Pepper: cytoscape app for protein complex expansion using protein-protein interaction networks.

UNLABELLED: We introduce Pepper (Protein complex Expansion using Protein-Protein intERactions), a Cytoscape app designed to identify protein complexes as densely connected subnetworks from seed lists of proteins derived from proteomic studies. Pepper identifies connected subgraph by using multi-objective optimization involving two functions: (i) the coverage, a solution must contain as many proteins from the seed as possible, (ii) the density, the proteins of a solution must be as connected as possible, using only interactions from a proteome-wide interaction network. Comparisons based on gold standard yeast and human datasets showed Pepper's integrative approach as superior to standard protein complex discovery methods. The visualization and interpretation of the results are facilitated by an automated post-processing pipeline based on topological analysis and data integration about the predicted complex proteins. Pepper is a user-friendly tool that can be used to analyse any list of proteins. AVAILABILITY: Pepper is available from the Cytoscape plug-in manager or online (http://apps.cytoscape.org/apps/pepper) and released under GNU General Public License version 3.

Outcomes of splenectomy in patients with common variable immunodeficiency (CVID): a survey of 45 patients.

Splenectomy has been used in patients with common variable immunodeficiency disorders (CVID), mainly in the context of refractory autoimmune cytopenia and suspected lymphoma, but there are understandable concerns about the potential of compounding an existing immunodeficiency. With increasing use of rituximab as an alternative treatment for refractory autoimmune cytopenia, the role of splenectomy in CVID needs to be re-examined. This retrospective study provides the largest cohesive data set to date describing the outcome of splenectomy in 45 CVID patients in the past 40 years. Splenectomy proved to be an effective long-term treatment in 75% of CVID patients with autoimmune cytopenia, even in some cases when rituximab had failed. Splenectomy does not worsen mortality in CVID and adequate immunoglobulin replacement therapy appears to play a protective role in overwhelming post-splenectomy infections. Future trials comparing the effectiveness and safety of rituximab and splenectomy are needed to provide clearer guidance on the second-line management of autoimmune cytopenia in CVID.

Faraday cup for commissioning and quality assurance for proton pencil beam scanning beams at conventional and ultra-high dose rates.

Recently, proton therapy treatments delivered with ultra-high dose rates have been of high scientific interest, and the Faraday cup (FC) is a promising dosimetry tool for such experiments. Different institutes use different FC designs, and either a high voltage guard ring, or the combination of an electric and a magnetic field is employed to minimize the effect of secondary electrons. The authors first investigate these different approaches for beam energies of 70, 150, 230 and 250 MeV, magnetic fields between 0 and 24 mT and voltages between -1000 and 1000 V. When applying a magnetic field, the measured signal is independent of the guard ring voltage, indicating that this setting minimizes the effect of secondary electrons on the reading of the FC. Without magnetic field, applying the negative voltage however decreases the signal by an energy dependent factor up to 1.3% for the lowest energy tested and 0.4% for the highest energy, showing an energy dependent response. Next, the study demonstrates the application of the FC up to ultra-high dose rates. FC measurements with cyclotron currents up to 800 nA (dose rates of up to approximately 1000 Gy s-1) show that the FC is indeed dose rate independent. Then, the FC is applied to commission the primary gantry monitor for high dose rates. Finally, short-term reproducibility of the monitor calibration is quantified within single days, showing a standard deviation of 0.1% (one sigma). In conclusion, the FC is a promising, dose rate independent tool for dosimetry up to ultra-high dose rates. Caution is however necessary when using a FC without magnetic field, as a guard ring with high voltage alone can introduce an energy dependent signal offset.

Technical Note: GATE-RTion: a GATE/Geant4 release for clinical applications in scanned ion beam therapy.

PURPOSE: GATE-RTion is a validated version of GATE for clinical use in the field of light ion beam therapy. This paper describes the GATE-RTion project and illustrates its potential through clinical applications developed in three European centers delivering scanned proton and carbon ion treatments. METHODS: GATE-RTion is a collaborative framework provided by the OpenGATE collaboration. It contains a validated GATE release based on a specific Geant4 version, a set of tools to integrate GATE into a clinical environment and a network for clinical users. RESULTS: Three applications are presented: Proton radiography at the Centre Antoine Lacassagne (Nice, France); Independent dose calculation for proton therapy at the Christie NHS Foundation Trust (Manchester, UK); Independent dose calculation for protons and carbon ions at the MedAustron Ion Therapy center (Wiener Neustadt, Austria). CONCLUSIONS: GATE-RTion builds the bridge between researchers and clinical users from the OpenGATE collaboration in the field of Light Ion Beam Therapy. The applications presented in three European facilities using three completely different machines (three different vendors, cyclotron- and synchrotron-based systems, protons, and carbon ions) demonstrate the relevance and versatility of this project.

