Interactive, Visual Simulation of a Spatio-Temporal Model of Gas Exchange in the Human Alveolus.

In interdisciplinary fields such as systems biology, good communication between experimentalists and theorists is crucial for the success of a project. Theoretical modeling in physiology usually describes complex systems with many interdependencies. On one hand, these models have to be grounded on experimental data. On the other hand, experimenters must be able to understand the interdependent complexities of the theoretical model in order to interpret the model's results in the physiological context. We promote interactive, visual simulations as an engaging way to present theoretical models in physiology and to make complex processes tangible. Based on a requirements analysis, we developed a new model for gas exchange in the human alveolus in combination with an interactive simulation software named Alvin. Alvin exceeds the current standard with its spatio-temporal resolution and a combination of visual and quantitative feedback. In Alvin, the course of the simulation can be traced in a three-dimensional rendering of an alveolus and dynamic plots. The user can interact by configuring essential model parameters. Alvin allows to run and compare multiple simulation instances simultaneously. We exemplified the use of Alvin for research by identifying unknown dependencies in published experimental data. Employing a detailed questionnaire, we showed the benefits of Alvin for education. We postulate that interactive, visual simulation of theoretical models, as we have implemented with Alvin on respiratory processes in the alveolus, can be of great help for communication between specialists and thereby advancing research.

Detection and fragmentation of doubly charged peptide ions in MALDI-Q-TOF-MS by ion mobility spectrometry for improved protein identification.

Today, bottom-up protein identification in MALDI-MS is based on employing singly charged peptide ions, which are predominantly formed in the ionization process. However, peptide mass fingerprinting (PMF) with subsequent tandem MS confirmation using these peptide ions is often hampered due to the lower quality of fragment ion mass spectra caused by the higher collision energy necessary for fragmenting singly protonated peptides. Accordingly, peptide ions of higher charge states would be of high interest for analytical purposes, but they are usually not detected in MALDI-MS experiments as they overlap with singly charged matrix clusters and peptide ions. However, when utilizing ion mobility spectrometry (IMS), doubly charged peptide ions can be actively used by separating them from the singly protonated peptides, visualized, and selectively targeted for tandem MS experiments. The generated peptide fragment ion spectra can be used for a more confident protein identification using PMF with tandem MS confirmation, as most doubly protonated peptide ions yield fragment ion mass spectra of higher quality compared to tandem mass spectra of the corresponding singly protonated precursor ions. Mascot protein scores can be increased by approximately 50% when using tandem mass spectra of doubly charged peptide ions, with ion scores up to six times higher compared with ion scores of tandem mass spectra from singly charged precursors.

The matrix-assisted laser desorption/ionisation in-source decay of peptides using ion mobility enabled quadrupole-time-of-flight mass spectrometry.

RATIONALE: In-source decay (ISD) matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry with a 1,5-diaminonaphthalene (1,5-DAN) matrix is used for the structural characterisation of peptides. However, MALDI spectra are intrinsically complicated by the presence of matrix ions, which interfere with the peptide fragments. This may cause false-positive results or reduced sequence coverage. This paper reports investigations of ISD processes in an intermediate pressure MALDI ion source and a protocol for the removal of interfering ions using ion mobility separation (IMS). METHODS: An intermediate pressure MALDI source of a Q-IMS-Q-TOF instrument (Synapt G2) has been employed for the ISD of selected peptides using a 1,5-DAN matrix. RESULTS: Successful coupling of the MALDI source tuned for ISD experiments using IMS is demonstrated. The IMS made it possible to remove interfering matrix ions effectively from the spectra and thus to increase the confidence of spectral interpretation. Extensive fragment series corresponding to N-Cα bond cleavages were observed under optimised conditions; on the other hand, weaker series of ions caused by peptide bond cleavages were prevalent for default conditions and/or the α-hydroxycinnamic acid matrix. CONCLUSIONS: Ion mobility has been used for the elimination of matrix ions. The technique has been applied to top-down sequencing of non-tryptic peptides, such as the human palmitoylated analogue of prolactin-releasing peptide used in recent obesity studies, and human and insect antimicrobial peptides.

