Vibrational circular dichroism of carbohydrate films formed from aqueous solutions

Vibrational circular dichroism (VCD) spectra in the entire 2000-900 cm(-1) region have been recorded, for the first time, for films of carbohydrates prepared from aqueous solutions. Eight different carbohydrates, alpha-D-glucopyranosyl-(1-->4)-D-glucose, cyclomaltohexaose, alpha-D-glucopyranosyl alpha-D-glucopyranoside, beta-D-glucopyranosyl-(1-->6)-D-glucose, beta-D-glucopyranosyl-(1-->4)-D-glucose, D-glucose, and both enantiomers of 6-deoxygalactose and of allose, were investigated. The VCD spectra obtained for films are found to be identical to the corresponding spectra obtained for aqueous solutions of carbohydrates. These measurements demonstrate several advantages of significant importance. The strong infrared absorption of water has prevented, in the past, the pursuit for routine applications of VCD in determining the structures of carbohydrates in aqueous solutions. This limitation is not present for film studies because water solvent is removed in the process of preparing the films. Also, strong infrared absorption of water at 1650 cm(-1) requires the use of very short-pathlength (6 microm) cells for measurements on aqueous solutions. This requirement and concomitant inconveniences (such as laborious assembling of a demountable liquid cell or purchasing an expensive variable pathlength liquid cell) have been eliminated for film measurements. The removal of interfering water absorption in film studies resulted in higher light throughput and better signal-to-noise ratios for VCD measurements. Another point of significance is that the amount of carbohydrate sample required for VCD measurements on films is approximately one to two orders of magnitude smaller than that required for corresponding VCD measurements on aqueous solutions. Since carbohydrate samples can now be studied as films, VCD spectroscopy becomes much more broadly applicable for carbohydrates than previously believed. The present work, in combination with other film measurements in our laboratory, indicate that VCD studies on films can be used more generally, providing a convenient and powerful approach for probing structural information for biologically important compounds.     

Validation of a metabolic model for tritium

The purpose of this paper is to validate a metabolic model describing the kinetics of tritium in man. The validation is based on measurements of background levels of loose and bound tritium in Italian subjects and their diets. Model predictions are compared with empirical measurements of tritium in human urine and tissue samples, and appear to be in close agreement.     

Monte Carlo dose in a prosthesis phantom based on exact geometry vs streak artefact contaminated CT data as benchmarked against Gafchromic film measurements

PurposeIn radiotherapy, accurate calculation of patient radiation dose is very important for good clinical outcome. In the presence of metallic implants, the dose calculation accuracy could be compromised by metal artefacts generated in computed tomography (CT) images of patients. This study investigates the influence of metal-induced CT artefacts on MC dose calculations in a pelvic prosthesis phantom.MethodsA pelvic phantom containing unilateral Ti prosthesis was CT-scanned and accurate Hounsfield unit (HU) values were assigned to known materials of the phantom as opposed to HU values produced through the artefact CT images of the phantom. Using the DOSXYZnrc MC code, dose calculations were computed in the phantom model constructed from the original CT images containing the artefacts and artefact-free images made from the exact geometry of the phantom with known materials. The dose calculations were benchmarked against Gafchromic EBT3 film measurements using 15 MeV electron and 10 MV photon beams.ResultsThe average deviations between film and MC dose data decreased from 3 ± 2% to 1 ± 1% and from about 6 ± 2% to 3 ± 1% for the artefact and artefact-free phantom models against film data for the electron and photon fields, respectively.ConclusionsFor the Ti prosthesis phantom, the presence of metal-induced CT artefacts could cause dose inaccuracies of about 3%. Construction of an artefact-free phantom model made from the exact geometry of the phantom with known materials to overcome the effect of artefacts is advantageous compared to using CT data directly of which the exact tissue composition is not well-known.     

