EmsB, a tandem repeated multi-loci microsatellite, new tool to investigate the genetic diversity of Echinococcus multilocularis

In order to explore the genetic diversity within Echinococcus multilocularis (E. multilocularis), the cestode responsible for the alveolar echinococcosis (AE) in humans, a microsatellite, composed of (CA) and (GA) repeats and designated EmsB, was isolated and characterized in view of its nature and potential field application. PCR-amplification with specific primers exhibited a high degree of size polymorphism between E. multilocularis and Echinococcus granulosus sheep (G1) and camel (G6) strains. Fluorescent-PCR was subsequently performed on a panel of E. multilocularis isolates to assess intra-species polymorphism level. EmsB provided a multi-peak profile, characterized by tandemly repeated microsatellite sequences in the E. multilocularis genome. This "repetition of repeats" feature provided to EmsB a high discriminatory power in that eight clusters, supported by bootstrap p-values larger than 95%, could be defined among the tested E. multilocularis samples. We were able to differentiate not only the Alaskan from the European samples, but also to detect different European isolate clusters. In total, 25 genotypes were defined within 37 E. multilocularis samples. Despite its complexity, this tandem repeated multi-loci microsatellite possesses the three important features for a molecular marker, i.e. sensitivity, repetitiveness and discriminatory power. It will permit assessing the genetic polymorphism of E. multilocularis and to investigate its spatial distribution in detail.

Discovery of insertion element ISCfe1: a new tool for Campylobacter fetus subspecies differentiation

The species Campylobacter fetus is divided into the subspecies C. fetus subsp. venerealis (CFV) and C. fetus subsp. fetus (CFF). CFV is the causative agent of bovine genital campylobacteriosis, a highly contagious venereal disease that may lead to serious reproductive problems, including sterility and abortion. In contrast, CFF can be isolated from the gastrointestinal tract of a wide range of host species, is associated with abortion in sheep and cattle, and can also be isolated from local and systemic infections in humans. Despite differences in host and niche preferences, microbiological differentiation of the two subspecies of C. fetus is extremely difficult. This study describes the identification of a new insertion element, ISCfe1, which is present exclusively in CFV strains, with highly conserved specific ISCfe1 insertion sites. The results are useful for identification and differentiation of the two C. fetus subspecies and will help in understanding the evolution and pathogenesis of C. fetus.

Follicle-forming cat thyroid cell lines synthesizing extracellular matrix and basal membrane components: a new tool for the study of thyroidal morphogenesis

Interactions between follicular epithelial cells and extracellular matrix (ECM) are supposed to play an important role in the development and maintenance of thyroid tissue architecture. In the present study we have therefore investigated the synthesis of ECM components by a feline thyroid cell line which is able to form follicle-like structures in vitro, and also in v-ras-transfected and control-transfected sublines. Transfections were performed by lipofection with pZSR (viral Harvey ras gene; neo) and pSV2-neo (control, neo only) plasmids. We have adapted a semisolid culture system composed exclusively of polymerized alginate and therefore devoid of ECM components. Feline cells embedded in alginate gels as single cells and cultured for up to 90 days formed cell clusters within 10 days. Follicle-like structures were formed in the original cell lines and also in the v-ras- and control-transfected cells. Differences in proliferation rates were observed, the v-ras-transfected cells growing up to two to three times faster than the non-transfected cells. Immunostaining was done using rabbit first antibodies directed against mouse collagen IV, human fibronectin, laminin (tumor Engelbreth-Holm-Swarm laminin), perlecan and other ECM components. For comparison, immunostaining was also performed on cryosections of nodular goiters of six hyperthyroid cats. The cell lines and their transfected clones stained strongly positive for collagen IV and fibronectin, and positively but less strongly for laminin and perlecan. The cat goiter tissue stained positively for collagen IV, laminin, perlecan, and fibronectin, and positive staining for S-laminin (containing the beta2-chain) was seen in blood vessel walls in this tissue. In conclusion, cat cell lines grow three-dimensionally in alginate beads over several weeks, they form follicle-like structures and express the same ECM components as the native cat goiter tissue. Transfection with v-ras does increase proliferation rate, but does not fundamentally alter formation of follicle-like structures and ECM expression. Alginate gel culture is a promising new tool for the study of follicular morphogenesis, polarity, the expression pattern of ECM components and of the interaction between thyrocytes and ECM. It avoids interference caused by gels composed of ECM components.

