ANALYSIS OF THE IN VIVO FUNCTION OF HASPIN KINASE USING ZEBRAFISH AS A MODEL SYSTEM: KNOCKDOWN AND KNOCKOUT APPROACHES

The Haspin gene encodes an atypical serine/threonine mitotic kinase first discovered in mouse spermatocytes and preferentially expressed in tissues with a high rate of proliferating cells. Haspin acts at metaphase by phosphorylating threonine 3 of histone H3 (H3Thr3PH) and this modification allows the recruitment of the chromosomal passenger complex, a key factor required to orchestrate different steps of mitosis. In human cells, HASPIN depletion causes a decrease in H3Thr3 levels, resulting in premature loss of sister chromatid cohesion and in defects in chromosome alignment at metaphase. Haspin has been found in all eukaryotic organisms; however, up to know, its role during animal embryonic development has never been investigated. We decided to investigate its function and expression during zebrafish embryonic development and, to this aim, we took advantage of a morpholino (MO)-mediated knockdown approach and of the CRISPR-Cas9 knockout strategy. We identified and cloned the zebrafish haspin ortholog, together with a previously unknown splicing isoform, and we clarified its expression pattern during embryogenesis and in some adult tissues. We demonstrated a relevant maternal contribution for the haspin transcript and important levels of zygotic expression in tissues with a high rate of proliferating cells, such as the developing brain and hematopoietic tissues. We also detected haspin transcript in the adult gonads and found that its expression is significantly switched on after injury during adult fin tissue regeneration. Interestingly, after Haspin functional inactivation using two different MOs, a translation blocking (ATG MO) and a splicing one, we demonstrated that Haspin is involved in H3Thr3PH also in zebrafish. Moreover, microinjection of the haspin ATG MO results in high embryo mortality and severe defects during epiboly stages, indicating important alterations in cellular rearrangements and movements. A haspin stable mutant line was generated by using the CRISPR-Cas9 technology: we isolated three different mutant haspin alleles, all causing the formation of premature stop codons. Although they do not show evident phenotypic alterations during embryogenesis, embryos carrying a homozygous genotype for these mutations are not able to reach the adulthood stage, showing a high rate of mortality in the first three weeks of larval development, indicating that Haspin is fundamental for larval survival and growth. To conclude, we clarified various aspects of haspin expression pattern during zebrafish development and in adult organs. Even though we were not able yet to unambiguously define the phenotypic effect of Haspin functional inactivation by using a MO-mediated approach, we paved the way for the analysis of the effect of a complete haspin gene knockout during zebrafish development by generating a haspin stable KO line and by showing that this null mutant allele significantly affects larval survival and growth

Long walk to genomics : history and current approaches to genome sequencing and assembly

Genomes represent the starting point of genetic studies. Since the discovery of DNA structure, scientists have devoted great efforts to determine their sequence in an exact way. In this review we provide a comprehensive historical background of the improvements in DNA sequencing technologies that have accompanied the major milestones in genome sequencing and assembly, ranging from early sequencing methods to Next-Generation Sequencing platforms. We then focus on the advantages and challenges of the current technologies and approaches, collectively known as Third Generation Sequencing. As these technical advancements have been accompanied by progress in analytical methods, we also review the bioinformatic tools currently employed in de novo genome assembly, as well as some applications of Third Generation Sequencing technologies and high-quality reference genomes

ADAP2 in heart development: a candidate gene for the occurrence of cardiovascular malformations in NF1 microdeletion syndrome

Background Cardiovascular malformations have a higher incidence in patients with NF1 microdeletion syndrome compared to NF1 patients with intragenic mutation, presumably owing to haploinsufficiency of one or more genes included in the deletion interval and involved in heart development. In order to identify which genes could be responsible for cardiovascular malformations in the deleted patients, we carried out expression studies in mouse embryos and functional studies in zebrafish. Methods and results The expression analysis of three candidate genes included in the NF1 deletion interval, ADAP2, SUZ12 and UTP6, performed by in situ hybridisation, showed the expression of ADAP2 murine ortholog in heart during fundamental phases of cardiac morphogenesis. In order to investigate the role of ADAP2 in cardiac development, we performed loss-of-function experiments of zebrafish ADAP2 ortholog, adap2, by injecting two different morpholino oligos (adap2-MO and UTR-adap2-MO). adap2-MOs-injected embryos (morphants) displayed in vivo circulatory and heart shape defects. The molecular characterisation of morphants with cardiac specific markers showed that the injection of adap2-MOs causes defects in heart jogging and looping. Additionally, morphological and molecular analysis of adap2 morphants demonstrated that the loss of adap2 function leads to defective valvulogenesis, suggesting a correlation between ADAP2 haploinsufficiency and the occurrence of valve defects in NF1-microdeleted patients. Conclusions Overall, our findings indicate that ADAP2 has a role in heart development, and might be a reliable candidate gene for the occurrence of cardiovascular malformations in patients with NF1 microdeletion and, more generally, for the occurrence of a subset of congenital heart defects

