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Invertebrates rely on Dicer to cleave viral double-stranded RNA (dsRNA), and Drosophila Dicer-2 distinguishes dsRNA substrates by their termini. Blunt termini promote processive cleavage, while 3' overhanging termini are cleaved distributively. To understand this discrimination, we used cryo-electron microscopy to solve structures of Drosophila Dicer-2 alone and in complex with blunt dsRNA. Whereas the Platform-PAZ domains have been considered the only Dicer domains that bind dsRNA termini, unexpectedly, we found that the helicase domain is required for binding blunt, but not 3' overhanging, termini. We further showed that blunt dsRNA is locally unwound and threaded through the helicase domain in an adenosine triphosphate-dependent manner. Our studies reveal a previously unrecognized mechanism for optimizing antiviral defense and set the stage for the discovery of helicase-dependent functions in other Dicers., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Loquacious-PD (Loqs-PD) is required for biogenesis of many endogenous siRNAs in Drosophila In vitro, Loqs-PD enhances the rate of dsRNA cleavage by Dicer-2 and also enables processing of substrates normally refractory to cleavage. Using purified components, and Loqs-PD truncations, we provide a mechanistic basis for Loqs-PD functions. Our studies indicate that the 22 amino acids at the C terminus of Loqs-PD, including an FDF-like motif, directly interact with the Hel2 subdomain of Dicer-2's helicase domain. This interaction is RNA-independent, but we find that modulation of Dicer-2 cleavage also requires dsRNA binding by Loqs-PD. Furthermore, while the first dsRNA-binding motif of Loqs-PD is dispensable for enhancing cleavage of optimal substrates, it is essential for enhancing cleavage of suboptimal substrates. Finally, our studies define a previously unrecognized Dicer interaction interface and suggest that Loqs-PD is well positioned to recruit substrates into the helicase domain of Dicer-2., Competing Interests: The authors declare no conflict of interest.

Purpose: Radiation therapy plays an important curative role for patients with locally advanced cervical cancer (LACC). There are no standards to define best practice. The purpose of this study was to develop a suite of radiation therapy key quality-of-care indicators (KQIs) for the curative management of LACC based on expert consensus., Methods and Materials: A modified Delphi method was used after identifying candidate KQIs. Round 1 involved surveying all Canadian gynecology radiation oncologists. The current and anticipated future (5 years) importance and current achievability of each KQI was ranked. Round 2 consisted of a facilitated face-to-face meeting with a smaller expert panel to discuss, revise, and develop consensus on the KQIs., Results: The literature review identified 83 candidate KQIs. Survey response was 71%. Round 2 yielded a final suite of 40 KQIs in the following categories: pretreatment assessment, external beam radiation therapy, brachytherapy, follow-up, and expertise/workload. A prominent theme was the importance of having KQIs to measure the current state, evolution, and future uptake of magnetic resonance-guided brachytherapy., Conclusions: To our knowledge, this is the first study establishing radiation therapy KQIs in LACC based on expert consensus. These KQIs should be used to guide programmatic direction and resource allocation to assure consistent and optimal patient care., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

Protein kinase RNA-activated (PKR) has long been known to be activated by viral double-stranded RNA (dsRNA) as part of the mammalian immune response. However, in mice PKR is also activated by metabolic stress in the absence of viral infection, and this requires a functional kinase domain, as well as a functional dsRNA-binding domain. The endogenous cellular RNA that potentially leads to PKR activation during metabolic stress is unknown. We investigated this question using mouse embryonic fibroblast cells expressing wild-type PKR (PKRWT) or PKR with a point mutation in each dsRNA-binding motif (PKRRM). Using this system, we identified endogenous RNA that interacts with PKR after induction of metabolic stress by palmitic acid (PA) treatment. Specifically, RIP-Seq analyses showed that the majority of enriched RNAs that interacted with WT PKR (≥twofold, false discovery rate ≤ 5%) were small nucleolar RNAs (snoRNAs). Immunoprecipitation of PKR in extracts of UV-cross-linked cells, followed by RT-qPCR, confirmed that snoRNAs were enriched in PKRWT samples after PA treatment, but not in the PKRRM samples. We also demonstrated that a subset of identified snoRNAs bind and activate PKR in vitro; the presence of a 5'-triphosphate enhanced PKR activity compared with the activity with a 5'-monophosphate, for some, but not all, snoRNAs. Finally, we demonstrated PKR activation in cells upon snoRNA transfection, supporting our hypothesis that endogenous snoRNAs can activate PKR. Our results suggest an unprecedented and unexpected model whereby snoRNAs play a role in the activation of PKR under metabolic stress.