Clinical outcome of patients with lymph node-positive prostate cancer following radical prostatectomy and extended sentinel lymph node dissection.

OBJECTIVE: This study sought to evaluate the clinical outcome after extended sentinel lymph node dissection (eSLND) and radical retropubic prostatectomy (RRP) in patients with clinically localized prostate cancer (PCa). SUBJECTS AND METHODS: From August 2002 until February 2011, a total of 819 patients with clinically localized PCa, confirmed by biopsy, were treated with RRP plus eSLND. Biochemical recurrence-free survival (RFS), cancer-specific survival (CSS), and overall survival (OS) were assessed with Kaplan-Meier curves. Various histopathological parameters were analyzed by univariate and multivariate analysis. RESULTS: The mean follow-up was 5.3 years. Lymph node (LN) metastases occurred in 140 patients. We removed an average of 10.9 LNs via eSLND from patients with pN1 PCa. Postoperatively, 121 pN1 patients temporarily received adjuvant androgen deprivation therapy. The mean survival periods for RFS, RFS after secondary treatment, CSS, and OS were 4.7, 7.0, 8.8, and 8.1 years, respectively. The cancer-specific death rate of the 140 pN1 patients was 13.6%. RFS, CSS, and OS were significantly correlated with pathological margin status, LN density, the total diameter of evident metastases, and membership in the subgroup 'micrometastases only'. CONCLUSION: Despite the presence of LN metastases, patients with a low nodal tumor burden demonstrate a remarkable clinical outcome after undergoing eSLND and RRP, thus suggesting a potential curative therapeutic approach.

Mortality of patients with hematological malignancy after admission to the intensive care unit.

BACKGROUND: The admission of patients with malignancies to an intensive care unit (ICU) still remains a matter of substantial controversy. The identification of factors that potentially influence the patient outcome can help ICU professionals make appropriate decisions. PATIENTS AND METHODS: 90 adult patients with hematological malignancy (leukemia 47.8%, high-grade lymphoma 50%) admitted to the ICU were analyzed retrospectively in this single-center study considering numerous variables with regard to their influence on ICU and day-100 mortality. RESULTS: The median simplified acute physiology score (SAPS) II at ICU admission was 55 (ICU survivors 47 vs. 60.5 for non-survivors). The overall ICU mortality rate was 45.6%. With multivariate regression analysis, patients admitted with sepsis and acute respiratory failure had a significantly increased ICU mortality (sepsis odds ratio (OR) 9.12, 95% confidence interval (CI) 1.1- 99.7, p = 0.04; respiratory failure OR 13.72, 95% CI 1.39-136.15, p = 0.025). Additional factors associated with an increased mortality were: high doses of catecholamines (ICU: OR 7.37, p = 0.005; day 100: hazard ratio (HR) 2.96, p < 0.0001), renal replacement therapy (day 100: HR 1.93, p = 0.026), and high SAPS II (ICU: HR 1.05, p = 0.038; day 100: HR 1.2, p = 0.027). CONCLUSION: The decision for or against ICU admission of patients with hematological diseases should become increasingly independent of the underlying malignant disease.

Determination of ¹5N-incorporation into plant proteins and their absolute quantitation: a new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions.

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

Sialome of a generalist lepidopteran herbivore: identification of transcripts and proteins from Helicoverpa armigera labial salivary glands.

