Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.

Virtual tissue alignment and cutting plane definition--a new method to obtain optimal longitudinal histological sections.

Histomorphometric evaluation of the buccal aspects of periodontal tissues in rodents requires reproducible alignment of maxillae and highly precise sections containing central sections of buccal roots; this is a cumbersome and technically sensitive process due to the small specimen size. The aim of the present report is to describe and analyze a method to transfer virtual sections of micro-computer tomographic (CT)-generated image stacks to the microtome for undecalcified histological processing and to describe the anatomy of the periodontium in rat molars. A total of 84 undecalcified sections of all buccal roots of seven untreated rats was analyzed. The accuracy of section coordinate transfer from virtual micro-CT slice to the histological slice, right-left side differences and the measurement error for linear and angular measurements on micro-CT and on histological micrographs were calculated using the Bland-Altman method, interclass correlation coefficient and the method of moments estimator. Also, manual alignment of the micro-CT-scanned rat maxilla was compared with multiplanar computer-reconstructed alignment. The supra alveolar rat anatomy is rather similar to human anatomy, whereas the alveolar bone is of compact type and the keratinized gingival epithelium bends apical to join the junctional epithelium. The high methodological standardization presented herein ensures retrieval of histological slices with excellent display of anatomical microstructures, in a reproducible manner, minimizes random errors, and thereby may contribute to the reduction of number of animals needed.

Proposal for a 10-high-power-fields scoring method for the assessment of tumor budding in colorectal cancer

Although tumor budding is linked to adverse prognosis in colorectal cancer, it remains largely unreported in daily diagnostic work due to the absence of a standardized scoring method. Our aim was to assess the inter-observer agreement of a novel 10-high-power-fields method for assessment of tumor budding at the invasive front and to confirm the prognostic value of tumor budding in our setting of colorectal cancers. Whole tissue sections of 215 colorectal cancers with full clinico-pathological and follow-up information were stained with cytokeratin AE1/AE3 antibody. Presence of buds was scored across 10-high-power fields at the invasive front by two pathologists and two additional observers were asked to score 50 cases of tumor budding randomly selected from the larger cohort. The measurements were correlated to the patient and tumor characteristics. Inter-observer agreement and correlation between observers' scores were excellent (P<0.0001; intraclass correlation coefficient=0.96). A test subgroup of 65 patients (30%) was used to define a valid cutoff score for high-grade tumor budding and the remaining 70% of the patients were entered into the analysis. High-grade budding was defined as an average of ≥10 buds across 10-high-power fields. High-grade budding was associated with a higher tumor grade (P<0.0001), higher TNM stage (P=0.0003), vascular invasion (P<0.0001), infiltrating tumor border configuration (P<0.0001) and reduced survival (P<0.0001). Multivariate analysis confirmed its independent prognostic effect (P=0.007) when adjusting for TNM stage and adjuvant therapy. Using 10-high-power fields for evaluating tumor budding has independent prognostic value and shows excellent inter-observer agreement. Like the BRE and Gleason scores in breast and prostate cancers, respectively, tumor budding could be a basis for a prognostic score in colorectal cancer.

Design-based stereology to quantify structural properties of artificial and natural snow using thin sections

The quantification of the structural properties of snow is traditionally based on model-based stereology. Model-based stereology requires assumptions about the shape of the investigated structure. Here, we show how the density, specific surface area, and grain boundary area can be measured using a design-based method, where no assumptions about structural properties are necessary. The stereological results were also compared to X-ray tomography to control the accuracy of the method. The specific surface area calculated with the stereological method was 19.8 ± 12.3% smaller than with X-ray tomography. For the density, the stereological method gave results that were 11.7 ± 12.1% larger than X-ray tomography. The statistical analysis of the estimates confirmed that the stereological method and the sampling used are accurate. This stereological method was successfully tested on artificially produced ice beads but also on several snow types. Combining stereology and polarisation microscopy provides a good estimate of grain boundary areas in ice beads and in natural snow, with some limitatio

Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra.

Divalent metal ion transporter 1 (DMT1) is a proton-coupled Fe(2+) transporter that is essential for iron uptake in enterocytes and for transferrin-associated endosomal iron transport in many other cell types. DMT1 dysfunction is associated with several diseases such as iron overload disorders and neurodegenerative diseases. The main objective of the present work is to develop and validate a fluorescence-based screening assay for DMT1 modulators. We found that Fe(2+) or Cd(2+) influx could be reliably monitored in calcium 5-loaded DMT1-expressing HEK293 cells using the FLIPR Tetra fluorescence microplate reader. DMT1-mediated metal transport shows saturation kinetics depending on the extracellular substrate concentration, with a K0.5 value of 1.4 µM and 3.5 µM for Fe(2+) and Cd(2+), respectively. In addition, Cd(2+) was used as a substrate for DMT1, and we find a Ki value of 2.1 µM for a compound (2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea) belonging to the benzylisothioureas family, which has been identified as a DMT1 inhibitor. The optimized screening method using this compound as a reference demonstrated a Z' factor of 0.51. In summary, we developed and validated a sensitive and reproducible cell-based fluorescence assay suitable for the identification of compounds that specifically modulate DMT1 transport activity.

