Atlas of Signaling for Interpretation of Microarray Experiments

Microarray-based expression profiling of living systems is a quick and inexpensive method to obtain insights into the nature of various diseases and phenotypes. A typical microarray profile can yield hundreds or even thousands of differentially expressed genes and finding biologically plausible themes or regulatory mechanisms underlying these changes is a non-trivial and daunting task. We describe a novel approach for systems-level interpretation of microarray expression data using a manually constructed “overview” pathway depicting the main cellular signaling channels (Atlas of Signaling). Currently, the developed pathway focuses on signal transduction from surface receptors to transcription factors and further transcriptional regulation of cellular “workhorse” proteins. We show how the constructed Atlas of Signaling in combination with an enrichment analysis algorithm allows quick identification and visualization of the main signaling cascades and cellular processes affected in a gene expression profiling experiment. We validate our approach using several publicly available gene expression datasets

Construction and characterization of a cyanobacterial bioreporter capable of assessing nitrate assimilatory capacity in freshwaters

The use of cyanobacterial whole-cell luminescent bioreporters has enhanced our ability to monitor nutrient availability in aquatic ecosystems. We have constructed a Synechocystis sp. strain PCC6803 bioluminescent reporter for the assessment of nitrate bioavailability. Specifically, a 380-base pair DNA fragment containing the NtcA/B-dependent nitrate/nitrite-activated nirA promoter (regulating expression of genes encoding nitrite reductase) was fused to the bacterial luciferase genes, luxAB, and introduced into Synechocystis by genetic transformation. Characterization of this strain, designated AND100, yielded dose-dependent increased bioluminescence coincident with increased nitrate added to the growth medium from 1 to 100 μM. Bioluminescence in response to nitrate addition was light dependent up to 50 μmol quanta m-2 s-1. Assessing environmental samples collected from oligotrophic Lake Superior, we demonstrated that the onset of luminescence coincided with the drawdown of nitrate by simultaneously monitoring nitrate depletion from reaction vessels. Nitrate in the Lake Superior samples was consistently underestimated by the bioreporter. Only by following amendment of these samples with phosphate and iron was total nitrate accurately reflected by the cyanobacterial bioreporter. Thus, strain AND 100 can be used to elucidate factors that constrain use of nitrate in freshwaters. This is pertinent to a system such as Lake Superior where the concentration of nitrate has increased 6-fold in the last century. Indeed, pilot experiments with the bioreporter suggest that nutrient co-limitation (P and Fe), as well as low light, may reduce the capacity for nitrate assimilation in field samples from Lake Superior. © 2005, by the American Society of Limnology and oceanography, Inc

Nitrate utilization by phytoplankton in Lake Superior is impaired by low nutrient (P, Fe) availability and seasonal light limitation - A cyanobacterial bioreporter study

We previously developed a luminescent Synechococystis sp. strain PCC 6803 cyanobacterial bioreporter that is used as a real-time whole-cell sensor to assess nitrate assimilatory capacity in freshwaters. Applying the bioreporter assay to Lake Superior, a system whose nitrate levels have increased 6-fold since 1900, we investigated factors that constrain nitrate utilization in this oligotrophic system. Clean sampling methods were used to collect water from Lake Superior during spring and summer 2004, and nitrate utilization was measured by monitoring bioreporter luminescence. Bioreporter response was monitored during experiments in which the lake water was amended with nutrients and incubated under light regimes simulating integrated spring and summer mixing depths. These studies demonstrated that nitrate utilization was enhanced at most stations following addition of phosphorus (P). Moreover, at many stations, addition of iron (Fe) enhanced the P effect. Strength-of-effect statistical analysis provided the individual contribution of P and Fe toward stimulating bioreporter response. In general, distance from shore and season were not good predictors of nitrate assimilatory capacity. Manipulation of light flux during bioreporter experiments also showed that light intensities experienced during spring mixing are likely insufficient to saturate the rate of nitrate utilization. Overall, these data suggest that P-limited algae are deficient in their ability to assimilate nitrate in Lake Superior. Furthermore, we suggest that a secondary limitation for Fe may occur that further constrains nitrate drawdown. Lastly, during spring, light fluxes are sufficiently low to prevent maximal nitrate utilization, even in the absence of nutrient limitation. © 2007 Phycological Society of America

Lake Superior Supports Novel Clusters of Cyanobacterial Picoplankton▿ †

Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the world's lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene and cpcBA PCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the “picocyanobacterial clade” consisting of freshwater and marine Synechococcus and Prochlorococcus. Two new groups of Synechococcus spp., the pelagic Lake Superior clusters I and II, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwater Synechococcus diversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, the cpcBA library from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of the cpcBA sequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 105 cells ml−1 and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer

Synthesis and variable coordination modes of a bis-thiophene-appended macrocycle in complex with cobalt(III)

The bis-thiophene appended cyclam derivative L (trans-6,13-dimethyl-6,13-bis(thiophene-3′-ylmethylamino)-1,4,8,11-tetraazacyclotetradecane) has been synthesised, characterised and complexed with Co. The crystal structure of the diprotonated ligand as its cyanoborohydride salt [HL][NCBH] is reported. Variable coordination modes to Co have been identified that find the ligand binding either as a tetradentate (cyclam-like) macrocycle in the structurally characterised complexes trans-[CoLCl]Cl and trans-[CoL(NCBH)(OH)]Cl or as a hexadentate in [CoL]Cl where both functionalised exocyclic amines coordinate in trans coordination sites. In this case, we have found that the structure of the hexadentate coordinated complex [CoL] is significantly tetragonally elongated due to steric effects of the thiophene rings and this also leads to a very large 500 mV anodic shift in the Co redox potential relative to the unsubstituted hexaamine complex of Co

Molecular Enumeration of an Ecologically Important Cyanophage in a Laurentian Great Lake ▿

Considerable research has shown that cyanobacteria and the viruses that infect them (cyanophage) are pervasive and diverse in global lake populations. Few studies have seasonally analyzed freshwater systems, and little is known about the bacterial and viral communities that coexist during the harsh winters of the Laurentian Great Lakes. Here, we employed quantitative PCR to estimate the abundance of cyanomyoviruses in this system, using the portal vertex g20 gene as a proxy for cyanophage abundance and to determine the potential ecological relevance of these viruses. Cyanomyoviruses were abundant in both the summer and the winter observations, with up to 3.1 × 106 copies of g20 genes ml−1 found at several stations and depths in both seasons, representing up to 4.6% of the total virus community. Lake Erie was productive during both our observations, with high chlorophyll a concentrations in the summer (up to 10.3 μg liter−1) and winter (up to 5.2 μg liter−1). Both bacterial and viral abundances were significantly higher during the summer than during the winter (P < 0.05). Summer bacterial abundances ranged from 3.3 × 106 to 1.6 × 107 ml−1 while winter abundances ranged between ∼3.4 × 105 and 1.2 × 106 ml−1. Total virus abundances were high during both months, with summer abundances significantly higher at most stations, ranging from 6.5 × 107 to 8.8 × 107 ml−1, and with winter abundances ranging from 3.4 × 107 to 6.6 × 107 ml−1. This work confirms that putative cyanomyoviruses are ubiquitous in both summer and winter months in this large freshwater lake system and that they are an abundant component of the virioplankton group