Publications by authors named "Christopher Wells"

71 Publications

Endoscopy training in the UK pre-COVID-19 environment: a multidisciplinary survey of endoscopy training and the experience of reciprocal feedback.

Frontline Gastroenterol 2022 22;13(1):39-44. Epub 2021 Feb 22.

Gastroenterology, North Tees and Hartlepool NHS Foundation Trust, Hartlepool, Hartlepool, UK.

Objective: Training in gastrointestinal endoscopy in the UK occurs predominantly in a real world one-to-one trainer to trainee interaction. Previous surveys have shown surgical and gastroenterology trainees have had mixed experiences of supervision and training, and no surveys have explored specifically the role of trainee to trainer feedback. This study aimed to explore the experience of training and of providing trainer feedback for all disciplines of endoscopy trainees.

Design/method: An online survey designed in collaboration with Joint Advisory Committee training committee and trainee representatives was distributed from January 2020 but was interrupted by the COVID-19 pandemic and hence terminated early.

Results: There were 129 responses, including trainees from all disciplines and regions, of which 86/129 (66.7%) rated the culture in their endoscopy units favourably-either good or excellent. 65/129 (50.4%) trainees reported having one or more training lists allocated per week, with 41/129 (31.8%) reporting only ad hoc lists. 100/129 (77.5%) respondents were given feedback and 97/129 (75.2%) were provided with learning points from the list. 65/129 (50.4%) respondents reported their trainer completed a direct observation of procedure or direct observation of polypectomies. 73/129 (56.6%) respondents reported that they felt able to give feedback to their trainer, with 88/129 (68.2%) feeling they could do this accurately. Barriers to trainer feedback cited included time constraints, lack of anonymity and concerns about affecting the trainer-trainee relationship.

Conclusion: Overall, the training environment has improved since previous surveys. There are still issues around interdisciplinary differences with some surgical trainees finding the training environment less welcoming, and trainee perceptions of hierarchical barriers and trainer responsiveness to feedback limiting the accuracy of their feedback.
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http://dx.doi.org/10.1136/flgastro-2020-101734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902317PMC
February 2021

The integration of personalized medicine into health systems: progress and a path forward.

Per Med 2021 09 21;18(6):527-531. Epub 2021 Oct 21.

Personalized Medicine Coalition, 1710 Rhode Island Avenue, NW, Washington, DC 20036, USA.

Tweetable abstract Fueled by technological advancements and the integration efforts of many pioneer health systems, personalized medicine is now being clinically implemented at measurable but incomplete levels system-wide.
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http://dx.doi.org/10.2217/pme-2021-0102DOI Listing
September 2021

Projected shifts in coral size structure in the Anthropocene.

Adv Mar Biol 2020 7;87(1):31-60. Epub 2020 Sep 7.

Australian Research Council Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, QLD, Australia.

Changes in the size structure of coral populations have major consequences for population dynamics and community function, yet many coral reef monitoring projects do not record this critical feature. Consequently, our understanding of current and future trajectories in coral size structure, and the demographic processes underlying these changes, is still emerging. Here, we provide a conceptual summary of the benefits to be gained from more comprehensive attention to the size of coral colonies in reef monitoring projects, and we support our argument through the use of case-history examples and a simplified ecological model. We neither seek to review the available empirical data, or to rigorously explore causes and implications of changes in coral size, we seek to reveal the advantages to modifying ongoing programs to embrace the information inherent in changing coral colony size. Within this framework, we evaluate and forecast the mechanics and implications of changes in the population structure of corals that are transitioning from high to low abundance, and from large to small colonies, sometimes without striking effects on planar coral cover. Using two coral reef locations that have been sampled for coral size, we use demographic data to underscore the limitations of coral cover in understanding the causes and consequences of long-term declining coral size, and abundance. A stage-structured matrix model is used to evaluate the demographic causes of declining coral colony size and abundance, particularly with respect to the risks of extinction. The model revealed differential effects of mortality, growth and fecundity on coral size distributions. It also suggested that colony rarity and declining colony size in association with partial tissue mortality and chronic declines in fecundity, can lead to a demographic bottleneck with the potential to prolong the existence of coral populations when they are characterized by mostly very small colonies. Such bottlenecks could have ecological importance if they can delay extinction and provide time for human intervention to alleviate the environmental degradation driving reductions in coral abundance.
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http://dx.doi.org/10.1016/bs.amb.2020.07.003DOI Listing
December 2020

Healthcare Resources Utilization and Costs of Patients with Non-IPF Progressive Fibrosing Interstitial Lung Disease Based on Insurance Claims in the USA.

Adv Ther 2020 07 21;37(7):3292-3298. Epub 2020 May 21.

Decision Resources Group, Burlington, USA.

Introduction: Idiopathic pulmonary fibrosis (IPF) is the classic progressive fibrosing interstitial lung disease (ILD), but some patients with ILDs other than IPF also develop a progressive fibrosing phenotype (PF-ILD). Information on use and cost of healthcare resources in patients with PF-ILD is limited.

