Subcutaneous extraskeletal osteosarcoma of the foot: A case report

Extraskeletal osteosarcoma (ESOS) is a rare variant of osteosarcoma which occurs exclusively in the soft tissue without any bone involvement. Subcutaneous ESOS, in particular, is very rare and is seen in <10% of cases. Here, we report a case of a subcutaneous tumor in the fourth web space of the left foot in a 73-year-old man. The diagnosis of ESOS was made on histology and by immunohistochemical reactivity to special AT-rich sequence-binding protein 2 (SATB2), which is a sensitive, nuclear marker of osteoblastic differentiation. We present this case because of its rarity and the use of SATB2 immunohistochemistry to confirm the diagnosis.

Sodium Alginate versus Plasma Thrombin Cell Blocks in Diagnostic Cytopathology: A Comparative Analysis

<b><i>Background:</i></b> Cell blocks (CBs) are an essential adjunct in cytopathology practice. The aim of this study was to compare 2 techniques of CB preparation – plasma thrombin (PT) method with sodium alginate (SA) method for overall cellularity, morphological preservation, obscuring artefacts, immunocytochemistry (ICC), suitability for molecular analysis, and cost of preparation. <b><i>Design:</i></b> A total of 80 fine-needle aspirates from various sites and serous effusion samples were included. Of these cases, by random selection, 40 each were prepared by PT method and SA methods, respectively. The haematoxylin-eosin-stained sections from the formalin-fixed, paraffin-embedded CBs from both methods were evaluated in a blinded fashion by 2 cytopathologists and scored for cellularity, artefacts, and morphological preservation and analysed by χ² test with Yates correction. We evaluated 6 cases from each method by ICC for a range of membrane, cytoplasmic and nuclear marker expression. DNA was extracted from four cases to evaluate their utility for molecular analysis. <b><i>Results:</i></b> CB sections from PT and SA techniques showed comparable cellularity and excellent cytomorphological preservation. Blue gel-like artefacts were common in the SA technique but did not interfere with morphological evaluation. ICC staining results were also similar. DNA yield and utility for PCR were also comparable. The SA-CB cost half that of PT-CB (USD 0.4 vs. USD 1). <b><i>Conclusion:</i></b> SA technique of CB preparation is an excellent low-cost alternative to PT method for CB preparation.

Combination of nuclear and mitochondrial markers as a useful tool to identify Ctenophthalmus species and subspecies (Siphonaptera: Ctenophthalmidae)

Ctenophthalmus is considered the largest genus within the Order Siphonaptera. From a morphological point of view, only males of this genus can be identified at species and subspecies levels using morphological keys, whereas there are no morphological criteria in order to classify females at these taxonomical levels. Furthermore, the amount of available molecular and phylogenetic data for this genus is quite scarce so far. The main objective of this work was to assess the utility of the combination of nuclear and mitochondrial markers with respect to their ability to differentiate among different subspecies within the Ctenophthalmus genus. With this purpose, we carried out a comparative morphological and molecular study of three different subspecies (Ctenophthalmus baeticus arvernus, Ctenophthalmus nobilis dobyi, and Ctenophthalmus andorrensis catalaniensis) in order to clarify and discuss its taxonomic status. In addition, our study complemented the molecular data previously provided for Ctenophthalmus baeticus boisseauorum and Ctenophthalmus apertus allani subspecies. We sequenced five different molecular markers: EF1-α, ITS1, ITS2, cox1, and cytb. Our results confirmed that morphological data by themselves are not able to discriminate among Ctenophthalmus female taxa; however, the combination of the nuclear marker EF1-α together with mtDNA markers cytb and cox1 constituted a useful taxonomical and phylogenetic tool to solve this issue. Based on these results, we consider that the use of this molecular approach should be gradually used within Ctenophthalmus genus in order to complement its classical taxonomy and clarifying the complex taxonomy of other congeneric species of fleas.

OSCAR: a framework to identify and quantify cells in densely packed three-dimensional biological samples

We have developed an Object Segmentation, Counter and Analysis Resource (OSCAR) that is designed specifically to quantify densely packed biological samples with reduced signal-to-background ratio. OSCAR uses as input three dimensional images reconstructed from confocal 2D sections stained with dies such as nuclear marker and immunofluorescence labeling against specific antibodies to distinguish the cell types of interest. Taking advantage of a combination of arithmetic, geometric and statistical algorithms, OSCAR is able to reconstruct the objects in the 3D space bypassing segmentation errors due to the typical reduced signal to noise ration of biological tissues imaged in toto. When applied to the zebrafish developing retina, OSCAR is able to locate and identify the fate of each nuclei as a cycling progenitor or a terminally differentiated cell, providing a quantitative characterization of the dynamics of the developing vertebrate retina in space and time with unprecedented accuracy.