Purpose: To develop expert consensus on a suite of reporting standards for HRQL outcomes of RCTs., Methods: A Task Force of The International Society of Quality of Life Research (ISOQOL) undertook a systematic review of the literature to identify candidate reporting standards for HRQL in RCTs. Subsequently, a web-based survey was circulated to the ISOQOL membership. Respondents were asked to rate candidate standards on a 4-point Likert scale based on their perceived value in reporting studies in which HRQL was a study outcome (primary or secondary). Results were synthesized into draft reporting guidelines, which were further reviewed by the membership to inform the final guidance., Results: Forty-six existing candidate standards for reporting HRQL results in RCTs were synthesized to produce a 40 item survey that was completed electronically by 161 respondents. The majority of respondents rated all 40 items to be either 'essential' or 'desirable' when HRQL was a primary RCT outcome. Ratings changed when HRQL was a secondary study outcome. Feedback on the survey findings resulted in the Task Force generalizing the guidance to include patient-reported outcomes (PROs). The final guidance, which recommends standards for use in reporting PROs generally, and more specifically, for PROs identified as primary study outcomes, was approved by the ISOQOL Board of Directors., Conclusions: ISOQOL has developed a suite of recommended standards for reporting PRO results of RCTs. Improved reporting of PROs will enable accurate interpretation of evidence to inform patient choice, aid clinical decision making, and inform health policy.

Purpose: To evaluate the technical quality of external beam radiotherapy for prostate cancer in Canada., Methods: This was a multi-institution, retrospective study of a random sample of patients undergoing radiotherapy (RT) for prostate cancer in Canada. Patterns of care were determined by abstracting details of the patients' management from original records. The quality of patient's technical care was measured against a previously published, comprehensive suite of quality indicators., Results: 32 of the 37 RT centres participated. The total study population of 810 patients included 25% low-risk, 44% intermediate-risk, and 28% high-risk cases. 649 received external beam RT (EBRT) only, for whom compliance with 12 indicators of the quality of pre-treatment assessment ranged from 56% (sexual function documented) to 96% (staging bone scan obtained in high-risk patients). Compliance with treatment-related indicators ranged from 78% (dose to prostate ≥74 Gy in intermediate risk patients not receiving hormone therapy) to 100% (3DCRT or IMRT plan). Compliance varied among centres; no centre demonstrated 100% compliance on all indicators and every centre was 100% compliant on at least some indicators. The number of assessment-related indicators (n=13) with which a given centre was 100% compliant ranged from 4 to 11 (median 7) and the number of the treatment-specific indicators (n=8) with which a given centre was 100% compliant ranged from 6 to 8 (median 8). ADT therapy was utilised in most high-risk cases (191, 92.3%)., Conclusions: While patterns of prostate cancer care in Canada vary somewhat, compliance on the majority of quality indicators is very high. However, all centres showed room for improvement on several indicators and few individual patients received care that met target benchmarks on all quality measures. This variation is particularly important for indicators such as delivered dose where impact on disease outcome is known to exist, and suggests that quality improvement programmes have the potential to further improve quality of care., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Adenosine deaminases that act on RNA (ADARs) deaminate adenosines in dsRNA to produce inosines. ADARs are essential in mammals and are particularly important in the nervous system. Altered levels of adenosine-to-inosine (A-to-I) editing are observed in several diseases. The extent to which an adenosine is edited depends on sequence context. Human ADAR2 (hADAR2) has 5' and 3' neighbor preferences, but which amino acids mediate these preferences, and by what mechanism, is unknown. We performed a screen in yeast to identify mutations in the hADAR2 catalytic domain that allow editing of an adenosine within a disfavored triplet. Binding affinity, catalytic rate, base flipping, and preferences were monitored to understand the effects of the mutations on ADAR reactivity. Our data provide information on the amino acids that affect preferences and point to a conserved loop as being of key importance. Unexpectedly, our data suggest that hADAR2's preferences derive from differential base flipping rather than from direct recognition of neighboring bases. Our studies set the stage for understanding the basis of altered editing levels in disease and for developing therapeutic reagents.