Methods: We used USA-based medical insurance claims (2014-2016) to assess use and cost of healthcare resources in PF-ILD. Patients with at least two ILD claims and at least one pulmonologist visit were considered to have ILD. Pulmonologist visit frequency was used as a proxy to identify PF-ILD (at least four visits in 2016, or at least three more visits in 2016 vs. 2014).

Results: Of 2517 patients with non-IPF ILD, 15% (n = 373) had PF-ILD. Mean annual medical costs associated with ILD claims were $35,364 in patients with non-IPF PF-ILD versus $20,211 in the non-IPF ILD population. In 2016, patients with non-IPF PF-ILD made more hospital ILD claims than patients with non-IPF ILD (10.5 vs. 4.7).

Conclusions: These findings suggest higher disease severity and overall healthcare use for patients with a non-IPF ILD manifesting a progressive fibrosing phenotype (non-IPF PF-ILD).
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http://dx.doi.org/10.1007/s12325-020-01380-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467408PMC
July 2020

A consensus on collaboration: reviewing the 15th Annual Personalized Medicine Conference at Harvard Medical School.

Per Med 2020 03 7;17(2):79-81. Epub 2020 Feb 7.

Vice President, Public Affairs, Personalized Medicine Coalition, 1710 Rhode Island Avenue, NW, Suite 700, Washington, DC 20036, USA.

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http://dx.doi.org/10.2217/pme-2020-0001DOI Listing
March 2020

Alternative transcription cycle for bacterial RNA polymerase.

Nat Commun 2020 01 23;11(1):448. Epub 2020 Jan 23.

Department of Biochemistry, Brandeis University, Waltham, MA, 02454, USA.

RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. Here we use single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remains bound to DNA and sometimes exhibits one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarts transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this secondary initiation in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.
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http://dx.doi.org/10.1038/s41467-019-14208-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978322PMC
January 2020

Out of the Blue: The Failure of the Introduced Sea Anemone (Dalyell, 1848) in Salem Harbor, Massachusetts.

Biol Bull 2019 12 9;237(3):283-291. Epub 2019 Dec 9.

Failed invasions can be a key component for understanding and controlling introduced populations because understanding mechanisms behind failures can improve effective controls. In 2000, the non-native sea anemone was first found in Salem, Massachusetts, and it recolonized each summer. No individuals of have been found after 2010, despite intensive search efforts. A mismatch between the species' thermal tolerance and winter water temperature is the most likely mechanism for this failed invasion. In both laboratory- and field-based temperature growth studies, began regressing at 11 °C, stopped asexually reproducing at 9 °C, and died by 4 °C. These temperatures are above the average winter sea surface temperature in the Gulf of Maine, therefore suggesting that requires a warm-water refuge. Another potential contributor to the disappearance of is low genetic diversity as a result of establishment of only females (likely clones) and no males.
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http://dx.doi.org/10.1086/705515DOI Listing
December 2019

Progressive fibrosing interstitial lung diseases: current practice in diagnosis and management.

Curr Med Res Opin 2019 11 2;35(11):2015-2024. Epub 2019 Aug 2.

Reference Center for Rare Pulmonary Diseases, Louis Pradel Hospital, Claude Bernard University Lyon 1 , Lyon , France.

Some patients with interstitial lung diseases (ILDs) other than idiopathic pulmonary fibrosis (IPF) develop a progressive fibrosing phenotype. We investigated the diagnosis and management of non-IPF ILDs using data from a survey of physicians and from US insurance claims. Pulmonologists, rheumatologists and internists in France, Germany, Italy, Japan, Spain, UK and US who had managed ≥10 patients with non-IPF ILDs in the past year, including those with progressive fibrosing ILDs, completed an online survey. Data on US insurance and prescription claims were obtained from a repository that aggregates data on claims routed from providers or pharmacies to payers. In May-June 2017, 243 pulmonologists, 203 rheumatologists and 40 internists completed an online survey. Respondents estimated that 18-32% of patients diagnosed with non-IPF ILDs develop progressive fibrosis and that time from symptom onset to death in these patients was 61-80 months. Drug treatment was given to 50-75% of patients with non-IPF progressive fibrosing ILDs. Reasons for patients not being treated included that physicians considered patients to have mild or slowly progressing disease, or did not believe that available treatments are effective or well tolerated. Corticosteroids were the preferred first-line treatment for all types of non-IPF ILD. There was considerable heterogeneity in preferences for second- and third-line treatments. US insurance claims data from 3823 patients indicated that, in 2016, 50-75% of patients with ILDs received drug treatment (mostly corticosteroids) for their ILD. Physicians estimate that 18-32% of patients diagnosed with non-IPF ILDs develop a progressive fibrosing phenotype and that these patients experience significant delays in the diagnosis of ILD and the detection of progressive fibrosis. Between 25% and 50% of patients with progressive fibrosing ILDs do not receive drug therapy. There is an unmet need for effective and well tolerated treatments for progressive fibrosing ILDs.
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http://dx.doi.org/10.1080/03007995.2019.1647040DOI Listing
November 2019

Certification of UK gastrointestinal endoscopists and variations between trainee specialties: results from the JETS e-portfolio.

