DNA end resection by Dna2–Sgs1–RPA and its stimulation by Top3–Rmi1 and Mre11–Rad50–Xrs2

The repair of DNA double-strand breaks (DSBs) by homologous recombination requires processing of broken ends. For repair to start, the DSB must first be resected to generate a 3′-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad51 (ref. 1). Genetic studies have implicated a multitude of proteins in the process, including helicases, nucleases and topoisomerases. Here we biochemically reconstitute elements of the resection process and reveal that it requires the nuclease Dna2, the RecQ-family helicase Sgs1 and the ssDNA-binding protein replication protein-A (RPA). We establish that Dna2, Sgs1 and RPA constitute a minimal protein complex capable of DNA resection in vitro. Sgs1 helicase unwinds the DNA to produce an intermediate that is digested by Dna2, and RPA stimulates DNA unwinding by Sgs1 in a species-specific manner. Interestingly, RPA is also required both to direct Dna2 nucleolytic activity to the 5′-terminated strand of the DNA break and to inhibit 3′ to 5′ degradation by Dna2, actions that generate and protect the 3′-ssDNA overhang, respectively. In addition to this core machinery, we establish that both the topoisomerase 3 (Top3) and Rmi1 complex and the Mre11–Rad50–Xrs2 complex (MRX) have important roles as stimulatory components. Stimulation of end resection by the Top3–Rmi1 heterodimer and the MRX proteins is by complex formation with Sgs1 (refs 5, 6), which unexpectedly stimulates DNA unwinding. We suggest that Top3–Rmi1 and MRX are important for recruitment of the Sgs1–Dna2 complex to DSBs. Our experiments provide a mechanistic framework for understanding the initial steps of recombinational DNA repair in eukaryotes

Grey-matter texture abnormalities and reduced hippocampal volume are distinguishing features of schizophrenia

Neurodevelopmental processes are widely believed to underlie schizophrenia. Analysis of brain texture from conventional magnetic resonance imaging (MRI) can detect disturbance in brain cytoarchitecture. We tested the hypothesis that patients with schizophrenia manifest quantitative differences in brain texture that, alongside discrete volumetric changes, may serve as an endophenotypic biomarker. Texture analysis (TA) of grey matter distribution and voxel-based morphometry (VBM) of regional brain volumes were applied to MRI scans of 27 patients with schizophrenia and 24 controls. Texture parameters (uniformity and entropy) were also used as covariates in VBM analyses to test for correspondence with regional brain volume. Linear discriminant analysis tested if texture and volumetric data predicted diagnostic group membership (schizophrenia or control). We found that uniformity and entropy of grey matter differed significantly between individuals with schizophrenia and controls at the fine spatial scale (filter width below 2 mm). Within the schizophrenia group, these texture parameters correlated with volumes of the left hippocampus, right amygdala and cerebellum. The best predictor of diagnostic group membership was the combination of fine texture heterogeneity and left hippocampal size. This study highlights the presence of distributed grey-matter abnormalities in schizophrenia, and their relation to focal structural abnormality of the hippocampus. The conjunction of these features has potential as a neuroimaging endophenotype of schizophrenia

DNA resection in eukaryotes: deciding how to fix the break

DNA double-strand breaks are repaired by different mechanisms, including homologous recombination and nonhomologous end-joining. DNA-end resection, the first step in recombination, is a key step that contributes to the choice of DSB repair. Resection, an evolutionarily conserved process that generates single-stranded DNA, is linked to checkpoint activation and is critical for survival. Failure to regulate and execute this process results in defective recombination and can contribute to human disease. Here, I review recent findings on the mechanisms of resection in eukaryotes, from yeast to vertebrates, provide insights into the regulatory strategies that control it, and highlight the consequences of both its impairment and its deregulation

Safety and Efficacy of Adalimumab in Patients with Noninfectious Uveitis in an Ongoing Open-Label Study: VISUAL III

PURPOSE: To evaluate safety and efficacy of adalimumab in patients with noninfectious intermediate, posterior, or panuveitis. DESIGN: Phase 3, open-label, multicenter clinical trial extension (VISUAL III). PARTICIPANTS: Adults meeting treatment failure (TF) criteria or who completed VISUAL I or II (phase 3, randomized, double-masked, placebo-controlled) without TF. METHODS: Patients received adalimumab 40 mg every other week. Interim follow-up data were described from VISUAL III weeks 0 through 78. MAIN OUTCOME MEASURES: Disease quiescence, steroid-free quiescence, active inflammatory chorioretinal/retinal vascular lesions, anterior chamber cell grade, vitreous haze grade, best-corrected visual acuity (BCVA), and corticosteroid dose. Binary data were reported using nonresponder imputation (NRI), continuous data using last observation carried forward and as-observed analysis, and corticosteroid dose using observed-case analysis. Adverse events (AEs) were reported from first adalimumab dose in VISUAL III through interim cutoff. RESULTS: Of 424 patients enrolled, 371 were included in intent-to-treat analysis. At study entry, 242 of 371 (65%) patients had active uveitis; 60% (145/242, NRI) achieved quiescence at week 78, and 66% (95/143, as-observed) of those were corticosteroid free. At study entry, 129 of 371 (35%) patients had inactive uveitis; 74% (96/129, NRI) achieved quiescence at week 78, and 93% (89/96, as-observed) of those were corticosteroid free. Inflammatory lesions, anterior chamber grade, and vitreous haze grade showed initial improvement followed by decline in patients with active uveitis and remained stable in patients with inactive uveitis. BCVA improved in patients with active uveitis from weeks 0 to 78 (0.27 to 0.14 logMAR; left and right eyes; as-observed) and remained stable in patients with inactive uveitis. Mean corticosteroid dose decreased from 13.6 mg/day (week 0) to 2.6 mg/day (week 78) in patients with active uveitis and remained stable in those with inactive uveitis (1.5-1.2 mg/day). AEs (424 events/100 patient-years) and serious AEs (16.5 events/100 patient-years) were comparable with previous VISUAL trials. CONCLUSIONS: Patients with active uveitis at study entry who received adalimumab therapy were likely to achieve quiescence, improve visual acuity, and reduce their daily uveitis-related systemic corticosteroid use. Most patients with inactive uveitis at study entry sustained quiescence without a systemic corticosteroid dose increase. No new safety signals were identified

Implementation of 3 T Lactate-Edited 3D 1H MR Spectroscopic Imaging with Flyback Echo-Planar Readout for Gliomas Patients

The purpose of this study was to implement a new lactate-edited 3D 1H magnetic resonance spectroscopic imaging (MRSI) sequence at 3 T and demonstrate the feasibility of using this sequence for measuring lactate in patients with gliomas. A 3D PRESS MRSI sequence incorporating shortened, high bandwidth 180° pulses, new dual BASING lactate-editing pulses, high bandwidth very selective suppression (VSS) pulses and a flyback echo-planar readout was implemented at 3 T. Over-prescription factor of PRESS voxels was optimized using phantom to minimize chemical shift artifacts. The lactate-edited flyback sequence was compared with lactate-edited MRSI using conventional elliptical k-space sampling in a phantom and volunteers, and then applied to patients with gliomas. The results demonstrated the feasibility of detecting lactate within a short scan time of 9.5 min in both phantoms and patients. Over-prescription of voxels gave less chemical shift artifacts allowing detection of lactate on the majority of the selected volume. The normalized SNR of brain metabolites using the flyback encoding were comparable to the SNR of brain metabolites using conventional phase encoding MRSI. The specialized lactate-edited 3D MRSI sequence was able to detect lactate in brain tumor patients at 3 T. The implementation of this technique means that brain lactate can be evaluated in a routine clinical setting to study its potential as a marker for prognosis and response to therapy

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

Evidence that MEK1 positively promotes interhomologue double-strand break repair

During meiosis there is an imperative to create sufficient crossovers for homologue segregation. This can be achieved during repair of programmed DNA double-strand breaks (DSBs), which are biased towards using a homologue rather than sister chromatid as a repair template. Various proteins contribute to this bias, one of which is a meiosis specific kinase Mek1. It has been proposed that Mek1 establishes the bias by creating a barrier to sister chromatid repair, as distinct from enforcing strand invasion with the homologue. We looked for evidence that Mek1 positively stimulates strand invasion of the homologue. This was done by analysing repair of DSBs induced by the VMA1-derived endonuclease (VDE) and flanked by directly repeated sequences that can be used for intrachromatid single-strand annealing (SSA). SSA competes with interhomologue strand invasion significantly more successfully when Mek1 function is lost. We suggest the increase in intrachromosomal SSA reflects an opportunistic default repair pathway due to loss of a MEK1 stimulated bias for strand invasion of the homologous chromosome. Making use of an inhibitor sensitive mek1-as1 allele, we found that Mek1 function influences the repair pathway throughout the first4–5 h of meiosis. Perhaps reflecting a particular need to create bias for successful interhomologue events before chromosome pairing is complete

Treatment strategies in primary vitreoretinal lymphoma: a 17-center European collaborative study.