Although the importance of insect saliva in insect-host plant interactions has been acknowledged, there is very limited information on the nature and complexity of the salivary proteome in lepidopteran herbivores. We inspected the labial salivary transcriptome and proteome of Helicoverpa armigera, an important polyphagous pest species. To identify the majority of the salivary proteins we have randomly sequenced 19,389 expressed sequence tags (ESTs) from a normalized cDNA library of salivary glands. In parallel, a non-cytosolic enriched protein fraction was obtained from labial salivary glands and subjected to two-dimensional gel electrophoresis (2-DE) and de novo peptide sequencing. This procedure allowed comparison of peptides and EST sequences and enabled us to identify 65 protein spots from the secreted labial saliva 2DE proteome. The mass spectrometry analysis revealed ecdysone, glucose oxidase, fructosidase, carboxyl/cholinesterase and an uncharacterized protein previously detected in H. armigera midgut proteome. Consistently, their corresponding transcripts are among the most abundant in our cDNA library. We did find redundancy of sequence identification of saliva-secreted proteins suggesting multiple isoforms. As expected, we found several enzymes responsible for digestion and plant offense. In addition, we identified non-digestive proteins such as an arginine kinase and abundant proteins of unknown function. This identification of secreted salivary gland proteins allows a more comprehensive understanding of insect feeding and poses new challenges for the elucidation of protein function.

Silencing NaTPI expression increases nectar germin, nectarins, and hydrogen peroxide levels and inhibits nectar removal from plants in nature.

Native flower visitors removed less nectar from trypsin proteinase inhibitor (TPI)-silenced Nicotiana attenuata plants (ir-pi) than from wild-type plants in four field seasons of releases, even when the nectar repellent, nicotine, was also silenced. Analysis of floral chemistry revealed no differences in the emission of the floral attractants benzylacetone and benzaldehyde or in the concentrations of nectar sugar and nicotine between wild-type and ir-pi flowers, suggesting that these two lines are equally able to attract insect visitors. TPI activity was found in all wild-type flower parts and was highest in anther heads, while TPI activity was not found in any parts of ir-pi flowers. The nectar of ir-pi flowers contained 3.6-fold more total proteins than the nectar of wild-type flowers. Proteomics analysis and hydrogen peroxide (H2O2) measurements revealed that ir-pi nectar contained more nectarins and nectar germin-like proteins and about 1.5-fold more H2O2 compared with wild-type nectar. Field experiments with wild-type flowers supplemented with a solution containing sugar and glucose oxidase demonstrated a causal association between the accumulation of H2O2 and the reduction in nectar removal. These results showed that silencing TPI expression increases the accumulation of nectar proteins and H2O2 levels, which in turn reduces nectar removal by native insect floral visitors. The effect of silencing TPIs on nectar protein accumulation suggests an endogenous regulatory function for TPIs in N. attenuata flowers. The repellency of H2O2 to floral visitors raises new questions about the qualities of nectar that make it attractive for pollinators.

Glucanases and chitinases as causal agents in the protection of Acacia extrafloral nectar from infestation by phytopathogens.

Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii x Nicotiana sanderae) contains "nectarins," proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function.

Chromatographic and electrophoretic resolution of proteins and protein complexes from the larval midgut microvilli of Manduca sexta.

The microvillar proteome of Manduca sexta larval midguts was analyzed by subjecting brush border membrane vesicles (BBMV) to two different two-dimensional approaches: (i) Anion exchange chromatography followed by SDS-PAGE and (ii) Blue Native-PAGE followed by SDS-PAGE. The first technique was superior to conventional 2-D gel electrophoresis in resolving the most abundant proteins associated with the midgut microvilli. Twenty of them were successfully identified as digestive enzymes, binding targets of the insecticidal Cry1A toxins from Bacillus thuringiensis (Bt), and signal transduction proteins. A homolog of the chlorophyllide A binding protein from the silkworm and several aminopeptidases N represent the most abundant proteins associated with the BBMV. The second technique revealed protein oligomeric complexes associated with midgut microvilli in vivo. Two such complexes contained subunits of the vacuolar ATP synthase complex, and one was an oligomer of the chlorophyllide A binding protein. An additional complex consisted of homo- or hetero-tetramers of three different aminopeptidases N (APNs). As APNs are well-known binding partners of Cry1A toxins, their quaternary structure has implications for Bt toxin mode of action. Both techniques provide a useful complement to conventional 2-D gel electrophoresis in analyzing the complex proteome of the microvillar membrane fraction.

A dual fluorescent/MALDI chip platform for analyzing enzymatic activity and for protein profiling.

