A detailed methodology for the long-term in vitro culture and analysis of three-dimensional, self-structuring bone models generated from cell lines or primary osteoblastic cell populations.

Background: There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models.Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). Methods: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including RT-qPCR, immunofluorescence staining and XRF, are described in detail. Results: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. Conclusions: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.

Improving on whole-brain radiotherapy in patients with large brain metastases: A planning study to support the AROMA clinical trial.

PURPOSE: To develop a novel dose-escalated volumetric modulated arc therapy (VMAT) strategy for patients with single or multiple large brain metastases which can deliver a higher dose to individual lesions for better local control (LC), and to compare dosimetry between whole brain radiotherapy (WBRT), hippocampal-sparing whole brain radiotherapy (HS-WBRT) and different VMAT-based focal radiotherapy approaches. METHODS AND MATERIALS: We identified 20 patients with one to ten brain metastases and at least one lesion larger than 15 cm3 who had received WBRT as part of routine care. For each patient, we designed and evaluated five radiotherapy treatment plans, including WBRT, HS-WBRT and three VMAT dosing models. A dose of 20 Gy in 5 fractions was prescribed to the whole brain or target volumes depending on the plan, with higher doses to smaller lesions and dose-escalated inner planning target volumes (DE-iPTV) in VMAT plans, respectively. Treatment plans were evaluated using the efficiency index, mean dose and D0.1 cc to the target volumes and organs at risk. RESULTS: Compared with WBRT, VMAT plans achieved a significantly more efficient dose distribution in brain lesions, especially with our DE-iPTV model, while minimising the dose to the normal brain and other organs at risks (OARs) (p < 0.05). CONCLUSIONS: VMAT plans obtained higher doses to brain metastases and minimised doses to OARs. Dose-escalated VMAT for larger lesions allows higher radiotherapy doses to be delivered to larger lesions while maintaining safe doses to OARs.

Visibility and attractiveness of Fritillaria (Liliaceae) flowers to potential pollinators.

Visual floral characters play an important role in shaping plant-pollinator interactions. The genus Fritillaria L. (Liliaceae), comprising approximately 140 species, is described as displaying a remarkable variety of flower colours and sizes. Despite this variation in visual floral traits of fritillaries, little is known about the potential role of these features in shaping plant-pollinator interactions. Here, we seek to clarify the role of visual attraction in species offering a robust food reward for pollinators early in the spring, which is the case for Fritillaria. We also searched for potential tendencies in the evolution of floral traits crucial for plant-pollinator communication. The generality of species with green and purple flowers may indicate an influence of environmental factors other than pollinators. The flowers of the studied species seem to be visible but not very visually attractive to potential pollinators. The food rewards are hidden within the nodding perianth, and both traits are conserved among fritillaries. Additionally, visual floral traits are not good predictors of nectar properties. When in the flowers, pollinators are navigated by nectar guides in the form of contrasting nectary area colouration. Flower colour does not serve as a phenotypic filter against illegitimate pollinators-red and orange bird-pollinated fritillaries are visible to bees.

Evolution of Bird and Insect Flower Traits in Fritillaria L. (Liliaceae).

Pollinators are often perceived as a primary selective agent influencing flower traits such as colour, size, and nectar properties. The genus Fritillaria L. (Liliaceae), comprising approximately 150 species, is described as generally insect pollinated. However, there are at least three exceptions: two hummingbird-pollinated North American species and one passerine-pollinated Asian species. Despite this variation in pollination, little is known about flower traits that may accompany this shift in fritillaries. In this study, we aimed to assess the attractiveness of the floral traits for (new) pollinators and track the evolution of flowers traits in the context of a shift in the principal pollinator. Therefore, we studied 14 flower traits related to the pollination in 60 Fritillaria species and traced the evolutionary trajectory of these traits. We used a phylogenetic tree of the genus, based on five DNA markers (matK, rpl16, and rbcL, 18S, and ITS) to reconstruct the ancestral state of studied flower traits. The results show that in bird-pollinated species several new traits evolved. For example, flower colouration, nectar sugar, and amino acid concentration and composition fulfil the criteria of ornithophilous flowers, although flower traits do not exclude insect pollinators in bird-pollinated fritillaries. Interestingly, we recorded potential reversals from bird to insect pollination. Our analysis, showing a broad study of flower traits among closely related species in the context of pollinator shift, serves as a starting point for future work exploring the genetic and physiological mechanisms controlling flower traits in the genus Fritillaria.

Functional Diversity of Nectary Structure and Nectar Composition in the Genus Fritillaria (Liliaceae).

Fritillaria is a genus consisting of 130 to 140 species of bulbous plants, native to temperate regions of the northern hemisphere. Generally viewed as an insect pollinated genus with the exception of two North American species, Fritillaria gentneri and F. recurva, which are described as hummingbird-pollinated and the Asian species, F. imperialis, described as passerine-pollinated. These pollinator shifts are possibly the result of adaptive changes to the structure and morphology of the nectary, as well as a change in the nectar concentration and composition. A study was conducted in a target group of 56 Fritillaria species, based on the morphology of their nectaries and nectar composition to assess the significance of pollination mode as well as its predisposition for the evolution of bird pollination. All species studied had nectaries located at their tepal base and produced nectar, but their size, shape, color, and composition all varied. Most fritillaries had hexose-rich nectar, in easily accessible and unprotected nectaries. Scanning electron microscope (SEM) analysis revealed that the surface of the nectaries of most Fritillaria species was flat and clearly distinct from that of the surrounding tissues, which might be regarded as an adaptation for insect-pollination. Nectaries of F. imperialis were considerably larger and had dilute nectar without sucrose, which was produced profusely, thereby fulfilling the criteria characteristic of ornithophilous flowers. The copious nectar of presumed hummingbird-pollinated species was rather balanced and of medium sugar concentration. Their large lanceolate nectaries contrasted sharply with the tessellated background of their tepals. These characters might indicate a mixed pollination system that engages both birds and insects. Floral anatomy and microstructure and nectar composition for Fritillaria species in subgenera Korolkowia and Liliorhiza are studied for the first time.

Evolutionary relationships in the medicinally important genus Fritillaria L. (Liliaceae).

Fritillaria (Liliaceae) is a genus of approximately 140 species of bulbous perennial plants that includes taxa of both horticultural and medicinal importance. As well as being commercially valuable, Fritillaria species have attracted attention because of their exceptionally large genome sizes, with all values recorded to date in excess of 30Gb. Despite such interest in the genus, phylogenetic relationships between the majority of species have remained untested. Here we present the first phylogenetic reconstruction of relationships to encompass most of the currently recognised species diversity in the genus. Three regions of the plastid genome were sequenced in 117 individuals of Fritillaria, representing 92 species (c. 66% of the genus) and in representatives of nine other genera of Liliaceae. Eleven low-copy nuclear gene regions were also screened in selected species for their potential utility. Phylogenetic analysis of a combined plastid dataset using maximum parsimony and Bayesian inference provided support for the monophyly of the majority of currently recognised subgenera. However, subgenus Fritillaria, which is by far the largest of the subgenera and includes the most important species used in traditional Chinese medicine, is found to be polyphyletic. Moreover, several taxa that were represented by multiple individuals show evidence of species non-monophyly. The Japanese endemic subgenus Japonica, which contains the species with the largest recorded genome size for any diploid plant, is resolved as sister to the predominantly Middle Eastern and Central Asian subgenus Rhinopetalum. Whilst relationships between most of the major Fritillaria lineages can now be resolved, our results also highlight the need for data from additional independently evolving loci; an endeavour that may be particularly challenging in light of the huge nuclear genomes found in these plants.