Soil CO2 flux and photoautotrophic community composition in high-elevation, 'barren' soil

Soil-dominated ecosystems, with little or no plant cover (i.e. deserts, polar regions, high-elevation areas and zones of glacial retreat), are often described as 'barren', despite their potential to host photoautotrophic microbial communities. In high-elevation, subnival zone soil (i.e. elevations higher than the zone of continuous vegetation), the structure and function of these photoautotrophic microbial communities remains essentially unknown. We measured soil CO₂ flux at three sites (above 3600 m) and used molecular techniques to determine the composition and distribution of soil photoautotrophs in the Colorado Front Range. Soil CO₂ flux data from 2002 and 2007 indicate that light-driven CO₂ uptake occurred on most dates. A diverse community of Cyanobacteria , Chloroflexi and eukaryotic algae was present in the top 2 cm of the soil, whereas these clades were nearly absent in deeper soils (2–4 cm). Cyanobacterial communities were composed of lineages most closely related to Microcoleus vaginatus and Phormidium murrayi , eukaryotic photoautotrophs were dominated by green algae, and three novel clades of Chloroflexi were also abundant in the surface soil. During the light hours of the 2007 snow-free measurement period, CO₂ uptake was conservatively estimated to be 23.7 g C m⁻² season⁻¹. Our study reveals that photoautotrophic microbial communities play an important role in the biogeochemical cycling of subnival zone soil.     

Identification of ATP binding residues of a protein from its primary sequence

Background  

One of the major challenges in post-genomic era is to provide functional annotations for large number of proteins arising from genome sequencing projects. The function of many proteins depends on their interaction with small molecules or ligands. ATP is one such important ligand that plays critical role as a coenzyme in the functionality of many proteins. There is a need to develop method for identifying ATP interacting residues in a ATP binding proteins (ABPs), in order to understand mechanism of protein-ligands interaction.     

A typical preparation of Francisella tularensis O-antigen yields a mixture of three types of saccharides

Tularemia is a severe infectious disease in humans caused by the Gram-negative bacterium Francisella tularensis (Ft). Because of its low infectious dose, high mortality rate, and the threat of its large-scale dissemination in weaponized form, development of vaccines and immunotherapeutics against Ft is essential. Ft lipopolysaccharide (LPS), which contains the linear graded-length saccharide component O-antigen (OAg) attached to a core oligosaccharide, has been reported as a protective antigen. Purification of LPS saccharides of defined length and composition is necessary to reveal the epitopes targeted by protective antibodies. In this study, we purified saccharides from LPS preparations from both the Ft subspecies holarctica live vaccine strain (LVS) and the virulent Ft subspecies tularensis SchuS4 strain using liquid chromatography. We then characterized the fractions using high-resolution mass spectrometry and tandem mass spectrometry. Three types of saccharides were observed in both the LVS and SchuS4 preparations: two consisting of OAg tetrasaccharide repeats attached to one of two core oligosaccharide variants and one consisting of tetrasaccharide repeats only (coreless). The coreless OAg oligosaccharides were shown to contain Qui4NFm (4,6-dideoxy-4-formamido-D-glucose) at the nonreducing end and QuiNAc (2-acetamido-2,6-dideoxy-O-D-glucose) at the reducing end. Purified homogeneous preparations of saccharides of each type will allow mapping of protective epitopes in Ft LPS.     

Integration of molecular genetics and proteomics with cell metabolism: how to proceed; how not to proceed!

There now exists a resurgence of interest in the role of intermediary metabolism in medicine; especially in relation to medical disorders. Coupled with this is the contemporary focus on molecular biology, genetics and proteomics and their integration into studies of regulation and alterations in cellular metabolism in health and disease. This is a marriage that has vast potential for elucidation of the factors and conditions that are involved in cellular metabolic and functional changes, which heretofore could not be addressed by the earlier generations of biochemists who established the major pathways of intermediary metabolism. The achievement of this present potential requires the appropriate application and interpretation of genetic and proteomic studies relating to cell metabolism and cell function. This requires knowledge and understanding of the principles, relationships, and methodology, such as biochemistry and enzymology, which are involved in the elucidation of cellular regulatory enzymes and metabolic pathways. Unfortunately, many and possibly most contemporary molecular biologists are not adequately trained and knowledgeable in these areas of cell metabolism. This has resulted in much too common inappropriate application and misinformation from genetic/proteomic studies of cell metabolism and function. This presentation describes important relationships of cellular intermediary metabolism, and provides examples of the appropriate and inappropriate application of genetics and proteomics. It calls for the inclusion of biochemistry, enzymology, cell metabolism and cell physiology in the graduate and postgraduate training of molecular biology and other biomedical researchers.     

Diversity and Distribution of Deep-Sea Shrimps in the Ross Sea Region of Antarctica

