Convergence of SIRT1 and ATR signaling to modulate replication origin dormancy.

During routine genome duplication, many potential replication origins remain inactive or 'dormant'. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that are distinguished from baseline (i.e. ordinarily active) origins by their preferential association with two phospho-isoforms of the helicase component MCM2. During normal unperturbed cell growth, baseline origins, but not dormant origins, associate with a form of MCM2 that is phosphorylated by DBF4-dependent kinase (DDK) on serine 139 (pS139-MCM2). This association facilitates the initiation of DNA replication from baseline origins. Concomitantly, SIRT1 inhibits Ataxia Telangiectasia and Rad3-related (ATR)-kinase-mediated phosphorylation of MCM2 on serine 108 (pS108-MCM2) by deacetylating the ATR-interacting protein DNA topoisomerase II binding protein 1 (TOPBP1), thereby preventing ATR recruitment to chromatin. In cells devoid of SIRT1 activity, or challenged by replication stress, this inhibition is circumvented, enabling ATR-mediated S108-MCM2 phosphorylation. In turn, pS108-MCM2 enables DDK-mediated phosphorylation on S139-MCM2 and facilitates replication initiation at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational MCM modifications that reflect a balance between SIRT1 activity and ATR signaling.

Rarity patterns of woody plant species are associated with life form and diversification rates in Pacific islands forests.

PREMISE: Rarity is a complex and central concept in ecology and conservation biology. Yet, it is still poorly understood why some species are rare and others common. Here, we aimed to understand the drivers of species rarity patterns in woody plant communities. METHODS: We analyzed the local abundance and landscape frequency of 121 woody plant species across 238 plots on American Samoa and Hawaiian islands. We first assessed whether taxonomy, life form (shrub, small tree, large tree), and dispersal syndrome (dispersed by animals or by other means) are associated with the rarity of species. We then analyzed phylogenetic patterns in plant rarity and tested whether rarity patterns are associated with species evolutionary distinctiveness and the number of species within genera and families. RESULTS: Large trees were less abundant but more frequent than shrub species. Animal-dispersed species tended to be less abundant than species dispersed by other means, while species frequency was not associated with dispersal syndromes. Relative frequency in Hawai'i exhibited a more robust phylogenetic signal than did abundance. Both evolutionary distinctiveness and taxa species richness were significantly associated with the frequency of shrub species in Hawai'i. CONCLUSIONS: Life form appears consistently associated with the rarity of species. High diversification rate is probably a key factor explaining landscape-scale rarity of native species on isolated archipelagos like Hawai'i. At the landscape scale, rarity appears to be inversely associated with evolutionary distinctiveness, but at the local scale, species abundance may be not associated with evolutionary distinctiveness.

Evaluation of the Impact of an Online Opioid Education Program for Acute Pain Management.

OBJECTIVE: The University of Washington instituted a policy requiring all credentialed clinicians who prescribe opioids to complete a one-time education activity about safe and responsible opioid prescribing. A scenario-based, interactive online learning module was developed for opioid management of acute pain in hospitalized adults. This study examined the impact of the education module on learners' knowledge, perceived competence, and use of guideline-adherent practices. METHODS: Clinicians who completed the education module participated in a voluntary de-identified online survey approximately six months after the learning activity. Survey questions were related to 1) the perception of improved knowledge; 2) impact on learner's use of three guideline-adherent practices; and 3) perceived competence in managing opioids for acute pain. Descriptive statistics were generated, and multiple linear regression models were used for analysis. RESULTS: Clinicians (N = 167) reported improvement in knowledge and perceived competence. Controlling for other aspects of knowledge evaluated, learning to construct a safe opioid taper plan for acute pain, distinguishing between short- and long-acting opioids, and safely initiating opioids for acute pain were significantly associated with increased self-reported likelihood of incorporating the Washington state Prescription Monitoring Program (P = 0.003), using multimodal analgesia (P = 0.022), and reducing the duration of opioids prescribed (P = 0.016). Only improvement in knowledge of how to construct a safe opioid taper plan was significantly associated with increased perceived competence (P = 0.002). CONCLUSIONS: Our findings suggest that this online education module about safe opioid prescribing for acute pain management was effective at improving knowledge, increasing the likelihood of using guideline-adherent clinical practices, and increasing perceived competence.

