Free energy calculations of the mutation of Ile96 -> Ala in barnase: contributions to the difference in stability

Free energy calculations were carried out to determine the relativeunfolding free energy of the Ile96 wild type and Ala96 mutantbarnases. The total calculated free energies suggest that substitutionof Ile96 with Ala destabilizes barnase by 3.9 kcal/mol, whichis in good agreement with the independently determined experimentalvalues of 4.0 and 3.3 kcal/mol and a previous simulation. However,a decomposition of the free energy finds the dominant contributionsto this free energy arising from the noncovalent Interactionsbetween the perturbed group and distant residues of barnasein the sequence and water molecules and only a very small contributionfrom covalent interactions. This is in contrast to the previoussimulation, using the dual topology methodology, which produceda decomposition with an {small tilde}60% free energy contributionfrom changes in covalent interactions. The use of the singletopology employed in the present calculations and the dual topologyemployed in the previous study are analyzed in order to understandthe contrast between the present results and the results ofthe previous study.     

Guarding against online threats: why entrepreneurs take protective measures

Information technology is becoming increasingly important for entrepreneurs. Protecting their technical infrastructure and stored data is, therefore, also becoming more vital. Nevertheless, research into the safety and security of entrepreneurs in general, and online threats targeted at entrepreneurs in particular, is still limited. This study investigates to what extent self-employed entrepreneurs protect themselves against online threats, and what motivates them to do so. Based on secondary analyses on data collected from 1622 Dutch entrepreneurs, we observe that the majority implement technical and personal coping measures. By adopting protection motivation theory as a theoretical basis for our study, we found that coping appraisal explains the adoption of protective measures. Entrepreneurs are likely to implement protective measures when they believe a measure is effective, when they are capable of using internet technology, when their attitude towards online protection is positive and when they believe they are responsible for their own online security. Although the secondary analysis provides some limitations, this study offers new insights into the usage of protective measures and the motivation for taking them. These insights can help to improve information security campaigns.     

Analysis of biofluids for biomarker research

While many of the developments made in high-throughput proteomics were originally applied to procaryotic and simple eucaryotic organisms, the analysis of biofluids became increasingly important as the prospect of using proteomics to discover novel biomarkers became realized. Biofluids have represented a unique challenge to proteomics as they are often present only in small amounts and, particularly in the cases of serum and plasma, can have protein concentration ranges that differ by ten orders of magnitude. While the discovery of authentic, clinically useful biomarkers in the proteomics era has been lacking, there have been a number of significant developments in the ability to more comprehensively characterize biofluid proteomes. The rapid pace of these developments suggest that the eventual discovery of clinically validate biomarkers using proteomic technologies still has a bright future.     

Stepwise mobilization of focused proteins in capillary isoelectric focusing mass spectrometry

A stepwise mobilization strategy has been developed for the elution of complex protein mixtures, separated by capillary isoelectric focusing (CIEF) for detection using on-line electrospray ionization mass spectrometry (ESI-MS). Carrier polyampholytes are used to establish a pH gradient as well as to control the electroosmotic flow arising from the use of uncoated fused-silica capillaries. Elution of focused protein zones is achieved by controlling the mobilization pressure and voltage, leaving the remaining protein zones focused inside the capillary. Protein zones are stepwise eluted from the capillary by changing the mobilization conditions. Stepwise mobilization improves separation resolution and simplifies coupling with multistage MS (i.e., MSn) analysis since it allows more effective temporal control of protein elution from the CIEF capillary. We also describe a modified configuration for coupling CIEF with ESI-MS using a coaxial sheath flow interface that facilitate the automation of on-line CIEF-ESI-MS analyses. The stepwise mobilization strategy is demonstrated for the analysis of standard protein mixtures and soluble E. coli lysate proteins using CIEF-ESI-MS. These results indicate that inlet pressure or voltage programming to control the elution of the protein zones from the capillary (i.e., gradient mobilization) may allow for the optimization of the mobilization conditions and provide higher resolution for CIEF separation of complex mixtures with on-line MS.     

Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics

In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-micron) C18 bonded particles provided a total peak capacity of approximately 1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10,000 psi. Large quantities of cellular proteolytic digests (e.g., 500 micrograms) could be loaded onto packed capillaries of 150-micron inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of > 1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.     

Design and performance of an ESI interface for selective external ion accumulation coupled to a Fourier transform ion cyclotron mass spectrometer

The coupling of Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) with electrospray ionization has advanced the analysis of large biopolymers and provided the basis for high-throughput protein characterization (e.g., for rapid "proteome" analyses). In this work, the combination of high-performance capillary liquid chromatography with FTICR mass spectrometry and external ion accumulation has been shown to increase both sensitivity and analysis duty cycle. Instrument versatility is further improved by ion preselection followed by ion accumulation in an external linear quadrupole ion trap. The interface was tested with a 3.5-T FTICR mass spectrometer and evaluated with a number of peptides and proteins whose molecular weights ranged from 500 to 66000. A significant increase in the sensitivity, duty cycle, and dynamic range over that of the previously used accumulated trapping was achieved, exhibiting a detection limit of approximately 10 zmol (approximately 6000 molecules) for smaller proteins such as cytochrome c. Capillary LC external accumulation interface with FTICR was successfully applied for the study of whole-proteome mouse tryptic digests.     

Three-dimensional time-resolved optical tomography of a conical breast phantom

A 32-channel time-resolved imaging device for medical optical tomography has been employed to evaluate a scheme for imaging the human female breast. The fully automated instrument and the reconstruction procedure have been tested on a conical phantom with tissue-equivalent optical properties. The imaging protocol has been designed to obviate compression of the breast and the need for coupling fluids. Images are generated from experimental data with an iterative reconstruction algorithm that employs a three-dimensional (3D) finite-element diffusion-based forward model. Embedded regions with twice the background optical properties are revealed in separate 3D absorption and scattering images of the phantom. The implications for 3D time-resolved optical tomography of the breast are discussed.     

Effect of pulse consumption on perceived flatulence and gastrointestinal function in healthy males

Despite their demonstrated health benefits, consumption of pulses in Western societies has traditionally been low. This is, in part, due to the perception that pulses cause flatulence and gastrointestinal upset. This randomized, double-blind placebo-controlled, cross-over study assessed the impact of 28 consecutive days consumption of 100 g dry weight Kabuli chickpeas, green Laird lentils, and green peas, in comparison to a potato control, on perceived flatulence, abdominal comfort, bowel movements and overall gastrointestinal function using a questionnaire with yes/no and visual analog rankings, in 21 healthy males between the ages of 19–40. Questionnaires were completed during three phases (PRE, EARLY, LATE) of each treatment period. Chi-square (occurrence) and Wilcoxon test (severity rank) analyses revealed minor changes in occurrence and/or severity of flatulence and abdominal comfort, but no changes in overall gastrointestinal function. These results demonstrate that pulses containing oligosaccharides are well tolerated with negligible perceived changes in flatulence and overall gastrointestinal function when incorporated into the diet of healthy adult males.     

Tunable high-frequency Gm-C filters

We consider known, modified, and new transistor circuits for transconductors and some theory on their application in integrated tunable Gm-C or transconductor-eapacitor filters for high frequencies. The circuits are balanced and mostly complementary. They use bipolar (NPN and VPNP) and/or CMOS transistors. The low delay and low input capacitance of the newer circuits makes them particularly suitable for very high frequencies, in the hundreds of megahertz. The relatively high power efficiency and low noise lead to a low power consumption in combination with a high signal-to-noise ratio. Their simplicity results in a small chip area and hence in low cost. The partly new theory is on noise, sensitivities, quality factors, linearization, power and power efficiencies