In vivo laser Doppler holography of the human retina

The eye offers a unique opportunity for non-invasive exploration of cardiovascular diseases. Optical angiography in the retina requires sensitive measurements, which hinders conventional full-field laser Doppler imaging schemes. To overcome this limitation, we used digital holography to perform laser Doppler perfusion imaging of the human retina in vivo with near-infrared light. Wideband measurements of the beat frequency spectrum of optical interferograms recorded with a 39 kHz CMOS camera are analyzed by short-time Fourier transformation. Power Doppler images and movies drawn from the zeroth moment of the power spectrum density reveal blood flows in retinal and choroidal vessels over 512 $\times$ 512 pixels covering 2.4 $\times$ 2.4 mm$^2$ on the retina with a 13 ms temporal resolution.Comment: 5 pages, 5 figure

Pulsatile microvascular blood flow imaging by short-time Fourier transform analysis of ultrafast laser holographic interferometry

We report on wide-field imaging of pulsatile microvascular blood flow in the exposed cerebral cortex of a mouse by holographic interferometry. We recorded interferograms of laser light backscattered by the tissue, beating against an off-axis reference beam with a 50 kHz framerate camera. Videos of local Doppler contrasts were rendered numerically by Fresnel transformation and short-time Fourier transform analysis. This approach enabled instantaneous imaging of pulsatile blood flow contrasts in superficial blood vessels over 256 x 256 pixels with a spatial resolution of 10 microns and a temporal resolution of 20 ms.Comment: 4 page

Aneuploidy Drives Genomic Instability in Yeast

Aneuploidy decreases cellular fitness, yet it is also associated with cancer, a disease of enhanced proliferative capacity. To investigate one mechanism by which aneuploidy could contribute to tumorigenesis, we examined the effects of aneuploidy on genomic stability. We analyzed 13 budding yeast strains that carry extra copies of single chromosomes and found that all aneuploid strains exhibited one or more forms of genomic instability. Most strains displayed increased chromosome loss and mitotic recombination, as well as defective DNA damage repair. Aneuploid fission yeast strains also exhibited defects in mitotic recombination. Aneuploidy-induced genomic instability could facilitate the development of genetic alterations that drive malignant growth in cancer

Теоремы сходимости и компактности для уравнений Бельтрами

Доведено ряд теорем збіжності та компактності класів регулярних розв'язків вироджених рівнянь Бельтрамі з обмеженнями інтегрального типу на дилатацію.A number of convergence and compactness theorems for classes of regular solutions of the degenerate Beltrami equations with restrictions of the integral type on a dilatation is proved

Funduscopy in Adult Zebrafish and Its Application to Isolate Mutant Strains with Ocular Defects

Funduscopy is one of the most commonly used diagnostic tools in the ophthalmic practice, allowing for a ready assessment of pathological changes in the retinal vasculature and the outer retina. This non-invasive technique has so far been rarely used in animal model for ophthalmic diseases, albeit its potential as a screening assay in genetic screens. The zebrafish (Danio rerio) is well suited for such genetic screens for ocular alterations. Therefore we developed funduscopy in adult zebrafish and employed it as a screening tool to find alterations in the anterior segment and the fundus of the eye of genetically modified adult animals. A stereomicroscope with coaxial reflected light illumination was used to obtain fundus color images of the zebrafish. In order to find lens and retinal alterations, a pilot screen of 299 families of the F3 generation of ENU-treated adult zebrafish was carried out. Images of the fundus of the eye and the anterior segment can be rapidly obtained and be used to identify alterations in genetically modified animals. A number of putative mutants with cataracts, defects in the cornea, eye pigmentation, ocular vessels and retina were identified. This easily implemented method can also be used to obtain fundus images from rodent retinas. In summary, we present funduscopy as a valuable tool to analyse ocular abnormalities in adult zebrafish and other small animal models. A proof of principle screen identified a number of putative mutants, making funduscopy based screens in zebrafish feasible

DNA Resection at Chromosome Breaks Promotes Genome Stability by Constraining Non-Allelic Homologous Recombination

DNA double-strand breaks impact genome stability by triggering many of the large-scale genome rearrangements associated with evolution and cancer. One of the first steps in repairing this damage is 5′→3′ resection beginning at the break site. Recently, tools have become available to study the consequences of not extensively resecting double-strand breaks. Here we examine the role of Sgs1- and Exo1-dependent resection on genome stability using a non-selective assay that we previously developed using diploid yeast. We find that Saccharomyces cerevisiae lacking Sgs1 and Exo1 retains a very efficient repair process that is highly mutagenic to genome structure. Specifically, 51% of cells lacking Sgs1 and Exo1 repair a double-strand break using repetitive sequences 12–48 kb distal from the initial break site, thereby generating a genome rearrangement. These Sgs1- and Exo1-independent rearrangements depend partially upon a Rad51-mediated homologous recombination pathway. Furthermore, without resection a robust cell cycle arrest is not activated, allowing a cell with a single double-strand break to divide before repair, potentially yielding multiple progeny each with a different rearrangement. This profusion of rearranged genomes suggests that cells tolerate any dangers associated with extensive resection to inhibit mutagenic pathways such as break-distal recombination. The activation of break-distal recipient repeats and amplification of broken chromosomes when resection is limited raise the possibility that genome regions that are difficult to resect may be hotspots for rearrangements. These results may also explain why mutations in resection machinery are associated with cancer