Objectives: To synthesize the results of studies including lymphovascular invasion (LVI) in the multivariate analyses of potential prostate cancer prognostic factors. To determine the role of LVI as an independent prognostic factor for biochemical recurrence in prostate cancer., Patients and Methods: We performed a comprehensive systematic literature review of studies examining the association between LVI in prostatectomy specimens and prostate cancer recurrence. Ovid MEDLINE, Embase, Web of Knowledge, Cochrane Database of Systematic Reviews, Database of Abstracts of Review of Effects (DARE) and Google Scholar were searched from January 2000 to February 2009. The primary outcome of interest was biochemical recurrence measured by serum prostate specific antigen (PSA)., Results: One thousand two hundred and forty-eight papers met our search criteria. Of these, 19 articles meeting our selection criteria reported results of a multivariate analysis to evaluate LVI as an independent prognostic factor of biochemical recurrence. Eleven (58%) of these studies concluded that LVI was an independent prognostic factor. Significant heterogeneity in the study population, disease characteristics and quality of the studies prevented meta-analysis of the results. In the nine studies in which the magnitude of independent association of LVI with recurrence was reported, it ranged from an odds ratio or relative risk of 1.37 to 4.39., Conclusions: The existing literature is conflicting and of insufficient homogeneity to definitively establish LVI as an important independent prognostic factor of biochemical recurrence in prostate cancer prostatectomy specimens. Additional adequately powered studies are required to determine the clinical value of reports of LVI involvement. In the meantime, the use of LVI status as an independent prognostic factor for clinical prognostication and medical decision making is not recommended., (© 2012 THE AUTHORS. BJU INTERNATIONAL © 2012 BJU INTERNATIONAL.)

Oncology health services research (HSR) is a broad, multidimensional field. Current areas of research foci may be informative. We searched Medline for oncology HSR papers published in English in a single year (2009). Abstracted data related to access, quality, cost, health/wellbeing, place on the cancer continuum and study design. Among 1113 papers, the most commonly studied HSR domain was quality-of-care (65%). Within the care continuum, 'treatment' received the greatest attention (37%), and 'prevention' the least (5%). More specifically, treatment-related quality-of-care was most often studied (23%). Breast cancer was the most common site focus (28%). Most studies were descriptive (75%), retrospective (35%) or cross-sectional (35%). These findings might inform the decisions of researchers or policy makers seeking to improve cancer health services delivery.

Purpose: Measurement and reporting of health-related quality of life (HRQL) data have evolved considerably over the past 10 years. Our goal was to identify the current barriers to, and enablers of, the effective translation of HRQL outcome data from randomized clinical trials by investigating physician attitudes, knowledge, and education needs., Methods: We undertook a mixed qualitative and quantitative study of 33 oncologists' attitudes and educational needs around the value, interpretation, and application of HRQL data from cancer clinical trials. The approach was designed to identify barriers and enablers relating to the characteristics of the knowledge itself, to the potential users of the knowledge, and to the environment in which the knowledge is used., Results: The majority of barriers and enablers identified were "second order", i.e., related to the understandability and generalizability of the data, its presentation, its accessibility within the medical literature, and its relevance to specific patient populations., Conclusions: Our results suggest knowledge translation (KT) of HRQL results would improve if the clinical trial HRQL data were easily accessible to clinicians, and presented in a comprehensible and clinically applicable format, which includes discussion of the relevance of the measurement domains and implications of the findings. We recommend that standards of clinical trial HRQL reporting be implemented in clinical journals.

Purpose: To assess the patterns of, and trends over time in, health-related quality of life (HRQL) reporting in randomized controlled trials (RCTs)., Methods: The English-language literature of RCTs published in 2002-2008 was identified using Medline, Embase, and Healthstar databases, in addition to the Cochrane Clinical Trials Registry. Eligible trials were phase III studies that included an HRQL outcome. Data were abstracted on eight outcomes derived from previously recommended quality standards for reporting HRQL, and on four outcomes describing how HRQL data are presented in RCT reports. Two readers examined each article; discrepancies were resolved through discussion and third review if required., Results: A sample of 794 RCTs was identified. HRQL was a primary outcome in 25.4% (200/794). One hundred and ten RCTs (14%) used "supplementary" reports (separate from the first publication) to report HRQL findings. The proportion of RCTs that met the eight quality indicators ranged from 15% (HRQL used in the calculation of sample size) to 81% (reporting instrument validity). RCTs with HRQL as a primary outcome or with a supplementary report had higher concordance on the quality measures. Reporting improved on many indicators over time. Substantive variation in how HRQL data are presented in RCTs was evident., Conclusions: Current practice of reporting HRQL outcomes in RCTs remains highly variable, both with regard to quality of reporting and the patterns of data analysis and presentation. This variation presents challenges for clinicians to apply these data in clinical practice. Consistent reporting practices, which are interpretable by clinicians, are required, as are processes to achieve this consistency in future reports.

