Deletion of \u3cem\u3eULS1\u3c/em\u3e Confers Damage Tolerance in \u3c/em\u3esgs1\u3c/em\u3e Mutants Through a Top3-dependent D-loop Mediated Fork Restart Pathway

Homologous recombination (HR)-based repair during DNA replication can apparently utilize several partially overlapping repair pathways in response to any given lesion. A key player in HR repair is the Sgs1-Top3-Rmi1 (STR) complex, which is critical for resolving X-shaped recombination intermediates formed following bypass of methyl methanesulfonate (MMS)-induced damage. STR mutants are also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU), but unlike MMS treatment, HU treatment is not accompanied by X-structure accumulation, and it is thus unclear how STR functions in this context. Here we provide evidence that HU-induced fork stalling enlists Top3 prior to recombination intermediate formation. The resistance of sgs1Δ mutants to HU is enhanced by the absence of the putative SUMO (Small Ubiquitin MOdifier)-targeted ubiquitin ligase, Uls1, and we demonstrate that Top3 is required for this enhanced resistance and for coordinated breaks and subsequent d-loop formation at forks stalled at the ribosomal DNA (rDNA) replication fork block (RFB). We also find that HU resistance depends on the catalytic activity of the E3 SUMO ligase, Mms21, and includes a rapid Rad51-dependent restart mechanism that is different from the slow Rad51-independent HR fork restart mechanism operative in sgs1Δ ULS1+ mutants. These data support a model in which repair of HU-induced damage in sgs1Δ mutants involves an error-prone break-induced replication pathway but, in the absence of Uls1, shifts to one that is higher-fidelity and involves the formation of Rad51-dependent d-loops

Radiation Shielding of Lunar Regolith/Polyethylene Composites and Lunar Regolith/Water Mixtures

Space radiation is a complex mixed field of ionizing radiation that can pose hazardous risks to sophisticated electronics and humans. Mission planning for lunar exploration and long duration habitat construction will face tremendous challenges of shielding against various types of space radiation in an attempt to minimize the detrimental effects it may have on materials, electronics, and humans. In late 2009, the Lunar Crater Observation and Sensing Satellite (LCROSS) discovered that water content in lunar regolith found in certain areas on the moon can be up to 5.6 +/-2.8 weight percent (wt%) [A. Colaprete, et. al., Science, Vol. 330, 463 (2010). ]. In this work, shielding studies were performed utilizing ultra high molecular weight polyethylene (UHMWPE) and aluminum, both being standard space shielding materials, simulated lunar regolith/ polyethylene composites, and simulated lunar regolith mixed with UHMWPE particles and water. Based on the LCROSS findings, radiation shielding experiments were conducted to test for shielding efficiency of regolith/UHMWPE/water mixtures with various percentages of water to compare relative shielding characteristics of these materials. One set of radiation studies were performed using the proton synchrotron at the Loma Linda Medical University where high energy protons similar to those found on the surface of the moon can be generated. A similar experimental protocol was also used at a high energy spalation neutron source at Los Alamos Neutron Science Center (LANSCE). These experiments studied the shielding efficiency against secondary neutrons, another major component of space radiation field. In both the proton and neutron studies, shielding efficiency was determined by utilizing a tissue equivalent proportional counter (TEPC) behind various thicknesses of shielding composite panels or mixture materials. Preliminary results from these studies indicated that adding 2 wt% water to regolith particles could increase shielding of the regolith materials by about 6%. The findings may be utilized to extend the possibilities of potential candidate materials for lunar habitat structures, will potentially impact the design criteria of future human bases on the moon, and provide some guidelines for future space mission planning with respect to radiation exposure and risks posed on astronauts

Resolution by Unassisted Top3 Points to Template Switch Recombination Intermediates during DNA Replication