Wavelet-denoising of electroencephalogram and the absolute slope method: a new tool to improve electroencephalographic localization and lateralization

OBJECTIVE: In ictal scalp electroencephalogram (EEG) the presence of artefacts and the wide ranging patterns of discharges are hurdles to good diagnostic accuracy. Quantitative EEG aids the lateralization and/or localization process of epileptiform activity. METHODS: Twelve patients achieving Engel Class I/IIa outcome following temporal lobe surgery (1 year) were selected with approximately 1-3 ictal EEGs analyzed/patient. The EEG signals were denoised with discrete wavelet transform (DWT), followed by computing the normalized absolute slopes and spatial interpolation of scalp topography associated to detection of local maxima. For localization, the region with the highest normalized absolute slopes at the time when epileptiform activities were registered (>2.5 times standard deviation) was designated as the region of onset. For lateralization, the cerebral hemisphere registering the first appearance of normalized absolute slopes >2.5 times the standard deviation was designated as the side of onset. As comparison, all the EEG episodes were reviewed by two neurologists blinded to clinical information to determine the localization and lateralization of seizure onset by visual analysis. RESULTS: 16/25 seizures (64%) were correctly localized by the visual method and 21/25 seizures (84%) by the quantitative EEG method. 12/25 seizures (48%) were correctly lateralized by the visual method and 23/25 seizures (92%) by the quantitative EEG method. The McNemar test showed p=0.15 for localization and p=0.0026 for lateralization when comparing the two methods. CONCLUSIONS: The quantitative EEG method yielded significantly more seizure episodes that were correctly lateralized and there was a trend towards more correctly localized seizures. SIGNIFICANCE: Coupling DWT with the absolute slope method helps clinicians achieve a better EEG diagnostic accuracy.

The EmsB tandemly repeated multilocus microsatellite: a new tool to investigate genetic diversity of Echinococcus granulosus sensu lato

Cystic echinococcosis (CE) is a widespread and severe zoonotic disease caused by infection with the larval stage of the eucestode Echinococcus granulosus sensu lato. The polymorphism exhibited by nuclear and mitochondrial markers conventionally used for the genotyping of different parasite species and strains does not reach the level necessary for the identification of genetic variants linked to restricted geographical areas. EmsB is a tandemly repeated multilocus microsatellite that proved its usefulness for the study of genetic polymorphisms within the species E. multilocularis, the causative agent of alveolar echinococcosis. In the present study, EmsB was used to characterize E. granulosus sensu lato samples collected from different host species (sheep, cattle, dromedaries, dogs, and human patients) originating from six different countries (Algeria, Mauritania, Romania, Serbia, Brazil, and the People's Republic of China). The conventional mitochondrial cox1 and nad1 markers identified genotypes G1, G3, G5, G6, and G7, which are clustered into three groups corresponding to the species E. granulosus sensu stricto, E. ortleppi, and E. canadensis. With the same samples, EmsB provided a higher degree of genetic discrimination and identified variations that correlated with the relatively small-scale geographic origins of the samples. In addition, one of the Brazilian single hydatid cysts presented a hybrid genotypic profile that suggested genetic exchanges between E. granulosus sensu stricto and E. ortleppi. In summary, the EmsB microsatellite exhibits an interesting potential for the elaboration of a detailed map of the distribution of genetic variants and therefore for the determination and tracking of the source of CE.

Fluorescent In Situ hybridization: a new tool for the direct identification and detection of F. psychrophilum

F. psychrophilum is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of Flavobacterium species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of F. psychrophilum infections in fish. We constructed a generic probe for the genus Flavobacterium ("Pan-Flavo") and two specific probes targeting F. psychrophilum based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different Flavobacterium and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the F. psychrophilum probe, respectively). FISH was able to detect F. psychrophilum in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of Flavobacterium spp. and F. psychrophilum.

