographies studied were designed at a feature spacing of 2 microm and all significantly reduced spore settlement compared to a smooth surface. An indirect correlation between spore settlement and a newly described engineered roughness index (ERI) was identified. ERI is a dimensionless ratio based on Wenzel's roughness factor, depressed surface fraction, and the degree of freedom of spore movement. Uniform surfaces of either 2 mum diameter circular pillars (ERI=5.0) or 2 microm wide ridges (ERI=6.1) reduced settlement by 36% and 31%, respectively. A novel multi-feature topography consisting of 2 mum diameter circular pillars and 10 microm equilateral triangles (ERI=8.7) reduced spore settlement by 58%. The largest reduction in spore settlement, 77%, was obtained with the Sharklet AF topography (ERI=9.5).     

Effect of surfactants on plasticizer biodegradation by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> ATCC 6633

The biodegradation of plasticizers has been previously shown to result in the accumulation of metabolites that are more toxic than the initial compound. The present work shows that the pattern of degradation of di-2-ethylhexyl adipate by Bacillus subtilis can be significantly altered by the presence of biosurfactants, such as surfactin, or synthetic surfactants, such as Pluronic L122. In particular, this work confirms that the monoester, mono-2-ethylhexyl adipate, is a metabolite in the breakdown of the plasticizer. This metabolite was proposed but not observed in earlier studies. Toxicity measurements showed it to be significantly more toxic than the plasticizer. Thus, the effect of the surfactants was to significantly increase the accumulation of one or both of the two most toxic metabolites; i.e., the monoester and 2-ethylhexanol. It was proposed that the most likely cause of the effect of the surfactants was the sequestering of these two metabolites into mixed micelles, resulting in their reduced availability for further degradation.     

Na,K-ATPase alpha2 and Ncx4a regulate zebrafish left-right patterning

A conserved molecular cascade involving Nodal signaling that patterns the laterality of the lateral mesoderm in vertebrates has been extensively studied, but processes involved in the initial break of left-right (LR) symmetry are just beginning to be explored. Here we report that Na,K-ATPase alpha2 and Ncx4a function upstream of Nodal signaling to regulate LR patterning in zebrafish. Knocking down Na,K-ATPase alpha2 and Ncx4a activity in dorsal forerunner cells (DFCs), which are precursors of Kupffer's vesicle (KV), is sufficient to disrupt asymmetric gene expression in the lateral plate mesoderm and randomize the placement of internal organs, indicating that the activity of Na,K-ATPase alpha2 and Ncx4a in DFCs/KV is crucial for LR patterning. High-speed videomicroscopy and bead implantation experiments show that KV cilia are immobile and the directional fluid flow in KV is abolished in Na,K-ATPase alpha2 and Ncx4a morphants, suggesting their essential role in KV ciliary function. Furthermore, we found that intracellular Ca(2+) levels are elevated in Na,K-ATPase alpha2 and Ncx4a morphants and that the defects in ciliary motility, KV fluid flow and placement of internal organs induced by their knockdown could be suppressed by inhibiting the activity of Ca(2+)/calmodulin-dependent protein kinase II. Together, our data demonstrate that Na,K-ATPase alpha2 and Ncx4a regulate LR patterning by modulating intracellular calcium levels in KV and by influencing cilia function, revealing a previously unrecognized role for calcium signaling in LR patterning.     

Effective killing of the human pathogen Candida albicans by a specific inhibitor of non-essential mitotic kinesin Kip1p

Kinesins from the bipolar (Kinesin-5) family are conserved in eukaryotic organisms and play critical roles during the earliest stages of mitosis to mediate spindle pole body separation and formation of a bipolar mitotic spindle. To date, genes encoding bipolar kinesins have been reported to be essential in all organisms studied. We report the characterization of CaKip1p, the sole member of this family in the human pathogenic yeast Candida albicans. C. albicans Kip1p appears to localize to the mitotic spindle and loss of CaKip1p function interferes with normal progression through mitosis. Inducible excision of CaKIP1 revealed phenotypes unique to C. albicans, including viable homozygous Cakip1 mutants and an aberrant spindle morphology in which multiple spindle poles accumulate in close proximity to each other. Expression of the C. albicans Kip1 motor domain in Escherichia coli produced a protein with microtubule-stimulated ATPase activity that was inhibited by an aminobenzothiazole (ABT) compound in an ATP-competitive fashion. This inhibition results in 'rigor-like', tight association with microtubules in vitro. Upon treatment of C. albicans cells with the ABT compound, cells were killed, and terminal phenotype analysis revealed an aberrant spindle morphology similar to that induced by loss of the CaKIP1 gene. The ABT compound discovered is the first example of a fungal spindle inhibitor targeted to a mitotic kinesin. Our results also show that the non-essential nature and implementation of the bipolar motor in C. albicans differs from that seen in other organisms, and suggest that inhibitors of a non-essential mitotic kinesin may offer promise as cidal agents for antifungal drug discovery.     

