CCDC80 AND CCDC80-L1: IDENTIFICATION AND FUNCTIONAL ANALYSIS OF TWO NOVEL GENES INVOLVED IN ZEBRAFISH (DANIO RERIO) DEVELOPMENT

The ccdc80 and ccdc80-like1 (ccdc80-l1) genes were isolated in zebrafish in silico, on the basis of their high aminoacidic sequence identity with the human CCDC80 protein (coiled-coil domain containing 80). In human, CCDC80 is involved in several carcinomas, and during recent years it has been proposed as an onco-suppressor gene, increasing the interest in the comprehension of its functions. During my Ph.D., I have been studying the expression patterns and the functions of its zebrafish homologs, in order to gain insights into the processes and the molecular mechanisms in which they are involved. I took advantage of zebrafish as a model system allowing the application of common genetic and biological experimental assays such as PCR, whole mount in situ hybridization and microinjection technique. I investigated ccdc80 and ccdc80-l1 functions during zebrafish embryonic development, finding that they show very different expression patterns and roles. ccdc80 is expressed in the notochord during somitogenesis up to 48 hour post fertilization (hpf). At this stage, it is expressed also in the heart. Instead, at the same developmental stages, ccdc80-l1 is expressed in cranial ganglia, adaxial cells, muscle pioneers and dorsal dermis. These patterns are suggestive of different roles, in fact, the genes are involved in distinct developmental processes, such as somitogenesis and axonal pathfinding, respectively. Functional analysis were obtained performing loss- and gain-of-function experiments. The results clearly demonstrated that manipulation of Ccdc80 protein levels during embryonic development, both increasing and decreasing them, leads to a severe impairment of somites, metameric structures from which several tissues derive, such as muscle. Nevertheless, this phenotype seemed to be recovered at 24 hpf, since at this stage somites no longer showed the same morphological alterations observed previously. By converse, notwithstanding its expression in muscular tissues, loss-of-ccdc80-l1 does not impair somites formation, leading instead to motoneurons axonal migrations defects, capable to affect embryonic motility. Given the external development of the zebrafish embryo, motility is required very soon for prey and escaping predators, and muscle and motoneurons are equally required for a proper motor behavior. Communication and interactions between these two tissues are fundamental for proper muscles innervation and transmission of nervous inputs to be translated in muscular contractions. ccdc80 and ccdc80-l1 may derive from an ancestor gene involved in these processes and, during evolution, may have developed different functions, but still bound to a key process for embryonic viability in the external environment

Comparison of anticoagulation quality between acenocoumarol and warfarin in patients with mechanical prosthetic heart valves: Insights from the nationwide PLECTRUM study

Vitamin K antagonists are indicated for the thromboprophylaxis in patients with mechanical prosthetic heart valves (MPHV). However, it is unclear whether some differences between acenocoumarol and warfarin in terms of anticoagulation quality do exist. We included 2111 MPHV patients included in the nationwide PLECTRUM registry. We evaluated anticoagulation quality by the time in therapeutic range (TiTR). Factors associated with acenocoumarol use and with low TiTR were investigated by multivariable logistic regression analysis. Mean age was 56.8 ± 12.3 years; 44.6% of patients were women and 395 patients were on acenocoumarol. A multivariable logistic regression analysis showed that patients on acenocoumarol had more comorbidities (i.e., ≥3, odds ratio (OR) 1.443, 95% confidence interval (CI) 1.081-1.927, p = 0.013). The mean TiTR was lower in the acenocoumarol than in the warfarin group (56.1 ± 19.2% vs. 61.6 ± 19.4%, p < 0.001). A higher prevalence of TiTR (<60%, <65%, or <70%) was found in acenocoumarol users than in warfarin ones (p < 0.001 for all comparisons). Acenocoumarol use was associated with low TiTR regardless of the cutoff used at multivariable analysis. A lower TiTR on acenocoumarol was found in all subgroups of patients analyzed according to sex, hypertension, diabetes, age, valve site, atrial fibrillation, and INR range. In conclusion, anticoagulation quality was consistently lower in MPHV patients on acenocoumarol compared to those on warfarin

ccdc80-l1 Is Involved in Axon Pathfinding of Zebrafish Motoneurons

Axon pathfinding is a subfield of neural development by which neurons send out axons to reach the correct targets. In particular, motoneurons extend their axons toward skeletal muscles, leading to spontaneous motor activity. In this study, we identified the zebrafish Ccdc80 and Ccdc80-like1 (Ccdc80-l1) proteins in silico on the basis of their high aminoacidic sequence identity with the human CCDC80 (Coiled-Coil Domain Containing 80). We focused on ccdc80-l1 gene that is expressed in nervous and non-nervous tissues, in particular in territories correlated with axonal migration, such as adaxial cells and muscle pioneers. Loss of ccdc80-l1 in zebrafish embryos induced motility issues, although somitogenesis and myogenesis were not impaired. Our results strongly suggest that ccdc80-l1 is involved in axon guidance of primary and secondary motoneurons populations, but not in their proper formation. ccdc80-l1 has a differential role as regards the development of ventral and dorsal motoneurons, and this is consistent with the asymmetric distribution of the transcript. The axonal migration defects observed in ccdc80-l1 loss-of-function embryos are similar to the phenotype of several mutants with altered Hedgehog activity. Indeed, we reported that ccdc80-l1 expression is positively regulated by the Hedgehog pathway in adaxial cells and muscle pioneers. These findings strongly indicate ccdc80-l1 as a down-stream effector of the Hedgehog pathway

