Morphological quantification of proliferation-to-invasion transition in tumor spheroids

BackgroundMetastasis determines the lethality of cancer. In most clinical cases, patients are able to live with tumor proliferation before metastasis. Thus, the transition from tumor proliferation to metastasis/invasion is essential. However, the mechanism is still unclear and especially, the proliferation-to-metastasis/invasion transition point has not been well defined. Therefore, quantitative characterization of this transition is urgently needed.MethodsWe have successfully developed a home-built living-cell incubation system combined with an inverted optical microscope, and a systematic, quantitative approach to describing the major characteristic morphological parameters for the identification of the critical transition points for tumor-cell spheroids in a collagen fiber scaffold.ResultsThe system focuses on in vitro tumor modeling, e.g. the development of tumor-cell spheroids in a collagen fiber scaffold and the monitoring of cell transition from proliferation to invasion. By applying this approach to multiple tumor spheroid models, such as U87 (glioma tumor), H1299 (lung cancer), and MDA-MB-231 (breast cancer) cells, we have obtained quantitative morphological references to evaluate the proliferation-to-invasion transition time, as well as differentiating the invasion potential of tumor cells upon environmental changes, i.e. drug application.ConclusionsOur quantitative approach provides a feasible clarification for the proliferation-to-invasion transition of in vitro tumor models (spheroids). Moreover, the transition time is a useful reference for the invasive potential of tumor cells.General significanceThis quantitative approach is potentially applicable to primary tumor cells, and thus has potential applications in the fields of cancer metastasis investigations and clinical diagnostics.     

Morphological quantification of filamentous fungal development using membrane immobilization and automatic image analysis

Mycelial morphology is a critically important process property in industrial fermentations of filamentous micro-organisms, as particular phenotypes are associated with maximum productivity. However, the accurate quantification of complex morphologies still represents a significant challenge in elucidating this relationship. A system has been developed for high-resolution characterisation of filamentous fungal growth on a solid substrate, using membrane immobilization and fully-automatic plug-ins developed for the public domain, Java-based, image-processing software, ImageJ. The system has been used to quantify the microscopic development of Aspergillus oryzae on malt agar, by measuring spore projected area and circularity, the total length of a hyphal element, the number of tips per element, and the hyphal growth unit. Two different stages of growth are described, from the swelling of a population of conidiospores up to fully developed, branched hyphae 24 h after inoculation. Spore swelling expressed as an increase in mean equivalent spore diameter was found to be approximately linear with time. Widespread germination of spores was observed by 8 h after inoculation. From approximately 12 h, the number of tips was found to increase exponentially. The specific growth rate of a population of hyphae was calculated as approximately 0.24–0.27 h⁻¹. A wide variation in growth kinetics was found within the population. The robustness of the image-analysis system was verified by testing the effect of small variations in the input data.     

Morphological quantification of pellets in Streptomyces hygroscopicus var. geldanus fermentation broths using a flatbed scanner

An image analysis technique has been developed to allow high throughput morphological characterisation of microbial fermentation broths containing spherical pellets greater than 100 m in diameter. Images of stained Streptomyces hygroscopicus var. geldanus culture samples at three different inoculum levels were captured using a flatbed scanner, at a resolution of 21 m per pixel (1200 dots per inch) and subsequently analysed leading to the generation of a morphological profile of each sample. The time taken for image capture and analysis of a prepared sample, containing approx. 2000 particles, was 3 min 6 s.     

Assessing the variation in individual frontal sinus outlines

It is often suggested that the frontal sinus morphology of no two individuals is alike, and that the configuration of the frontal sinus is as unique to an individual as his or her fingerprints. However, no empirical, quantitative testing of the uniqueness of frontal sinus outlines has ever been performed. Such testing is necessary for frontal sinus identifications to be admissible in many courts. This study investigated frontal sinus outline variability using elliptic Fourier analysis (EFA), a geometric morphometric approach that fits a closed curve to an ordered set of data points, generating a set of coefficients that can be used to reproduce the outline. Two-dimensional representations of 808 frontal sinuses (as seen in posterior-anterior cranial radiographs) were digitized, and differences in their shapes were assessed quantitatively by comparing the Euclidean distances between EFA-generated outlines. Results show that Euclidean distances between outlines of different individuals are significantly larger than those between replicates of the same individual, and typicalities show that the probability of finding two different individuals with Euclidean distances less that that between a particular case's replicate is very small. Thus, there is a quantifiable and significant difference between the shapes of individuals' frontal sinus outlines.     

