Part II: defining and quantifying individual and co-cultured intracellular proteomes of two thermophilic microorganisms by GeLC-MS2 and spectral counting

The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms, no doubt harbors technologically important enzymes and other thermostable biomolecules that, to date, have been characterized only to a limited extent. In the first of a two-part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS² analysis and prediction servers suggested probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments, all including the C₁₈ bead technique, demonstrated that two levels of sample purification were necessary to effectively desalt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described, as enzymatic and putative uncharacterized proteins, anticipate consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion, and 11 contained transmembrane domain stretches suggested by Phobius and transmembrane hidden Markov model. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study, involved acetone precipitation followed by the C₁₈ bead method in which 2.4% (63 proteins) of the predicted proteome was identified, including proteins suggested to have secretion and transmembrane moieties.     

A hard microflow cytometer using groove-generated sheath flow for multiplexed bead and cell assays

With a view toward developing a rugged microflow cytometer, a sheath flow system was micromachined in hard plastic (polymethylmethacrylate) for analysis of particles and cells using optical detection. Six optical fibers were incorporated into the interrogation region of the chip, in which hydrodynamic focusing narrowed the core stream to ∼35 μm × 40 μm. The use of a relatively large channel at the inlet as well as in the interrogation region (375 μm × 125 μm) successfully minimized the risk of clogging. The device could withstand pressures greater than 100 psi without leaking. Assays using both coded microparticles and cells were demonstrated using the microflow cytometer. Multiplexed immunoassays detected nine different bacteria and toxins using a single mixture of coded microspheres. A549 cancer cells processed with locked nucleic acid probes were evaluated using fluorescence in situ hybridization.     

Simultaneous XAFS measurements of multiple samples

A four‐channel ionization chamber has been designed, constructed and tested. This ionization chamber allows X‐ray absorption spectra to be collected in transmission from up to four samples simultaneously. This results in spectra that are free of systematic uncertainty in relative energy alignment introduced by scan‐to‐scan stability of the monochromator or of numerical uncertainty associated with a post‐processing alignment algorithm, allowing, in a single shot, an absolute measure of edge shift between four samples of different valence. As four samples can be measured in parallel, the time expended over the course of an experiment to cycle the measurement environment between its rest state and the measurement condition is substantially reduced. The ionization chamber is simple in design and could be implemented at virtually any XAFS beamline with a horizontal fan of radiation such as that provided by a bend magnet or wiggler.     

Coherent X-ray diffraction investigation of twinned microcrystals

Coherent X-ray diffraction has been used to study pseudo-merohedrally twinned manganite microcrystals. The analyzed compositions were Pr(5/8)Ca(3/8)MnO(3) and La(0.275)Pr(0.35)Ca(3/8)MnO(3). The prepared loose powder was thermally attached to glass (and quartz) capillary walls by gentle heating to ensure positional stability during data collection. Many diffraction data sets were recorded and some of them were split as expected from the main observed twin law: 180° rotation around [101]. The peak splitting was measured with very high precision owing to the high-resolution nature of the diffraction data, with a resolution (Δd/d) better than 2.0 × 10(-4). Furthermore, when these microcrystals are illuminated coherently, the different crystallographic phases of the structure factors induce interference in the form of a speckle pattern. The three-dimensional speckled Bragg peak intensity distribution has been measured providing information about the twin domains within the microcrystals. Research is ongoing to invert the measured patterns. Successful phase retrieval will allow mapping out the twin domains and twin boundaries which play a key role in the physical properties.     

Comparing discrete-event simulation and system dynamics: users' perceptions

This paper is the first of its type in that it provides an empirical study comparing the two simulation approaches of discrete-event simulation (DES) and system dynamics (SD). Prior comparison work is limited and mostly based on the authors' personal opinions. In the present work, the comparison is based on managers' (executive MBA students) perceptions of two simulation models of the same problem, one in DES and one in SD. The study found that there is no significant difference from the users' point of view between DES and SD in terms of model understanding and model usefulness. Some minor differences were found in terms of complexity and validity of the models, and the model results. The implications of our findings regarding model understanding, model complexity, model validity, model usefulness and model results are discussed.     

Chemical vapor detection using single-walled carbon nanotubes

Single-walled carbon nanotubes possess unique properties that make them a potentially ideal material for chemical sensing. However, their extremely small size also presents technical challenges for realizing a practical sensor technology. In this tutorial review we explore the transduction physics by which the presence of molecular adsorbates is converted into a measurable electronic signal, and we identify solutions to the problems such as nanotube device fabrication and large, low-frequency noise that have inhibited commercial sensor development. Finally, we examine strategies to provide the necessary chemical specificity to realize a nanotube-based detection system for trace-level chemical vapor detection.     

