Spiking of serum specimens with exogenous reporter peptides for mass spectrometry based protease profiling as diagnostic tool

Serum is a difficult matrix for the identification of biomarkers by mass spectrometry (MS). This is due to high-abundance proteins and their complex processing by a multitude of endogenous proteases making rigorous standardisation difficult. Here, we have investigated the use of defined exogenous reporter peptides as substrates for disease-specific proteases with respect to improved standardisation and disease classification accuracy. A recombinant N-terminal fragment of the Adenomatous Polyposis Coli (APC) protein was digested with trypsin to yield a peptide mixture for subsequent Reporter Peptide Spiking (RPS) of serum. Different preanalytical handling of serum samples was simulated by storage of serum samples for up to 6 h at ambient temperature, followed by RPS, further incubation under standardised conditions and testing for stability of protease-generated MS profiles. To demonstrate the superior classification accuracy achieved by RPS, a pilot profiling experiment was performed using serum specimens from pancreatic cancer patients (n = 50) and healthy controls (n = 50). After RPS six different peak categories could be defined, two of which (categories C and D) are modulated by endogenous proteases. These latter are relevant for improved classification accuracy as shown by enhanced disease-specific classification from 78% to 87% in unspiked and spiked samples, respectively. Peaks of these categories presented with unchanged signal intensities regardless of preanalytical conditions. The use of RPS generally improved the signal intensities of protease-generated peptide peaks. RPS circumvents preanalytical variabilities and improves classification accuracies. Our approach will be helpful to introduce MS-based proteomic profiling into routine laboratory testing.     

Methods to reduce interference effects in thermal conversion elemental analyzer/continuous flow isotope ratio mass spectrometry delta18O measurements of nitrogen-containing compounds

On-line determination of the oxygen isotopic composition (delta(18)O value) in organic and inorganic samples is commonly performed using a thermal conversion elemental analyzer (TC-EA) linked to a continuous flow isotope ratio mass spectrometry (IRMS) system. Accurate delta(18)O analysis of N-containing compounds (like nitrates) by TC-EA-IRMS may be complicated because of interference of the N(2) peak on the m/z 30 signal of the CO peak. In this study we evaluated the effectiveness of two methods to overcome this interference which do not require any hardware modifications of standard TC-EA-IRMS systems. These methods were (1) reducing the amount of N(2) introduced into the ion source through He dilution of the N(2) peak and (2) an improved background correction on the CO m/z 30 sample peak integration.Our results show that He dilution is as effective as diverting the N(2) peak in order to eliminate this interference. We conclude that the He-dilution technique is a viable method for the delta(18)O analysis of nitrates and other N-containing samples (which are not routinely measured using He dilution) using TC-EA-IRMS, since it can easily be programmed in the standard software of IRMS systems. With the He-dilution technique delta(18)O values of the nitrate isotope standards USGS34, IAEA-N3 and USGS35 were measured using the shortest possible traceability chain to the VSMOW-SLAP scale, and the results were -28.1 +/- 0.1 per thousand, +25.5 +/- 0.1 per thousand and +57.5 +/- 0.2 per thousand, respectively. An improved background correction was also an effective method, but required manual correction of the raw data.     

New entries toward 3,3-difluoropiperidines

Difluoropiperidines attract considerable interest from organic and medicinal chemists, but their synthesis is often problematic. This paper describes a new synthetic pathway toward valuable 3,3-difluoropiperidines starting from suitable delta-chloro-alpha,alpha-difluoroimines. The latter imines can be synthesized via electrophilic fluorination of the corresponding delta-chloroimines using NFSI (N-fluorodibenzenesulfonimide) in acetonitrile. After hydride reduction of the imino bond and subsequent intramolecular substitution of the chloride atom, new 3,3-difluoropiperidines were obtained in good yields. In addition, this methodology was applied to establish the first synthesis of N-protected 3,3-difluoropipecolic acid, a new fluorinated amino acid.     