Genome structure impacts molecular evolution at the AvrLm1 avirulence locus of the plant pathogen Leptosphaeria maculans

Leptosphaeria maculans, a dothideomycete fungus causing stem canker on oilseed rape, develops gene-for-gene interactions with its host plants. It has the ability to rapidly adapt to selection pressure exerted by cultivars harbouring novel resistance genes as exemplified recently by the 3-year evolution towards virulence at the AvrLm1 locus in French populations. The AvrLm1 avirulence gene was recently cloned and shown to be a solo gene within a 269 kb non-coding, heterochromatin-like region. Here we describe the sequencing of the AvrLm1 genomic region in one avirulent and two virulent isolates to investigate the molecular basis of evolution towards virulence at the AvrLm1 locus. For these virulent isolates, the gain of virulence was linked to a 260 kb deletion of a chromosomal segment spanning AvrLm1 and deletion breakpoints were identical or similar. Among the 460 isolates analysed from France, Australia and Mexico, a similar large deletion was apparent in > 90% of the virulent isolates. Deletion breakpoints were also strongly conserved in most of the virulent isolates, which led to the hypothesis that a unique deletion event leading to the avrLm1 virulence has diffused in pathogen populations. These data finally suggest that retrotransposons are key drivers in genome evolution and adaptation to novel selection pressure in L. maculans.     

A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway

The signalling pathway for the hepatocyte growth factor receptor, Met/HGF-R, is hijacked by the bacterial surface protein InlB to induce Listeria monocytogenes entry into non-phagocytic cells. We previously showed that Listeria invades host cells by interacting with specialized microdomains of the host plasma membrane called lipid rafts. In this study, we analysed in living cells signalling events that are crucial for Listeria entry using a fluorescence resonance energy transfer-based microscopic method. Phosphoinositide (PI) 3-kinase activity and Rac1 signalling induced by Listeria interacting with epithelial cells were monitored as well as signalling induced by soluble InlB and the Met natural ligand HGF. We found that InlB and HGF induced similar kinetics of PI 3-kinase and Rac1 activation. PI 3-kinase activation was upstream and independent of Rac1 activation. Cholesterol-depletion experiments were performed to address the role of lipid rafts in Met signalling. The amount of 3'-phosphoinositides produced by PI 3-kinase was not affected by cholesterol depletion, while their membrane dynamic was cholesterol-dependent. Rac1 activation, downstream from PI 3-kinase, was cholesterol-dependent suggesting that the spatial distribution of 3'-phosphoinositides within membrane microdomains is critical for Rac1 activation and consequently for F-actin assembly at bacterial entry site.     

An evolutionary method for learning HMM structure: prediction of protein secondary structure

Background  

The prediction of the secondary structure of proteins is one of the most studied problems in bioinformatics. Despite their success in many problems of biological sequence analysis, Hidden Markov Models (HMMs) have not been used much for this problem, as the complexity of the task makes manual design of HMMs difficult. Therefore, we have developed a method for evolving the structure of HMMs automatically, using Genetic Algorithms (GAs).     

Cell migration patterns and ongoing somatic mutations in the progression of follicular lymphoma

Follicular lymphoma (FL) constitutes the neoplastic equivalent of germinal center B-cells. Like its physiological counterpart, FL grows in (atypical) follicular structures, the formation of which is as yet poorly understood. Recent data indicate that in early tumour stages, neoplastic FL cells home to and colonise reactive germinal centers. Laser microdissection (LMD) and micromanipulation techniques now allow for the molecular genetic analysis of single cell mutation patterns in FL. The purpose of the present study was the analysis of the sequence and order of somatic mutations in FL, i.e. the influence of the germinal center microenvironment on the clonal evolution in different grades of FL. By generating phylogenetic trees as calculated from tumour cell sequences, the clonal evolution from a putative progenitor cell was elucidated and finally, the tumour cell migration pattern in disease progression was assessed by analyzing biopsies at different time points in relapsed tumours. Four patients suffering from FL were included in the study. A primary FL grade 1 showed clustering of genetically related subclones in distinct follicles. A moderate interfollicular exchange of tumour cells was detected. Three cases of FL grade 2 were found to show decreased subclonal clustering in follicles and an increase in the interfollicular migration. Accumulations of replacement mutations in antigen binding domains (CDR) and silent mutations in non-antigen binding domains (FR), respectively, indicating antigen influence on hypermutation were only found in the case of FL grade 1. Our conclusion is that the microenvironment in germinal centers exercises influence on clonal evolution and tumour cell distribution patterns in FL. With increasing histologic grade during disease progression, a reduced intraclonal diversity and selection of subclones also occurs outside the setting of transformation to high-grade lymphoma. Antigen-dependent hypermutations were only seen in FL grade 1, while in progressed FL, random mutation patterns and a decrease of clonal diversity were found.