DNA replication stress restricts ribosomal DNA copy number

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number

Rif1 maintains telomeres and mediates DNA repair by encasing DNA ends

In yeast, Rif1 is part of the telosome, where it inhibits telomerase and checkpoint signaling at chromosome ends. In mammalian cells, Rif1 is not telomeric, but it suppresses DNA end resection at chromosomal breaks, promoting repair by nonhomologous end joining (NHEJ). Here, we describe crystal structures for the uncharacterized and conserved ∼125-kDa N-terminal domain of Rif1 from Saccharomyces cerevisiae (Rif1-NTD), revealing an α-helical fold shaped like a shepherd's crook. We identify a high-affinity DNA-binding site in the Rif1-NTD that fully encases DNA as a head-to-tail dimer. Engagement of the Rif1-NTD with telomeres proved essential for checkpoint control and telomere length regulation. Unexpectedly, Rif1-NTD also promoted NHEJ at DNA breaks in yeast, revealing a conserved role of Rif1 in DNA repair. We propose that tight associations between the Rif1-NTD and DNA gate access of processing factors to DNA ends, enabling Rif1 to mediate diverse telomere maintenance and DNA repair functions

Arrival time correction for dynamic susceptibility contrast MR permeability imaging in stroke patients.

To determine if applying an arrival time correction (ATC) to dynamic susceptibility contrast (DSC) based permeability imaging will improve its ability to identify contrast leakage in stroke patients for whom the shape of the measured curve may be very different due to hypoperfusion.A technique described in brain tumor patients was adapted to incorporate a correction for delayed contrast delivery due to perfusion deficits. This technique was applied to the MRIs of 9 stroke patients known to have blood-brain barrier (BBB) disruption on T1 post contrast imaging. Regions of BBB damage were compared with normal tissue from the contralateral hemisphere. Receiver operating characteristic (ROC) analysis was performed to compare the detection of BBB damage before and after ATC.ATC improved the area under the curve (AUC) of the ROC from 0.53 to 0.70. The sensitivity improved from 0.51 to 0.67 and the specificity improved from 0.57 to 0.66. Visual inspection of the ROC curve revealed that the performance of the uncorrected analysis was worse than random guess at some thresholds.The ability of DSC permeability imaging to identify contrast enhancing tissue in stroke patients improved considerably when an ATC was applied. Using DSC permeability imaging in stroke patients without an ATC may lead to false identification of BBB disruption

Immunomodulating activities of a natural α1 -acid glycoprotein and its carbohydrate chains attached to the Protein-Free Polymer

Immunomodulating effects of a neoglycoconjugate created on the basis of α1 -acid glycoprotein (AGP) carbohydrate chains and synthetic protein-free carrier have been investigated. It was demonstrated that this pseudo-AGP suppressed PHA- or anti-CD3 antibody-induced lymphocyte proliferation in a dosedependent manner. Pseudo-AGP revealed a similar antiproliferative effect as the natural AGP samples. Stimulation of the LPS-induced proinflammatory cytokine production by m ononuclear cells treated with both natural and pseudo-AGP has been also demonstrated. These data show that carbohydrate chains of AGP play a crucial role in the studied biological effect realization

The top row of images show a stroke on DWI/ADC which has some enhancement on post contrast T1 imaging.

<p>The bottom row shows the perfusion deficit on a TTP map, the permeability image when not corrected for arrival time, and the permeability image after arrival time correction. The green circles show corresponding areas of contrast leakage on the T1 post contrast and ATC permeability images. (DWI = diffusion weighted image, ADC = apparent diffusion coefficient, PWI = perfusion weighted image, TTP = time to peak, ATC = arrival time corrected).</p

The receiver-operator characteristic (ROC) curves, which demonstrate the ability to correctly identify enhancing tissue, are plotted before and after arrival time correction (ATC).

<p>Prior to correction the technique performs worse than random chance at some thresholds due to perfusion deficits.</p

The two graphs show the ΔR2* for non-enhancing (control) and enhancing hypoperfused regions before and after arrival time correction (ATC).

<p>In the first graph, due to even a small delay in time-to-peak, the control signal appears to approach baseline faster thus obscuring the phenomenon being measured. However, in the second graph, after the ATC has been applied to the control, it becomes evident that the enhancing region signal is approaching the baseline faster due to the T1 effect of contrast accumulation in the parenchyma.</p