Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase

We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energy-transfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z' factors.     

Toxicological screening with formula-based metabolite identification by liquid chromatography/time-of-flight mass spectrometry

An analytical procedure was evaluated for the comprehensive toxicological screening of drugs, metabolites, and pesticides in 1-mL urine samples by TurboIon spray liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) in the positive ionization mode and continuous mass measurement. The substance database consisted of exact monoisotopic masses for 637 compounds, of which an LC retention time was available for 392. A macroprogram was refined for extracting the data into a legible report, utilizing metabolic patterns and preset identification criteria. These criteria included +/-30 ppm mass tolerance, a +/-0.2-min window for absolute retention time, if available, and a minimum area count of 500. The limit of detection, determined for 90 compounds, was <0.1 mg/L for 73% of the compounds studied and >1.0 mg/L for 6% of the compounds. For method comparisons, 50 successive autopsy urine samples were analyzed by this method, and the results confirmed by gas chromatography/mass spectrometry (GC/MS). Findings for parent drugs were consistent with both methods; in addition, LC/TOFMS regularly revealed apparently correct findings for metabolites not shown by GC/MS. Mean and median mass accuracy by LC/TOFMS was 7.6 and 5.4 ppm, respectively. The procedure proved well-suited for tentative identification without reference substances. The few false positives emphasized the fact that all three parameters, exact mass, retention time, and metabolite pattern, are required for unequivocal identification.     

Selection of NF membrane to improve quality of chemically treated surface water

The requirement for higher quality drinking water necessitates the application of more efficient water treatment techniques. Nanofiltration is one promising option for enhanced water treatment, for example, in enhanced organic matter removal. The characteristics of different nanofiltration membranes vary remarkably, and the selection of a membrane has to be made according to the requirements of an application. In this study six nanofiltration membranes (NF70, NF255, NTR-7450, NTR-7410, Desal-5 and TFC-S) were evaluated in improving the quality of chemically pre-treated surface water in a pilot-scale process. The results indicate that the membrane with high organics removal and slightly reduced ion removal characteristics (NF255) performed best in terms of product water quality as well as membrane productivity and fouling. The most permeable membrane (NTR-7410) suffered intensive fouling and insufficient product water quality. An interesting finding was that the permeates of all the tested membranes possessed a significant potential for microbial growth, despite the low nutrient contents.     

Estimation of tree crown volume from airborne lidar data using computational geometry

Although simple geometrical shapes are commonly used to describe tree crowns, computational geometry enables calculation of the individual crown properties directly from airborne lidar point clouds. Our objective was to calculate crown volumes (CVs) using this technique and validate the results by comparing them with field-measured values and modelled ellipsoidal crowns. The CVs of standing trees were obtained by measuring the crown radii at different heights, integrating the obtained crown profiles as solids of revolution, and finally averaging the volumes obtained from the four separate profiles. With the lidar data, the CVs were extracted using 3D alpha shape and 3D convex hull techniques. Crown base heights (CBHs) were also estimated from the lidar data and used to exclude echoes from the understory, which was also done using field-based CBHs to exclude this error source. The results show that the field-measured CVs had a high correlation with lidar-based estimates (best R ² = 0.83), but the lidar-based estimates were generally smaller than the field values. The best correspondence (root mean square difference (RMSD) = 45.0%, average difference = –24.7%) was obtained using the convex hull of the point data and field-measured CBH. The CBHs were consistently overestimated (RMSD = 37.3%; average difference = –20.0%), especially in spruces with long crowns. Thus using lidar-based CBH also increased the inaccuracy of the CV estimates. While the underestimation of CV is mainly explained by the inadequate number of echoes from the lower regions of the crowns, the CVs obtained from the lidar were better than those obtained with ellipsoids fitted by using general models for crown dimensions. The utility of the estimated CVs in the prediction of stem diameter is also demonstrated.     

Towards Micromechanical Radio: Overtone Excitations of a Microresonator Through the Nonlinearities of the Second and Third Order

A micromechanical resonator with eigenfrequencies in the megahertz-range is excited by signals having frequencies from tens of megahertz to gigahertz. The high-frequency excitation voltage is downconverted to mechanical force at the lower resonance frequency by the second-order force-voltage nonlinearity. The conversion is either assisted by additional local-oscillator signal or it is intrinsic due to an amplitude-modulated (AM) input signal. A circuit-simulator model is tested against measurements and an excellent agreement and thorough interpretation of the results is found. The third-order intercept point is measured and simulated to study the strength of the capacitive third-order nonlinearity. Finally, various nonlinear contributions are compared and further improvements for the device are suggested based on the simulations.     