The Strengths and Weaknesses of Current US Policy to Address Pain.

Pain is a significant public health problem that needs policy at the national and local level to resolve incidents of insufficient, ineffective, and disparate pain treatment while limiting the risk of inadvertently increasing the use of treatment such as opioids that can result in public harm.The National Pain Strategy serves as the first comprehensive approach to address pain and provides a roadmap with substantial broad and specific policy implications. Although much has been accomplished to date, transitions in political power, available data and funding, and the current opioid epidemic continue to have an impact on implementation of the National Pain Strategy.A sustained, coordinated effort with multipronged policies in many forms on both federal and state levels via regulations, laws, and guidelines is warranted. However, research is needed to evaluate the impact and potential unintended consequences of increased legislation and regulation. Nevertheless, policy related to the management of pain may provide the path to new treatments and models of care to reduce the impact of pain as a public health crisis in this country.

Integrated pain care models and the importance of aligning stakeholder values.

Sustained widespread deployment of clinically and cost-effective models of integrated pain care could be bolstered by optimally aligning shared stakeholder values.

Integrative epigenomic analyses of small cell lung cancer cells demonstrates the clinical translational relevance of gene body methylation.

DNA methylation is a key regulator of gene expression and a clinical therapeutic predictor. We examined global DNA methylation beyond the generally used promoter areas in human small cell lung cancer (SCLC) and find that gene body methylation is a robust positive predictor of gene expression. Combining promoter and gene body methylation better predicts gene expression than promoter methylation alone including genes involved in the neuroendocrine classification of SCLC and the expression of therapeutically relevant genes including MGMT, SLFN11, and DLL3. Importantly, for super-enhancer (SE) covered genes such as NEUROD1 or MYC, using H3K27ac and NEUROD1, ASCL1, and POU2F3 ChIP-seq data, we show that genic methylation is inversely proportional to expression, thus providing a new approach to identify potential SE regulated genes involved in SCLC pathogenesis. To advance SCLC transitional research, these data are integrated into our web portal (https://discover.nci.nih.gov/SclcCellMinerCDB/) for open and easy access to basic and clinical investigators.

The Test Re-Test Reliability of A Novel Single Leg Hop Test (T-Drill Hop Test).

BACKGROUND: Functional training and testing are an important part of a comprehensive rehabilitation program stressing the neuromuscular system in ways that simulate athletic performance to help determine criteria for return to sport. There are numerous single leg hop tests that have been used for these purposes, however, the validity and clinical relevance has been questioned. Many of the functional performance tests assess only the sagittal plane or forward direction and may only partially assess a person's athletic abilities. There is a need for reliable and valid functional tests to assess in a multi-directional manner. PURPOSE/HYPOTHESIS: The purpose of this study is to determine the test re-test reliability of a novel multi-directional timed single leg hop test (T-Drill Hop Test) for use in rehabilitation and performance assessments. STUDY DESIGN: Cross-sectional reliability study. METHODS: Fifty healthy recreationally active college age subjects, (23 males and 27 females) between the ages of 18 and 35, (mean age 23.48 with SD 3.82) consented to perform the test. The subjects hopped along a 10ft. x 10ft. "T" shaped course. Subjects performed two timed maximum effort trials of the T-Drill Hop Test on each leg with an interval of 3-7 days between the two testing days. Intraclass Correlation Coefficients (ICC) were calculated to determine intersession reliability. RESULTS: The inter-rater reliability (ICC's) for the entire group of 50 subjects ranged between .98 and 1.00 suggesting excellent reliability. The bilateral comparison, utilizing paired t-tests, of the T-Drill Hop Test demonstrated no significant differences between the time scores for the dominant and non-dominant legs for either males or females (p>.05). CONCLUSION: This study demonstrates the T-Drill Hop Test has excellent test re-test reliability. These results are important prior to validation and utilization as a clinical functional performance test. LEVELS OF EVIDENCE: Level 2.