Influence of the machine structure on micro milling process

To achieve accurate machining using the micro milling process, precise tools (diameter range 0.2 mm–2 mm) are used. Because of the special properties of the micro milling tools and the related machine structures, it is not enough to assign the classical technique in macro milling process, e.g. stability prediction respecting only the dynamics of the tool/workpiece, directly to analyze the micro milling process. It is proved to be required also to take characteristics of the entire machine structure into account. Therefore, the mechanical vibration research of the micro milling machine structure is necessary, and subsequently the modeling of the structure as a multiple body system is described. The identification of the natural frequencies of a micro milling machine structure by means of the classical modal analysis is shown, and the influence of the structure is verified by comparing the surface roughness of the manufactured workpiece in experimental testings. (© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim)     

In situ monitoring of hydrogen loss during pyrolysis of wood by neutron imaging

Hydrogen is an element of fundamental importance for energy but hard to quantify in bulk materials. Neutron radiography was used to map in situ loss of elemental hydrogen from beech tree wood samples during pyrolysis. The samples consisted of three wood cylinders (finished dowel or cut branch) of approximately 1 cm in length. The samples were pyrolyzed under vacuum in a furnace vessel that was placed inside a cold neutron imaging beamline using a temperature ramp of 5 °C/min from ambient up to 400 °C. Neutron radiographs with exposures of 30 s were sequentially recorded with a charge-coupled device over the course of the experiment. Relative absorbance/scattering of the neutron beam by each sample was based on intensity (or brightness) values as a function of pixel position. The much larger neutron cross section for hydrogen compared to carbon and oxygen enables almost direct conversion of neutron attenuation into sample hydrogen content for each time step during the pyrolysis experiment. Target and vessel temperatures were recorded concurrently with collection of the radiographs so that changes could be directly correlated to different states of pyrolysis. The most visible change appeared at the initial phase of the 400 °C plateau as evidenced by strong hydrogen loss and primarily diametric shrinking of the samples. The loss of elemental hydrogen between initial and final states of pyrolysis was estimated to be about 70%.     

Experimental investigation of the auto-ignition of a transient propane Jet-in-Hot-Coflow

Auto-ignition is a complex process which is extremely sensitive to boundary conditions such as local temperature, mixture or strain rate and occurs on very short time-scales. Therefore, measurement techniques with high spatio-temporal resolution have to be applied to test cases with well-defined boundary conditions in order to generate high-quality validation data for numerical simulations. In the current paper, the auto-ignition of a transient propane jet-in-hot coflow was studied with high-speed OH* chemiluminescence imaging and high-speed Rayleigh scattering for the simultaneous determination of mixture fraction, mixture temperature and scalar dissipation rate immediately prior to the onset of auto-ignition. A variation of the coflow temperature showed a pronounced temperature dependence of the auto-ignition location and time, and the temperature sensitivity was higher than for a comparable methane test case from the literature. This is explained by the lower sensitivity of propane ignition delay times to the local strain rate in comparison to methane. The Rayleigh measurements however showed that the formation mechanism of auto-ignition kernels is similar for propane and methane. Ignition kernels were found to form upstream of bulges of the inflowing jet at locations with locally low scalar dissipation rate.     

Plastics of the Future? The Impact of Biodegradable Polymers on the Environment and on Society

In recent years the littering of plastics and the problems related to their persistence in the environment have become a major focus in both research and the news. Biodegradable polymers like poly(lactic acid) are seen as a suitable alternative to commodity plastics. However, poly(lactic acid) is basically non‐degradable in seawater. Similarly, the degradation rate of other biodegradable polymers also crucially depends on the environments they end up in, such as soil or marine water, or when used in biomedical devices. In this Minireview, we show that biodegradation tests carried out in artificial environments lack transferability to real conditions and, therefore, highlight the necessity of environmentally authentic and relevant field‐testing conditions. In addition, we focus on ecotoxicological implications of biodegradable polymers. We also consider the social aspects and ask how biodegradable polymers influence consumer behavior and municipal waste management. Taken together, this study is intended as a contribution towards evaluating the potential of biodegradable polymers as alternative materials to commodity plastics.     