Electronic detectors for electron microscopy

Electron microscopy (EM) is an important tool for high-resolution structure determination in applications ranging from condensed matter to biology. Electronic detectors are now used in most applications in EM as they offer convenience and immediate feedback that is not possible with film or image plates. The earliest forms of electronic detector used routinely in transmission electron microscopy (TEM) were charge coupled devices (CCDs) and for many applications these remain perfectly adequate. There are however applications, such as the study of radiation-sensitive biological samples, where film is still used and improved detectors would be of great value. The emphasis in this review is therefore on detectors for use in such applications. Two of the most promising candidates for improved detection are: monolithic active pixel sensors (MAPS) and hybrid pixel detectors (of which Medipix2 was chosen for this study). From the studies described in this review, a back-thinned MAPS detector appears well suited to replace film in for the study of radiation-sensitive samples at 300 keV, while Medipix2 is suited to use at lower energies and especially in situations with very low count rates. The performance of a detector depends on the energy of electrons to be recorded, which in turn is dependent on the application it is being used for; results are described for a wide range of electron energies ranging from 40 to 300 keV. The basic properties of detectors are discussed in terms of their modulation transfer function (MTF) and detective quantum efficiency (DQE) as a function of spatial frequency.     

An automatic gas counter for quantitative micro-determinations of tritium in biological material

An automatic microcounter for quantitative determinations of tritium has been developed. It has a background of 0.5 cpm and a constant and quench-free efficiency of 57.4 ± 2.1% ($\text{E}{}^2\text{b}$ 6600). Radioactivities of 2 pCi can be measured with a standard deviation of ±10% using a 1-hr counting time. It is particularly adapted for tritium microassay from biological material in which tritium is converted to a gaseous state by a single step combustion and reduction method, utilizing a mixture of metallic zinc and anhydrous sodium carbonate. Samples weighing up to 2 mg can be measured using sample ampules made of Pyrex glass with outer diameter of 5 mm and length of 30 mm. The material costs are extremely low and one technician can load and scal over 100 samples in a day. The apparatus may have significance as an additional instrument beside liquid scintillation counters in the region of radioactivities below 20 pCi. The method has been successfully used in investigations concerning steroid metabolism in rat adrenal cortex and testis. Specific activities have been determined from tissue samples weighing 1 μg and less.     

A dosimetry method in the transverse plane of HDR Ir-192 brachytherapy source using gafchromic EBT2 film

Radiochromic film dosimetry is increasingly used in brachytherapy applications for its higher resolution ability as compared to other experimental methods. The present study was aimed to assess the accuracy and suitability of use of the improved radiochromic film model, Gafchromic EBT2, to evaluate the dose distribution in the transverse plane of microselectron HDR ¹⁹²Ir source.A specially designed and locally fabricated Polymethyl methacrylate (PMMA) phantom was used in this work for the experimental measurement of dose distribution around the source in its transverse plane. The AAPM TG-43U1 recommended radial dose function, g (r), and dose rate constant, Λ, for the source were measured using Gafchromic EBT2 film and thermoluminescent dosimeters (TLD). The EBT2 film measured dosimetric quantities were validated against their values obtained from the TLD measurements and previously published values for the same source available in literature.The dose rate constant and radial dose function for microselectron HDR ¹⁹²Ir source obtained from Gafchromic EBT2 film measurements are in agreement with their TLD measured results within 3.9% and 2.8% respectively. They also agree within the accepted range of uncertainty with their experimental and Monte Carlo calculated results reported in literature.This work demonstrates the suitability of using Gafchromic EBT2 film dosimetry in characterization of dose distribution in the transverse plane of HDR Ir-192 source. This is a more efficient method than TLD dosimetry at discrete and distant positions. Relative to TLD dosimetry, it is found to be better reproducible, easy to use and a less expensive method of dosimetry.     

Tritium partitioning and isotope effects in adenosylcobalamin-dependent glutamate mutase.

Tritiated adenosylcobalamin, labeled at the exchangeable position, has been used to investigate the partitioning of tritium between substrate and product in the reaction catalyzed by glutamate mutase. The isotope partitions between glutamate and methylaspartate in nearly 1:1 ratio, regardless of the direction in which the overall reaction is proceeding. This is consistent with a free-energy profile in which the interconversion of the intermediate glutamyl and methylaspartyl radicals is rapid relative to the transfer of tritium from 5'-deoxyadenosine to either substrate or product. Initial velocity measurements have been used to measure the tritium isotope effects for the transfer of tritium from adenosylcobalamin to product in each direction. The isotope effect is 21 for the formation of glutamate and 19 for the formation of methylasparate. The large magnitude of these isotope effects makes it likely that the rate-determining step may be altered by the substitution of tritium for hydrogen in the reaction. The results of these experiments are compared with previous isotope effect measurements made on other adenosylcobalamin-dependent enzymes.     