Haspin regulates Ras localization to promote Cdc24-driven mitotic depolarization

Cell polarization is of paramount importance for proliferation, differentiation, development, and it is altered during carcinogenesis. Polarization is a reversible process controlled by positive and negative feedback loops. How polarized factors are redistributed is not fully understood and is the focus of this work. In Saccharomyces cerevisiae, mutants defective in haspin kinase exhibit stably polarized landmarks and are sensitive to mitotic delays. Here, we report a new critical role for haspin in polarisome dispersion; failure to redistribute polarity factors, in turn, leads to nuclear segregation defects and cell lethality. We identified a mitotic role for GTP-Ras in regulating the local activation of the Cdc42 GTPase, resulting in its dispersal from the bud tip to a homogeneous distribution over the plasma membrane. GTP-Ras2 physically interacts with Cdc24 regulateing its mitotic distribution. Haspin is shown to promote a mitotic shift from a bud tip-favored to a homogenous PM fusion of Ras-containing vesicles. In absence of haspin, active Ras is not redistributed from the bud tip; Cdc24 remains hyperpolarized promoting the activity of Cdc42 at the bud tip, and the polarisome fails to disperse leading to erroneously positioned mitotic spindle, defective nuclear segregation, and cell death after mitotic delays. These findings describe new functions for key factors that modulate cell polarization and mitotic events, critical processes involved in development and tumorigenesis

The balance of power: accretion and feedback in stellar mass black holes

In this review we discuss the population of stellar-mass black holes in our galaxy and beyond, which are the extreme endpoints of massive star evolution. In particular we focus on how we can attempt to balance the available accretion energy with feedback to the environment via radiation, jets and winds, considering also possible contributions to the energy balance from black hole spin and advection. We review quantitatively the methods which are used to estimate these quantities, regardless of the details of the astrophysics close to the black hole. Once these methods have been outlined, we work through an outburst of a black hole X-ray binary system, estimating the flow of mass and energy through the different accretion rates and states. While we focus on feedback from stellar mass black holes in X-ray binary systems, we also consider the applicability of what we have learned to supermassive black holes in active galactic nuclei. As an important control sample we also review the coupling between accretion and feedback in neutron stars, and show that it is very similar to that observed in black holes, which strongly constrains how much of the astrophysics of feedback can be unique to black holes.Comment: To be published in Haardt et al. Astrophysical Black Holes. Lecture Notes in Physics. Springer 201

Observation of hard scattering in photoproduction events with a large rapidity gap at HERA

Events with a large rapidity gap and total transverse energy greater than 5 GeV have been observed in quasi-real photoproduction at HERA with the ZEUS detector. The distribution of these events as a function of the $\gamma p$ centre of mass energy is consistent with diffractive scattering. For total transverse energies above 12 GeV, the hadronic final states show predominantly a two-jet structure with each jet having a transverse energy greater than 4 GeV. For the two-jet events, little energy flow is found outside the jets. This observation is consistent with the hard scattering of a quasi-real photon with a colourless object in the proton.Comment: 19 pages, latex, 4 figures appended as uuencoded fil

Search for a W' boson decaying to a bottom quark and a top quark in pp collisions at sqrt(s) = 7 TeV

Results are presented from a search for a W' boson using a dataset corresponding to 5.0 inverse femtobarns of integrated luminosity collected during 2011 by the CMS experiment at the LHC in pp collisions at sqrt(s)=7 TeV. The W' boson is modeled as a heavy W boson, but different scenarios for the couplings to fermions are considered, involving both left-handed and right-handed chiral projections of the fermions, as well as an arbitrary mixture of the two. The search is performed in the decay channel W' to t b, leading to a final state signature with a single lepton (e, mu), missing transverse energy, and jets, at least one of which is tagged as a b-jet. A W' boson that couples to fermions with the same coupling constant as the W, but to the right-handed rather than left-handed chiral projections, is excluded for masses below 1.85 TeV at the 95% confidence level. For the first time using LHC data, constraints on the W' gauge coupling for a set of left- and right-handed coupling combinations have been placed. These results represent a significant improvement over previously published limits.Comment: Submitted to Physics Letters B. Replaced with version publishe

Search for the standard model Higgs boson decaying into two photons in pp collisions at sqrt(s)=7 TeV

A search for a Higgs boson decaying into two photons is described. The analysis is performed using a dataset recorded by the CMS experiment at the LHC from pp collisions at a centre-of-mass energy of 7 TeV, which corresponds to an integrated luminosity of 4.8 inverse femtobarns. Limits are set on the cross section of the standard model Higgs boson decaying to two photons. The expected exclusion limit at 95% confidence level is between 1.4 and 2.4 times the standard model cross section in the mass range between 110 and 150 GeV. The analysis of the data excludes, at 95% confidence level, the standard model Higgs boson decaying into two photons in the mass range 128 to 132 GeV. The largest excess of events above the expected standard model background is observed for a Higgs boson mass hypothesis of 124 GeV with a local significance of 3.1 sigma. The global significance of observing an excess with a local significance greater than 3.1 sigma anywhere in the search range 110-150 GeV is estimated to be 1.8 sigma. More data are required to ascertain the origin of this excess.Comment: Submitted to Physics Letters