Covalently Binding of Bovine Serum Albumin to Unsaturated Poly(Globalide‐Co‐ε‐Caprolactone) Nanoparticles by Thiol‐Ene Reactions

When nanoparticles (NPs) are introduced to a biological fluid, different proteins (and other biomolecules) rapidly get adsorbed onto their surface, forming a protein corona capable of giving to the NPs a new “identity” and determine their biological fate. Protein–nanoparticle conjugation can be used in order to promote specific interactions between living systems and nanocarriers. Non‐covalent conjugates are less stable and more susceptible to desorption in biological media, which makes the development of engineered nanoparticle surfaces by covalent attachment an interesting topic. In this work, the surface of poly(globalide‐co‐ε‐caprolactone) (PGlCL) nanoparticles containing double bonds in the main polymer chain is covalently functionalized with bovine serum albumin (BSA) by thiol‐ene chemistry, producing conjugates which are resistant to dissociation. The successful formation of the covalent conjugates is confirmed by flow cytometry (FC) and fluorescence correlation spectroscopy (FCS). Transmission electron microscopy (TEM) allows the visualization of the conjugate formation, and the presence of a protein layer surrounding the NPs can be observed. After conjugation with BSA, NPs present reduced cell uptake by HeLa and macrophage RAW264.7 cells, in comparison to uncoated NP. These results demonstrate that it is possible to produce stable conjugates by covalently binding BSA to PGlCL NP through thiol‐ene reaction.     

Assessing the Predictive Capabilities of Combustion LES as Applied to the Sydney Flame Series

Large Eddy Simulation (LES) and flamelet-based combustion models were applied to four bluff-body stabilized nonpremixed and partially premixed flames selected from the Sydney flame series, based on Masri’s bluff-body test rig (University of Sydney). Three related non-reacting flow cases were also investigated to assess the performance of the LES solver. Both un-swirled and swirled cases were studied exhibiting different flow features, such as recirculation, jet precessing and vortex breakdown. Due to various fuel compositions, flow rates and swirl numbers, the combustion characteristics of the flames varied greatly. On six meshes with different blocking structure and mesh sizes, good prediction of flow and scalar fields using LES/flamelet approaches and known fuel and oxidizer mass fluxes was achieved. The accuracy of predictions was strongly influenced by the combustion model used. All flames were calculated using at least two modeling strategies. Starting with calculations of isothermal flow cases, simple single flamelet based calculations were carried out for the corresponding reacting cases. The combustion models were then adjusted to fit the requirements of each flame. For all flame calculations good agreement of the main flow features with the measured data was achieved. For purely nonpremixed flames burning attached to the bluff-body’s outer edge, flamelet modeling including strain rate effects provided good results for the flow field and for most scalars. The prediction of a partially premixed swirl flame could only be achieved by applying a flamelet-based progress variable approach.     

Flexibility is the key to tuning the transport properties of fluorinated imide-based ionic liquids

Ionic liquids are becoming increasingly popular for practical applications such as biomass processing and lithium-ion batteries. However, identifying ionic liquids with optimal properties for specific applications by trial and error is extremely inefficient since there are a vast number of potential candidate ions. Here we combine experimental and computational techniques to determine how the interplay of fluorination, flexibility and mass affects the transport properties of ionic liquids with the popular imide anion. We observe that fluorination and flexibility have a large impact on properties such as viscosity, whereas the influence of mass is negligible. Using targeted modifications, we show that conformational flexibility provides a significant contribution to the success of fluorination as a design element. Contrary to conventional wisdom, fluorination by itself is thus not a guarantor for beneficial properties such as low viscosity.

The interplay of fluorination, flexibility, and mass affects the transport properties of imide ionic liquids. Here we show how the combination of experimental and theoretical techniques can disentangle such confounding variables.