Elongation as a factor in artefacts of humans and other animals: an Acheulean example in comparative context

Elongation is a commonly found feature in artefacts made and used by humans and other animals and can be analysed in comparative study. Whether made for use in hand or beak, the artefacts have some common properties of length, breadth, thickness and balance point, and elongation can be studied as a factor relating to construction or use of a long axis. In human artefacts, elongation can be traced through the archaeological record, for example in stone blades of the Upper Palaeolithic (traditionally regarded as more sophisticated than earlier artefacts), and in earlier blades of the Middle Palaeolithic. It is now recognized that elongation extends to earlier Palaeolithic artefacts, being found in the repertoire of both Neanderthals and more archaic humans. Artefacts used by non-human animals, including chimpanzees, capuchin monkeys and New Caledonian crows show selection for diameter and length, and consistent interventions of modification. Both chimpanzees and capuchins trim side branches from stems, and appropriate lengths of stave are selected or cut. In human artefacts, occasional organic finds show elongation back to about 0.5 million years. A record of elongation achieved in stone tools survives to at least 1.75 Ma (million years ago) in the Acheulean tradition. Throughout this tradition, some Acheulean handaxes are highly elongated, usually found with others that are less elongated. Finds from the million-year-old site of Kilombe and Kenya are given as an example. These findings argue that the elongation need not be integral to a design, but that artefacts may be the outcome of adjustments to individual variables. Such individual adjustments are seen in animal artefacts. In the case of a handaxe, the maker must balance the adjustments to achieve a satisfactory outcome in the artefact as a whole. It is argued that the need to make decisions about individual variables within multivariate objects provides an essential continuity across artefacts made by different species.     

Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD₇₅₀) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD₇₅₀ measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth.     

Design and clinical use of a rotational phantom for dosimetric verification of IMRT/VMAT treatments

PurposeTo describe the design and clinical use of a rotational phantom for dosimetric verification of IMRT/VMAT treatment plans using radiochromic film.MethodsA solid water cylindrical phantom was designed with separable upper and lower halves and rests on plastic bearings allowing for 360° rotation about its central axis. The phantom accommodates a half sheet of radiochromic film, and by rotating the cylinder, the film can be placed in any plane between coronal and sagittal. Calculated dose planes coinciding with rotated film measurements are exported by rotating the CT image and dose distribution within the treatment planning system. The process is illustrated with 2 rotated film measurements of an SRS treatment plan involving 4 separate targets. Additionally, 276 patient specific QA measurements were obtained with the phantom and analyzed with a 2%/2 mm gamma criterion.ResultsThe average 2%/2 mm gamma passing rate for all 276 plans was 99.3%. Seventy-two of the 276 plans were measured with the plane of the film rotated between the coronal and sagittal planes and had an average passing rate of 99.4%.ConclusionsThe rotational phantom allows for accurate film measurements in any plane. With this technique, regions of a dose distribution which might otherwise require multiple sagittal or coronal measurements can be verified with as few as a single measurement. This increases efficiency and, in combination with the high spatial resolution inherent to film dosimetry, makes the rotational technique an attractive option for patient-specific QA.     

Investigations on ultrathin cryosections from cryoprotected and non-cryoprotected tissues by electron energy loss spectroscopy: Advantages and limits of application

The use of ultrathin cryosections for ultraimmunohistochemical investigations has gained in importance as technical and methodological improvements have been achieved. The well-known disadvantages of the synthetic resin technique (e.g. antigen denaturation, washing effects) have made it necessary to look for a more gentle method. In fact, material thus prepared offers the advantage that artefacts caused by dehydration and embedding in synthetic resin can be avoided. Besides, it also has a higher antigenicity compared with material prepared by means of the synthetic resin technique.Due to the different steps of incubation one has to face loss of substance and orientation when employing cryoprotected samples. Therefore it would be best to employ non-cryoprotected samples but this is nearly impossible in the case of ultraimmunohistochemical investigations because recrystallization of ice in the tissue leads to destruction of the ultrastructure.In order to quantify the extent of the changes we have compared the elemental distribution for several light elements of cryoprotected material, which was prepared following the Tokuyasu-technique, with non-cryoprotected samples. The latter were frozen in cryogen and the ultrathin sections were carefully freeze-dried. Although the loss caused by washing is low for membrane-linked structures, cryoprotected samples lose on average 50% of the analysed elements in the course of preparation in comparison with unprotected and only freeze-dried samples.     