The evolutionarily conserved Sgs1/Top3/Rmi1 (STR) complex plays vital roles in DNA replication and repair. One crucial activity of the complex is dissolution of toxic X-shaped recombination intermediates that accumulate during replication of damaged DNA. However, despite several years of study the nature of these X-shaped molecules remains debated. Here we use genetic approaches and two-dimensional gel electrophoresis of genomic DNA to show that Top3, unassisted by Sgs1 and Rmi1, has modest capacities to provide resistance to MMS and to resolve recombination-dependent X-shaped molecules. The X-shaped molecules have structural properties consistent with hemicatenane-related template switch recombination intermediates (Rec-Xs) but not Holliday junction (HJ) intermediates. Consistent with these findings, we demonstrate that purified Top3 can resolve a synthetic Rec-X but not a synthetic double HJ in vitro. We also find that unassisted Top3 does not affect crossing over during double strand break repair, which is known to involve double HJ intermediates, confirming that unassisted Top3 activities are restricted to substrates that are distinct from HJs. These data help illuminate the nature of the X-shaped molecules that accumulate during replication of damaged DNA templates, and also clarify the roles played by Top3 and the STR complex as a whole during the resolution of replication-associated recombination intermediates

A Multiwavelength Classification and Study of Red Supergiant Candidates in NGC 6946

We have combined resolved stellar photometry from Hubble Space Telescope (\emph{HST}), \emph{Spitzer}, and \emph{Gaia} to identify red supergiant (RSG) candidates in NGC~6946, based on their colors, proper motions, visual morphologies, and spectral energy distributions. We start with a large sample of 17,865 RSG candidates based solely on \emph{HST} near-infrared photometry. We then chose a small sample of 385 of these candidates with Spitzer matches for more detailed study. Using evolutionary models and isochrones, we isolate a space where RSGs would be found in our photometry catalogs. We then visually inspect each candidate and compare to Gaia catalogs to identify and remove foreground stars. As a result, we classify 95 potential RSGs, with 40 of these being in our highest-quality sample. We fit the photometry of the populations of stars in the regions surrounding the RSGs to infer their ages. Placing our best candidate RSG stars into three age bins between 1 and 30 Myr, we find 27.5\% of the candidates falling between 1-10 Myr, 37.5\% between 10-20 Myr, and 35\% 20-30 Myr. A comparison of our results to the models of massive star evolution shows some agreement between model luminosities and the luminosities of our candidates for each age. Three of our candidates appear significantly more consistent with binary models than single-star evolution models.Comment: 32 pages, 18 figures, 4 table

Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci

<p>Abstract</p> <p>Background</p> <p>Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF), that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts.</p> <p>Results</p> <p>We show that mouse embryo fibroblasts (MEFs) and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells.</p> <p>Conclusions</p> <p>In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types) to become immortalized and transformed, compared to human cells.</p

Cdc13 OB2 Dimerization Required for Productive Stn1 Binding and Efficient Telomere Maintenance

SummaryCdc13 is an essential yeast protein required for telomere length regulation and genome stability. It does so via its telomere-capping properties and by regulating telomerase access to the telomeres. The crystal structure of the Saccharomyces cerevisiae Cdc13 domain located between the recruitment and DNA binding domains reveals an oligonucleotide-oligosaccharide binding fold (OB2) with unusually long loops extending from the core of the protein. These loops are involved in extensive interactions between two Cdc13 OB2 folds leading to stable homodimerization. Interestingly, the functionally impaired cdc13-1 mutation inhibits OB2 dimerization. Biochemical assays indicate OB2 is not involved in telomeric DNA or Stn1 binding. However, disruption of the OB2 dimer in full-length Cdc13 affects Cdc13-Stn1 association, leading to telomere length deregulation, increased temperature sensitivity, and Stn1 binding defects. We therefore propose that dimerization of the OB2 domain of Cdc13 is required for proper Cdc13, Stn1, Ten1 (CST) assembly and productive telomere capping

The relative importance of graft surveillance and warfarin therapy in infrainguinal prosthetic bypass failure