The Two Different Isoforms of the RSC Chromatin Remodeling Complex Play Distinct Roles in DNA Damage Responses

The RSC chromatin remodeling complex has been implicated in contributing to DNA double-strand break (DSB) repair in a number of studies. Both survival and levels of H2A phosphorylation in response to damage are reduced in the absence of RSC. Importantly, there is evidence for two isoforms of this complex, defined by the presence of either Rsc1 or Rsc2. Here, we investigated whether the two isoforms of RSC provide distinct contributions to DNA damage responses. First, we established that the two isoforms of RSC differ in the presence of Rsc1 or Rsc2 but otherwise have the same subunit composition. We found that both rsc1 and rsc2 mutant strains have intact DNA damage-induced checkpoint activity and transcriptional induction. In addition, both strains show reduced non-homologous end joining activity and have a similar spectrum of DSB repair junctions, suggesting perhaps that the two complexes provide the same functions. However, the hypersensitivity of a rsc1 strain cannot be complemented with an extra copy of RSC2, and likewise, the hypersensitivity of the rsc2 strain remains unchanged when an additional copy of RSC1 is present, indicating that the two proteins are unable to functionally compensate for one another in DNA damage responses. Rsc1, but not Rsc2, is required for nucleosome sliding flanking a DNA DSB. Interestingly, while swapping the domains from Rsc1 into the Rsc2 protein does not compromise hypersensitivity to DNA damage suggesting they are functionally interchangeable, the BAH domain from Rsc1 confers upon Rsc2 the ability to remodel chromatin at a DNA break. These data demonstrate that, despite the similarity between Rsc1 and Rsc2, the two different isoforms of RSC provide distinct functions in DNA damage responses, and that at least part of the functional specificity is dictated by the BAH domains

A Simple Model for the Influence of Meiotic Conversion Tracts on GC Content

A strong correlation between GC content and recombination rate is observed in many eukaryotes, which is thought to be due to conversion events linked to the repair of meiotic double-strand breaks. In several organisms, the length of conversion tracts has been shown to decrease exponentially with increasing distance from the sites of meiotic double-strand breaks. I show here that this behavior leads to a simple analytical model for the evolution and the equilibrium state of the GC content of sequences devoid of meiotic double-strand break sites. In the yeast Saccharomyces cerevisiae, meiotic double-strand breaks are practically excluded from protein-coding sequences. A good fit was observed between the predictions of the model and the variations of the average GC content of the third codon position (GC3) of S. cerevisiae genes. Moreover, recombination parameters that can be extracted by fitting the data to the model coincide with experimentally determined values. These results thus indicate that meiotic recombination plays an important part in determining the fluctuations of GC content in yeast coding sequences. The model also accounted for the different patterns of GC variations observed in the genes of Candida species that exhibit a variety of sexual lifestyles, and hence a wide range of meiotic recombination rates. Finally, the variations of the average GC3 content of human and chicken coding sequences could also be fitted by the model. These results suggest the existence of a widespread pattern of GC variation in eukaryotic genes due to meiotic recombination, which would imply the generality of two features of meiotic recombination: its association with GC-biased gene conversion and the quasi-exclusion of meiotic double-strand breaks from coding sequences. Moreover, the model points out to specific constraints on protein fragments encoded by exon terminal sequences, which are the most affected by the GC bias

Classification and evolutionary history of the single-strand annealing proteins, RecT, Redβ, ERF and RAD52

BACKGROUND: The DNA single-strand annealing proteins (SSAPs), such as RecT, Redβ, ERF and Rad52, function in RecA-dependent and RecA-independent DNA recombination pathways. Recently, they have been shown to form similar helical quaternary superstructures. However, despite the functional similarities between these diverse SSAPs, their actual evolutionary affinities are poorly understood. RESULTS: Using sensitive computational sequence analysis, we show that the RecT and Redβ proteins, along with several other bacterial proteins, form a distinct superfamily. The ERF and Rad52 families show no direct evolutionary relationship to these proteins and define novel superfamilies of their own. We identify several previously unknown members of each of these superfamilies and also report, for the first time, bacterial and viral homologs of Rad52. Additionally, we predict the presence of aberrant HhH modules in RAD52 that are likely to be involved in DNA-binding. Using the contextual information obtained from the analysis of gene neighborhoods, we provide evidence of the interaction of the bacterial members of each of these SSAP superfamilies with a similar set of DNA repair/recombination protein. These include different nucleases or Holliday junction resolvases, the ABC ATPase SbcC and the single-strand-binding protein. We also present evidence of independent assembly of some of the predicted operons encoding SSAPs and in situ displacement of functionally similar genes. CONCLUSIONS: There are three evolutionarily distinct superfamilies of SSAPs, namely the RecT/Redβ, ERF, and RAD52, that have different sequence conservation patterns and predicted folds. All these SSAPs appear to be primarily of bacteriophage origin and have been acquired by numerous phylogenetically distant cellular genomes. They generally occur in predicted operons encoding one or more of a set of conserved DNA recombination proteins that appear to be the principal functional partners of the SSAPs