Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state¹ and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish^2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.     

The chewing lice (Phthiraptera: Ischnocera,Amblycera) of the great cormorant (Phalacrocorax carbo)

The Great Cormorant is a widespread bird species with almost worldwide distribution. Accordingly, its general biology has been investigated thoroughly. Less well known, however, are the chewing lice that live inside the plumage of this diving bird. We examined the two known species of Great Cormorant chewing lice, Eidmanniella pellucida (Rudow, 1869) (Amblycera: Menoponidae) and Pectinopygus gyricornis (Denny, 1842) (Ischnocera: Philopteridae). Taking advantage of the autofluorescence of the cuticle, confocal laser scanning microscopy (CLSM) was used to explore the external morphology of all developmental stages of P. gyricornis. Morphometric analyses revealed a standard increase in body size from first larval instar to the adult. In addition, all instars exhibited increasing body segment differentiation, especially in the abdomen and the head. A total of 277 individuals of Pectinopygus gyricornis and 2 individuals of Eidmanniella pellucida were collected from eleven Great Cormorants from Mecklenburg-Western Pomerania, Germany, in 2015.     

Static and rotational intensity modulated techniques for head-neck cancer radiotherapy: A planning comparison

PurposeTo compare helical Tomotherapy (HT), two volumetric-modulated arc techniques and conventional fixed-field intensity modulated techniques (S-IMRT) for head-neck (HN) cancers.Methods and materialsEighteen HN patients were considered. Four treatment plans were generated for each patient: HT, S-IMRT optimised with Eclipse treatment planning system and two volumetric techniques using Elekta–Oncentra approach (VMAT) and Varian-RapidArc (RA), using two full arcs. All techniques were optimised to simultaneously deliver 66Gy to PTV1 (GTV and enlarged nodes) and 54Gy to PTV2 (subclinical and electively treated nodes). Comparisons were assessed on several dosimetric parameters and, secondarily, on planned MUs and delivery time.ResultsConcerning PTV coverage, significantly better results were found for HT and RA. HT significantly improved the target coverage both compared to S-IMRT and VMAT. No significant differences were found between S-IMRT and volumetric techniques in terms of dose homogeneity. For OARs, all the techniques were able to satisfy all hard constraints; significantly better results were found for HT, especially in the intermediate dose range (15–30 Gy). S-IMRT reached a significantly better OARs sparing with respect to VMAT and RA. No significant differences were found for body mean dose, excepting higher values of V5–V10 for HT. A reduction of planned MUs and delivery treatment time was found with volumetric techniques.ConclusionsThe objectives of satisfying target coverage and sparing of critical structures were reached with all techniques. S-IMRT techniques were found more advantageous compared to RA and VMAT for OARs sparing. HT reached the best overall treatment plan quality.     

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).^1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.^3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.^4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.^18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),^8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),^9,10 and Bacteroidetes (Bac303).¹¹ In addition, specific probes binding to Streptococcus mutans (MUT590)^12,13 and Porphyromonas gingivalis (POGI)^13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.^5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations^3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.