BackgroundWe sought to describe modes of failure and associated limb loss after infrainguinal polytetrafluoroethylene bypass grafting in patients lacking a saphenous venous conduit and to define specific clinical or hemodynamic factors prognostic for bypass failure.MethodsWe identified 121 patients (mean age, 67 years; 90 men and 31 women) with determinable outcomes (minimum follow-up, 2 months; mean, 17 months) after 130 prosthetic infrainguinal bypasses between 1997 and 2005. Ischemic presentation was rest pain in 52%, tissue loss in 34%, and disabling claudication and/or popliteal aneurysm in 14%, with 24% of patients requiring a redo bypass. Distal targets were the above-knee (n = 44), distal popliteal (n = 27), or tibial/pedal (n = 59) arteries. Sixty-six (77%) of the below-knee (BK) target (distal popliteal or tibial) bypasses had distal anastomotic adjuncts (vein cuff or patch). Duplex graft surveillance was performed at 1, 4, and 7 months after surgery and twice yearly thereafter, with recording of midgraft velocities and imaging encompassing inflow and outflow vessels. Arteriography and open/endovascular intervention was performed for stenoses identified by duplex scanning (peak systolic velocity >300 cm/s; velocity ratio >3.5). An attempt was made to salvage occluded grafts by using catheter-directed thrombolysis or open techniques. Eighty-six patients (74% of BK bypasses) were placed on chronic warfarin therapy with a target international normalized ratio range between 2 and 3. Prognostic factors were identified by using univariate statistics and multivariate logistic regression analysis.ResultsThree-year primary, assisted, and secondary patency rates were 39%, 43%, and 59%, respectively, for all bypasses, with no difference noted between above-knee and BK grafts (P = .5). At 3 years, freedom from limb loss was 75%, and patient survival was only 70%, with no adverse effect on survival imparted by amputation. Sixty-nine total adverse events occurred as a result of thrombotic occlusion (n = 51), duplex scan–detected stenosis (n = 13), or graft infection (n = 5). Forty-nine percent of all initial graft occlusions eventually led to amputation. Twenty-three grafts (27% of 86 patients) in patients maintained on chronic warfarin were subtherapeutic at the time of occlusion. Use of a distal anastomotic adjunct with BK bypasses reduced graft thrombosis (35% with vs 60% without) but did not impart a significant patency advantage (P = .07). Multivariate analysis revealed low graft flow (midgraft velocity ≤45 cm/s; odds ratio [OR], 6.1; 95% confidence interval [CI], 1.9-19.2), use of warfarin (OR, 8.4; 95% CI, 2.1-34.5), and therapeutic warfarin (OR, 24.6; 95% CI, 5.7-106) to be independently predictive for bypass patency. Graft patency was maintained in 89% of grafts remaining therapeutic on warfarin compared with only 55% of subtherapeutic or nonanticoagulated grafts (P < .001). Low-flow grafts (n = 61) occluded more frequently than higher-flow grafts (46% vs 13%; P < .001). Therapeutic warfarin augmented the patency of low-flow (P < .001) but not high-flow (P = .15) grafts.ConclusionsLow graft flow was a more common mode of prosthetic bypass failure than development of duplex scan–detected stenotic lesions during follow-up. Early duplex scanning may be more important for characterizing midgraft velocity and related thrombotic potential and selecting patients for chronic anticoagulation. Maintenance of therapeutic warfarin is paramount in optimizing prosthetic bypass patency and limb preservation

Outcome of carotid stent-assisted angioplasty versus open surgical repair of recurrent carotid stenosis

