Deficiency in origin licensing proteins impairs cilia formation: implications for the aetiology of meier-gorlin syndrome

Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA-mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency

Uncoupling of Satellite DNA and Centromeric Function in the Genus Equus

In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs

The mechanism of DNA unwinding by the eukaryotic replicative helicase

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication

Genome-wide evolutionary and functional analysis of the Equine Repetitive Element 1: an insertion in the myostatin promoter affects gene expression

BACKGROUND: In mammals, an important source of genomic variation is insertion polymorphism of retrotransposons. These may acquire a functional role when inserted inside genes or in their proximity. The aim of this work was to carry out a genome wide analysis of ERE1 retrotransposons in the horse and to analyze insertion polymorphism in relation to evolution and function. The effect of an ERE1 insertion in the promoter of the myostatin gene, which is involved in muscle development, was also investigated. RESULTS: In the horse population, the fraction of ERE1 polymorphic loci is related to the degree of similarity to their consensus sequence. Through the analysis of ERE1 conservation in seven equid species, we established that the level of identity to their consensus is indicative of evolutionary age of insertion. The position of ERE1s relative to genes suggests that some elements have acquired a functional role. Reporter gene assays showed that the ERE1 insertion within the horse myostatin promoter affects gene expression. The frequency of this variant promoter correlates with sport aptitude and racing performance. CONCLUSIONS: Sequence conservation and insertion polymorphism of ERE1 elements are related to the time of their appearance in the horse lineage, therefore, ERE1s are a useful tool for evolutionary and population studies. Our results suggest that the ERE1 insertion at the myostatin locus has been unwittingly selected by breeders to obtain horses with specific racing abilities. Although a complex combination of environmental and genetic factors contributes to athletic performance, breeding schemes may take into account ERE1 insertion polymorphism at the myostatin promoter. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12863-015-0281-1) contains supplementary material, which is available to authorized users

DERMATOSI E DERMATITI NON INFETTIVE

Capitolo IV, inserito nel libro summenzionato, dove sono state indicate tutte le dermatosi e dermatiti non infettive nell'ambito della terapia in patologia vulvo-vestibolo-vaginale. Per ognuna delle malattie sono indicate le caratteristiche cliniche e la terapia

[Vulvovaginal candidiasis: a therapeutic approach].

The vulvovaginal candidiasis represents, after the bacterial vaginosis, the most frequent cause of vaginal affection. It is esteemed that around the 75% of the women of reproductive age suffered from an episode of vulvovaginitis from candida and 40-45% have had more episodes, of which 10-20% in complicated form. The kind of candida more frequently isolated in the vagina of symptomatic women is the Candida albicans: in the 10-20% of the cases the agent is present in absence of symptomatology, and we can almost consider it a saprophytic. On the other hand, always with greater frequency fetterses can be isolated of not albicans Candida, particularly the tropicalis and the glabrata kind, usually resistant to the common therapies. The classification of the vulvovaginal candidiasis proposed by Sobel, and by now universally approved, foresees 2 clinical forms of vulvovaginal candidiasis, the vulvovaginitis from not complicated candida (VVC) and the vulvovaginitis from complicated candida (VVCC): different for pathogenesis, elapsed clinical, symptomatology and frequency. They have to be considered in the substance 2 different nosological entities, and they request a diagnostic approach and a well different therapeutic appointment. In this study we will shortly reassume the principal characteristics of it, detaining us on the most recent acquisitions in theme of therapy. The base medicines of ac. boric, to parity of effectiveness, seem to introduce the most contained cost and the best compliance, and they offer him to a complementary use or, in some cases, alternative to the more you consolidate therapies with azoli

Efficacy of yeast starters to drive and improve Picual, Manzanilla and Kalamàta table olive fermentation

BACKGROUND: Table olive fermentation is an unpredictable process and frequently performed using traditional practices often inadequate to obtain products with acceptable quality and safety standards. In the present study, the efficacy of selected yeast strains as starters to drive fermentations of green and black table olives by the Greek method was investigated. Pilot-scale production by spontaneous fermentation as a control, olives started with previously selected Saccharomyces cerevisiae strains and fermentation driven by commercial S. cerevisiae baker's yeast strain were carried out for each of Manzanilla, Picual and Kalamàta table olive cultivars. RESULTS: Time of fermentation was significantly shortened to 40 days to complete the transformation process for all three tested cultivars. Inoculated table olives were enhanced in their organoleptic and nutritional properties in comparison with corresponding samples obtained by spontaneous fermentation. The use of starters was also able to improve safety traits of table olives in terms of biogenic amine reduction as well as absence of undesired microorganisms at the end of the process. CONCLUSIONS: Autochthonous, but also non-autochthonous, yeasts can be used to start and control table olive fermentations and can significantly improve quality and safety aspects of table olives produced by many smallholder farmers. © 2018 Society of Chemical Industry

The Unique DNA Sequences Underlying Equine Centromeres

Centromeres are highly distinctive genetic loci whose function is specified largely by epigenetic mechanisms. Understanding the role of DNA sequences in centromere function has been a daunting task due to the highly repetitive nature of centromeres in animal chromosomes. The discovery of a centromere devoid of satellite DNA in the domestic horse consolidated observations on the epigenetic nature of centromere identity, showing that entirely natural chromosomes could function without satellite DNA cues. Horses belong to the genus Equus which exhibits a very high degree of evolutionary plasticity in centromere position and DNA sequence composition. Examination of horses has revealed that the position of the satellite-free centromere is variable among individuals. Analysis of centromere location and composition in other Equus species, including domestic donkey and zebras, confirms that the satellite-less configuration of centromeres is common in this group which has undergone particularly rapid karyotype evolution. These features have established the equids as a new mammalian system in which to investigate the molecular organization, dynamics and evolutionary behaviour of centromeres