A study of lateral fall-off (penumbra) optimisation for pencil beam scanning (PBS) proton therapy.

The lateral fall-off is crucial for sparing organs at risk in proton therapy. It is therefore of high importance to minimize the penumbra for pencil beam scanning (PBS). Three optimisation approaches are investigated: edge-collimated uniformly weighted spots (collimation), pencil beam optimisation of uncollimated pencil beams (edge-enhancement) and the optimisation of edge collimated pencil beams (collimated edge-enhancement). To deliver energies below 70 MeV, these strategies are evaluated in combination with the following pre-absorber methods: field specific fixed thickness pre-absorption (fixed), range specific, fixed thickness pre-absorption (automatic) and range specific, variable thickness pre-absorption (variable). All techniques are evaluated by Monte Carlo simulated square fields in a water tank. For a typical air gap of 10 cm, without pre-absorber collimation reduces the penumbra only for water equivalent ranges between 4-11 cm by up to 2.2 mm. The sharpest lateral fall-off is achieved through collimated edge-enhancement, which lowers the penumbra down to 2.8 mm. When using a pre-absorber, the sharpest fall-offs are obtained when combining collimated edge-enhancement with a variable pre-absorber. For edge-enhancement and large air gaps, it is crucial to minimize the amount of material in the beam. For small air gaps however, the superior phase space of higher energetic beams can be employed when more material is used. In conclusion, collimated edge-enhancement combined with the variable pre-absorber is the recommended setting to minimize the lateral penumbra for PBS. Without collimator, it would be favourable to use a variable pre-absorber for large air gaps and an automatic pre-absorber for small air gaps.

Validating a Monte Carlo approach to absolute dose quality assurance for proton pencil beam scanning.

For radiotherapy, it is crucial to guarantee that the delivered dose matches the planned dose. Therefore, patient specific quality assurance (QA) of absolute dose distributions is necessary. Here, we investigate the potential of replacing patient specific QA for pencil beam scanned proton therapy with Monte Carlo simulations. First, the set-up of the automated Monte Carlo model is presented with an emphasis on the absolute dose validation. Second, the absolute dose results obtained from the Monte Carlo simulation for a comprehensive set of patient fields are compared to patient specific QA measurements. Absolute doses measured with the Farmer chamber are shown to be 1.4% higher than the doses measured with the Semiflex chamber. For single energy layers, Monte Carlo simulated doses are 2.1% ± 0.4% lower than the ones measured with the ionization chamber and 1.1% ± 1.0% lower than measurements compared to patient field verification measurements. After rescaling to account for this 1.1% discrepancy, 98 fields (94.2%) agree within 2% to measurements, the maximum difference being 2.3%. In conclusion, an automated, easy-to-use Monte Carlo calculation system has been set up. This system reproduced patient specific QA results over a wide range of cases, showing that the time consuming measurements could be reduced or even replaced using Monte Carlo simulations without jeopardizing treatment quality.

Two Extremely Thermostable Xylanases of the Hyperthermophilic Bacterium Thermotoga maritima MSB8.

During growth with xylose or xylan as the source of carbon, xylanase production by Thermotoga maritima MSB8 was enhanced about 10-fold compared with growth with glucose or starch. Two extremely thermostable endoxylanases (1,4-(beta)-d-xylan-xylanohydrolase, EC 3.2.1.8), designated XynA and XynB, were identified and purified from cells of this organism. XynA and XynB occurred as proteins with apparent molecular masses of about 120 and 40 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximum activity at the optimal pH (pH 6.2 and pH 5.4 for XynA and XynB, respectively) was measured at about 92(deg)C for XynA (10-min assay) and at about 105(deg)C for XynB (5-min assay). XynB activity was stimulated twofold by the addition of 500 mM NaCl, while XynA displayed maximum activity without the addition of salt. Both xylanases were tolerant of relatively high salt concentrations. At 2 M (about 12% wt/vol) NaCl, XynA and XynB retained 49 and 65%, respectively, of their maximum activities. In contrast to XynB, XynA was able to adsorb to microcrystalline cellulose. Antibodies raised against a recombinant truncated XynA protein cross-reacted with XynB, indicating that the enzymes may have sequence or structural similarities. Part of the xylanase activity appeared to be associated with the outer membrane of T. maritima cells, since more than 40% of the total xylanase activity present in the crude cellular extract was found in the membrane fraction after high-speed centrifugation. Most of the membrane-bound activity appeared to be due to the 120-kDa xylanase XynA.

