TRF2 controls telomeric nucleosome organization in a cell cycle phase-dependent manner

Mammalian telomeres stabilize chromosome ends as a result of their assembly into a peculiar form of chromatin comprising a complex of non-histone proteins named shelterin. TRF2, one of the shelterin components, binds to the duplex part of telomeric DNA and is essential to fold the telomeric chromatin into a protective cap. Although most of the human telomeric DNA is organized into tightly spaced nucleosomes, their role in telomere protection and how they interplay with telomere-specific factors in telomere organization is still unclear. In this study we investigated whether TRF2 can regulate nucleosome assembly at telomeres.By means of chromatin immunoprecipitation (ChIP) and Micrococcal Nuclease (MNase) mapping assay, we found that the density of telomeric nucleosomes in human cells was inversely proportional to the dosage of TRF2 at telomeres. This effect was not observed in the G1 phase of the cell cycle but appeared coincident of late or post-replicative events. Moreover, we showed that TRF2 overexpression altered nucleosome spacing at telomeres increasing internucleosomal distance. By means of an in vitro nucleosome assembly system containing purified histones and remodeling factors, we reproduced the short nucleosome spacing found in telomeric chromatin. Importantly, when in vitro assembly was performed in the presence of purified TRF2, nucleosome spacing on a telomeric DNA template increased, in agreement with in vivo MNase mapping.Our results demonstrate that TRF2 negatively regulates the number of nucleosomes at human telomeres by a cell cycle-dependent mechanism that alters internucleosomal distance. These findings raise the intriguing possibility that telomere protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome organization

Phenylephrine-induced epistaxis in a six-year-old Quarter horse with nephrosplenic entrapment

Left dorsal displacement of the large colon is a common cause of colic in horses. Treatment consists of surgery, rolling the horse under general anesthesia or intravenous administration of phenylephrine. Treatment with phenylephrine, an alpha 1-adrenergic drug, is often associated with sweating and trembling. Especially in horses of more than 15 years old, fatal hemorrhage may occur due to hemothorax or hemoperitoneum. Therefore, phenylephrine treatment is generally not given in horses over 15 years of age. In this report, severe epistaxis in a six-year-old Quarter horse is described after intravenous administration of 22.5 mu g/kg BW phenylephrine, and it is highlighted that hemorrhage may also occur in younger horses

Successful surgical repair of a temporal and parietal bone fracture with associated traumatic brain injury in a fourteen-day-old foal = Succesvolle chirurgische behandeling van een os temporale en parietale fractuur met bijhorende hersenschade bij een twee weken oud veulen

A fourteen-day-old warmblood colt presented with severe neurological signs of unknown origin. The foal was recumbent and comatose, and showed spastic motion of the head. Computed tomography (CT) examination revealed multiple impression fractures to the left parietal and temporal bones, as well as traumatic brain injury. Three fragments of the temporal bone were dislocated into the cranial vault. The foal was surgically treated within 24 hours of presentation: the fragments were elevated and the largest fragment was fixated with an L-shaped titanium Matrix Midface 0.8 mm reconstruction plate. The brain injury resolved with medical treatment and supportive care. Apart from a localized wound abscess that was drained, postoperative healing was uneventful and the foal regained the ability to ambulate 18 days after surgery. Follow-up CT imaging documented good fracture healing and a favorable development of the soft tissue injuries. Seven months after surgery, the foal was fully recovered and showed no neurological signs

Evaluation of the pocH-100iV DIFF hematology analyzer for use in horses and cattle

The results of the analysis of equine and bovine blood samples with the automated pocH-100iV DIFF hematology analyzer were compared with the results obtained with reference methods or other analyzers (Vet ABC, Coulter Counter ZF, Coulter LH 750 analyzer). For equine blood and most parameters in bovine blood good to excellent correlations between methods and analyzers were obtained. For bovine blood good to poor correlations and significant differences were obtained between the pocH-100iV DIFF and other methods or analyzers mainly for hematocrit and hemoglobin determinations and platelet counts. Overall the pocH-100iV DIFF seems to be a reliable and user-friendly analyzer

arrEYE : a customized platform for high-resolution copy number analysis of coding and noncoding regions of known and candidate retinal dystrophy genes and retinal noncoding RNAs

Purpose: Our goal was to design a customized microarray, arrEYE, for high-resolution copy number variant (CNV) analysis of known and candidate genes for inherited retinal dystrophy (iRD) and retina expressed noncoding RNAs (ncRNAs). Methods: arrEYE contains probes for the full genomic region of 106 known iRD genes, including those implicated in retinitis pigmentosa (RP) (the most frequent iRD), cone rod dystrophies, macular dystrophies, and an additional 60 candidate iRD genes and 196 ncRNAs. Eight CNVs in iRD genes identified by other techniques were used as positive controls. The test cohort consisted of 57 patients with autosomal dominant, X-linked, or simplex RP. Results: In an RP patient, a novel heterozygous deletion of exons 7 and 8 of the HGSNAT gene was identified: c.634-408_820+338delins AGAATATG, p.(G1u2 I 2Glyfs*2). A known variant was found on the second allele: c.1843G>A, p.(A1a615Thr). Furthermore, we expanded the allelic spectrum of USH2A and RCBTB1 with novel CNVs. Conclusion: The arrEYE platform revealed subtle single-exon to larger CNVs in iRD genes that could be characterized at the nucleotide level, facilitated by the high resolution of the platform. We report the first CNV in HGSNAT that, combined with another mutation, leads to RP, further supporting its recently identified role in nonsyndromic iRD

Hidden genetic variation in LCA9-associated congenital blindness explained by 5′UTR mutations and copy-number variations of NMNAT1

Leber congenital amaurosis (LCA) is a severe autosomal-recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9-associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5'UTR variant, c.-70A>T. Moreover, an adjacent 5'UTR variant, c.-69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5'UTR variants resulted in decreased NMNAT1 mRNA abundance in patients' lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE-1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu-rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1-associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat-mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness