A comparative ultrastructural study on the nanoscale extrusomes of tintinnids (Alveolata, Ciliophora, Spirotricha) and their phylogenetic significance.

The capsules, putative extrusomes in tintinnid ciliates, are known since 1971. Based on their ultrastructure, shape, and size, five capsule types were distinguished and suggested to be of phylogenetic significance. However, detailed morphometric data and transmission electron micrographs are lacking to verify former conclusions. In the current study, comprehensive analyses of transmission electron microscopic data were performed, investigating 14 species from 13 genera and more than seven families collected in European coastal waters and in the Northeast Pacific. Our data suggest two main capsule types (large and ampulliform vs small and ellipsoidal/ovoidal) each including two subtypes characterised by their internal structures. Species groupings inferred from the capsule (sub-)types emerge also as closely related in gene trees. Additionally, the ampulliform type unites the Undellidae, Xystonellidae, and Tintinnid clade 2, while the shared possession of the small ellipsoidal type proposes a close relationship of Tintinnid clade 11 with the Rhabdonellidae and Cyttarocylididae. Thus, the capsules provide promising features to shed light on several unresolved evolutionary relationships among tintinnid genera and families; yet, information on capsules is still missing for many monophyletic groupings. Finally, we provide the first ultrastructural clues for the extrusive character of these organelles.

A light and electron microscopical study on the resting cyst of the tintinnid Schmidingerella (Alveolata, Ciliophora) including a phylogeny-aware comparison.

Resting cysts protect ciliates against adverse environmental conditions. The morphology and ultrastructure of resting cysts has been described in very few Oligotrichea, a group of mainly marine planktonic ciliates. The present study provides the first ultrastructural data for loricate choreotrichids, applying light and electron microscopy on the cysts of the tintinnid Schmidingerella meunieri (Kofoid and Campbell, 1929) Agatha and Strüder-Kypke, 2012. The morphology of live cysts and the wall ultrastructure of cryofixed cysts were morphometrically analysed. The resting cyst is roughly flask-shaped, broadening to a slightly concave, laterally protruding anterior plate. An emergence pore closed by a skull cap-shaped papula is directed to the bottom of the lorica on the opposite side of the cyst. The cyst wall consists of an ectocyst, mesocyst, and endocyst differing in thickness, structure, and nitrogen concentration as revealed by conventional transmission electron microscopy, electron energy loss spectroscopy, and electron spectroscopic imaging. The cysts of S. meunieri belong to the kinetosome-resorbing type, which also occurs in the majority of hypotrich ciliates. Two main features (flask-shape and presence of an emergence pore) are shared with the closely related aloricate choreotrichids and oligotrichids, distinguishing the Oligotrichea from the hypotrich and the more distantly related euplotid ciliates.

Somatic infraciliature in tintinnid ciliates (Alveolata, Ciliophora, Spirotricha): An ultrastructural comparison.

A recent ultrastructural study on the tintinnid ciliate Schmidingerella meunieri displayed unique types of somatic kinetids. The dikinetids (paired basal bodies) have, besides kinetodesmal fibrils and transverse ribbons, some special features, that is, overlapping postciliary ribbons and three extraordinary microtubular ribbons, which together form a conspicuous network in the ciliated anterior cell portion. The distribution of this feature among tintinnids is studied in chemically fixed and ultrathin-sectioned specimens from six genera and five families collected in European coastal waters. The taxa are scattered across the molecular tree. Actually, the somatic kinetids of these six genera share the special features discovered in S. meunieri. Accordingly, the overlapping postciliary ribbons and the three extraordinary ribbons were already present in the early stages of tintinnid evolution, namely in the last common ancestor of tintinnids with hard loricae. Owing to the lack of ultrastructural data in the basally branching Tintinnidiidae with their soft loricae and in aloricate choreotrichids other than the aberrant strobilidiids, the first appearance of the structures is still uncertain. The related oligotrichids do not possess overlapping postciliary ribbons, but show electron-dense material at the sites where the ribbons I-III originate in tintinnids. None of these features is found in any other spirotrich ciliate.

A comparative ultrastructural study of tintinnid loricae (Alveolata, Ciliophora, Spirotricha) and a hypothesis on their evolution.

Tintinnid ciliates build loricae, whose structure, shape, and size still largely represent the basis for taxonomy and classification, although genetic analyses demonstrated their limited utility for inferring evolutionary relationships. The textures of the lorica walls, however, result from the chemical and physical properties of the forming material, which is supposed to be rather conserved in closely related taxa, viz., congeners and confamilial genera. Within a particular texture, small deviations in the chemical composition might affect the wall's stickiness and accordingly its capability to adhere foreign particles, explaining the intertwining of tintinnids with hyaline and agglutinated loricae in phylogenetic inferences. In a comprehensive comparative study, the lorica textures were electron microscopically and morphometrically analyzed in 21 species from 17 genera and more than nine families together with literature data. Most species were investigated for the first time, and the taxa cover a substantial portion of the molecular genealogy. The phylogeny-aware analysis of the lorica-related features provides a preliminary hypothesis on lorica evolution. Eventually, this conspectus suggests the dominance of hard lorica walls with an alveolar texture and proposes different modes of lorica formation.