IMPORTANCE: The best treatment option for primary vitreoretinal lymphoma (PVRL) without signs of central nervous system lymphoma (CNSL) involvement determined on magnetic resonance imaging or in cerebrospinal fluid is unknown. OBJECTIVE: To evaluate the outcomes of treatment regimens used for PVRL in the prevention of subsequent CNSL. DESIGN, SETTING, AND PARTICIPANTS: A retrospective cohort study was conducted at 17 referral ophthalmologic centers in Europe. We reviewed clinical, laboratory, and imaging data on 78 patients with PVRL who did not have CNSL on presentation between January 1, 1991, and December 31, 2012, with a focus on the incidence of CNS manifestations during the follow-up period. INTERVENTIONS: The term extensive treatment was used for various combinations of systemic and intrathecal chemotherapy, whole-brain radiotherapy, and peripheral blood stem cell transplantation. Therapy to prevent CNSL included ocular radiotherapy and/or ocular chemotherapy (group A, 31 patients), extensive systemic treatment (group B, 21 patients), and a combination of ocular and extensive treatment (group C, 23 patients); 3 patients did not receive treatment. A total of 40 patients received systemic chemotherapy. MAIN OUTCOMES AND MEASURES: Development of CNSL following the diagnosis of PVRL relative to the use or nonuse of systemic chemotherapy and other treatment regimens. RESULTS: Overall, CNSL developed in 28 of 78 patients (36%) at a median follow-up of 49 months. Specifically, CNSL developed in 10 of 31 (32%) in group A, 9 of 21 (43%) in group B, and 9 of 23 (39%) in group C. The 5-year cumulative survival rate was lower in patients with CNSL (35% [95% CI, 50% to 86%]) than in patients without CNSL (68% [95% CI, 19% to 51%]; P = .003) and was similar among all treatment groups (P = .10). Adverse systemic effects occurred in 9 of 40 (23%) patients receiving systemic chemotherapy; the most common of these effects was acute renal failure. CONCLUSIONS AND RELEVANCE: In the present series of patients with isolated PVRL, the use of systemic chemotherapy was not proven to prevent CNSL and was associated with more severe adverse effects compared with local treatment

Clinical features and outcomes of patients with tubercular uveitis treated with antitubercular therapy in the collaborative ocular tuberculosis study (COTS)-1

IMPORTANCE Eradication of systemic tuberculosis (TB) has been limited by neglected populations and the HIV pandemic. Whereas ocular TB often presents as uveitis without any prior evidence of systemic TB, the existing uncertainty in the diagnosis of TB uveitis may perpetuate missed opportunities to address systemic TB. OBJECTIVE To examine the clinical features of TB uveitis and the associations with response to antitubercular therapy (ATT). DESIGN, SETTING, AND PARTICIPANTS This retrospective multinational cohort study included patients from 25 ophthalmology referral centers diagnosed with TB uveitis and treated with ATT from January 1, 2004, through December 31, 2014, with a minimum follow-up of 1 year. MAIN OUTCOMES AND MEASURES Treatment failure, defined as a persistence or recurrence of inflammation within 6 months of completing ATT, inability to taper oral corticosteroids to less than 10mg/d or topical corticosteroid drops to less than 2 drops daily, and/or recalcitrant inflammation necessitating corticosteroid-sparing immunosuppressive therapy. RESULTS A total of 801 patients (1272 eyes) were studied (mean [SD] age, 40.5 [14.8] years; 413 [51.6%] male and 388 [48.4%] female; 577 [73.6%] Asian). Most patients had no known history (498 of 661 [75.3%]) of systemic TB. Most patients had bilateral involvement (471 of 801 [58.8%]). Common clinical signs reported include vitreous haze (523 of 1153 [45.4%]), retinal vasculitis (374 of 874 [42.8%]), and choroidal involvement (419 of 651 [64.4%]). Treatment failure developed in 102 of the 801 patients (12.7%). On univariate regression analysis, the hazard ratios (HRs) associated with intermediate uveitis (HR, 2.21; 95%CI, 1.07-4.55; P = .03), anterior uveitis (HR, 2.68; 95%CI, 1.32-2.35; P = .006), and panuveitis (HR, 3.28; 95%CI, 1.89-5.67; P < .001) were significantly higher compared with posterior distribution. The presence of vitreous haze had a statistically significant association (HR, 1.95; 95%CI, 1.26-3.02; P = .003) compared with absence of vitreous haze. Bilaterality had an associated HR of 1.50 (95%CI, 0.96-2.35) compared with unilaterality (HR, 1 [reference]), although this finding was not statistically significant (P = .07). On multivariate Cox proportional hazards regression analysis, the presence of vitreous haze had an adjusted HR of 2.98 (95%CI, 1.50-5.94; P = .002), presence of snow banking had an adjusted HR of 3.71 (95%CI, 1.18-11.62; P = .02), and presence of choroidal involvement had an adjusted HR of 2.88 (95%CI, 1.22-6.78; P = .02). CONCLUSIONS AND RELEVANCE A low treatment failure rate occurred in patients with TB uveitis treated with ATT. Phenotypes and test results are studied whereby patients with panuveitis having vitreous and choroidal involvement had a higher risk of treatment failure. These findings are limited by retrospectivemethods. A prospectively derived composite clinical risk score might address this diagnostic uncertainty through holistic and standardized assessment of the combinations of clinical features and investigation results that may warrant diagnosis of TB uveitis and treatment with ATT