Dynamics of replication origin over-activation.

Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We determined DNA synthesis patterns in cancer cells undergoing partial genome re-replication due to perturbed regulatory interactions (re-replicating cells). These cells exhibited slow replication, increased frequency of replication initiation events, and a skewed initiation pattern that preferentially reactivated early-replicating origins. Unlike in cells exposed to replication stress, which activated a novel group of hitherto unutilized (dormant) replication origins, the preferred re-replicating origins arose from the same pool of potential origins as those activated during normal growth. Mechanistically, the skewed initiation pattern reflected a disproportionate distribution of pre-replication complexes on distinct regions of licensed chromatin prior to replication. This distinct pattern suggests that circumventing the strong inhibitory interactions that normally prevent excess DNA synthesis can occur via at least two pathways, each activating a distinct set of replication origins.

BAMscale: quantification of next-generation sequencing peaks and generation of scaled coverage tracks.

BACKGROUND: Next-generation sequencing allows genome-wide analysis of changes in chromatin states and gene expression. Data analysis of these increasingly used methods either requires multiple analysis steps, or extensive computational time. We sought to develop a tool for rapid quantification of sequencing peaks from diverse experimental sources and an efficient method to produce coverage tracks for accurate visualization that can be intuitively displayed and interpreted by experimentalists with minimal bioinformatics background. We demonstrate its strength and usability by integrating data from several types of sequencing approaches. RESULTS: We have developed BAMscale, a one-step tool that processes a wide set of sequencing datasets. To demonstrate the usefulness of BAMscale, we analyzed multiple sequencing datasets from chromatin immunoprecipitation sequencing data (ChIP-seq), chromatin state change data (assay for transposase-accessible chromatin using sequencing: ATAC-seq, DNA double-strand break mapping sequencing: END-seq), DNA replication data (Okazaki fragments sequencing: OK-seq, nascent-strand sequencing: NS-seq, single-cell replication timing sequencing: scRepli-seq) and RNA-seq data. The outputs consist of raw and normalized peak scores (multiple normalizations) in text format and scaled bigWig coverage tracks that are directly accessible to data visualization programs. BAMScale also includes a visualization module facilitating direct, on-demand quantitative peak comparisons that can be used by experimentalists. Our tool can effectively analyze large sequencing datasets (~ 100 Gb size) in minutes, outperforming currently available tools. CONCLUSIONS: BAMscale accurately quantifies and normalizes identified peaks directly from BAM files, and creates coverage tracks for visualization in genome browsers. BAMScale can be implemented for a wide set of methods for calculating coverage tracks, including ChIP-seq and ATAC-seq, as well as methods that currently require specialized, separate tools for analyses, such as splice-aware RNA-seq, END-seq and OK-seq for which no dedicated software is available. BAMscale is freely available on github (https://github.com/ncbi/BAMscale).

The RepID-CRL4 ubiquitin ligase complex regulates metaphase to anaphase transition via BUB3 degradation.

The spindle assembly checkpoint (SAC) prevents premature chromosome segregation by inactivating the anaphase promoting complex/cyclosome (APC/C) until all chromosomes are properly attached to mitotic spindles. Here we identify a role for Cullin-RING ubiquitin ligase complex 4 (CRL4), known for modulating DNA replication, as a crucial mitotic regulator that triggers the termination of the SAC and enables chromosome segregation. CRL4 is recruited to chromatin by the replication origin binding protein RepID/DCAF14/PHIP. During mitosis, CRL4 dissociates from RepID and replaces it with RB Binding Protein 7 (RBBP7), which ubiquitinates the SAC mediator BUB3 to enable mitotic exit. During interphase, BUB3 is protected from CRL4-mediated degradation by associating with promyelocytic leukemia (PML) nuclear bodies, ensuring its availability upon mitotic onset. Deficiencies in RepID, CRL4 or RBBP7 delay mitotic exit, increase genomic instability and enhance sensitivity to paclitaxel, a microtubule stabilizer and anti-tumor drug.