Endosc Int Open 2019 Apr 4;7(4):E551-E560. Epub 2019 Apr 4.

Joint Advisory Group on Gastrointestinal Endoscopy, Royal College of Physicians, London, UK.

In the UK, endoscopy certification is administered by the Joint Advisory Group on Gastrointestinal Endoscopy (JAG). Since 2011, certification for upper and lower gastrointestinal endoscopy has been awarded via a national (JETS) e-portfolio to the main training specialties of: gastroenterology, gastrointestinal surgeons (GS) and non-medical endoscopists (NME). Trends in endoscopy certification and differences between trainee specialties were analyzed. This prospective UK-wide observational study identified trainees awarded gastroscopy, sigmoidoscopy, colonoscopy (provisional and full) certification between June 2011 - 2017. Trends in certification, procedures and time-to-certification, and key performance indicators (KPIs) in the 3-month pre- and post-certification period were compared between the three main training specialties. Three thousand one hundred fifty-seven endoscopy-related certifications were awarded to 1928 trainees from gastroenterology (52.3 %), GS (28.4 %) and NME (16.5  %) specialties. During the study period, certification numbers increased for all modalities and specialties, particularly NME trainees. For gastroscopy and colonoscopy, procedures-to-certification were lowest for GS (  < 0.001), whereas time-to-certification was consistently shortest in NMEs (  < 0.001). A post-certification reduction in mean cecal intubation rate (95.2 % to 93.8 %,  < 0.001) was observed in colonoscopy, and D2 intubation (97.6 % to 96.2 %,  < 0.001) and J-maneuver (97.3 % to 95.8 %,  < 0.001) in gastroscopy. Overall, average pre- and post-certification KPIs still exceeded national minimum standards. There was an increase in PDR for NMEs after provisional colonoscopy certification but a decrease in PDR for GS trainees after sigmoidoscopy and full colonoscopy certification. Despite variations among trainee specialties, average pre- and post-certification KPIs for certified trainees met national standards, suggesting that JAG certification is a transparent benchmark which adequately safeguards competency in endoscopy training.
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http://dx.doi.org/10.1055/a-0839-4476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449159PMC
April 2019

The Facility-Level HIV Treatment Cascade: Using a Population Health Tool in Health Care Facilities to End the Epidemic in New York State.

Open Forum Infect Dis 2018 Oct 26;5(10):ofy254. Epub 2018 Oct 26.

New York State Department of Health AIDS Institute, New York, New York.

Background: The HIV treatment cascade is a tool for characterizing population-level gaps in HIV care, yet most adaptations of the cascade rely on surveillance data that are ill-suited to drive quality improvement (QI) activities at the facility level. We describe the adaptation of the cascade in health care organizations and report its use by HIV medical providers in New York State (NYS).

Methods: As part of data submissions to the NYS Department of Health, sites that provide HIV medical care in NYS developed cascades using facility-generated data. Required elements included data addressing identification of people living with HIV (PLWH) receiving any service at the facility, linkage to HIV medical care, prescription of antiretroviral therapy (ART), and viral suppression (VS). Sites also submitted a methodology report summarizing how cascade data were collected and an improvement plan identifying care gaps.

Results: Two hundred twenty-two sites submitted cascades documenting the quality of care delivered to HIV patients presenting for HIV- or non-HIV-related services during 2016. Of 101 341 PLWH presenting for any medical care, 75 106 were reported as active in HIV programs, whereas 21 509 had no known care status. Sites reported mean ART prescription and VS rates of 94% and 80%, respectively, and 60 distinct QI interventions.

Conclusions: Submission of facility-level cascades provides data on care utilization among PLWH that cannot be assessed through traditional HIV surveillance efforts. Moreover, the facility-level cascade represents an effective tool for identifying care gaps, focusing data-driven improvement efforts, and engaging frontline health care providers to achieve epidemic control.
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http://dx.doi.org/10.1093/ofid/ofy254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202506PMC
October 2018

Correction: Changes in scoring of Direct Observation of Procedural Skills (DOPS) forms and the impact on competence assessment.

Endoscopy 2018 08 26;50(8):C9. Epub 2018 Jul 26.

Joint Advisory Group on Gastrointestinal Endoscopy, Royal College of Physicians, London, United Kingdom.

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http://dx.doi.org/10.1055/a-0658-2732DOI Listing
August 2018

Changes in scoring of Direct Observation of Procedural Skills (DOPS) forms and the impact on competence assessment.

Endoscopy 2018 08 3;50(8):770-778. Epub 2018 Apr 3.

Joint Advisory Group on Gastrointestinal Endoscopy, Royal College of Physicians, London, United Kingdom.

Background: Direct Observation of Procedural Skills (DOPS) is an established competence assessment tool in endoscopy. In July 2016, the DOPS scoring format changed from a performance-based scale to a supervision-based scale. We aimed to evaluate the impact of changes to the DOPS scale format on the distribution of scores in novice trainees and on competence assessment.

