Expression of sept3, sept5a and sept5b in the Developing and Adult Nervous System of the Zebrafish (Danio rerio)

Septins are a highly conserved family of small GTPases that form cytoskeletal filaments. Their cellular functions, especially in the nervous system, still remain largely enigmatic, but there are accumulating lines of evidence that septins play important roles in neuronal physiology and pathology. In order to further dissect septin function in the nervous system a detailed temporal resolved analysis in the genetically well tractable model vertebrate zebrafish (Danio rerio) is crucially necessary. To close this knowledge gap we here provide a reference dataset describing the expression of selected septins (sept3, sept5a and sept5b) in the zebrafish central nervous system. Strikingly, proliferation zones are devoid of expression of all three septins investigated, suggesting that they have a role in post-mitotic neural cells. Our finding that three septins are mainly expressed in non-proliferative regions was further confirmed by double-stainings with a proliferative marker. Our RNA in situ hybridization (ISH) study, detecting sept3, sept5a and sept5b mRNAs, shows that all three septins are expressed in largely overlapping regions of the developing brain. However, the expression of sept5a is much more confined compared to sept3 and sept5b. In contrast, the expression of all the three analyzed septins is largely similar in the adult brain

Fiji macro 3D ART VeSElecT: 3D automated reconstruction tool for vesicle structures of electron tomograms

Automatic image reconstruction is critical to cope with steadily increasing data from advanced microscopy. We describe here the Fiji macro 3D ART VeSElecT which we developed to study synaptic vesicles in electron tomograms. We apply this tool to quantify vesicle properties (i) in embryonic Danio rerio 4 and 8 days past fertilization (dpf) and (ii) to compare Caenorhabditis elegans N2 neuromuscular junctions (NMJ) wild-type and its septin mutant (unc-59(e261)). We demonstrate development-specific and mutant-specific changes in synaptic vesicle pools in both models. We confirm the functionality of our macro by applying our 3D ART VeSElecT on zebrafish NMJ showing smaller vesicles in 8 dpf embryos then 4 dpf, which was validated by manual reconstruction of the vesicle pool. Furthermore, we analyze the impact of C. elegans septin mutant unc-59(e261) on vesicle pool formation and vesicle size. Automated vesicle registration and characterization was implemented in Fiji as two macros (registration and measurement). This flexible arrangement allows in particular reducing false positives by an optional manual revision step. Preprocessing and contrast enhancement work on image-stacks of 1nm/pixel in x and y direction. Semi-automated cell selection was integrated. 3D ART VeSElecT removes interfering components, detects vesicles by 3D segmentation and calculates vesicle volume and diameter (spherical approximation, inner/outer diameter). Results are collected in color using the RoiManager plugin including the possibility of manual removal of non-matching confounder vesicles. Detailed evaluation considered performance (detected vesicles) and specificity (true vesicles) as well as precision and recall. We furthermore show gain in segmentation and morphological filtering compared to learning based methods and a large time gain compared to manual segmentation. 3D ART VeSElecT shows small error rates and its speed gain can be up to 68 times faster in comparison to manual annotation. Both automatic and semi-automatic modes are explained including a tutorial

Differential expression of transposable elements in the medaka melanoma model

Malignant melanoma incidence is rising worldwide. Its treatment in an advanced state is difficult, and the prognosis of this severe disease is still very poor. One major source of these difficulties is the high rate of metastasis and increased genomic instability leading to a high mutation rate and the development of resistance against therapeutic approaches. Here we investigate as one source of genomic instability the contribution of activation of transposable elements (TEs) within the tumor. We used the well-established medaka melanoma model and RNA-sequencing to investigate the differential expression of TEs in wildtype and transgenic fish carrying melanoma. We constructed a medaka-specific TE sequence library and identified TE sequences that were specifically upregulated in tumors. Validation by qRT- PCR confirmed a specific upregulation of a LINE and an LTR element in malignant melanomas of transgenic fish

Workflow of vesicle annotation using 3D ART VeSElecT.

<p>First, the automated registration macro is used, which scales the tomogram via user input of the pixel size and applies various filters in the preprocessing step. Afterwards the foreground is separated, the user semi-automatically selects an area of interest, and the macro applies the watershed algorithm for vesicle segmentation and registration. Second, an optional manual proof-reading step can be applied here, if necessary. Finally, the automatic measurement macro is used to extract results using certain characteristics. All manual steps are colored in yellow, semi-automated steps are in turquoise, automated steps are in blue.</p

Measurement of inner and outer synaptic vesicle diameter.