Fluid supported lipid bilayers containing monosialoganglioside GM1: a QCM-D and FRAP study

In an effort to use model fluid membranes for immunological studies, we compared the formation of planar phospholipid bilayers supported on silicon dioxide surfaces with and without incorporation of glycolipids as the antigen for in situ antibody binding. Dynamic light scattering measurements did not differentiate the hydrodynamic volumes of extruded small unilamellar vesicles (E-SUVs) containing physiologically relevant concentrations (0.5-5 mol%) of monosialoganglioside GM1 (GM1) from exclusive egg yolk L-alpha-phosphatidylcholine (egg PC) E-SUVs. However, quantifiable differences in deposition mass and dissipative energy loss emerged in the transformation of 5 mol% GM1/95 mol% egg PC E-SUVs to planar supported lipid bilayers (PSLBs) by vesicle fusion on thermally evaporated SiO2, as monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. Compared to the 100 mol% egg PC bilayers on the same surface, E-SUVs containing 5 mol% GM1 reached a approximately 12% higher mass and a lower dissipative energy loss during bilayer transformation. PSLBs with 5 mol% GM1 are approximately 18% heavier than 100 mol% egg PC and approximately 11% smaller in projected area per lipid, indicating an increased rigidity and a tighter packing. Subsequent binding of polyclonal immunoglobulin G anti-GM1 to the PSLBs was performed in situ and showed specificity. The anti-GM1 to GM1 ratios at equilibrium were roughly proportional to the concentrations of anti-GM1 administered in the solution. Fluorescence recovery after photobleaching was utilized to verify the retained, albeit reduced lateral fluidity of the supported membranes. Five moles percentage of GM1 membranes (GM1 to PC ratio approximately 1:19) decorated with 1 mol% N-(Texas Red sulfonyl)-1,2-dihexadecanoyl-sn-glycerol-3-phosphoethanolamine (Texas Red DHPE) exhibited an approximately 16% lower diffusion coefficient of 1.32+/-0.06 microm2/s, compared to 1.58+/-0.04 microm2/s for egg PC membranes without GM1 (p<0.01). The changes in vesicle properties and membrane lateral fluidity are attributed to the interactions of GM1 with itself and GM1 with other membrane lipids. This system allows for molecules of interest such as GM1 to exist on a more biologically relevant surface than those used in conventional methods such as ELISA. Our analysis of rabbit serum antibodies binding to GM1 demonstrates this platform can be used to test for the presence of anti-lipid antibodies in serum.     

The detection of phenotypic differences in the metabolic plasma profile of three strains of Zucker rats at 20 weeks of age using ultra-performance liquid chromatography/orthogonal acceleration time-of-flight mass spectrometry

Analysis of biological fluids using ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) (metabonomics) can allow new insights to be gained into disease processes, with advances in chromatographic techniques enabling the detection of thousands of metabolites. In this work metabonomics has been used to investigate the metabolic processes involved in type II diabetes in the Zucker obese rat. Plasma was analyzed from three different strains, the Zucker (fa/fa) obese, Zucker lean and the lean/(fa) obese cross. Using UPLC/MS, ca. 10,000 ions were detected due to the narrow peak widths and excellent peak shapes achieved with this technology. Confidence in the chromatographic performance was demonstrated by the use of quality control standards. The positive and negative ion total ion chromatograms obtained from the three strains were readily distinguishable using multivariate statistical analysis. The greatest difference was observed between the Zucker lean and Zucker lean/(fa) rats compared to the Zucker (fa/fa) obese rats. Positive ions m/z 220 (4.36 min), 282(3.78 min), 359 (5.33 min) and 405 (7.77 min) were elevated in the plasma derived from Zucker lean rats whilst ions m/z 385 (6.80 min) and 646 (4.36 min) were at a lower concentration compared to the plasma from the Zucker (fa/fa) obese animals. Negative ions elevated in the Zucker lean rats included m/z 212 (2.30 min), 514 (2.85 min), 295 (4.39 min), 329 (3.11 min), 343 (2.86 min) and 512 (2.86 min) with ions m/z 538 (4.18 min), 568 (4.18 min), 568 (5.09 min) and 612 (4.30 min) being raised in the samples derived from Zucker (fa/fa) obese animals. The ion m/z 514 (3.85 min) was found to correspond to taurocholate, providing further support for an involvement of taurine metabolism in diabetes.