Anisotropy-based sensing with reference fluorophores

We describe a new approach to fluorescence sensing based on measurements of steady-state anisotropies in the presence of reference fluorophores with known anisotropies. The basic concept is that the anisotropy of a mixture reflects a weighted average of the anisotropies of the emitting species. By use of reference fluorophores the starting anisotropy can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the analyte result in changes in anisotropy. A wide dynamic range of anisotropies is available because of the freedom to select high or low starting values. Anisotropy-based sensing was demonstrated for pH using 6-carboxyfluorescein and for protein affinity or immunoassay using an oriented film with high anisotropy and a protein labeled with a metal-ligand complex. The latter measurements were performed with a simple light-emitting diode excitation source without an excitation polarizer. The sensitive range of the assay can be adjusted by changing the intensity of the reference fluorophore. Anisotropy-based sensing can have numerous applications in clinical and analytical chemistry.     

New generation sequencers as a tool for genotyping of highly polymorphic multilocus MHC system

Accurate genotyping of complex systems, such as the major histocompatibility complex (MHC) often requires simultaneous analysis of multiple co-amplifying loci. Here we explore the utility of the massively parallel 454 sequencing method as a universal tool for genotyping complex MHC systems in nonmodel vertebrates. The power of this approach stems from the use of tagged polymerase chain reaction (PCR) primers to identify individual amplicons which can be simultaneously sequenced to the arbitrarily chosen coverage. However, the error-prone sequencing technology poses considerable challenges as it may be difficult to discriminate between sequencing errors and true rare alleles; due to complex nature of artefacts and errors, efficient quality control is required. Nevertheless, our study demonstrates the parallel 454 sequencing can be an efficient genotyping platform for MHC and provides an alternative to classical genotyping methods. We introduced procedures to identify the threshold that can be used to reduce number of genotyping errors by eliminating most of artefactual alleles (AA) representing PCR or sequencing errors. Our procedures are based on two expectations: first, that AA should be relatively rare, both overall and on per-individual basis, and second, that most AA result from errors introduced to sequences of true alleles. In our data set, alleles with an average per-individual frequency below 3% most likely represented artefacts. This threshold will vary in other applications according to the complexity of the genotyped system. We strongly suggest direct assessment of genotyping error in every experiment by running a fraction of duplicates: individuals amplified in independent PCRs.     

NPQ(T): a chlorophyll fluorescence parameter for rapid estimation and imaging of non‐photochemical quenching of excitons in photosystem‐II‐associated antenna complexes

In photosynthesis, light energy is absorbed by light‐harvesting complexes and used to drive photochemistry. However, a fraction of absorbed light is lost to non‐photochemical quenching (NPQ) that reflects several important photosynthetic processes to dissipate excess energy. Currently, estimates of NPQ and its individual components (q_E, q_I, q_Z and q_T) are measured from pulse‐amplitude‐modulation (PAM) measurements of chlorophyll fluorescence yield and require measurements of the maximal yield of fluorescence in fully dark‐adapted material (F_m), when NPQ is assumed to be negligible. Unfortunately, this approach requires extensive dark acclimation, often precluding widespread or high‐throughput use, particularly under field conditions or in imaging applications, while introducing artefacts when F_m is measured in the presence of residual photodamaged centres. To address these limitations, we derived and characterized a new set of parameters, NPQ_(T), and its components that can be (1) measured in a few seconds, allowing for high‐throughput and field applications; (2) does not require full relaxation of quenching processes and thus can be applied to photoinhibited materials; (3) can distinguish between NPQ and chloroplast movements; and (4) can be used to image NPQ in plants with large leaf movements. We discuss the applications benefits and caveats of both approaches.     

Motion Analysis in the Assessment of Surgical Skill

Abstract

Manual skill is now widely recognised as an important aspect of training in surgery. However, measurement of the skill of a surgeon has in the past been rather subjective in nature, relying on the judgement of experts in the analysis of videotapes. Objective measurements can be made by analysing the velocities of a surgeon's hands during a procedure. In particular, we have found that the number of movements made during a typical procedure will decrease as the surgeon's skill increases. Velocity traces display purposeful movements corrupted by uncorrelated noise from sources such as hand tremor and measurement artefacts. However, we have found that it is possible to filter the noise effectively. Furthermore, we have shown that the skill measurement obtained by counting movements is highly robust to over or under filtering.     