Although decapod crustaceans are widespread in the oceans, only Natantia (shrimps) are common in the Antarctic. Because remoteness, depth and ice cover restrict sampling in the South Ocean, species distribution modelling is a useful tool for evaluating distributions. We used physical specimen and towed camera data to describe the diversity and distribution of shrimps in the Ross Sea region of Antarctica. Eight shrimp species were recorded: Chorismus antarcticus; Notocrangon antarcticus; Nematocarcinus lanceopes; Dendrobranchiata; Pasiphaea scotiae; Pasiphaea cf. ledoyeri; Petalidium sp., and a new species of Lebbeus. For the two most common species, N. antarcticus and N. lanceopes, we used maximum entropy modelling, based on records of 60 specimens and over 1130 observations across 23 sites in depths from 269 m to 3433 m, to predict distributions in relation to environmental variables. Two independent sets of environmental data layers at 0.05° and 0.5° resolution respectively, showed how spatial resolution affected the model. Chorismus antarcticus and N. antarcticus were found only on the continental shelf and upper slopes, while N. lanceopes, Lebbeus n. sp., Dendrobranchiata, Petalidium sp., Pasiphaea cf. ledoyeri, and Pasiphaea scotiae were found on the slopes, seamounts and abyssal plain. The environmental variables that contributed most to models for N. antarcticus were depth, chlorophyll-a concentration, temperature, and salinity, and for N. lanceopes were depth, ice concentration, seabed slope/rugosity, and temperature. The relative ranking, but not the composition of these variables changed in models using different spatial resolutions, and the predicted extent of suitable habitat was smaller in models using the finer-scale environmental layers. Our modelling indicated that shrimps were widespread throughout the Ross Sea region and were thus likely to play important functional role in the ecosystem, and that the spatial resolution of data needs to be considered both in the use of species distribution models.     

Direct analysis of sialylated or sulfated glycosphingolipids and other polar and neutral lipids using TLC-MS interfaces

Gangliosides and sulfatides (STs) are acidic glycosphingolipids (GSLs) that have one or more sialic acids or sulfate substituents, in addition to neutral sugars, attached to the C-1 hydroxyl group of the ceramide long chain base. TLC is a widely employed and convenient technique for separation and characterization of GSLs. When TLC is directly coupled to MS, it provides both the molecular mass and structural information without further purification. Here, after development of the TLC plates, the structural analyses of acidic GSLs, including gangliosides and STs, were investigated using the liquid extraction surface analysis (LESA™) and CAMAG TLC-MS interfaces coupled to an ESI QSTAR Pulsar i quadrupole orthogonal TOF mass spectrometer. Coupling TLC with ESI-MS allowed the acquisition of high resolution mass spectra of the acidic GSLs with high sensitivity and mass accuracy, without the loss of sialic acid residues that frequently occurs during low-pressure MALDI MS. These systems were then applied to the analysis of total lipid extracts from bovine brain. This allowed profiling of many different lipid classes, not only gangliosides and STs, but also SMs, neutral GSLs, and phospholipids.     

Techniques for the objective detection of pattern in chlorophyll samples at different temporal scales: an example using data from Lough Hyne, a seasonally stratified inlet

Chlorophyll concentrations in coastal systems are frequently variable to the extent that identifying the scales where pattern occurs is very difficult. Judgements on the temporal structure of data sets are frequently rather subjective. By examining the temporal structure of chlorophyll variation in Lough Hyne with hierarchical techniques, it was possible to identify the important temporal scales objectively. Suggestions can also be made about appropriate sampling programmes for similar coastal systems. There was significant variation in measured chlorophyll concentrations between seasons and between months within seasons. High chlorophyll concentrations were more likely during spring and autumn, as would be predicted from the seasonal cycle of stratification in the lough. Seasonality could also be detected in the tidal inflow to the lough from adjacent coastal waters. More intensive sampling during the summer did not reveal any temporal structure in surface water samples. However, there were 14 day periodicities associated with measurements of depth integrated chlorophyll, oxygen, salinity, water column stability and attenuation coefficient. It is suggested that these periodicities are consistent with spring neap tidal forcing. No interannual variation in chlorophyll concentrations was detected. Examination of the power in the analysis of variance suggested that the monthly sampling frequency was unlikely to have detected differences of less than 145% between the means of pairs of years. A sampling interval of less than a week would be needed to have confidence in detecting differences of 50% between annual means.     

Characterization of Glycoproteoforms of Integrins α2 and β1 in Megakaryocytes in the Occurrence of JAK2V617F Mutation-Induced Primary Myelofibrosis

Primary myelofibrosis (PMF) is a neoplasm prone to leukemic transformation, for which limited treatment is available. Among individuals diagnosed with PMF, the most prevalent mutation is the JAK2V617F somatic point mutation that activates the Janus kinase 2 (JAK2) enzyme. Our earlier reports on hyperactivity of β1 integrin and enhanced adhesion activity of the α2β1 complex in JAK2V617F megakaryocytes (MKs) led us to examine the new hypothesis that this mutation leads to posttranslational modification via changes in glycosylation. Samples were derived from immunoprecipitation of MKs obtained from Vav1-hJAK2^V617F and WT mice. Immunoprecipitated fractions were separated by SDS-PAGE and analyzed using LC-MS/MS techniques in a bottom-up glycoproteomics workflow. In the immunoprecipitate, glycopeptiforms corresponding to 11 out of the 12 potential N-glycosylation sites of integrin β1 and to all nine potential glycosylation sites of integrin α2 were observed. Glycopeptiforms were compared across WT and JAK2V617F phenotypes for both integrins. The overall trend observed is that JAK2V617F mutation in PMF MKs leads to changes in β1 glycosylation; in most cases, it results in an increase in the integrated area of glycopeptiforms. We also observed that in mutated MKs, changes in integrin α2 glycosylation were more substantial than those observed for integrin β1 glycosylation, a finding that suggests that altered integrin α2 glycosylation may also affect activation. Additionally, the identification of proteins associated to the cytoskeleton that were co-immunoprecipitated with integrins α2 and β1 demonstrated the potential of the methodology employed in this study to provide some insight, at the peptide level, into the consequences of integrin activation in MKs. The extensive and detailed glycosylation patterns we uncovered provide a basis for future functional studies of each site in control cells as compared to JAK2V617F-mutated cells. Data are available via ProteomeXchange with identifier PXD030550.