Maturation of the gastric microvasculature in Xenopus laevis (Lissamphibia, Anura) occurs at the transition from the herbivorous to the carnivorous lifestyle, predominantly by intussuceptive microvascular growth (IMG): a scanning electron microscope study of microvascular corrosion casts and correlative light microscopy.

The microvascular bed of the stomach of Xenopus laevis and the changes it undergoes when the herbivorous tadpole becomes a carnivorous adult were studied by scanning electron microscopy of vascular corrosion casts and light microscopy of stained tissue sections. In tadpoles an upper and a lower gastric artery supplied, and upper, middle and lower medial and lateral gastric veins drained the vertically extending stomach. During metamorphosis, the stomach gained a horizontal cranio-caudal extension and vessels accordingly become dorsal and ventral gastric arteries, and anterior, middle and posterior gastric veins, respectively. Up to stage 64 (late climax) mucosal capillaries formed a polygonal network of wide immature-looking capillaries ensheathing gastric glands in a basket-like manner. From stage 64 onwards, blood vessels of the stomach appeared mature, revealed a clear hierarchy and were correlated closely with the histomorphology of the stomach, which had also gained the adult pattern. Within the gastric mucosa, ascending arterioles branched in a fountain-like pattern into wide subepithelial capillaries establishing a centripetal blood flow along the gastric glands, which makes an ultrashort control loop of glandular cells within the branched tubular gastric glands very unlikely. Formation of the stomach external muscular layer started at stage 57 when smooth muscle cells locally formed a single longitudinal and one-to-two single circular layers. Abundant signs of intussusceptive microvascular growth and rare vascular sprouts in vascular corrosion casts indicated that the larval-to-adult microvascular pattern formation of the stomach of Xenopus laevis Daudin occurs predominantly by non-sprouting angiogenesis.

HER2 is unlikely to be involved in directly regulating angiogenesis in human breast cancer.

Angiogenesis is a fundamental component of oncogenesis. Angiogenic factors such as vascular endothelial growth factor (VEGF) and platelet derived-endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) are generated from tumor cells to provide tumor growth and are thought to be regulated via the HER2 oncogene, whose amplification is the most common genetic alteration in breast cancer. The present study aimed to evaluate the immunoreactivity of angiogenic factors (VEGF, PD-ECGF/TP) and microvessel density (MVD) via epidermal growth factor receptor (EGFR) and HER2, and to correlate their expression with clinicopathologic features. Two hundred one invasive human breast cancer specimens were tested immunohistochemically for the expression of these proteins. In addition, MVD was examined using computerized image analysis. VEGF could be an additional interesting prognostic variable, as it was significantly associated with tumor grade (P=0.002), stage (P=0.018), and negative estrogen receptor status (P=0.011). EGFR was significantly related to invasive ductal carcinoma (P=0.030), tumor grade (P=0.009), VEGF expression (P=0.013), PD-ECGF/TP expression (P=0.024), and MVD (P=0.050). The finding that VEGF is not correlated to MVD does not rule out a crucial role of VEGF as a key factor in angiogenesis. HER2 could not be correlated to MVD, VEGF expression, or PD-ECGF/TP expression, indicating that this factor is unlikely to be involved in directly regulating angiogenesis, whereas the significant correlations between EGFR and histologic tumor type, tumor grade, the angiogenic factors VEGF and PD-ECGF/TP, and MVD point out that EGF is the major modulating growth factor for angiogenesis in breast cancer.

How a highly complex three-dimensional network of blood vessels regresses: the gill blood vascular system of tadpoles of Xenopus during metamorphosis. A SEM study on microvascular corrosion casts.

The morphology and vasculature of filter plates and filter rows of tadpoles of Xenopus laevis Daudin were studied from prometamorphosis (stage 56) to metamorphic climax (stage 62) by scanning electron microscopy (SEM) of (i) critical point-dried specimens and (ii) microvascular corrosion casts. The number and frequencies of the highest branching orders of venous filter row vessels, which reach their maximum (6) in stage 58, as well as vessel diameters, interbranching distances, and branching angles were measured. Branching orders continuously decrease until stage 62, so that only two are left. Vessel (luminal) diameters increased from 16.9 +/- 0.65 microm (SEM) (stage 56) to 19.3 +/- 0.61 microm (stage 58) (branching order 0) and then decreased to 16.7 +/- 0.91 microm (stage 62). Interbranching distances were largest in stage 59 (57.5 +/- 2.07 microm (branching order 0). Branching angles were highest in stage 62 (91.4 degrees +/- 8.82 degrees; branching order 2) and lowest in stage 56 (76.0 degrees +/- 4.64 degrees; branching order 4). From the morphology of cast vessels we conclude that vascular regression is organized by progressive "fusion" of opposite vessels in the highest branching orders ("miniaturization") until only a single capillary loop connects the terminal arteriole and the main filter fold venule. With obliteration of the single capillary loop, the regression of the gill vasculature is terminated. It is noted that the "fusion" of gill vessels is reminiscent of "reversed" intussusceptive microvascular growth.