Multi-scale cellular imaging of DNA double strand break repair

Live-cell and high-resolution fluorescence microscopy are powerful tools to study the organization and dynamics of DNA double-strand break repair foci and specific repair proteins in single cells. This requires specific induction of DNA double-strand breaks and fluorescent markers to follow the DNA lesions in living cells. In this review, where we focused on mammalian cell studies, we discuss different methods to induce DNA double-strand breaks, how to visualize and quantify repair foci in living cells., We describe different (live-cell) imaging modalities that can reveal details of the DNA double-strand break repair process across multiple time and spatial scales. In addition, recent developments are discussed in super-resolution imaging and single-molecule tracking, and how these technologies can be applied to elucidate details on structural compositions or dynamics of DNA double-strand break repair.</p

Slc2a10 knock-out mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicated GLUT10 in the transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations did not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in mouse. Gulo;Slc2a10 double knock-out mice showed mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 double knock-out mice did not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. Transforming growth factor beta signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 double knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined

VE-cadherin and claudin-5: it takes two to tango

Endothelial barrier function requires the adhesive activity of VE-cadherin and claudin-5, which are key components of adherens and tight endothelial junctions, respectively. Emerging evidence suggests that VE-cadherin controls claudin-5 expression by preventing the nuclear accumulation of FoxO1 and -catenin, which repress the claudin-5 promoter. This indicates that a crosstalk mechanism operates between these junctional structures

Absence of cardiovascular manifestations in a haploinsufficient TGFBR1 mouse model

Loeys-Dietz syndrome (LDS) is an autosomal dominant arterial aneurysm disease belonging to the spectrum of transforming growth factor beta (TGF beta)-associated vasculopathies. In its most typical form it is characterized by the presence of hypertelorism, bifid uvula/cleft palate and aortic aneurysm and/or arterial tortuosity. LDS is caused by heterozygous loss of function mutations in the genes encoding TGF beta receptor 1 and 2 (TGFBR1 and -2), which lead to a paradoxical increase in TGF beta signaling. To address this apparent paradox and to gain more insight into the pathophysiology of aneurysmal disease, we characterized a new Tgfbr1 mouse model carrying a p.Y378* nonsense mutation. Study of the natural history in this model showed that homozygous mutant mice die during embryonic development due to defective vascularization. Heterozygous mutant mice aged 6 and 12 months were morphologically and (immuno) histochemically indistinguishable from wild-type mice. We show that the mutant allele is degraded by nonsense mediated mRNA decay, expected to result in haploinsufficiency of the mutant allele. Since this haploinsufficiency model does not result in cardiovascular malformations, it does not allow further study of the process of aneurysm formation. In addition to providing a comprehensive method for cardiovascular phenotyping in mice, the results of this study confirm that haploinsuffciency is not the underlying genetic mechanism in human LDS

Musical Women and Identity-Building in Early Independent Mexico (1821-1854)

This thesis investigates music in Mexico City, with an emphasis on women's relationship to Romanticism, education, consumption, domestic music-making and public performance. During the first decades after independence in 1821, Mexicans began the process of constructing an identity, which musically speaking meant an expansion of the secular musical world. Such construction involved the development of internal activity alongside a conditional receptivity to external influence in the form of the visits of Italian opera companies such as those of Manuel García and Max Maretzek, and travelling virtuosi such as pianist virtuoso Henri Herz, who brought new repertoire and performance practices to Mexican theatres and homes. As consumers and as musicians, women were at the centre of such developments. In Mexico, both European music and that of local musicians was disseminated by means of ladies’ journals and imported and locally-printed sheet music by foreign and Mexican composers, in order to supply a growing home market for amateurs. Abundant surviving repertoire for the home, the widespread availability of musical instruction as revealed through advertisements, and witness accounts of soirées and concerts in the theatre reveal a budding musical world that has hitherto been overlooked and which occurred during a period generally deemed of little importance in Mexican musical history. By investigating a key period in the social-cultural history of Mexican music, this thesis crafts a narrative of intersections between the musical life of Mexican women and the incipient construction of a musical-cultural identity

Perinatal Exogenous Nitric Oxide in Fawn-Hooded Hypertensive Rats Reduces Renal Ribosomal Biogenesis in Early Life