Variation and Diagnostic Power of the Internal Transcribed Spacer 2 in Mediterranean and Atlantic Eolid Nudibranchs (Mollusca, Gastropoda)

Mediterranean marine biodiversity is still underestimated especially for groups such as nudibranchs. The identification of nudibranchs taxa is challenging because few morphological characters are available and among them chromatic patterns often do not align with species delimitation. Molecular assessments helped unveiling cryptic diversity within nudibranchs and have been mostly based on mitochondrial markers. Fast evolving nuclear markers are much needed to complement phylogenetic and systematic assessments at the species and genus levels. Here, we assess the utility of the nuclear Internal Transcribed Spacer 2 (ITS2) to delimit species in the eolid nudibranchs using both primary and secondary structures. Comparisons between the variation observed at the ITS2 and at the two commonly used mitochondrial markers (COI and 16S) on 14 eolid taxa from 10 genera demonstrate the ability of ITS2 to detect congeneric, closely related, species. While ITS2 has been fruitfully used in several other mollusc taxa, this study represents the first application of this nuclear marker in nudibranchs.

Molecular and Chemical Markers to Illustrate the Complex Diversity of the Genus Lupinus (Fabaceae)

The potential of secondary metabolites as systematic markers to get new insights in an intricate phylogeny of a recent evolutionary radiation is explored. A chemosystematic study of the genus Lupinus (Fabaceae) was performed, using quinolizidine (QA) and piperidine alkaloids (ammodendrine) as diagnostic characters. Seven major QA and the piperidine alkaloid ammodendrine were found to be the most frequent compounds. Two groups were supported according to their geographic origin: an Old World/Atlantic American group and a West New World group and this pattern is concordant with molecular data (here, based on an original barcode approach using the nuclear marker ITS). However, QA profiles are less informative at the species level. Despite a lack of resolution within the two groups, the alkaloid profiles agree with well supported clades based on DNA molecular characters. The combined use of chemical and barcode genetic markers represents a viable alternative for separating recent evolutionary lineages to a first approximation without having to resort to an expensive and sophisticated molecular arsenal such as next generation sequencing.

Two centuries from species discovery to diagnostic characters: molecular and morphological evidence for narrower species limits in the widespread SW Australian Anarthria gracilis complex (Restionaceae s.l./Anarthriaceae, Poales)

Background The extreme southwest of Australia is a biodiversity hotspot region that has a Mediterranean-type climate and numerous endemic plant and animal species, many of which remain to be properly delimited. We refine species limits in Anarthria, a Western Australian endemic genus characterised by the occurrence of the greatest number of plesiomorphic character states in the restiid clade of Poales. In contrast to many other groups of wind-pollinated Australian Poales, Anarthria was traditionally viewed as having well-established species limits. All six currently recognised species, which are conspicuous members of some Western Australian plant communities, were described in the first half of the 19th century. They are traditionally distinguished from each other mainly using quantitative characters. Methods We examined extensive existing herbarium specimens and made new collections of Anarthria in nature. Scanning electron microscopy and light microscopy were used to study leaf micromorphology. Molecular diversity of Anarthria was examined using a plastid (trnL-F) and a low-copy nuclear marker (at103). This is the first study of species-level molecular diversity in the restiid clade using a nuclear marker. Results Material historically classified as Anarthria gracilis R.Br. actually belongs to three distinct species, A. gracilis s.str., A. grandiflora Nees and A. dioica (Steud.) C.I.Fomichev, each of which forms a well-supported clade in phylogenetic analyses. Both segregate species were described in the first half of the 19th century but not recognised as such in subsequent taxonomic accounts. Anarthria dioica was first collected in 1826, then wrongly interpreted as a species of Juncus (Juncaceae) and described as Juncus dioicus. We provide a formal transfer of the name to Anarthria and for the first time report its clear and qualitative diagnostic characters: an extremely short leaf ligule and distinctive pattern of leaf epidermal micromorphology. A long ligule is present in A. gracilis s.str. and A. grandiflora. These species differ from each other in leaf lamina morphology and anatomy and have mostly non-overlapping distribution ranges. The narrower definition of species provides a basis for future phylogeographic analyses in Anarthria. Our study highlights a need for more extensive use of nuclear DNA markers in Restionaceae. The use of the low copy nuclear marker at103 allowed a clade comprising all three ligulate species of Anarthria to be recognised. The ligule character is used here for the first time in the taxonomy of Anarthria and merits special attention in studies of other restiids. In general, our study uncovered a superficially hidden but, in reality, conspicuous diversity in a common group of wind-pollinated plants in the southwest of Western Australia.

DNA specificity in two Hedysarum sections, Hedysarum and Multicaulia (Siberia, Russia) inferred from ETS sequence data

Based on sequence data of the nuclear ETS markers of 20 species, the relationships of sections Hedysarum and Multicaulia were assessed. Phylogenetic analysis was performed on the complete sequences using the Neighbor-Joining methods. The nuclear marker ETS is investigated for the first time in the most species excluding H. hedysaroides, H. inundatum, H. ferganense, and H. neglectum. The length of sequences varies from 349 to 357 nucleotides. It is noteworthy that ETS data significantly discriminate H. ferganense from both sections, Hedysarum and Multicaulia. Moreover, our results did not support the subsectional division of Multicaulia section. Therefore, the ETS sequence data obtained in our study for 20 taxa of Hedysarum evidence the nonmonophyly of section Multicaulia.