Methods: We performed a prospective, multicenter (n = 276), observational study of formative DOPS assessments in endoscopy trainees with ≤ 100 lifetime procedures. DOPS were submitted in the 6-months before July 2016 (old scale) and after (new scale) for gastroscopy (n = 2998), sigmoidoscopy (n = 1310), colonoscopy (n = 3280), and polypectomy (n = 631). Scores for old and new DOPS were aligned to a 4-point scale and compared.

Results: 8219 DOPS (43 % new and 57 % old) submitted for 1300 trainees were analyzed. Compared with old DOPS, the use of the new DOPS was associated with greater utilization of the lowest score (2.4 % vs. 0.9 %;  < 0.001), broader range of scores, and a reduction in competent scores (60.8 % vs. 86.9 %;  < 0.001). The reduction in competent scores was evident on subgroup analysis across all procedure types ( < 0.001) and for each quartile of endoscopy experience. The new DOPS was superior in characterizing the endoscopy learning curve by demonstrating progression of competent scores across quartiles of procedural experience.

Conclusions: Endoscopy assessors applied a greater range of scores using the new DOPS scale based on degree of supervision in two cohorts of trainees matched for experience. Our study provides construct validity evidence in support of the new scale format.
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http://dx.doi.org/10.1055/a-0576-6667DOI Listing
August 2018

The N54- Mutant Has Decreased Affinity for and Suggests a Mechanism for Coupling Heterotrimeric G Protein Nucleotide Exchange with Subunit Dissociation.

J Pharmacol Exp Ther 2018 05 28;365(2):219-225. Epub 2018 Feb 28.

Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina.

Ser54 of G binds guanine nucleotide and Mg as part of a conserved sequence motif in GTP binding proteins. Mutating the homologous residue in small and heterotrimeric G proteins generates dominant-negative proteins, but by protein-specific mechanisms. For , this results from persistent binding of to , whereas for small GTP binding proteins and this results from persistent binding to guanine nucleotide exchange factor or receptor. This work examined the role of interactions in mediating the properties of the Ser54-like mutants of G subunits. Unexpectedly, WT- or N54- coexpressed with -adrenergic receptor in human embryonic kidney 293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism, but WT- was more effective than the mutant. One explanation for this result would be that , like Ser47 , blocks receptor activation by sequestering ; implying that N54- has reduced affinity for since it was less effective at blocking IP3 production. This possibility was more directly supported by the observation that WT- was more effective than the mutant in inhibiting activation of phospholipase C2. Further, in vitro synthesized N54- bound biotinylated- with lower apparent affinity than did WT- The Cys54 mutation also decreased binding but less effectively than N54- Substitution of the conserved Ser in with Cys or Asn increased binding, with the Cys mutant being more effective. This suggests that Ser54 of is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins.
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http://dx.doi.org/10.1124/jpet.117.245779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870480PMC
May 2018

Individual marking of soft-bodied subtidal invertebrates in situ - A novel staining technique applied to the giant plumose anemone Metridium farcimen (Tilesius, 1809).

PLoS One 2017 21;12(11):e0188263. Epub 2017 Nov 21.

Biology Department, University of Washington, Seattle, WA, United States of America.

The ability to recognize individuals and track growth over time is crucial to population dynamics research as well as studies of animal behavior. Invertebrates are particularly difficult to track as they often molt, have regenerative capabilities, or lack hard parts to attach markers. We tested, in laboratory and field studies, a new way of marking sea anemones (order Actiniaria) by injection of three vital stains (i.e., neutral red, methylene blue, and fluorescein). Neutral red and methylene blue did not affect growth or survival, but fluorescein was lethal at high concentrations. Marked individuals could be identified up to seven months after injection with neutral red, six weeks with methylene blue, and three days with low concentrations of fluorescein. Neutral red could be used for long-term monitoring of growth and survival in the field, and in combination with methylene blue could be used to mark individuals in distinguishable patterns for short-term studies such as examining predator-prey interactions, movement of individuals, and recruitment survival.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188263PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697843PMC
December 2017

Gβγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes.

J Biol Chem 2017 07 17;292(29):12165-12177. Epub 2017 May 17.

Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232-6600. Electronic address:

G-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gβγ-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of Gβγ subunits with the soluble -ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gβγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, Gβγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled Gβγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with Gβγ for SNARE-binding sites in lipid environments. Mutant Gβγ subunits that were previously shown to be more efficacious at inhibiting Ca-triggered exocytotic release than wild-type Gβγ were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant Gβγ subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on Gβ and Gγ shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In fusion assays using full-length t- and v-SNAREs embedded in liposomes, Gβγ inhibited Ca/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gβγ-SNARE interaction and show that the target of Gβγ, downstream of VGCC, is the membrane-embedded SNARE complex.
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http://dx.doi.org/10.1074/jbc.M116.773523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519367PMC
July 2017

The Unintended Consequences of Over-Reducing Cardiopulmonary Bypass Circuit Prime Volume.

Ann Thorac Surg 2017 Jun 21;103(6):1842-1848. Epub 2016 Dec 21.

SpecialtyCare, Nashville, Tennessee.

