A lightweight,flow-based toolkit for parallel and distributed bioinformatics pipelines

Background  

Bioinformatic analyses typically proceed as chains of data-processing tasks. A pipeline, or 'workflow', is a well-defined protocol, with a specific structure defined by the topology of data-flow interdependencies, and a particular functionality arising from the data transformations applied at each step. In computer science, the dataflow programming (DFP) paradigm defines software systems constructed in this manner, as networks of message-passing components. Thus, bioinformatic workflows can be naturally mapped onto DFP concepts.     

Phoenixin-14 stimulates differentiation of 3T3-L1 preadipocytes via cAMP/Epac-dependent mechanism

Phoenixin-14 (PNX) is a newly discovered peptide produced by proteolytic cleavage of the small integral membrane protein 20 (Smim20). Previous studies showed that PNX is involved in controlling reproduction, pain, anxiety and memory. Furthermore, in humans, PNX positively correlates with BMI suggesting a potential role of PNX in controlling fat accumulation in obesity. Since the influence of PNX on adipose tissue formation has not been so far demonstrated, we investigated the effects of PNX on proliferation and differentiation of preadipocytes using 3T3-L1 and rat primary preadipocytes. We detected Smim20 and Gpr173 mRNA in 3T3-L1 preadipocytes as well as in rat primary preadipocytes. Furthermore, we found that PNX peptide is produced and secreted from 3T3-L1 and rat primary adipocytes. PNX increased 3T3-L1 preadipocytes proliferation and viability. PNX stimulated the expression of adipogenic genes (Pparγ, C/ebpβ and Fabp4) in 3T3-L1 adipocytes. 3T3-L1 preadipocytes differentiated in the presence of PNX had increased lipid content. Stimulation of cell proliferation and differentiation by PNX was also confirmed in rat preadipocytes. PNX failed to induce AKT phosphorylation, however, PNX increased cAMP levels in 3T3-L1 cells. Suppression of Epac signalling attenuated PNX-induced Pparγ expression without affecting cell proliferation. Our data show that PNX stimulates differentiation of 3T3-L1 and rat primary preadipocytes into mature adipocytes via cAMP/Epac-dependent pathway. In conclusion our data shows that phoenixin promotes white adipogenesis, thereby may be involved in controlling body mass regulation.     

Detergent-based but gel-free method allows identification of several hundred membrane proteins in single LC-MS runs

Detergents are indispensable solubilizing agents in the purification and analysis of membrane proteins. For mass spectrometric identification of proteins, it is essential that detergents are removed prior to analysis, necessitating an in-gel digestion step. Here, we report a procedure that allows use of detergents and in-solution digestion of proteins. Crude membrane preparations from mouse brain were solubilized with Triton X-100, CHAPS, or SDS, and the detergents were depleted from the membrane proteins using a desalting column equilibrated with 8 M urea. Following digestion with endoproteinase Lys-C, the resulting peptides were analyzed by LC-MS/MS on Linear ion trap-Orbitrap instrument. Applying stringent identification criteria, in single-LC-MS-runs, 1059 +/- 108 proteins, including 797 +/- 43 membrane proteins, were mapped from mouse brain. The identified proteins represented a broad spectrum of neurotransmitter receptors and other ion channels. The general applicability of the method is demonstrated by profiling of membrane proteins from four other mouse organs. Single-run analyses of eye, liver, spleen, and skeletal muscle allowed identification of 522 +/- 9, 610 +/- 7, 777 +/- 8, and 307 +/- 7 membrane proteins. Our results demonstrate that membrane proteins can be analyzed as efficiently as soluble proteins.     

Continuous transformation of benzaldehyde to benzyl alcohol by Rhodotorula mucilaginosa immobilized in an ultrafiltration cell

Microbiological transformation of benzaldehyde accomplished by the fungus Rhodotorula mucilaginosa immobilized in the ultrafiltration cell was studied. A polysulfone membrane formed on a sintered PVC support was used for the separation of the transformation product from the cellular material. Kinetic investigations have led to results which are typical of continuously fed stirred tank reactors (CFSTR)-the value of the maximum reaction rate (V(max)) and apparent Michaelis constant (K'(m)) are practically independent of the substrate retention time (calculated in terms of the flow intensity value). A strong relationship was found to occur between V(max) and biomass concentration in the reactor. Study of the apparent enzyme stability shows that the decrease in the biocatalyst activity is chiefly caused by penetration of the cells through the membrane. The experimental results were approximated in terms of the adopted mathematical model. Based on this model, the half-lives (t(1/2)) of enzyme activities were determined. The t(1/2) value varies from 35 to 82 days and depends both on the permeate flux through the membrane and on the separation properties of the membrane.     

Accumulation of cesium and radiocesium in forest litter in selected regions of poland and its influence on litter-to-mushroom transfer factor

The aim of this work was to check whether the stable cesium content in forest litter affects the value of radiocesium from litter-to-mushroom transfer factorTf or not. Total cesium in litter, measured by AAS, varied from 0.1–2.7 μg/g. These data, combined with earlier results for mushrooms, showed no simple correlation forTf. More complex relationships provided very high correlation coefficients, but their validity needs further investigation.     

