A manipulative study of macroinvertebrate grazers in Hong Kong streams: do snails compete with insects?

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cAMP inhibits migration,ruffling and paxillin accumulation in focal adhesions of pancreatic ductal adenocarcinoma cells: Effects of PKA and EPAC

We demonstrated that increasing intracellular cAMP concentrations result in the inhibition of migration of PANC-1 and other pancreatic ductal adenocarcinoma (PDAC) cell types. The rise of cAMP was accompanied by rapid and reversible cessation of ruffling, by inhibition of focal adhesion turnover and by prominent loss of paxillin from focal adhesions. All these phenomena develop rapidly suggesting that cAMP effectors have a direct influence on the cellular migratory apparatus. The role of two primary cAMP effectors, exchange protein activated by cAMP (EPAC) and protein kinase A (PKA), in cAMP-mediated inhibition of PDAC cell migration and migration-associated processes was investigated. Experiments with selective activators of EPAC and PKA demonstrated that the inhibitory effect of cAMP on migration, ruffling, focal adhesion dynamics and paxillin localisation is mediated by PKA, whilst EPAC potentiates migration.     

An Automated Live Imaging Platform for Studying Merozoite Egress-Invasion in Malaria Cultures

Most cases of severe and fatal malaria are caused by the intraerythrocytic asexual reproduction cycle of Plasmodium falciparum. One of the most intriguing and least understood stages in this cycle is the brief preinvasion period during which dynamic merozoite–red-cell interactions align the merozoite apex in preparation for penetration. Studies of the molecular mechanisms involved in this process face formidable technical challenges, requiring multiple observations of merozoite egress-invasion sequences in live cultures under controlled experimental conditions, using high-resolution microscopy and a variety of fluorescent imaging tools. Here we describe a first successful step in the development of a fully automated, robotic imaging platform to enable such studies. Schizont-enriched live cultures of P. falciparum were set up on an inverted stage microscope with software-controlled motorized functions. By applying a variety of imaging filters and selection criteria, we identified infected red cells that were likely to rupture imminently, and recorded their coordinates. We developed a video-image analysis to detect and automatically record merozoite egress events in 100% of the 40 egress-invasion sequences recorded in this study. We observed a substantial polymorphism of the dynamic condition of pre-egress infected cells, probably reflecting asynchronies in the diversity of confluent processes leading to merozoite release.     

Habitat associations of Eurasian Skylarks Alauda arvensis breeding on Irish farmland and implications for agri-environment planning

Capsule Skylarks breeding in Ireland prefer extensive grassland habitats and almost completely avoid tillage habitats.

Aims To describe the distribution and habitat use of breeding Skylarks in Ireland, particularly in lowland agricultural habitats, and to use this information to inform conservation measures for this species.

Methods Countryside Bird Survey (CBS) and Farmland Bird Project (FBP) data were examined to determine large-scale (national) distribution and habitat selection, in addition to smaller-scale (farm- and field-level) habitat use. The CBS is a national breeding bird monitoring scheme involving 397 1-km squares. The FBP collected detailed bird and habitat data from 122 farms.

Results CBS and FBP data both showed significant regional differences in breeding Skylark densities, with the highest relative abundances in the northwest and west. Dry grassland/grass moor habitats supported the highest densities of breeding Skylarks in the CBS, which were significantly higher than in improved grassland or tillage. At the farm-level, Skylark numbers were positively related to wetland habitats but negatively associated with trees in field boundaries, dense ground vegetation and overall density of farm boundaries. At the field-scale, larger fields and unimproved grasslands were preferred.

Conclusion Agri-environment measures tailored to region-specific requirements and to the relatively local habitat preferences of target species are required if population declines of species of conservation concern, including Skylarks, are to be reversed.     

EVOLUTION OF SEX DETERMINATION SYSTEMS WITH HETEROGAMETIC MALES AND FEMALES IN SILENE

The plant genus Silene has become a model for evolutionary studies of sex chromosomes and sex‐determining mechanisms. A recent study performed in Silene colpophylla showed that dioecy and the sex chromosomes in this species evolved independently from those in Silene latifolia, the most widely studied dioecious Silene species. The results of this study show that the sex‐determining system in Silene otites, a species related to S. colpophylla, is based on female heterogamety, a sex determination system that is unique among the Silene species studied to date. Our phylogenetic data support the placing of S. otites and S. colpophylla in the subsection Otites and the analysis of ancestral states suggests that the most recent common ancestor of S. otites and S. colpophylla was most probably dioecious. These observations imply that a switch from XX/XY sex determination to a ZZ/ZW system (or vice versa) occurred in the subsection Otites. This is the first report of two different types of heterogamety within one plant genus of this mostly nondioecious plant family.     

Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases

Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these potential functions, the authors designed and used a protease substrate fluorescence resonance energy transfer (FRET) library comprising 115 short d- and l-amino-acid-containing fluorogenic substrates as a tool to generate proteolytic profiles for a wide range of bacteria. Bacterial specificity of the d-amino acid substrates was confirmed using enzymes isolated from both eukaryotic and prokaryotic organisms. Interestingly, bacterial proteases that are known to be involved in housekeeping and nutrition, but not in virulence, were able to degrade substrates in which a d-amino acid was present. Using our FRET peptide library and culture supernatants from a total of 60 different bacterial species revealed novel, bacteria-specific, proteolytic profiles, although in-species variation was observed for Pseudomonas aeruginosa, Porphyromonas gingivalis, and Staphylococcus aureus. Overall, the specific characteristic of our substrate peptide library makes it a rapid tool to high-throughput screen for novel substrates to detect bacterial proteolytic activity.     

Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells

Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathway in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting β-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting β-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.     

Laboratory and microcosm experiments testing the toxicity of chlorinated hydrocarbons on a cyanobacterium strain (Synechococcus PCC 6301) and on natural phytoplankton assemblages

In the last few years, halogenated hydrocarbons have been detected in the soil, in the aquatic environment, in organisms, and even in drinking water. The toxic effects of three chlorinated aliphatic hydrocarbons (trichloroethylene, tetrachloroethylene and tetrachloroethane) were studied in laboratory experiments (using the cyanobacterium Synecococcus elongatus PCC 6301 as test organism) and in field-like circumstances (natural phytoplankton assemblages enclosed in microcosms). The results of the laboratory experiments showed that all of the tested compounds significantly inhibited the growth of the cultures within the first 4 h. Enzymatic changes of the treated cultures suggested that oxidative stress occured—all of the three compounds caused an increase in the activity of peroxidases and superoxide dismutase, and also increased the levels of lipid peroxidation. Observed changes in microcosms were comparable with the results of the laboratory experiments: the number of individuals and chlorophyll contents decreased in the treated assemblages. The elevated levels of peroxidation on the second day in the assemblages treated with tetrachloroethane and tetrachloroetylene suggest that oxidative stress could occur in field conditions. One of the most important findings is the decrease in species number. Our results showed that cryptomonads, some green algae species and the cyanobacterium Limnothrix gradually disappeared from the treated beakers during the experiment.     

Imidazo[4,5-b]pyridine inhibitors of B-Raf kinase

This Letter details the synthesis and evaluation of imidazo[4,5-b]pyridines as inhibitors of B-Raf kinase. These compounds bind in a DFG-in, αC-helix out conformation of B-Raf, which is a binding mode associated with significant kinase selectivity. Structure–activity relationship studies involved optimization of the ATP-cleft binding region of these molecules, and led to compound 23, an inhibitor with excellent enzyme/cell potency, and kinase selectivity.