Background: The Society of Thoracic Surgeon blood conservation guidelines recommend minimizing cardiopulmonary bypass circuit prime volume (PV) as an integral, evidence-based (Class I, Level A) blood conservation strategy. We used a large, multiinstitutional registry to evaluate the effectiveness of restricting cardiopulmonary bypass PV on intraoperative red blood cell (RBC) transfusion.

Methods: We reviewed 47,273 isolated coronary artery bypass graft procedures performed among 189 institutions between April 2012 and May 2015. The primary outcome was intraoperative transfusion of at least 1 unit RBC; the secondary outcome was intraoperative transfusion of 4 or more units RBC. We estimated the adjusted odds of each transfusion type using separate multivariable logistic regression models that controlled for 13 confounding factors. The effect of PV on odds of transfusion was modeled using restricted cubic splines to assess possible nonlinearity.

Results: We found a statistically significant nonlinear pattern in the relation between PV and odds of transfusion of both 1 or more units RBC (χ = 116.3, df = 4, p < 0.001) and 4 or more units RBC (χ = 25.9, df = 4, p < 0.001). The lowest probability of transfusion of 1 or more units RBC was estimated at a ratio of PV to estimated blood volume of 0.152 (p < 0.001).

Conclusions: Efforts to minimize PV below 15% of a patient's estimated blood volume do not protect patients from intraoperative RBC transfusion, and may increase exposure. Prime volume can affect both patient morbidity and the economic impact associated with blood utilization. Further studies on the effect of PV on blood transfusion are warranted.
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http://dx.doi.org/10.1016/j.athoracsur.2016.09.099DOI Listing
June 2017

Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia.

Sci Adv 2016 Oct 12;2(10):e1600760. Epub 2016 Oct 12.

Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.; Ludwig Institute for Cancer Research, University of Oxford, Oxford OX3 7DQ, U.K.

Bromodomains (BRDs) have emerged as compelling targets for cancer therapy. The development of selective and potent BET (bromo and extra-terminal) inhibitors and their significant activity in diverse tumor models have rapidly translated into clinical studies and have motivated drug development efforts targeting non-BET BRDs. However, the complex multidomain/subunit architecture of BRD protein complexes complicates predictions of the consequences of their pharmacological targeting. To address this issue, we developed a promiscuous BRD inhibitor [bromosporine (BSP)] that broadly targets BRDs (including BETs) with nanomolar affinity, creating a tool for the identification of cellular processes and diseases where BRDs have a regulatory function. As a proof of principle, we studied the effects of BSP on leukemic cell lines known to be sensitive to BET inhibition and found, as expected, strong antiproliferative activity. Comparison of the modulation of transcriptional profiles by BSP after a short exposure to the inhibitor resulted in a BET inhibitor signature but no significant additional changes in transcription that could account for inhibition of other BRDs. Thus, nonselective targeting of BRDs identified BETs, but not other BRDs, as master regulators of context-dependent primary transcription response.
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http://dx.doi.org/10.1126/sciadv.1600760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061470PMC
October 2016

Nascent RNA length dictates opposing effects of NusA on antitermination.

Nucleic Acids Res 2016 06 28;44(11):5378-89. Epub 2016 Mar 28.

Department of Microbiology and Immunobiology, Boston, MA 02115, USA

The NusA protein is a universally conserved bacterial transcription elongation factor that binds RNA polymerase (RNAP). When functioning independently, NusA enhances intrinsic termination. Paradoxically, NusA stimulates the function of the N and Q antiterminator proteins of bacteriophage λ. The mechanistic basis for NusA's functional plasticity is poorly understood. Here we uncover an effect of nascent RNA length on the ability of NusA to collaborate with Q. Ordinarily, Q engages RNAP during early elongation when it is paused at a specific site just downstream of the phage late-gene promoter. NusA facilitates this engagement process and both proteins remain associated with the transcription elongation complex (TEC) as it escapes the pause and transcribes the late genes. We show that the λ-related phage 82 Q protein (82Q) can also engage RNAP that is paused at a promoter-distal position and thus contains a nascent RNA longer than that associated with the natively positioned TEC. However, the effect of NusA in this context is antagonistic rather than stimulatory. Moreover, cleaving the long RNA associated with the promoter-distal TEC restores NusA's stimulatory effect. Our findings reveal a critical role for nascent RNA in modulating NusA's effect on 82Q-mediated antitermination, with implications for understanding NusA's functional plasticity.
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http://dx.doi.org/10.1093/nar/gkw198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914094PMC
June 2016

Bacterial RNA polymerase can retain σ70 throughout transcription.

Proc Natl Acad Sci U S A 2016 Jan 5;113(3):602-7. Epub 2016 Jan 5.

Department of Biochemistry, Brandeis University, Waltham, MA 02454;

Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. During each of these stages, RNA polymerase (RNAP) function is regulated by RNAP-associated protein factors. In bacteria, RNAP-associated σ factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary σ factor in Escherichia coli, σ(70), can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of σ(70) retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ(70)-RNAP complex during initiation from the λ PR' promoter and throughout the elongation of a long (>2,000-nt) transcript. Our results provide direct measurements of the fraction of actively transcribing complexes with bound σ(70) and the kinetics of σ(70) release from actively transcribing complexes. σ(70) release from mature elongation complexes was slow (0.0038 s(-1)); a substantial subpopulation of elongation complexes retained σ(70) throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ(70) manifest enhanced recognition of a promoter-like pause element positioned hundreds of nucleotides downstream of the promoter. Together, the results provide a quantitative framework for understanding the postinitiation roles of σ(70) during transcription.
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http://dx.doi.org/10.1073/pnas.1513899113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725480PMC
January 2016

Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance.