A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons

A close to native structure of bulk biological specimens can be imaged by cryo-electron microscopy of vitreous sections (CEMOVIS). In some cases structural information can be combined with X-ray data leading to atomic resolution in situ. However, CEMOVIS is not routinely used. The two critical steps consist of producing a frozen section ribbon of a few millimeters in length and transferring the ribbon onto an electron microscopy grid. During these steps, the first sections of the ribbon are wrapped around an eyelash (unwrapping is frequent). When a ribbon is sufficiently attached to the eyelash, the operator must guide the nascent ribbon. Steady hands are required. Shaking or overstretching may break the ribbon. In turn, the ribbon immediately wraps around itself or flies away and thereby becomes unusable. Micromanipulators for eyelashes and grids as well as ionizers to attach section ribbons to grids were proposed. The rate of successful ribbon collection, however, remained low for most operators. Here we present a setup composed of two micromanipulators. One of the micromanipulators guides an electrically conductive fiber to which the ribbon sticks with unprecedented efficiency in comparison to a not conductive eyelash. The second micromanipulator positions the grid beneath the newly formed section ribbon and with the help of an ionizer the ribbon is attached to the grid. Although manipulations are greatly facilitated, sectioning artifacts remain but the likelihood to investigate high quality sections is significantly increased due to the large number of sections that can be produced with the reported tool.

The Forensic Reference Phantom-a new tool for quality assurance of attenuation measurements in forensic radiology

Introduction The purpose of this paper is to present the technical specifications of the Forensic Reference Phantom (FRP), to test its behavior relative to organic test materials, and discuss potential applications of the phantom in forensic radiology. Materials and method The FRP prototype is made of synthetic materials designed to simulate the computed tomography (CT) attenuation of water. It has six bore holes that accommodate multiuse containers. These containers were filled with test materials and scanned at 80 kVp, 120 kVp, and 140 kVp. X-ray attenuation was measured by two readers. Intra- and inter-reader reliability was assessed using the intra-class correlation coefficient (ICC). Significance levels between mean CT numbers at 80 kVp, 120 kVp, and 140 kVp were assessed with the Friedman-test. The T-test was used to assess significance levels between the FRP and water. Results Overall mean CT numbers ranged from −3.0–3.7HU for the FRP; −1000.3–−993.5HU for air; −157.7– −108.1HU for oil; 35.5–42.0HU for musle tissue; and 1301.5–2354.8HU for cortical bone. Inter-reader and intra-reader reliability were excellent (ICC>0.994; and ICC=0.999 respectively). CT numbers were significantly different at different energy levels. There was no significant difference between the attenuation of the FRP and water. Conclusions The FRP is a new tool for quality assurance and research in forensic radiology. The mean CT attenuation of the FRP is equivalent to water. The phantom can be scanned during routine post-mortem CT to assess the composition of unidentified objects. In addition, the FRP may be used to investigate new imaging algorithms and scan protocols in forensic radiology.

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.

Ceramic foam plates: a new tool for processing fresh radical prostatectomy specimens

Procurement of fresh tissue of prostate cancer is critical for biobanking and generation of xenograft models as an important preclinical step towards new therapeutic strategies in advanced prostate cancer. However, handling of fresh radical prostatectomy specimens has been notoriously challenging given the distinctive physical properties of prostate tissue and the difficulty to identify cancer foci on gross examination. Here, we have developed a novel approach using ceramic foam plates for processing freshly cut whole mount sections from radical prostatectomy specimens without compromising further diagnostic assessment. Forty-nine radical prostatectomy specimens were processed and sectioned from the apex to the base in whole mount slices. Putative carcinoma foci were morphologically verified by frozen section analysis. The fresh whole mount slices were then laid between two ceramic foam plates and fixed overnight. To test tissue preservation after this procedure, formalin-fixed and paraffin-embedded whole mount sections were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry, fluorescence, and silver in situ hybridization (FISH and SISH, respectively). There were no morphological artifacts on H&E stained whole mount sections from slices that had been fixed between two plates of ceramic foam, and the histological architecture was fully retained. The quality of immunohistochemistry, FISH, and SISH was excellent. Fixing whole mount tissue slices between ceramic foam plates after frozen section examination is an excellent method for processing fresh radical prostatectomy specimens, allowing for a precise identification and collection of fresh tumor tissue without compromising further diagnostic analysis.