Latency and mode of error detection in a process industry

Licensee event reports (LERs) from an industry provide important information feedback about safety to the industry itself, the regulators and to the public. LERs from four nuclear power reactors were analyzed to find out about detection times, mode of detection and qualitative differences in reports from different reactors. The reliability of the codings was satisfactory and measured as the covariance between the ratings from two independent judges. The results showed differences in detection time across the reactors. On the average about 10% of the errors remained undetected for 100 weeks or more, but the great majority of errors were detected soon after their first appearance in the plant. On the average 40% of the errors were detected in regular tests and 40% through alarms. Operators found about 16% of the errors through noticing something abnormal in the plant. The remaining errors were detected in other ways. There were qualitative differences between the LERs from the different reactors reflecting the different conditions in the plants. The number of reports differed by a magnitude 1:2 between the different plants. However, a greater number of LERs can indicate both higher safety standards (e.g. a greater willingness to report all possible events to be able to learn from them) and lower safety standards (e.g. reporting as few events as possible to make a good impression). It was pointed out that LERs are indispensable in order to maintain safety of an industry and that the differences between plants found in the analyses of this study indicate how error reports can be used to initiate further investigations for improved safety.     

Stability and functionality of freeze-dried probiotic Bifidobacterium cells during storage in juice and milk

We investigated the stability of differently produced (variables being fermentation time, pH during drying, and cryoprotectant) freeze-dried Bifidobacterium animalis subsp. lactis E-2010 (Bb-12) cells in fruit juice and low-fat milk. In addition, the effect of the food matrix on the acid and bile tolerance of the cells was studied. Cells produced in different ways had comparable stability in milk, whereas in juice, sucrose-protected cells survived better than reconstituted skim milk-protected cells. The acid and bile tolerance was better in cells added to milk compared with those in phosphate buffered saline or juice. Despite good culturable stability in milk the acid and bile tolerance of cells decreased during the storage. Apparently, culturable stability data alone do not give an accurate enough prediction of the probiotic functionality in adverse conditions (e.g. survival in acid and bile stress). When choosing a cryoprotectant for a probiotic also the stability in target food applications should be considered.     

From geotechnical maps to three-dimensional models

It is generally preferable to store geotechnical and geological investigation material for possible future use. Old material may be even more valuable than new results in some cases. If the location data for site investigations cannot be identified reliably, it is usually advisable to refrain from using the data at all. Quaternary maps compiled by the Geological Survey of Finland are available for the entire country. Some municipalities in areas where foundation conditions play a prominent part in the safety and economy of building have worked up their own foundation survey materials into regional geotechnical maps. The use of geotechnical data for planning has always required a three-dimensional viewpoint. 3D analysis requires a joint GIS-type database. These still generally consist of binary files, although a relational database solution is recommended for the management of large bodies of data in particular. The general principle as regards old investigations is that their costs have already been covered, whereas overwise separate search and maintenance costs will be involved. Data users are themselves responsible for the results of older site investigations. One important feature is that geotechnical data can be combined with information obtained from other systems.     

Improving the Accuracy of Structural Hot‐spot Stress Approach

A method for extending the applicability of the structural hot‐spot stress method for fatigue assessment of welded structures is discussed. The structural hot‐spot stress method for plate structures, as currently presented in commonly used design guidance documents, cannot account for the effect of weld size, and load carrying fillet welds are assessed using a different S‐N curve as compared to non‐load carrying fillet welds. This paper presents a proposal to linearize the local stress distribution through the plate thickness in the plane of the weld toe. This proposal considers the forces transmitted by the weld itself. A bilinear stress distribution, which partially captures the local effect of the weld, is derived from the non‐linear stress distribution. The non‐linear distribution is based on equilibrium and the stress in the fillet weld. A simple procedure is presented to determine the bilinear curve from the nominal weld stress. This is a great advantage in finite element analysis when only nominal base plate stress and nominal weld stresses need to be determined. When compared to the more conventional structural stress approach, the new method also has the advantage that extrapolation is not required. The same weld stresses can also be used in the analysis of the root cracks. The proposed method was scaled to correspond to the traditional structural hot‐spot stress method using detailed linear elastic fracture mechanics simulations. The method is only applied to fully load‐carrying welds here but can also be used for partial load‐carrying welds. A symmetric splice plate having a fully loaded fillet weld is presented as an example case. The influence of base plate thickness is studied as well.