Humeral outlines in some hominoid primates and in Plio-Pleistocene hominids

A method of drawing outlines of the distal end of the humerus is presented and carried out on some pongids (Pan troglodytes, Pan paniscus, Pongo pygmaeus), on modern man, and on some casts of Plio-Pleistocene hominids. It appears that these outlines are good indicators of the overall morphology and permit the distinguishing of the different hominoids. For example, the morphology of the pillars surrounding the fossa olecrani is useful for this purpose. In modern man, the lateral pillar is quadrangular, contrasting with the triangular medial one. In pongids, both of them are triangular; however, it is possible to note differences between Pongo and Pan. In the South Asian ape, there is a stronger anteroposterior flattening of the pillars as well as the diaphysis. The similarity of the shape of the pillars might be considered as a result of an adaptation to suspension. The differences might be due to different weights of the animals. Plio-Pleistocene hominids are variable with regard to the morphology of this region. For example, Gombore IB 7594 is similar to Homo. KNM ER 739 exhibits features intermediate between hominids and pongids. Finally, AL 288.1M is closer to pongids. These results confirm a previous anatomical work.     

Exploring artificial cranial deformation using elliptic Fourier analysis of Procrustes aligned outlines

The anatomical effects of artificial cranial deformation on the face and the base have been subject to various metric approaches, including standard linear as well as finite element techniques, and have produced controversial results (Antón [1989] Am. J. Phys. Anthropol. 79:253-267; Kohn et al. [1993] Am. J. Phys. Anthropol. 90:147-158). It can be argued that diverging observations partly result from methodological constraints. The present study compares samples of intentionally deformed and undeformed human crania, using elliptic Fourier analysis (EFA), a morphometric approach which has been shown to be particularly appropriate for characterizing the shape of two-dimensional outlines and associated shape changes. We improve the standard EFA approach by adding a preliminary orientation of the outlines following the rotation parameters of a Procrustes superimposition, using multiple homologous landmarks called control points. The results confirm that circumferentially deformed skulls exhibit modifications of the basioccipital region, together with increased anterior and inferior facial projection. However, the degree to which basioccipital flattening is modified in circumferentially deformed Peruvians was found to be less marked than changes observed in the face. Some of the modifications observed here can be related to morphological trends existing in the population from which our sample was taken. The observation of other modifications may be subject to methodological constraints of standard morphometric approaches.     

Method for analyzing outlines with an application to recent Japanese crania

If the visual image and quantitative treatment of a form could be merged, the combination could become a powerful analytical method in morphology. A method is presented for creating an averaged outline. In this method, since the averaged outline is produced statistically, hypothesis testing can be performed on the visual image of the outlines. Intra- and intergroup variation of the outlines can then be assessed by taking allometry into account. This method is applied to the analysis of sexual differences in the midsagittal outline of recent (1880s-1920s) Japanese crania. The averaged outlines of both sexes showed subtle features that could not be detected by traditional measuring techniques. Because the roundness, flatness, or protrusion of specific aspects of the morphology are clearly apparent, sexual differences in this sample could be identified. Changes resulting from allometry did not seem to be present in either sex. The shape distance (DIR) introduced here matched well the visual impression derived from comparisons of the Japanese sample with a Jomon (Neolithic) male and the Broken Hill specimen.     

Adaptive modifications of carapace outlines in the Cytheroidea (Ostracoda: Crustacea)

Shell outlines of 202 extant cytheroidean ostracods were analysed in dorsal, lateral, and posterior views by elliptic Fourier analysis. The results obtained confirm that the exterior morphology is related to ecological factors as well as phylogenetic constraints. Phytal species living on tall seagrass and benthic species burrowing in sediments are characterized and differentiated from the species crawling on sediments by the presence of slender shells with tapered venters. With reference to reconstruction of ancestral state of outline traits on the molecular phylogeny, the hypothetical ancestor of cytheroidean ostracods is presumed to have had an average-shaped shell. Morphological plasticity of the shell outline was observed in many families. The phytal species living on tall seagrass appear to have evolved convergently with species from other habitats, acquiring slim shell outlines during the Cenozoic period. The present analysis also reveals the phylogenetic constraints on the morphological evolution of the Trachyleberididae in their adaptation to a burrowing habit.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 810–821.     

Quantitative assessment of interproximal wear facet outlines for the association of isolated molars

The determination of the minimum number of individuals can be very challenging, especially in an assemblage of fragmentary bones and isolated teeth. Similarities in tooth morphology, degree of wear, and interproximal wear facets (IPWF) are generally used to associate isolated teeth qualitatively. However, no quantitative method has yet been established for an objective identification and matching of isolated tooth crowns. In this study, we analyze the IPWF morphology of adjacent mandibular molars (17 M(1)/M(2) pairs), applying both qualitative and quantitative methods to test a reproducible approach for crown association. The surfaces of distal (for M(1)) and mesial (for M(2)) IPWF were surface-scanned and digitally selected. Three-dimensional (3D) and two-dimensional (2D) outlines of IPWF were analyzed using elliptic Fourier analysis (EFA) and geometric morphometrics methods (GMM). Additionally, teeth were qualitatively associated by visual evaluation of the IPWF outline and by physical matching. Unsatisfactory results with less than 50% of tooth pairs correctly associated were obtained by using both methods, shape analysis (digital approach) and the visual evaluation (qualitative assessment) of the IPWF outline. The physical matching of the crowns showed highly variable accuracy ranging between 53% and 77%. The quantitative form-space analysis of 2D IPWF outlines provided the best results (82% of correctly associated teeth), but no statistically significant differences were recorded when compared with the manual matching. Since three tooth pairs out of 17 could not be quantitatively associated, we suggest that the quantitative analysis of IPWF should be used only in addition with other approaches.     