An experimentally validated finite element method for augmented vertebral bodies

Background Finite element models of augmented vertebral bodies require a realistic modelling of the cement infiltrated region. Most methods published so far used idealized cement shapes or oversimplified material models for the augmented region. In this study, an improved, anatomy-specific, homogenized finite element method was developed and validated to predict the apparent as well as the local mechanical behavior of augmented vertebral bodies. Methods Forty-nine human vertebral body sections were prepared by removing the cortical endplates and scanned with high-resolution peripheral quantitative CT before and after injection of a standard and a low-modulus bone cement. Forty-one specimens were tested in compression to measure stiffness, strength and contact pressure distributions between specimens and loading-plates. From the remaining eight, fourteen cylindrical specimens were extracted from the augmented region and tested in compression to obtain material properties. Anatomy-specific finite element models were generated from the CT data. The models featured element-specific, density-fabric-based material properties, damage accumulation, real cement distributions and experimentally determined material properties for the augmented region. Apparent stiffness and strength as well as contact pressure distributions at the loading plates were compared between simulations and experiments. Findings The finite element models were able to predict apparent stiffness (R2 > 0.86) and apparent strength (R2 > 0.92) very well. Also, the numerically obtained pressure distributions were in reasonable quantitative (R2 > 0.48) and qualitative agreement with the experiments. Interpretation The proposed finite element models have proven to be an accurate tool for studying the apparent as well as the local mechanical behavior of augmented vertebral bodies.

The 'triple contrast' method in experimental wound ballistics and backspatter analysis.

In practical forensic casework, backspatter recovered from shooters' hands can be an indicator of self-inflicted gunshot wounds to the head. In such cases, backspatter retrieved from inside the barrel indicates that the weapon found at the death scene was involved in causing the injury to the head. However, systematic research on the aspects conditioning presence, amount and specific patterns of backspatter is lacking so far. Herein, a new concept of backspatter investigation is presented, comprising staining technique, weapon and target medium: the 'triple contrast method' was developed, tested and is introduced for experimental backspatter analysis. First, mixtures of various proportions of acrylic paint for optical detection, barium sulphate for radiocontrast imaging in computed tomography and fresh human blood for PCR-based DNA profiling were generated (triple mixture) and tested for DNA quantification and short tandem repeat (STR) typing success. All tested mixtures yielded sufficient DNA that produced full STR profiles suitable for forensic identification. Then, for backspatter analysis, sealed foil bags containing the triple mixture were attached to plastic bottles filled with 10 % ballistic gelatine and covered by a 2-3-mm layer of silicone. To simulate backspatter, close contact shots were fired at these models. Endoscopy of the barrel inside revealed coloured backspatter containing typable DNA and radiographic imaging showed a contrasted bullet path in the gelatine. Cross sections of the gelatine core exhibited cracks and fissures stained by the acrylic paint facilitating wound ballistic analysis.

Measurement of dijet cross sections in pp collisions at 7 TeV centre-of-mass energy using the ATLAS detector

Double-differential dijet cross-sections measured in pp collisions at the LHC with a 7TeV centre-of-mass energy are presented as functions of dijet mass and half the rapidity separation of the two highest-pT jets. These measurements are obtained using data corresponding to an integrated luminosity of 4.5 fb−1, recorded by the ATLAS detector in 2011. The data are corrected for detector effects so that cross-sections are presented at the particle level. Cross-sections are measured up to 5TeV dijet mass using jets reconstructed with the anti-kt algorithm for values of the jet radius parameter of 0.4 and 0.6. The cross-sections are compared with next-to-leading-order perturbative QCD calculations by NLOJet++ corrected to account for non-perturbative effects. Comparisons with POWHEG predictions, using a next-to-leading-order matrix element calculation interfaced to a partonshower Monte Carlo simulation, are also shown. Electroweak effects are accounted for in both cases. The quantitative comparison of data and theoretical predictions obtained using various parameterizations of the parton distribution functions is performed using a frequentist method. In general, good agreement with data is observed for the NLOJet++ theoretical predictions when using the CT10, NNPDF2.1 and MSTW 2008 PDF sets. Disagreement is observed when using the ABM11 and HERAPDF1.5 PDF sets for some ranges of dijet mass and half the rapidity separation. An example setting a lower limit on the compositeness scale for a model of contact interactions is presented, showing that the unfolded results can be used to constrain contributions to dijet production beyond that predicted by the Standard Model.