Array2BIO: from microarray expression data to functional annotation of co-regulated genes

Background  

There are several isolated tools for partial analysis of microarray expression data. To provide an integrative, easy-to-use and automated toolkit for the analysis of Affymetrix microarray expression data we have developed Array2BIO, an application that couples several analytical methods into a single web based utility.     

The relationship between the soil water storage and water-use efficiency of seven energy crops

The aim of this work was to determine two types of photosynthetic water-use efficiency in order to examine their utility as selection criteria for tolerance of energy crops to soil water deficit. Furthermore, effects of crop cultivation on soil water content and storage were investigated. Seven energy crops were examined: miscanthus, prairie cordgrass, willow, thorn-free rose, Virginia mallow, Bohemian knotweed, and topinambour. The highest values of instantaneous (WUE) and intrinsic (WUE_i) water-use efficiencies were found for miscanthus and prairie cordgrass. The reduction of WUE and/or WUE_i was caused mainly by a rapid rise in the transpiration rate and a greater stomatal conductance, respectively. Principal component analysis showed that neither WUE nor WUE_i could be recommended as universal selection criteria for the drought tolerance in different energy crops. The proper localization of soil with a good supply of water is most the important condition for energy crop plantations.     

Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism

Background

It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein—fibrinogen and fibrin that forms a plasma clot.

Methods

The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism (VTE). We used a multiple enzyme digestion filter aided sample preparation, a multienzyme digestion (MED) FASP method combined with LC–MS/MS analysis performed on a Proxeon Easy-nLC System coupled to the Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results.

Results

Our proteomic approach revealed 476 proteins repeatedly identified in the plasma fibrin clots from patients with VTE including extracellular vesicle-derived proteins, lipoproteins, fibrinolysis inhibitors, and proteins involved in immune responses. The MED FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single step digestion. Peptide fractionation with a pipet-tip strong anion exchange (SAX) protocol increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC–MS/MS.

Conclusions

The MED FASP method combined with a label-free quantification is an excellent proteomic approach for the analysis of fibrin clots prepared ex vivo from citrated plasma of patients with prior VTE.

     

Distribution,habitat affinities and phenology of the <Emphasis Type="Italic">Micrargus herbigradus</Emphasis>-species group (Araneae: Linyphiidae) in Poland

We review the known information on the distribution and habitat affinities of the Micrargus herbigradus-species group in Poland. The analysis is based on a thorough literature survey, our own materials, and verification of some older collections. We give new diagnostic drawings and review the characters that are useful in identification of species within the group. Three species are present in Poland: M. herbigradus (Blackwall, 1854), M. apertus (O.-P. Cambridge, 1870) and M. georgescuae Millidge, 1976. The latter is recorded for the first time in the country, and we add numerous new localities for the two former species. Micrargus herbigradus is common and widespread in Poland, living in various habitats, with only a slight preference to forests. In contrast, M. apertus is widely distributed but rarely found, while its affinity to forests is the highest within the group. The records of this species are most numerous in lowland forests (up to c. 300 m a.s.l), but it can also be found at higher altitudes. M. georgescuae is found only in montane habitats, both in the Sudetes and the Carpathian Mountains, from above 650 m a.s.l. The adults of all three species occur the whole year round, but seem to be most abundant in May and June.     

Quantification of the N-glycosylated Secretome by Super-SILAC During Breast Cancer Progression and in Human Blood Samples