Sexual selection in hermit crabs: a review and outlines of future research

The information currently available on sexual selection in hermit crabs is reviewed to identify the role of males and females before, during and after mating. According to this information, possible mechanisms of male–male competition, female choice and/or sexual conflict are suggested. Important male components that may affect mating success include dragging the female shell, rotations of the female's shell and male cheliped palpations, and male size and/or shell characteristics (species and size). Possible female determinants of male mating/fertilization success include size (as an indicator of egg production capacity), signalling of sexual receptivity to males, delay from mate guarding to copulation and mating duration. Avenues for deeper exploration in males include the role of the number and morphometry of male sexual tubes during sperm transfer, and whether ejaculate size and sperm number can be adjusted with variable situations of sperm competition intensity and risk. In females it would be interesting to investigate the chemical and behavioural mechanisms affecting spermatophore breakage for sperm release and the variable duration from sperm transfer to spawning. Given these possibilities, and that sperm is externally deposited on the female's body but inside her shell (except for those species that do not use shells, e.g. Birgus , or species where shells are rather small and do not cover the body totally, e.g. Parapagurus ), we conclude that hermit crabs are unique subjects for separating male and female effects, particularly with respect to the applicability of current ideas in sexual selection such as female choice and sexual conflict. Some practical ideas are provided to disentangle both hypotheses using these animals.     

Micro-RNA quantification using DNA polymerase and pyrophosphate quantification

A rapid quantification method for micro-RNA based on DNA polymerase activity and pyrophosphate quantification has been developed. The tested micro-RNA serves as the primer, unlike the DNA primer in all DNA sequencing methods, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five femtomoles of the synthetic RNA could be detected. In 20–100 μg RNA samples purified from SiHa cells, the measurement was done using the proposed assay in which hsa-miR-16 and hsa-miR-21 are 0.34 fmol/μg RNA and 0.71 fmol/μg RNA, respectively. This simple and inexpensive assay takes less than 5 min after total RNA purification and preparation. The quantification is not affected by the pre-miRNA which cannot serve as the primer for the DNA synthesis in this assay. This assay is general for the detection of the target RNA or DNA with a known matched DNA template probe, which could be widely used for detection of small RNA, messenger RNA, RNA viruses, and DNA. Therefore, the method could be widely used in RNA and DNA assays.     

Protein quantification

Protein quantification is an integral part of any investigation related to protein isolation, purification, characterization, and analysis. Although the methods considered in this lecture have multi-year history and applied widely in the laboratory practice, there are some crucial points, which must be taken into consideration while choosing the method permitting reliably and with a high specificity and reproducibility to quantify protein.     

Optically eliminating the visible outlines of pores in intact polycarbonate (Nuclepore) filters

Under the microscope, Nuclepore filters display pore outlines. The polycarbonate material has two refractive indices (n = 1.584 and 1.616), which polarize transmitted light into two sets of rays at right angles to one another. This birefringence was used to eliminate the image of the pore outlines by the use of a specially made mounting medium with n20D = 1.584 in combination with polarized light: in a filter preparation of human leukocytes mounted in a medium with one matching refractive index and focused in polarized light, pore outlines were not visible. The preparation of the matching mounting medium, its use and its properties are described and discussed.     

The terminology and classification of steles: Historical perspective and the outlines of a system

Discussion focuses on the history of classification of the steles of vascular plants, from Jeffrey (1898) to the present. Recent stelar classifications are largely based (1) on Jeffrey’s system (protostele vs. siphonostele, the latter with ectophloic vs. amphiphloic types), (2) on the system developed by the early British morphologists, notably Brebner (1902), Gwynne-Vaughan (1901), Tansley (1907-08), and Tansley and Lulham (1902) (protostele vs. medullated protostele vs. solenostele vs. dictyostele vs. eustele, with various subtypes), or (3) on a combination of the preceding two systems, the British usually superimposed upon the Jeffreyian, although generally erratically so. An extended classification of three main stelar types and 25 stelar subtypes derived from Brebner (1902), Gwynne-Vaughan (1901), Jeffrey (1898), Tansley (1907-08), and others is presented in outline form with accompanying etymology, synonymy, examples, and with detailed notes on controversial taxa, classificatory rationale, and historical aspects. Recognized are (1) “protostele” (divided into “ectophloic,” “endophloic,” and “amphiphloic,” with 7, 1, and 1 subtypes respectively), (2) “siphonostele” (divided into “ectophloic” with 2 subtypes and “amphiphloic,” the latter divided into “solenostele” and “dictyostele” with 3 subtypes each, plus “siphonostele incertae sedis”), and (3) “eustele” (divided into “eustele sensu stricto” with 4 subtypes, “pseudosiphonostele,” “reduced eustele,” and “polycyclic eustele”).