Isolation and analysis of a gene encoding alpha-glucuronidase, an enzyme with a novel primary structure involved in the breakdown of xylan.

This is the first report describing the analysis of a gene encoding an alpha-glucuronidase, an enzyme essential for the complete breakdown of substituted xylans. A DNA fragment that carries the gene for alpha-glucuronidase was isolated from chromosomal DNA of the hyperthermophilic bacterium Thermotoga maritima MSB8. The alpha-glucuronidase gene (aguA) was identified and characterized with the aid of nucleotide sequence analysis, deletion experiments and expression studies in Escherichia coli, and the start of the coding region was defined by amino-terminal sequencing of the purified recombinant enzyme. The aguA gene encodes a 674-amino-acid, largely hydrophilic polypeptide with a calculated molecular mass of 78593 Da. The alpha-glucuronidase of T. maritima has a novel primary structure with no significant similarity to any other known amino acid sequence. The recombinant enzyme was purified to homogeneity as judged by SDS-PAGE. Gel filtration analysis at low salt concentrations revealed a high apparent molecular mass (> 630 kDa) for the recombinant enzyme, but the oligomeric structure changed upon variation of the ionic strength or the pH, yielding hexameric and/or dimeric forms which were also enzymatically active. The enzyme hydrolysed 2-O-(4-O-methyl-alpha-D-glucopyranosyluronic acid)-D-xylobiose (MeGlcAX2) to xylobiose and 4-O-methylglucuronic acid. The K(m) for MeGlcAX2 was 0.95 mM. The pH optimum was 6.3. Maximum activity was measured at 85 degrees C, about 25 degrees C or more above the values reported for all other alpha-glucuronidases known to date. When incubated at 55-75 degrees C, the enzyme suffered partial inactivation, but thereafter the residual activity remained nearly constant for several days.

Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway.

A chromosomal fragment has been identified in a gene bank from Escherichia coli, which augmented the yield of cysteine in an industrial production strain. Subcloning and genetic analysis showed that an open reading frame coding for a product of 299 amino acids (Orf299) was responsible. Orf299 was synthesized in the T7 polymerase/promoter system and exhibited the properties of an integral membrane protein. Mutational interruption of orf299 did not cause a distinct phenotype; however, transformants overexpressing orf299 had lost the ability to grow in minimal medium unless it was supplemented with a source of reduced sulphur compounds, and they excreted considerable amounts of cysteine and O-acetyl-L-serine, especially in the presence of thiosulphate. Most of the cysteine was found to be masked in 2-methyl-2,4-thiazolidinedicarboxylic acid. N-acetyl-L-serine was also present in the medium, but it is open to question whether it represents a primary excretion product. Measurement of the induction status of the cysteine regulon by means of a cysK'-'lacZ gene fusion demonstrated that the regulon is not induced upon growth in the presence of a poor sulphur source and that the introduction of a constitutive cysB allele alleviates this deficiency. The results indicate that orf299 codes for an export pump for different metabolites of the cysteine pathway. Its relation to other efflux systems and the physiological role are discussed.

Identification of a novel cellulose-binding domain within the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima.

A segment of Thermotoga maritima strain MSB8 chromosomal DNA was isolated which encodes an endo-1,4-beta-D-xylanase, and the nucleotide sequence of the xylanase gene, designated xynA, was determined. With a half-life of about 40 min at 90 degrees C at the optimal pH of 6.2, purified recombinant XynA is one of the most thermostable xylanases known. XynA is a 1059-amino-acid (approximately 120 kDa) modular enzyme composed of an N-terminal signal peptide and five domains, in the order A1-A2-B-C1-C2. By comparison with other xylanases of family 10 of glycosyl hydrolases, the central approximately 340-amino-acid part (domain B) of XynA represents the catalytic domain. The N-terminal approximately 150-amino-acid repeated domains (A1-A2) have no significant similarity to the C-terminal approximately 170-amino-acid repeated domains (C1-C2). Cellulose-binding studies with truncated XynA derivatives and hybrid proteins indicated that the C-terminal repeated domains mediate the binding of XynA to microcrystalline cellulose and that C2 alone can also promote cellulose binding. C1 and C2 did not share amino acid sequence similarity with any other known cellulose-binding domain (CBD) and thus are CBDs of a novel type. Structurally related protein segments which are probably also CBDs were found in other multidomain xylanolytic enzymes. Deletion of the N-terminal repeated domains or of all the non-catalytic domains resulted in substantially reduced thermostability while a truncated xylanase derivative lacking the C-terminal tandem repeat was as thermostable as the full-length enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)