Cells secrete a large number of proteins to communicate with their surroundings. Furthermore, plasma membrane proteins and intracellular proteins can be released into the extracellular space by regulated or non-regulated processes. Here, we profiled the supernatant of 11 cell lines that are representative of different stages of breast cancer development by specifically capturing N-glycosylated peptides using the N-glyco FASP technology. For accurate quantification we developed a super-SILAC mix from several labeled breast cancer cell lines and used it as an internal standard for all samples. In total, 1398 unique N-glycosylation sites were identified and quantified. Enriching for N-glycosylated peptides focused the analysis on classically secreted and membrane proteins. N-glycosylated secretome profiles correctly clustered the different cell lines to their respective cancer stage, suggesting that biologically relevant differences were detected. Five different profiles of glycoprotein dynamics during cancer development were detected, and they contained several proteins with known roles in breast cancer. We then used the super-SILAC mix in plasma, which led to the quantification of a large number of the previously identified N-glycopeptides in this important body fluid. The combination of quantifying the secretome of cancer cell lines and of human plasma with a super-SILAC approach appears to be a promising new approach for finding markers of disease.There has been a long-standing interest in applying proteomics to the cancer field (1). Technological advances in liquid chromatography-mass spectrometry (LC-MS) have made it feasible to profile the proteome of cancer cells to great depth (2, 3) and these developments now allow studying protein expression on a systems wide level (4). Analyses of intracellular proteins provide data on what is occurring at the intracellular level in terms of biochemical processes, signaling pathways and cellular structure. However, from a clinical perspective, focusing on proteins that are secreted by these cells is very appealing for diagnostic purposes, as they may filtrate into the peripheral blood (5). This is advantageous because peripheral blood is an easily accessible source whereas tissue biopsies are invasive and they are generally only taken when a medical condition is already suspected. Blood itself is a very complex fluid whose proteome is extremely challenging to analyze because of its very high dynamic range (6–8). Furthermore, a tumor in the initial stages would not be expected to secrete large amounts of proteins and these proteins would be severely diluted in the total blood volume (9). Therefore, discovery of biomarkers by direct analysis of blood plasma has been very difficult so far (10). A more straightforward approach would be the analysis of proteins secreted from homogeneous cell populations (11–14). Consequently, the conditioned medium of cell lines has extensively been used for the analysis of secreted cancer proteins (15). The secretome contains proteins that are actively secreted through classical and nonclassical routes but also proteins that are shed from the plasma membrane by various sheddases (12). Secretome studies are generally performed using serum-free media to reduce the initial protein contents. Further precautions are taken to minimize the contamination of intracellular proteins arising from dead cells that release their contents. Despite these caveats, the totality of proteins that are found in the conditioned medium has been referred to as the “secretome” (13).During cancer development, the invasive capacity of the cells increases progressively. Cancer cells lose cell-cell adhesion which allows eventual release of the cell from the surrounding tissue and may facilitate metastasis to other organs. The extracellular matrix is an important factor in this process as it plays a significant role in regulating numerous cellular functions like adhesion, cell shape, migration, proliferation, polarity, differentiation and apoptosis (16, 17). Many components of the extracellular matrix change in expression during cancer development. Therefore, these changes would likely be reflected in the protein contents of the secretome.Here, we set out to profile the proteins that are secreted by breast cancer cell lines from different stages by MS-based proteomics methods. For several reasons, we focused on N-glycosylated proteins as an appropriate handle to probe proteins that could be of clinical interest. First, proteins that use the classical secretion pathway or are shed from the membrane are typically N-glycosylated because they have passed through the endoplasmic reticulum (ER)¹ and Golgi system (18). Second, glycosylation may enhance the stability of the protein and protect it from proteolytic degradation (19), which would increase the likelihood of detection away from the place where the protein was produced or secreted. Third, glycosylation has a direct relationship to cancer development (20, 21). Fourth, almost all of the currently used protein biomarkers are in fact glycoproteins, such as carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), and prostate-specific antigen (PSA) (22). Finally, glycoproteins have themselves been used as therapeutic targets in cancer. These include ErbB2, targeted by trastuzumab and VEGF-A, targeted by bevacizumab (23).Experimentally, a prime advantage of targeting glycosylation is the fact that glycopeptides or glycoproteins can be efficiently enriched over nonglycosylated molecules. In proteomics, enrichment targeted to N-glycosylation has typically been performed using hydrazide chemistry (24–26) or lectin based enrichment (27, 28). Our group has previously used the ‘filter aided sample preparation’ (FASP) as a basis of N-glycopeptide enrichment (29). The filter membrane in FASP can be employed to physically retain mixtures of lectins, which do not need to be coupled to beads. N-glycopeptides are first bound to the lectins and in a subsequent step simultaneously deglycosylated and released from the lectins. The complexity of the sample is thereby reduced to a level where extensive fractionation is dispensable and the highly enriched fraction of previously N-glycosylated peptides can readily be analyzed in a single high-resolution LC-MS run. We have used N-glyco-FASP to determine N-glycosylation sites in several mouse tissues (29) and in evolutionary distant model organisms (30). Here we adapted the method to supernatants of cell lines and we used the latest generation of Orbitrap analyzers for MS detection. Furthermore, to allow accurate quantification of differences in abundance levels between different secretomes, we spiked an internal standard of a super-SILAC mix (31) containing the conditioned medium of three heavy stable isotope labeled cell lines into all the conditioned medium samples. We collected the conditioned medium from a panel of eleven breast cell lines that were representative of five different cancer stages, from healthy to metastatic cells. The method was further applied to the analysis of blood plasma to verify its applicability in a body fluid context.     