Analysis of microsatellite instability and loss of heterozygosity in breast cancer with the use of a well characterized multiplex system

Analysis of microsatellite instability (MI) and loss of heterozygosity (LOH) is recommended for screening patients with sporadic and hereditary malignancies. This study shows an application of a fluorescent hexaplex PCR system for microsatellite typing on A.L.F. DNA Sequencer (Pharmacia Biotech). This technique detects changes in microsatellites providing a time-efficient, reliable and accurate method for MI and LOH analyses. The Fragment Manager software was used for automated size calculation and quantitation of DNA fragments, enabling rapid and precise measurement of allelic ratios. We examined 70 breast cancer and 70 control DNA specimens, classified all the patterns of microsatellite alterations, and set up MI and LOH assessment criteria for the automated multiplex fluorescent method.     

Bio-oxidation of tripropylene glycol under aerobic conditions

Aerobic biodegradation of tripropylene glycol (PG3) was investigated under the conditions of the OECD screening test 301E and the Continuous Flow Activated Sludge Simulation test (CFAS). A modified two-chamber facility with a denitrification stage was used for the CFAS test. Primary PG3 biodegradation was measured by the HPLC with fluorimetric detection and analyte derivatisation. Metabolites were identified by LC-MS with electrospray ionisation and GC-MS with electron impact ionisation, as well as semiquantitatively determined by the LC-MS technique. PG3 was found to be inherently biodegradable and it exhibits a strong poisonous effect on activated sludge after exceeding the threshold concentration (10 mg l⁻¹). Metabolite accumulation onto the activated sludge is probably responsible for this poisonous effect. Probable biotransformation products of tripropylene glycol under the aerobic conditions include metabolites with a single terminal aldehyde or a ketone group and metabolites with two terminal aldehyde or ketone groups. Their concentration rises at the end of the OECD screening test.     

Thermolytic release of covalently linked DNA oligonucleotides and their conjugates from controlled-pore glass at near neutral pH

The functionalization of long chain alkylamine controlled-pore glass (CPG) with a 3-hydroxypropyl-(2-cyanoethyl)thiophosphoryl linker and its conversion to the support 7 has led to the synthesis of DNA oligonucleotides and their 3'- or (3',5')-conjugates. Indeed, CPG support 7 has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20-mers. Unlike conventional succinylated CPG supports, this distinctively functionalized support allows oligonucleotide deprotection and removal of the deprotection side products to proceed without releasing the oligonucleotide into the aqueous milieu. When freed from deprotection side products, the DNA oligonucleotide is thermolytically released from the support within 2 h under nearly neutral conditions (pH 7.2, 90 degrees C). The quality of these oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated CPG supports in terms of shorter than full length oligonucleotide contaminants and overall yields. The versatility of the thermolytic CPG support 7 is further demonstrated by the synthesis of a DNA oligonucleotide (20-mer) and its conjugation with an azido and alkynyl groups at both 5'-and 3'-termini, respectively. The functionality of the (3',5')-heteroconjugated oligonucleotide 18 is verified by its circularization to the DNA oligonucleotide 19 under "click" chemistry conditions.     

Fibroblast-like synoviocytes from rheumatoid arthritis patients express functional IL-15 receptor complex: endogenous IL-15 in autocrine fashion enhances cell proliferation and expression of Bcl-x(L) and Bcl-2

The hallmarks of rheumatoid arthritis (RA) are leukocytic infiltration of the synovium and expansiveness of fibroblast-like synoviocytes (FLS). The abnormal proliferation of FLS and their resistance to apoptosis is mediated, at least in part, by present in RA joints proinflammatory cytokines and growth factors. Because IL-15 exerts properties of antiapoptotic and growth factors, and is produced by RA FLS, we hypothesized that IL-15 participates in RA FLS activation. To test this hypothesis, we first examined whether RA FLS express chains required for high affinity functional IL-15R. Indeed, RA FLS express IL-15Ralpha at mRNA and protein levels. Moreover, we confirmed the presence of IL-2Rbeta and common gamma-chains. Interestingly, TNF-alpha or IL-1beta triggered significant elevation of IL-15Ralpha chain at mRNA and protein levels. Next, we investigated the effects of exogenous or endogenous IL-15 on Bcl-2 and Bcl-x(L) expression, FLS proliferation, and apoptosis. Exogenous IL-15 enhanced RA FLS proliferation and increased the level of mRNA-encoding Bcl-x(L). To test the role of endogenous IL-15 in the activation of RA FLS, an IL-15 mutant/Fcgamma2a protein exerting properties of specific antagonist to the IL-15Ralpha chain was used. We found that blocking IL-15 biological activities using this protein substantially reduced endogenous expression of Bcl-2 and Bcl-x(L), and RA FLS proliferation that was reflected by increased apoptosis. Thus, we have demonstrated that a distinctive phenotype of RA FLS, i.e., persistent activation, proliferation, and resistance to apoptosis, is related to the autocrine activation of IL-15Rs by FLS-derived IL-15.     

Occurrence of adhesin genes in coagulase-negative Staphylococcus aureus strains

Sixty fenotypically coagulase-negative Staphylococcus aureus strains were screened for the presence of six adhesion genes. The strains were isolated from varied clinical samples and varied patients in 16 medical centers, in majority from the region of Gdansk. Multiplex PCR in two primer sets was used for detection of the following genes: bbp (bone binding protein), cna (collagen binding protein), ebp (elastin binding protein)and fnbB (fibronectin B binding protein), fib (fibrinogen bindng protein) and clfA (clunmping factor A). More than half (57%) of the examined population harbored four genes: fnbB,fib, cna i clfA. All of the strains were found to be clfA positive and 90% of them were positive for fnbB, 90% for fib and 67% for cna. All of these genes were significantly more common in MRSA than in MSSA. The particular genes were occurred in strains derived from varied clinical samples.