Sci Adv 2015 Nov 13;1(10):e1500723. Epub 2015 Nov 13.

Target Discovery Institute, University of Oxford, NDM Research Building, Roosevelt Drive, Oxford OX3 7FZ, UK. ; Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK.

Mammalian SWI/SNF [also called Brg/Brahma-associated factors (BAFs)] are evolutionarily conserved chromatin-remodeling complexes regulating gene transcription programs during development and stem cell differentiation. BAF complexes contain an ATP (adenosine 5'-triphosphate)-driven remodeling enzyme (either BRG1 or BRM) and multiple protein interaction domains including bromodomains, an evolutionary conserved acetyl lysine-dependent protein interaction motif that recruits transcriptional regulators to acetylated chromatin. We report a potent and cell active protein interaction inhibitor, PFI-3, that selectively binds to essential BAF bromodomains. The high specificity of PFI-3 was achieved on the basis of a novel binding mode of a salicylic acid head group that led to the replacement of water molecules typically maintained in other bromodomain inhibitor complexes. We show that exposure of embryonic stem cells to PFI-3 led to deprivation of stemness and deregulated lineage specification. Furthermore, differentiation of trophoblast stem cells in the presence of PFI-3 was markedly enhanced. The data present a key function of BAF bromodomains in stem cell maintenance and differentiation, introducing a novel versatile chemical probe for studies on acetylation-dependent cellular processes controlled by BAF remodeling complexes.
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http://dx.doi.org/10.1126/sciadv.1500723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4681344PMC
November 2015

Generation of a Selective Small Molecule Inhibitor of the CBP/p300 Bromodomain for Leukemia Therapy.

Cancer Res 2015 Dec 9;75(23):5106-5119. Epub 2015 Nov 9.

Nuffield Department of Clinical Medicine, University of Oxford, Target Discovery Institute (TDI), Roosevelt Drive, Oxford OX3 7BN, UK.

The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9(+) acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-0236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948672PMC
December 2015

The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo.

Elife 2015 Sep 15;4. Epub 2015 Sep 15.

Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States.

The σ subunit of bacterial RNA polymerase (RNAP) confers on the enzyme the ability to initiate promoter-specific transcription. Although σ factors are generally classified as initiation factors, σ can also remain associated with, and modulate the behavior of, RNAP during elongation. Here we establish that the primary σ factor in Escherichia coli, σ(70), can function as an elongation factor in vivo by loading directly onto the transcription elongation complex (TEC) in trans. We demonstrate that σ(70) can bind in trans to TECs that emanate from either a σ(70)-dependent promoter or a promoter that is controlled by an alternative σ factor. We further demonstrate that binding of σ(70) to the TEC in trans can have a particularly large impact on the dynamics of transcription elongation during stationary phase. Our findings establish a mechanism whereby the primary σ factor can exert direct effects on the composition of the entire transcriptome, not just that portion that is produced under the control of σ(70)-dependent promoters.
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http://dx.doi.org/10.7554/eLife.10514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604602PMC
September 2015

CBP30, a selective CBP/p300 bromodomain inhibitor, suppresses human Th17 responses.

Proc Natl Acad Sci U S A 2015 Aug 10;112(34):10768-73. Epub 2015 Aug 10.

Botnar Research Institute, Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Science, University of Oxford, Oxford OX3 7LD, United Kingdom;

Th17 responses are critical to a variety of human autoimmune diseases, and therapeutic targeting with monoclonal antibodies against IL-17 and IL-23 has shown considerable promise. Here, we report data to support selective bromodomain blockade of the transcriptional coactivators CBP (CREB binding protein) and p300 as an alternative approach to inhibit human Th17 responses. We show that CBP30 has marked molecular specificity for the bromodomains of CBP and p300, compared with 43 other bromodomains. In unbiased cellular testing on a diverse panel of cultured primary human cells, CBP30 reduced immune cell production of IL-17A and other proinflammatory cytokines. CBP30 also inhibited IL-17A secretion by Th17 cells from healthy donors and patients with ankylosing spondylitis and psoriatic arthritis. Transcriptional profiling of human T cells after CBP30 treatment showed a much more restricted effect on gene expression than that observed with the pan-BET (bromo and extraterminal domain protein family) bromodomain inhibitor JQ1. This selective targeting of the CBP/p300 bromodomain by CBP30 will potentially lead to fewer side effects than with the broadly acting epigenetic inhibitors currently in clinical trials.
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http://dx.doi.org/10.1073/pnas.1501956112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4553799PMC
August 2015

Quantitative EEG and Current Source Density Analysis of Combined Antiepileptic Drugs and Dopaminergic Agents in Genetic Epilepsy: Two Case Studies.