Nitric oxide (NO) is known to depress ribosome biogenesis in vitro. In this study we analyzed the influence of exogenous NO on ribosome biogenesis in vivo using a proven antihypertensive model of perinatal NO administration in genetically hypertensive rats. Fawn-hooded hypertensive rat (FHH) dams were supplied with the NO-donor molsidomine in drinking water from 2 weeks before to 4 weeks after birth, and the kidneys were subsequently collected from 2 day, 2 week, and 9 to 10-month-old adult offspring. Although the NO-donor increased maternal NO metabolite excretion, the NO status of juvenile renal (and liver) tissue was unchanged as assayed by EPR spectroscopy of NO trapped with iron-dithiocarbamate complexes. Nevertheless, microarray analysis revealed marked differential up-regulation of renal ribosomal protein genes at 2 days and down-regulation at 2 weeks and in adult males. Such differential regulation of renal ribosomal protein genes was not observed in females. These changes were confirmed in males at 2 weeks by expression analysis of renal ribosomal protein L36a and by polysome profiling, which also revealed a down-regulation of ribosomes in females at that age. However, renal polysome profiles returned to normal in adults after early exposure to molsidomine. No direct effects of molsidomine were observed on cellular proliferation in kidneys at any age, and the changes induced by molsidomine in renal polysome profiles at 2 weeks were absent in the livers of the same rats. Our results suggest that the previously found prolonged antihypertensive effects of perinatal NO administration may be due to epigenetically programmed alterations in renal ribosome biogenesis during a critical fetal period of renal development, and provide a salient example of a drug-induced reduction of ribosome biogenesis that is accompanied by a beneficial long-term health effect in both males and females

Comparison of high-and low-let radiation-induced dna double-strand break processing in living cells

High-linear-energy-transfer (LET) radiation is more lethal than similar doses of low-LET radiation types, probably a result of the condensed energy deposition pattern of high-LET radiation. Here, we compare high-LET α-particle to low-LET X-ray irradiation and monitor double-strand break (DSB) processing. Live-cell microscopy was used to monitor DNA double-strand breaks (DSBs), marked by p53-binding protein 1 (53BP1). In addition, the accumulation of the endogenous 53BP1 and replication protein A (RPA) DSB processing proteins was analyzed by immunofluorescence. In contrast to α-particle-induced 53BP1 foci, X-ray-induced foci were resolved quickly and more dynamically as they showed an increase in 53BP1 protein accumulation and size. In addition, the number of individual 53BP1 and RPA foci was higher after X-ray irradiation, while focus intensity was higher after α-particle irradiation. Interestingly, 53BP1 foci induced by α-particles contained multiple RPA foci, suggesting multiple individual resection events, which was not observed after X-ray irradiation. We conclude that high-LET α-particles cause closely interspaced DSBs leading to high local concentrations of repair proteins. Our results point toward a change in DNA damage processing toward DNA end-resection and homologous recombination, possibly due to the depletion of soluble protein in the nucleoplasm. The combination of closely interspaced DSBs and perturbed DNA damage processing could be an explanation for the increased relative biological effectiveness (RBE) of high-LET α-particles compared to X-ray irradiation

Surface plasmon modes of a single silver nanorod: an electron energy loss study

We present an electron energy loss study using energy filtered TEM of spatially resolved surface plasmon excitations on a silver nanorod of aspect ratio 14.2 resting on a 30 nm thick silicon nitride membrane. Our results show that the excitation is quantized as resonant modes whose intensity maxima vary along the nanorod's length and whose wavelength becomes compressed towards the ends of the nanorod. Theoretical calculations modelling the surface plasmon response of the silver nanorod-silicon nitride system show the importance of including retardation and substrate effects in order to describe accurately the energy dispersion of the resonant modes.Comment: 9 pages, 6 figure

Individualized early death and long-term survival prediction after stereotactic radiosurgery for brain metastases of non-small cell lung cancer:Two externally validated nomograms

Introduction Commonly used clinical models for survival prediction after stereotactic radiosurgery (SRS) for brain metastases (BMs) are limited by the lack of individual risk scores and disproportionate prognostic groups. In this study, two nomograms were developed to overcome these limitations. Methods 495 patients with BMs of NSCLC treated with SRS for a limited number of BMs in four Dutch radiation oncology centers were identified and divided in a training cohort (n = 214, patients treated in one hospital) and an external validation cohort n = 281, patients treated in three other hospitals). Using the training cohort, nomograms were developed for prediction of early death (<3 months) and long-term survival (>12 months) with prognostic factors for survival. Accuracy of prediction was defined as the area under the curve (AUC) by receiver operating characteristics analysis for prediction of early death and long term survival. The accuracy of the nomograms was also tested in the external validation cohort. Results Prognostic factors for survival were: WHO performance status, presence of extracranial metastases, age, GTV largest BM, and gender. Number of brain metastases and primary tumor control were not prognostic factors for survival. In the external validation cohort, the nomogram predicted early death statistically significantly better (p < 0.05) than the unfavorable groups of the RPA, DS-GPA, GGS, SIR, and Rades 2015 (AUC = 0.70 versus range AUCs = 0.51–0.60 respectively). With an AUC of 0.67, the other nomogram predicted 1 year survival statistically significantly better (p < 0.05) than the favorable groups of four models (range AUCs = 0.57–0.61), except for the SIR (AUC = 0.64, p = 0.34). The models are available on www.predictcancer.org. Conclusion The nomograms predicted early death and long-term survival more accurately than commonly used prognostic scores after SRS for a limited number of BMs of NSCLC. Moreover these nomograms enable individualized probability assessment and are easy into use in routine clinical practice