Being able to rapidly and sensitively detect specific enzymatic products is important when screening biological samples for enzymatic activity. We present a simple method for assaying protease activity in the presence of protease inhibitors (PIs) by measuring tryptic peptide accumulation on copolymer pMALDI target chips using a dual fluorescence/MALDI-TOF-MS read-out. The small platform of the chip accommodates microliter amounts of sample and allows for rapid protein digestion. Fluorescamine labeling of tryptic peptides is used to indicate the proteolytic activity and is shown to be an affordable, simple process, yielding a strong fluorescence signal with a low background. Subsequent MALDI-TOF-MS analysis, performed in the same sample well, or in a parallel well without adding fluorescamine, detects the specific tryptic peptides and provides confidence in the assay. The dual read-out method was applied to screen the inhibition activity of plant PIs, components of plant defense against herbivores and pathogens. Extracts of PIs from Solanum nigrum and trypsin were applied together to a pMALDI chip on which a suitable substrate was adsorbed. The fluorescence and MALDI-TOF-MS signal decrease were associated with the inhibitory effect of the PIs on trypsin. The developed platform can be modified to screen novel protease inhibitors, namely, those potentially useful for treating or preventing infection by viruses, including HIV and hepatitis C.

Molecular design of specific metal-binding peptide sequences from protein fragments: theory and experiment.

A novel strategy is presented for designing peptides with specific metal-ion chelation sites, based on linking computationally predicted ion-specific combinations of amino acid side chains coordinated at the vertices of the desired coordination polyhedron into a single polypeptide chain. With this aim, a series of computer programs have been written that 1) creates a structural combinatorial library containing Zi-(X)n-Zj sequences (n=0-14; Z: amino acid that binds the metal through the side chain; X: any amino acid) from the existing protein structures in the non-redundant Protein Data Bank; 2) merges these fragments into a single Z1-(X)n1 -Z2-(X)n2 -Z3-(X)n3 -...-Zj polypeptide chain; and 3) automatically performs two simple molecular mechanics calculations that make it possible to estimate the internal strain in the newly designed peptide. The application of this procedure for the most M2+-specific combinations of amino acid side chains (M: metal; see L. Rulísek, Z. Havlas J. Phys. Chem. B 2003, 107, 2376-2385) yielded several peptide sequences (with lengths of 6-20 amino acids) with the potential for specific binding with six metal ions (Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+). The gas-phase association constants of the studied metal ions with these de novo designed peptides were experimentally determined by MALDI mass spectrometry by using 3,4,5-trihydroxyacetophenone as a matrix, whereas the thermodynamic parameters of the metal-ion coordination in the condensed phase were measured by isothermal titration calorimetry (ITC), chelatometry and NMR spectroscopy methods. The data indicate that some of the computationally predicted peptides are potential M2+-specific metal-ion chelators.

Metal-chelating plastic MALDI (pMALDI) chips for the enhancement of phosphorylated-peptide/protein signals.

A disposable polymeric pMALDI array with a universal metal cation-chelatable surface for pretreatment/signal enhancement of phosphoproteins and/or phosphopeptides in complex samples was developed. Acrylic acid N-hydroxysuccinimide ester and methyl methacrylate monomers were copolymerized in thin layer molds in a 1:13.3 molar ratio and subsequently treated with Nalpha,Nalpha-bis(carboxymethyl)-l-lysine to obtain a structured planar MALDI array. The prepared NTA pMALDI chip array was activated with metal cations (e.g., Ga(III), Ni(II)), and the selectivities for phosphopeptides (e.g., trypsin-digested alpha-casein (alpha-Cas), and phospho-angiotensin II (p-Ang)) were evaluated using MALDI-TOF/MS. The highest selectivity for proteins was observed for the Ni(II)-NTA chip. The p-Ang was enriched in the presence of BSA tryptic peptides ca. 5 times and represented the major peak after sample adsorption/washing on Ga(III)-NTA chip. The performance of the Ga(III)-chip, tested on alpha-Cas tryptic digest, is fully comparable to commercial systems. Additionally, higher MW peptides and limited methionine oxidation were observed with the chip. A combination of selective absorption of phosphoproteins on Ni(II)-chips and the further enrichment of digested phosphopeptides on the Ga(III)-chip can prove to be very useful for fast identification of unknown proteins using MALDI-TOF/MS.

Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. VII. Changes in the plant's proteome.

When Manduca sexta attacks Nicotiana attenuata, fatty acid-amino acid conjugates (FACs) in the larvae's oral secretions (OS) are introduced into feeding wounds. These FACs trigger a transcriptional response that is similar to the response induced by insect damage. Using two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time of flight, and liquid chromatography-tandem mass spectrometry, we characterized the proteins in phenolic extracts and in a nuclear fraction of leaves elicited by larval attack, and/or in leaves wounded and treated with OS, FAC-free OS, and synthetic FACs. Phenolic extracts yielded approximately 600 protein spots, many of which were altered by elicitation, whereas nuclear protein fractions yielded approximately 100 spots, most of which were unchanged by elicitation. Reproducible elicitor-induced changes in 90 spots were characterized. In general, proteins that increased were involved in primary metabolism, defense, and transcriptional and translational regulation; those that decreased were involved in photosynthesis. Like the transcriptional defense responses, proteomic changes were strongly elicited by the FACs in OS. A semiquantitative reverse transcription-PCR approach based on peptide sequences was used to compare transcript and protein accumulation patterns for 17 candidate proteins. In six cases the patterns of elicited transcript accumulation were consistent with those of elicited protein accumulation. Functional analysis of one of the identified proteins involved in photosynthesis, RuBPCase activase, was accomplished by virus-induced gene silencing. Plants with decreased levels of RuBPCase activase protein had reduced photosynthetic rates and RuBPCase activity, and less biomass, responses consistent with those of herbivore-attacked plants. We conclude that the response of the plant's proteome to herbivore elicitation is complex, and integrated transcriptome-proteome-metabolome analysis is required to fully understand this ubiquitous ecological interaction.

Boron-doped diamond microelectrodes for use in capillary electrophoresis with electrochemical detection.

The fabrication and characterization of boron-doped diamond microelectrodes for use in electrochemical detection coupled with capillary electrophoresis (CE-EC) is discussed. The microelectrodes were prepared by coating thin films of polycrystalline diamond on electrochemically sharpened platinum wires (76-, 25-, and 10-microm diameter), using microwave-assisted chemical vapor deposition (CVD). The diamond-coated wires were attached to copper wires (current collectors), and several methods were explored to insulate the cylindrical portion of the electrode: nail polish, epoxy, polyimide, and polypropylene coatings. The microelectrodes were characterized by scanning electron microscopy, Raman spectroscopy, and cyclic voltammetry. They exhibited low and stable background currents and sigmoidally shaped voltammetric curves for Ru(NH3)6(3+/2+) and Fe(CN)6(3-/4-) at low scan rates. The microelectrodes formed with the large diameter Pt and sealed in polypropylene pipet tips were employed for end-column detection in CE. Evaluation of the CE-EC system and the electrode performance were accomplished using a 10 mM phosphate buffer, pH 6.0, run buffer, and a 30-cm-long fused-silica capillary (75-microm i.d.) with dopamine, catechol, and ascorbic acid serving as test analytes. The background current (approximately 100 pA) and noise (approximately 3 pA) were measured at different detection potentials and found to be very stable with time. Reproducible separation (elution time) and detection (peak current or area) of dopamine, catechol, and ascorbic acid were observed with response precisions of 4.1% or less. Calibration curves constructed from the peak area were linear over 4 orders of magnitude, up to a concentration between 0.1 and 1 mM. Mass limits of detection for dopamine and catechol were 1.7 and 2.6 fmol, respectively (S/N = 3). The separation efficiency was approximately 33,000, 56,000, and 98,000 plates/m for dopamine, catechol, and ascorbic acid, respectively. In addition, the separation and detection of 1- and 2-naphthol in 160 mM borate buffer, pH 9.2, was investigated. Separation of these two analytes was achieved with efficiencies of 118,000 and 126,000 plates/m, respectively.