<p>In order to get an approximate value of the discrepancies between vesicle diameters of manual and automated measurement we applied the Fiji measurement tool. Fig 3 (A) shows the inner diameter of a vesicle that was annotated by 3D ART VeSElecT, (B) shows the outer diameter. Fig 3 (C) gives the results of the discrepancy of inner and outer diameter of all measured vesicles shown as a histogram (number of measurements = 80).</p

Tabular schedule showing required time for semi-automated reconstruction.

<p>Tabular schedule showing required time for semi-automated reconstruction.</p

Feasibility of the chick chorioallantoic membrane model for preclinical studies on tumor radiofrequency ablation

Abstract Background We evaluated the feasibility of a chick chorioallantoic membrane (CAM) tumor model for preclinical research on tumor radiofrequency ablation (RFA). Methods Fertilized chicken eggs were incubated and divided into five cohorts: RFA for 30 s (n = 5), RFA for 60 s (n = 5), RFA for 120 s (n = 4), sham (n = 8), and controls (n = 6). Xenografting using pancreatic neuroendocrine tumor cells of the BON-1 cell line was performed on embryonic day (ED) 8. The RFA was performed on ED 12. Survival, stereomicroscopic observations, and histological observations using hematoxylin–eosin (H&E) and Ki67 staining were evaluated. Results The survival rates in the 30-s, 60-s, and 120-s, sham and control cohort were 60%, 60%, 0%, 100%, and 50%, respectively. Signs of bleeding and heat damage were common findings in the evaluation of stereomicroscopic observations. Histological examination could be performed in all but one embryo. Heat damage, bleeding, thrombosis, and leukocyte infiltration and hyperemia were regular findings in H&E-stained cuts. A complete absence of Ki67 staining was recorded in 33.3% and 50% of embryos in the 30-s and 60-s cohorts that survived until ED 14, respectively. Conclusions The CAM model is a feasible and suiting research model for tumor RFA with many advantages over other animal models. It offers the opportunity to conduct in vivo research under standardized conditions. Further studies are needed to optimize this model for tumor ablations in order to explore promising but unrefined strategies like the combination of RFA and immunotherapy. Relevance statement The chick chorioallantoic membrane model allows in vivo research on tumor radiofrequency ablation under standardized conditions that may enable enhanced understanding on combined therapies while ensuring animal welfare in concordance with the “Three Rs.” Key points • The chorioallantoic membrane model is feasible and suiting for tumor radiofrequency ablation. • Radiofrequency ablation regularly achieved reduction but not eradication of Ki67 staining. • Histological evaluation showed findings comparable to changes in humans after RFA. • The chorioallantoic membrane model can enable studies on combined therapies after optimization. Graphical Abstrac

Isolation of native EVs from primary biofluids—Free‐flow electrophoresis as a novel approach to purify ascites‐derived EVs

Abstract Although extracellular vesicles (EVs) have been extensively characterized, efficient purification methods, especially from primary biofluids, remain challenging. Here we introduce free‐flow electrophoresis (FFE) as a novel approach for purifying EVs from primary biofluids, in particular from the peritoneal fluid (ascites) of ovarian cancer patients. FFE represents a versatile, fast, matrix‐free approach for separating different analytes with inherent differences in charge density and/or isoelectric point (pI). Using a series of buffered media with different pH values allowed us to collect 96 fractions of ascites samples. To characterize the composition of the individual fractions, we used state‐of‐the‐art methods such as nanoflow and imaging flow cytometry (nFCM and iFCM) in addition to classical approaches. Of note, tetraspanin‐positive events measured using nFCM were enriched in a small number of distinct fractions. This observation was corroborated by Western blot analysis and electron microscopy, demonstrating only minor contamination with soluble proteins and lipid particles. In addition, these gently purified EVs remain functional. Thus, FFE represents a new, efficient and fast method for separating native and highly purified EVs from complicated primary samples

Independent origin of XY and ZW sex determination mechanisms in Mosquitofish sister species

International audienceFish are known for the outstanding variety of their sex determination mechanisms and sex chromosome systems. The western () and eastern mosquitofish () are sister species for which different sex determination mechanisms have been described: ZZ/ZW for and XX/XY for Here, we carried out restriction-site associated DNA (RAD-) and pool sequencing (Pool-seq) to characterize the sex chromosomes of both species. We found that the ZW chromosomes of females and the XY chromosomes of males correspond to different linkage groups, and thus evolved independently from separate autosomes. In interspecific hybrids, the Y chromosome is dominant over the W chromosome, and X is dominant over Z. In , we identified a candidate region for the Y-linked melanic pigmentation locus, a rare male phenotype that constitutes a potentially sexually antagonistic trait and is associated with other such characteristics, , large body size and aggressive behavior. We developed a SNP-based marker in the Y-linked allele of (), which was linked to melanism in all tested populations. This locus represents an example for a color locus that is located in close proximity to a putative sex determiner, and most likely substantially contributed to the evolution of the Y