Precise measurement of the thermal and stellar 54^{54}Fe(n,γn, \gamma)55^{55}Fe cross sections via AMS

The detection of long-lived radionuclides through ultra-sensitive single atom counting via accelerator mass spectrometry (AMS) offers opportunities for precise measurements of neutron capture cross sections, e.g. for nuclear astrophysics. The technique represents a truly complementary approach, completely independent of previous experimental methods. The potential of this technique is highlighted at the example of the $^{54}$Fe($n, \gamma$)$^{55}$Fe reaction. Following a series of irradiations with neutrons from cold and thermal to keV energies, the produced long-lived $^{55}$Fe nuclei ($t_{1/2}=2.744(9)$ yr) were analyzed at the Vienna Environmental Research Accelerator (VERA). A reproducibility of about 1% could be achieved for the detection of $^{55}$Fe, yielding cross section uncertainties of less than 3%. Thus, the new data can serve as anchor points to time-of-flight experiments. We report significantly improved neutron capture cross sections at thermal energy ($\sigma_{th}=2.30\pm0.07$ b) as well as for a quasi-Maxwellian spectrum of $kT=25$ keV ($\sigma=30.3\pm1.2$ mb) and for $E_n=481\pm53$ keV ($\sigma= 6.01\pm0.23$ mb). The new experimental cross sections have been used to deduce improved Maxwellian average cross sections in the temperature regime of the common $s$-process scenarios. The astrophysical impact is discussed using stellar models for low-mass AGB stars

Neutron Resonance Spectrometry of 99-Tc from 3 eV to 150 keV.

Abstract not availableJRC.D-Institute for Reference Materials and Measurements (Geel

Resonance shape analysis of neutron capture measurements from xenon spherical samples

Techniques have been developed in the frame of the CONRAD Nuclear data analysis code in order to take into account the multiple scattering correction for capture yields. The resonance shape analysis of these capture data have to account for these corrections during the fitting procedure. Analytical formulae and Monte Carlo simulations can be performed through Multiple-Scattering-Correction module in the CONRAD code in order to estimate the differential and total capture yields. Monte Carlo method accounts for usual biasing techniques (implicite capture and Russian roulette). This experimental correction procedure is checked against other codes like REFIT and SAMMY which are extensively used for cylindrical sample analysis. In this work, xenon spherical samples have been studied. Capture and transmission measurements of gaseous samples have been performed at the GELINA facility of the Institute for Reference Materials and Measurements (IRMM) by P. Mutti, F. Corvi and A. Brusegan. For the capture measurements, spherical samples with diameter of 8 cm were used. Due to the spherical shape of the sample, equivalent cylinder (h ≈ 5.33 cm) was taken into account in SAMMY or REFIT analysis (spherical model is not implemented in these codes). These results are compared with analysis involving multiple scattering correction for spherical targets available in CONRAD

s-ccdc80 is involved in axon guidance of zebrafish primary motoneurons

The similar to ccdc80 (s-ccdc80) gene was in silico identified on the basis of its high nuclear sequence identity (56,6%) with zebrafish ccdc80 (coiled-coil domain containing 80). The analysis of s-ccdc80 temporal/spatial expression by means of RT-PCR and whole mount in situ hybridization revealed its diffuse expression at early developmental stages and in epiboly. During somitogenesis up to 48 hours post fertilization, s-ccdc80 is expressed in specific territories such as cranial ganglia, ventral portion of somites, migrating adaxial cells and muscle pioneers, dorsal dermis and caudal vein plexus region. s-ccdc80 knock-down through the injection of a specific morpholino did not lead to morphological body plan defects nor alteration of the expression of several myogenic markers, though motility resulted affected. Thus, we decided to investigate the motoneuronal development in knocked-down embryos, also bearing in mind s-ccdc80 expression in territories involved in motoneurons patterning and axonal pathfinding: muscle pioneers and adaxial cells. s-ccdc80 knock-down led to axonal outgrowth defects of two primary motoneurons (CaP and Mip), that branched excessively and followed aberrant pathways. Our results strongly suggest that s-ccdc80 is involved in the guidance of the axonal extension of primary motoneurons but not in their proper formation. Furthermore, s-ccdc80 expression seemed to be positively regulated by the Hedgehog pathway