Bacteriophage types of methicillin--resistant Staphylococcus aureus strains isolated in 2005-2006 in the Gdańsk area and their resistance to other antibiotics

The phage type of 38 methicillin--resistant Staphylococcus aureus strains isolated from clinical samples in 5 hospitals in the Gdańsk area in 2005-2007 was determined and antibiotic resistance of obtained phage types was analysed. Phage typing was performed using set of 10 phages as following: MR8, MR12, MR25, 30, 33, 38, M3, M5, 622, 56B. Drug resistance was determined by the disc--diffusion method. There were 12 phage types observed. The most frequent (28.9%) was 56B type which was present in 3 hospitals and in one hospital was found to be predominated. The studied strains were most frequent (23.7%) resistant to erythromycin, clindamycin, ciprofloxacin, trimethoprim/sulfamethoxazole, tetracycline and gentamicin. This resistance pattern predominated in strains belonging to 56B type.     

Characterization and potential evolutionary impact of transposable elements in the genome of Cochliobolus heterostrophus

Background

Cochliobolus heterostrophus is a dothideomycete that causes Southern Corn Leaf Blight disease. There are two races, race O and race T that differ by the absence (race O) and presence (race T) of ~ 1.2-Mb of DNA encoding genes responsible for the production of T-toxin, which makes race T much more virulent than race O. The presence of repetitive elements in fungal genomes is considered to be an important source of genetic variability between different species.

Results

A detailed analysis of class I and II TEs identified in the near complete genome sequence of race O was performed. In total in race O, 12 new families of transposons were identified. In silico evidence of recent activity was found for many of the transposons and analyses of expressed sequence tags (ESTs) demonstrated that these elements were actively transcribed. Various potentially active TEs were found near coding regions and may modify the expression and structure of these genes by acting as ectopic recombination sites. Transposons were found on scaffolds carrying polyketide synthase encoding genes, responsible for production of T-toxin in race T. Strong evidence of ectopic recombination was found, demonstrating that TEs can play an important role in the modulation of genome architecture of this species. The Repeat Induced Point mutation (RIP) silencing mechanism was shown to have high specificity in C. heterostrophus, acting only on transposons near coding regions.

Conclusions

New families of transposons were identified. In C. heterostrophus, the RIP silencing mechanism is efficient and selective. The co-localization of effector genes and TEs, therefore, exposes those genes to high rates of point mutations. This may accelerate the rate of evolution of these genes, providing a potential advantage for the host. Additionally, it was shown that ectopic recombination promoted by TEs appears to be the major event in the genome reorganization of this species and that a large number of elements are still potentially active. So, this study provides information about the potential impact of TEs on the evolution of C. heterostrophus.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-536) contains supplementary material, which is available to authorized users.     

Identification of Kernel Proteins Associated with the Resistance to Fusarium Head Blight in Winter Wheat (Triticum aestivum L.)

Numerous potential components involved in the resistance to Fusarium head blight (FHB) in cereals have been indicated, however, our knowledge regarding this process is still limited and further work is required. Two winter wheat (Triticum aestivum L.) lines differing in their levels of resistance to FHB were analyzed to identify the most crucial proteins associated with resistance in this species. The presented work involved analysis of protein abundance in the kernel bulks of more resistant and more susceptible wheat lines using two-dimensional gel electrophoresis and mass spectrometry identification of proteins, which were differentially accumulated between the analyzed lines, after inoculation with F. culmorum under field conditions. All the obtained two-dimensional patterns were demonstrated to be well-resolved protein maps of kernel proteomes. Although, 11 proteins were shown to have significantly different abundance between these two groups of plants, only two are likely to be crucial and have a potential role in resistance to FHB. Monomeric alpha-amylase and dimeric alpha-amylase inhibitors, both highly accumulated in the more resistant line, after inoculation and in the control conditions. Fusarium pathogens can use hydrolytic enzymes, including amylases to colonize kernels and acquire nitrogen and carbon from the endosperm and we suggest that the inhibition of pathogen amylase activity could be one of the most crucial mechanisms to prevent infection progress in the analyzed wheat line with a higher resistance. Alpha-amylase activity assays confirmed this suggestion as it revealed the highest level of enzyme activity, after F. culmorum infection, in the line more susceptible to FHB.     

Characterization and a point mutational approach of a psychrophilic lipase from an arctic bacterium,Bacillus pumilus

A bacterium with lipolytic activity was isolated from the Chukchi Sea within the Arctic Ocean. The lipase BpL5 from the isolate, Bacillus pumilus ArcL5, belongs to subfamily 4 of lipase family I. The optimum pH and temperature of the recombinant enzyme BpL5, as expressed in Escherichia coli, were 9.0 and 20 °C, respectively. The enzyme retained 85 % of its activity at 5 °C. There was a significant difference between temperatures for maximal activity (20 °C) and for protein denaturation (approx. 45 °C). The enzyme preferred middle-chain (C8) p-nitrophenyl substrates. Two mutants, S139A and S139Y, were rationally designed based on the 3D-structure model, and their activities were compared with that of the wild type. The both mutants showed significantly improved activity against tricaprylin.