Surgical repair of large cyclodialysis clefts.

PURPOSE: To describe a new surgical technique to effectively close large (>180 degrees) cyclodialysis clefts. METHODS: Our method involves the use of procedures commonly associated with repair of retinal detachment and complex cataract extraction: phacoemulsification with placement of a capsular tension ring followed by pars plana vitrectomy and gas tamponade with light cryotherapy. We also used anterior segment optical coherence tomography (OCT) as a noninvasive mechanism to determine the extent of the clefts and compared those results with ultrasound biomicroscopy (UBM) and gonioscopy. RESULTS: This technique was used to repair large cyclodialysis clefts in 4 eyes. All 4 eyes had resolution of hypotony and improvement of visual acuity. One patient had an intraocular pressure spike requiring further surgical intervention. Anterior segment OCT imaging in all 4 patients showed a more extensive cleft than UBM or gonioscopy. CONCLUSIONS: This technique is effective in repairing large cyclodialysis clefts. Anterior segment OCT more accurately predicted the extent of each cleft, while UBM and gonioscopy both underestimated the size of the cleft.

Evaluation of Automated Teleretinal Screening Program for Diabetic Retinopathy.

IMPORTANCE: Diabetic retinopathy is a leading cause of blindness, but its detrimental effects are preventable with early detection and treatment. Screening for diabetic retinopathy has the potential to increase the number of cases treated early, especially in populations with limited access to care. OBJECTIVE: To determine the efficacy of an automated algorithm in interpreting screening ophthalmoscopic photographs from patients with diabetes compared with a reading center interpretation. DESIGN, SETTING, AND PARTICIPANTS: Retrospective cohort analysis of 15,015 patients with type 1 or 2 diabetes in the Harris Health System in Harris County, Texas, who had undergone a retinal screening examination and nonmydriatic fundus photography via the Intelligent Retinal Imaging System (IRIS) from June 2013 to April 2014 were included. The IRIS-based interpretations were compared with manual interpretation. The IRIS algorithm population statistics were calculated. MAIN OUTCOMES AND MEASURES: Sensitivity and false-negative rate of the IRIS computer-based algorithm compared with reading center interpretation of the same images. RESULTS: A total of 15 015 consecutive patients (aged 18-98 years); mean 54.3 years with known type 1 or 2 diabetes underwent nonmydriatic fundus photography for a diabetic retinopathy screening examination. The sensitivity of the IRIS algorithm in detecting sight-threatening diabetic eye disease compared with the reading center interpretation was 66.4% (95% CI, 62.8%-69.9%) with a false-negative rate of 2%. The specificity was 72.8% (95% CI, 72.0%-73.5%). In a population where 15.8% of people with diabetes have sight-threatening diabetic eye disease, the IRIS algorithm positive predictive value was 10.8% (95% CI, 9.6%-11.9%) and the negative predictive value was 97.8% (95% CI, 96.8%-98.6%). CONCLUSIONS AND RELEVANCE: In this large urban setting, the IRIS computer algorithm-based screening program had a high sensitivity and a low false-negative rate, suggesting that it may be an effective alternative to conventional reading center image interpretation. The IRIS algorithm shows promise as a screening program, but algorithm refinement is needed to achieve better performance. Further studies of patient safety, cost-effectiveness, and widespread applications of this type of algorithm should be pursued to better understand the role of teleretinal imaging and automated analysis in the global health care system.