Provenance d’artefacts en rhyolite corse : évaluation des méthodes d’analyse géochimique

Through the analysis of flint and obsidian artefacts, numerous studies have shown the links that existed between Corsica and Sardinia during Neolithic and Chalcolithic. However, we have almost no information about the movements and exchanges that took place in Corsica itself. Rhyolite, a siliceous volcanic rock, has been exploited in Corsica and disseminated throughout the territory from the early Neolithic. Up to now, only one major study has been conducted in the North of the island, in order to identify, by petrographic analysis, the raw material deposits of these rhyolite artefacts. Petrography is a destructive method; it is thus difficult to apply it to large series of archaeological objects. Our aim was therefore to investigate less invasive analytical methods, based on the geochemistry of rocks, in order to conduct future studies based on these rhyolite remains. We analysed 34 geological samples from four different sources, as well as 31 archaeological samples unearthed at three different archaeological areas. We show that EDXRF is an effective method to discriminate sources but also to characterise artefacts using nondestructive protocol. LA-ICP-MS encounters issues due to heterogeneity of the studied rocks. However, we show that by adjusting our testing protocol this method can complement the EDXRF analysis, which is ineffective on objects that are too thin.     

Cryo-electron microscopy of vitreous sections

Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.     

Two-dimensional crystals of apoferritin

A simple 2D crystallization method using unfolded protein film as a supporting film of crystals was described, which allows modification of protein surfaces by injecting chemical reagents into the subphase after the crystal formation. As an example, glutaraldehyde was used to cross-link adjacent proteins and then stabilize protein crystals. The second layer of other proteins can also be formed on the apoferritin array using cross-linkers.The array of apoferritin is not only beneficial for electron crystallography but also for practical applications. For example, apoferritin produces a mineral core with a size which can be adjusted by the size to the cavity (i.e. 6 nm). Fabrication of such a small size of well defined fine particles is currently not easy using physical or chemical procedures. Using apoferritin, however, it is easy to produce uniform fine particles. If the core is designed to add interesting properties such as magnetism it is possible to make the highest class of magnetic film with ferritin 2D crystals.Basic researches toward practical applications of 2D protein crystal is now under way in various fields. The well defined size and function of protein molecules will benefit to many applications. The function and crystalline order can be designed by site-directed mutagenesis with the development of protein engineering.     

Detection of microbial concentration in ice-cream using the impedance technique

The detection of microbial concentration, essential for safe and high quality food products, is traditionally made with the plate count technique, that is reliable, but also slow and not easily realized in the automatic form, as required for direct use in industrial machines. To this purpose, the method based on impedance measurements represents an attractive alternative since it can produce results in about 10 h, instead of the 24–48 h needed by standard plate counts and can be easily realized in automatic form. In this paper such a method has been experimentally studied in the case of ice-cream products. In particular, all main ice-cream compositions of real interest have been considered and no nutrient media has been used to dilute the samples. A measurement set-up has been realized using benchtop instruments for impedance measurements on samples whose bacteria concentration was independently measured by means of standard plate counts. The obtained results clearly indicate that impedance measurement represents a feasible and reliable technique to detect total microbial concentration in ice-cream, suitable to be implemented as an embedded system for industrial machines.     

Detection and correction of probe-level artefacts on microarrays

ABSTRACT: BACKGROUND: A recent large-scale analysis of Gene Expression Omnibus (GEO) data found frequent evidence for spatial defects in a substantial fraction of Affymetrix microarrays in the GEO. Nevertheless, in contrast to quality assessment, artefact detection is not widely used in standard gene expression analysis pipelines. Furthermore, although approaches have been proposed to detect diverse types of spatial noise on arrays, the correction of these artefacts is mostly left to either summarization methods or the corresponding arrays are completely discarded. RESULTS: We show that state-of-the-art robust summarization procedures are vulnerable to artefacts on arrays and cannot appropriately correct for these. To address this problem, we present a simple approach to detect artefacts with high recall and precision, which we further improve by taking into account the spatial layout of arrays. Finally, we propose two correction methods for these artefacts that either substitute values of defective probes using probeset information or filter corrupted probes. We show that our approach can identify and correct defective probe measurements appropriately and outperforms existing tools. CONCLUSIONS: While summarization is insufficient to correct for defective probes, this problem can be addressed in a straightforward way by the methods we present for identification and correction of defective probes. As these methods output CEL files with corrected probe values that serve as input to standard normalization and summarization procedures, they can be easily integrated into existing microarray analysis pipelines as an additional pre-processing step. An R package is freely available from http://www.bio.ifi.lmu.de/artefact-correction.