Clin EEG Neurosci 2015 Jul 17;46(3):256-62. Epub 2014 Oct 17.

Emory Neurophysiology Institute, Los Angeles, CA, USA

Two adolescent females with absence epilepsy were classified, one as attention deficit and the other as bipolar disorder. Physical and cognitive exams identified hypotension, bradycardia, and cognitive dysfunction. Their initial electroencephalograms (EEGs) were considered slightly slow, but within normal limits. Quantitative EEG (QEEG) data included relative theta excess and low alpha mean frequencies. A combined treatment of antiepileptic drugs with a catecholamine agonist/reuptake inhibitor was sequentially used. Both patients' physical and cognitive functions improved and they have remained seizure free. The clinical outcomes were correlated with statistically significant changes in QEEG measures toward normal Z-scores in both anterior and posterior regions. In addition, low resolution electromagnetic tomography (LORETA) Z-scored source correlation analyses of the initial and treated QEEG data showed normalized patterns, supporting a neuroanatomic resolution. This study presents preliminary evidence for a neurophysiologic approach to patients with absence epilepsy and comorbid disorders and may provide a method for further research.
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http://dx.doi.org/10.1177/1550059414532253DOI Listing
July 2015

Discovery and optimization of small-molecule ligands for the CBP/p300 bromodomains.

J Am Chem Soc 2014 Jul 19;136(26):9308-19. Epub 2014 Jun 19.

Department of Chemistry, University of Oxford , South Parks Road, Oxford OX1 3TA, U.K.

Small-molecule inhibitors that target bromodomains outside of the bromodomain and extra-terminal (BET) sub-family are lacking. Here, we describe highly potent and selective ligands for the bromodomain module of the human lysine acetyl transferase CBP/p300, developed from a series of 5-isoxazolyl-benzimidazoles. Our starting point was a fragment hit, which was optimized into a more potent and selective lead using parallel synthesis employing Suzuki couplings, benzimidazole-forming reactions, and reductive aminations. The selectivity of the lead compound against other bromodomain family members was investigated using a thermal stability assay, which revealed some inhibition of the structurally related BET family members. To address the BET selectivity issue, X-ray crystal structures of the lead compound bound to the CREB binding protein (CBP) and the first bromodomain of BRD4 (BRD4(1)) were used to guide the design of more selective compounds. The crystal structures obtained revealed two distinct binding modes. By varying the aryl substitution pattern and developing conformationally constrained analogues, selectivity for CBP over BRD4(1) was increased. The optimized compound is highly potent (Kd = 21 nM) and selective, displaying 40-fold selectivity over BRD4(1). Cellular activity was demonstrated using fluorescence recovery after photo-bleaching (FRAP) and a p53 reporter assay. The optimized compounds are cell-active and have nanomolar affinity for CBP/p300; therefore, they should be useful in studies investigating the biological roles of CBP and p300 and to validate the CBP and p300 bromodomains as therapeutic targets.
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http://dx.doi.org/10.1021/ja412434fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183655PMC
July 2014

Effects of disturbance associated with seismic exploration for oil and gas reserves in coastal marshes.

Environ Manage 2014 Jul 1;54(1):30-50. Epub 2014 May 1.

U.S. Geological Survey, National Wetlands Research Center, 700 Cajundome Blvd., Lafayette, LA, 70506, USA,

Anthropogenic disturbances in wetland ecosystems can alter the composition and structure of plant assemblages and affect system functions. Extensive oil and gas extraction has occurred in wetland habitats along the northern Gulf of Mexico coast since the early 1900s. Activities involved with three-dimensional (3D) seismic exploration for these resources cause various disturbances to vegetation and soils. We documented the impact of a 3D seismic survey in coastal marshes in Louisiana, USA, along transects established before exploration began. Two semi-impounded marshes dominated by Spartina patens were in the area surveyed. Vegetation, soil, and water physicochemical data were collected before the survey, about 6 weeks following its completion, and every 3 months thereafter for 2 years. Soil cores for seed bank emergence experiments were also collected. Maximum vegetation height at impact sites was reduced in both marshes 6 weeks following the survey. In one marsh, total vegetation cover was also reduced, and dead vegetation cover increased, at impact sites 6 weeks after the survey. These effects, however, did not persist 3 months later. No effects on soil or water properties were identified. The total number of seeds that germinated during greenhouse studies increased at impact sites 5 months following the survey in both marshes. Although some seed bank effects persisted 1 year, these effects were not reflected in standing vegetation. The marshes studied were therefore resilient to the impacts resulting from 3D seismic exploration because vegetation responses were short term in that they could not be identified a few months following survey completion.
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http://dx.doi.org/10.1007/s00267-014-0274-2DOI Listing
July 2014

Dual kinase-bromodomain inhibitors for rationally designed polypharmacology.

Nat Chem Biol 2014 Apr 2;10(4):305-12. Epub 2014 Mar 2.

1] Nuffield Department of Clinical Medicine, University of Oxford, Structural Genomics Consortium, Oxford, UK. [2] Nuffield Department of Clinical Medicine, University of Oxford, Target Discovery Institute (TDI), Oxford, UK.

Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multitarget profile has, however, necessitated the application of combination therapies, which can pose major clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as new targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase-bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348, which are clinical PLK1 and JAK2-FLT3 kinase inhibitors, respectively, is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a new strategy for rational single-agent polypharmacological targeting. Furthermore, structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase-bromodomain inhibitors.
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http://dx.doi.org/10.1038/nchembio.1471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3998711PMC
April 2014

Response to letter to the editor.

Pain 2014 May 7;155(5):1045-1046. Epub 2014 Feb 7.

Departments of Anesthesiology and Neurology and Center for Human Experimental Therapeutics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA University of Rochester, Rochester, NY, USA Stanford University, Palo Alto, CA, USA Johns Hopkins University, Baltimore, MD, USA Oregon Health and Science University, Portland, OR, USA Northwestern University, Chicago, IL, USA University of Wisconsin, Madison, WI, USA University of Kiel, Kiel, Germany Schmerzfachpraxis, Krefeld, Germany University of Washington, Seattle, WA, USA Universität Heidelberg, Mannheim, Germany Pain Matters, Liverpool, UK.

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http://dx.doi.org/10.1016/j.pain.2014.01.029DOI Listing
May 2014

Modulation of neurotransmission by GPCRs is dependent upon the microarchitecture of the primed vesicle complex.

J Neurosci 2014 Jan;34(1):260-74

Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607 and Department of Pharmacology, Vanderbilt University Medical School, Nashville, Tennessee 37232.

G(i/o)-protein-coupled receptors (GPCRs) ubiquitously inhibit neurotransmission, principally via Gβγ, which acts via a number of possible effectors. GPCR effector specificity has traditionally been attributed to Gα, based on Gα's preferential effector targeting in vitro compared with Gβγ's promiscuous targeting of various effectors. In synapses, however, Gβγ clearly targets unique effectors in a receptor-dependent way to modulate synaptic transmission. It remains unknown whether Gβγ specificity in vivo is due to specific Gβγ isoform-receptor associations or to spatial separation of distinct Gβγ pathways through macromolecular interactions. We thus sought to determine how Gβγ signaling pathways within axons remain distinct from one another. In rat hippocampal CA1 axons, GABA(B) receptors (GABA(B)Rs) inhibit presynaptic Ca(2+) entry, and we have now demonstrated that 5-HT(1B) receptors (5-HT(1B)Rs) liberate Gβγ to interact with SNARE complex C terminals with no effect on Ca(2+) entry. Both GABA(B)Rs and 5-HT(1B)Rs inhibit Ca(2+)-evoked neurotransmitter release, but 5-HT(1B)Rs have no effect on Sr(2+)-evoked release. Sr(2+), unlike Ca(2+), does not cause synaptotagmin to compete with Gβγ binding to SNARE complexes. 5-HT(1B)Rs also fail to inhibit release following cleavage of the C terminus of the SNARE complex protein SNAP-25 with botulinum A toxin. Thus, GABA(B)Rs and 5-HT(1B)Rs both localize to presynaptic terminals, but target distinct effectors. We demonstrate that disruption of SNARE complexes and vesicle priming with botulinum C toxin eliminates this selectivity, allowing 5-HT(1B)R inhibition of Ca(2+) entry. We conclude that receptor-effector specificity requires a microarchitecture provided by the SNARE complex during vesicle priming.
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http://dx.doi.org/10.1523/JNEUROSCI.3633-12.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3866488PMC
January 2014

Maintenance of muscle myosin levels in adult C. elegans requires both the double bromodomain protein BET-1 and sumoylation.

Biol Open 2013 Dec 15;2(12):1354-63. Epub 2013 Dec 15.

Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK.

Attenuation of RAS-mediated signalling is a conserved process essential to control cell proliferation, differentiation, and apoptosis. Cooperative interactions between histone modifications such as acetylation, methylation and sumoylation are crucial for proper attenuation in C. elegans, implying that the proteins recognising these histone modifications could also play an important role in attenuation of RAS-mediated signalling. We sought to systematically identify these proteins and found BET-1. BET-1 is a conserved double bromodomain protein that recognises acetyl-lysines on histone tails and maintains the stable fate of various lineages. Unexpectedly, adults lacking both BET-1 and SUMO-1 are depleted of muscle myosin, an essential component of myofibrils. We also show that this muscle myosin depletion does not occur in all animals at a specific time, but rather that the penetrance of the phenotype increases with age. To gain mechanistic insights into this process, we sought to delay the occurrence of the muscle myosin depletion phenotype and found that it requires caspase activity and MEK-dependent signalling. We also performed transcription profiling on these mutants and found an up-regulation of the FGF receptor, egl-15, a tyrosine kinase receptor acting upstream of MEK. Consistent with a MEK requirement, we could delay the muscle phenotype by systemic or hypodermal knock down of egl-15. Thus, this work uncovered a caspase- and MEK-dependent mechanism that acts specifically on ageing adults to maintain the appropriate net level of muscle myosin.
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http://dx.doi.org/10.1242/bio.20136007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3863420PMC
December 2013
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