AbstractPurposeWe compared outcome and durability of carotid stent-assisted angioplasty (CAS) with open surgical repair (ie, repeat carotid endarterectomy [CEA]) to treat recurrent carotid stenosis (RCS).MethodsA retrospective review of anatomic and neurologic outcomes was carried out after 27 repeat CEA procedures (1993-2002) and 52 CAS procedures (1997-2002) performed to treat high-grade internal carotid artery (ICA) RCS after CEA. The incidence of intervention because of symptomatic RCS was similar (repeat CEA, 63%; CAS, 60%), but the interval from primary CEA to repeat intervention was greater (P < .05) in the repeat CEA group (83 ± 15 months) compared with the CAS group (50 ± 8 months). In the CAS group, 17 of 52 arteries (33%) were judged not to be surgical candidates because of surgically inaccessible high lesions (n = 8), medical comorbid conditions (n = 4), neck irradiation (n = 3), or previous surgery with cranial nerve deficit or stroke (n = 2). Three patients who underwent repeat CEA had lesions not appropriate for treatment with CAS.ResultsOverall 30-day morbidity was similar after CAS (12%; death due to ipsilateral intracranial hemorrhage, 1; nondisabling stroke, 1; reversible neurologic deficits or transient ischemic attack, 2; access site complication, 2) and repeat CEA (11%; no death; nondisabling stroke, 1; reversible cranial nerve injury, 1; cervical hematoma, 1). Combined stroke and death rate was 3.7% for repeat CEA and 5.7% for CAS (P > .1). All duplex ultrasound scans obtained within 3 months after CEA and CAS demonstrated patent ICA and velocity spectra of less than 50% stenosis. During follow-up, no repeat CEA (mean, 39 months) or CAS (mean, 26 months) repair demonstrated ICA occlusion, but two patients (8%) who underwent repeat CEA and 4 patients (8%) who underwent CAS required balloon or stent angioplasty because of 80% RCS. At last follow-up, no patient had ipsilateral stroke and all ICA remain patent. At duplex scanning, stenosis-free (<50% diameter reduction) ICA patency at 36 months was 75% after repeat CEA and 57% after CAS (P = .26, log-rank test).ConclusionsCarotid angioplasty for treatment of high-grade stenotic ICA after CEA resulted in similar anatomic and neurologic outcomes compared with open surgical repair. Most lesions are amenable to endovascular therapy, and CAS enabled treatment in patients judged not to be suitable surgical candidates. Duplex scanning surveillance after repeat CEA or CAS is recommended, because stenosis can recur after either secondary procedure

Plaque excision with the Silverhawk catheter: Early results in patients with claudication or critical limb ischemia

ObjectiveThis study was conducted to detail the early experience after infrainguinal atherectomy using the Silverhawk plaque excision catheter for the treatment of symptomatic peripheral vascular disease.MethodsA prospective database was established in August 2004 in which data for operations, outcomes, and follow-up were recorded for patients undergoing percutaneous plaque excision for peripheral arterial occlusive disease. Society for Vascular Surgery (SVS) ischemia scores and femoropopliteal TransAtlantic Inter-Society Consensus (TASC) criteria were assigned. A follow-up protocol included duplex ultrasound surveillance at 1, 3, and 6 months and then yearly thereafter. Standard statistical analyses were performed.ResultsDuring a 17-month period, 66 limbs of 60 patients (37 men [61.7%]) underwent 70 plaque excisions (four repeat procedures). Indications included tissue loss based on SVS ischemia at grades 5 and 6 (25/70), rest pain at grade 4 (22/70), and claudication at grades 2 to 3 (23/70). The mean lesion length was 8.8 ± 0.7 cm. The technical success rate was 87.1% (61/70). Adjunctive treatment was required in 17 procedures (24.3%), consisting of 14 balloon angioplasties and three stents. Femoropopliteal TASC criteria included 5 TASC A lesions, 14 TASC B lesions, 32 TASC C lesions, and 19 TASC D lesions. Although 17 plaque excisions included a tibial vessel, no patient underwent isolated tibial atherectomy. The mean increase in ankle-brachial index was 0.27 ± 0.04 and in toe pressure, 20.3 ± 6.9 mm Hg. Mean duplex ultrasound follow-up was 5.2 months (range, 1 to 17 months). One-year primary, primary assisted, and secondary patency was 61.7%, 64.1%, and 76.4%, respectively. Restenosis or occlusion developed in 12 patients (16.7%) and was detected at a mean of 2.8 ± 0.7 months. Restenosis or occlusion was significantly more common (P < .05) in patients with TASC C and D lesions compared with patients with TASC A and B lesions. Six (8.3%) of 12 patients underwent reintervention on the basis of duplex ultrasound surveillance results. Four (33.3%) of 12 patients experienced reocclusion during the same hospitalization, and amputation and open revascularization were required in two patients each.ConclusionsPercutaneous plaque excision is a viable treatment option for lower extremity revascularization. Outcomes are related to ischemia and lesion severity. Patency and limb salvage rates are equivalent to other endovascular modalities