Purpose: To develop a set of indicators of the quality of radiotherapy (RT) for localized prostate cancer., Methods and Materials: Following a comprehensive review of the literature to identify candidate quality indicators, we utilized a modified Delphi technique to develop a set of indicators of the quality of RT for localized prostate cancer. The first Delphi round consisted of an online survey in which radiation oncologists were asked to rate the importance of the candidate quality indicators. The second round was a face-to-face meeting of a smaller group of radiation oncologists to discuss, rate, and rank a final set of quality indicators., Results: The literature review identified 57 candidate quality indicators. After the two rounds of the Delphi process, a final set of 25 indicators was agreed upon. The set includes quality indicators covering all aspects of prostate cancer radical RT management: pre-treatment assessment, external beam RT, brachytherapy, androgen deprivation therapy, and follow-up., Conclusions: This new set of quality indicators is more comprehensive than others described in the literature, and can be applied to patterns of care studies that assess the quality of RT for prostate cancer. The process used to develop this set of indicators can be readily adapted for use in other contexts., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

ADARs are a family of enzymes, present in all animals, that convert adenosine to inosine within double-stranded RNA (dsRNA). Inosine and adenosine have different base-pairing properties, and thus, editing alters RNA structure, coding potential and splicing patterns. The first ADAR substrates identified were edited in codons, and ADARs were presumed to function primarily in proteome diversification. Although this is an important function of ADARs, especially in the nervous system, editing in coding sequences is rare compared to editing in noncoding sequences. Introns and untranslated regions of mRNA are the primary noncoding targets, but editing also occurs in small RNAs, such as miRNAs. Although the role of editing in noncoding sequences remains unclear, ongoing research suggests functions in the regulation of a variety of post-transcriptional processes., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Although much has been learned about short noncoding RNAs, long noncoding transcripts are largely uncharacterized. Here, we describe Caenorhabditis elegans rncs-1, a highly base-paired, 800-nucleotide noncoding RNA expressed in hypodermis and intestine. Transcription of rncs-1 is modulated in response to food supply. Although highly double-stranded, we show that rncs-1 RNA is not a substrate for Dicer because of branched structures at its termini. However, rncs-1 RNA inhibits Dicer cleavage of a second dsRNA in vitro, presumably by competition. We validate this observation in vivo by demonstrating that mRNA levels of several Dicer-regulated genes vary with changes in rncs-1 expression. Certain viruses express dsRNA to compete with cellular dsRNA-mediated pathways, and our data suggest that rncs-1 provides a cellular correlate of this phenomenon.

Many biochemical and biophysical analyses of enzymes require quantities of protein that are difficult to obtain from expression in an endogenous system. To further complicate matters, native adenosine deaminases that act on RNA (ADARs) are expressed at very low levels, and overexpression of active protein has been unsuccessful in common bacterial systems. Here we describe the plasmid construction, expression, and purification procedures for ADARs overexpressed in the yeast Saccharomyces cerevisiae. ADAR expression is controlled by the Gal promoter, which allows for rapid induction of transcription when the yeast are grown in media containing galactose. The ADAR is translated with an N-terminal histidine tag that is cleaved by the tobacco etch virus protease, generating one nonnative glycine residue at the N-terminus of the ADAR protein. ADARs expressed using this system can be purified to homogeneity, are highly active in deaminating RNA, and are produced in quantities (from 3 to 10mg of pure protein per liter of yeast culture) that are sufficient for most biophysical studies.

We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1.

Adenosine deaminases that act on RNA (ADARs) constitute a family of RNA-editing enzymes that convert adenosine to inosine within double-stranded regions of RNA. We previously developed a method to identify inosine-containing RNAs and used it to identify five ADAR substrates in Caenorhabditis elegans. Here we use the same method to identify five additional C. elegans substrates, including three mRNAs that encode proteins known to affect neuronal functions. All 10 of the C. elegans substrates are edited in long stem-loop structures located in noncoding regions, and thus contrast with previously identified substrates of other organisms, in which ADARs target codons. To determine whether editing in noncoding regions was a conserved ADAR function, we applied our method to poly(A)+ RNA of human brain and identified 19 previously unknown ADAR substrates. The substrates were strikingly similar to those observed in C. elegans, since editing was confined to 3' untranslated regions, introns, and a noncoding RNA. Also similar to what was found in C. elegans, 15 of the 19 substrates were edited in repetitive elements. The identities of the newly identified ADAR substrates suggest that RNA editing may influence many biologically important processes, and that for many metazoa, A-to-I conversion in coding regions may be the exception rather than the rule.

ADARs are RNA editing enzymes that target double-stranded regions of nuclear-encoded RNA and viral RNA. These enzymes are particularly abundant in the nervous system, where they diversify the information encoded in the genome, for example, by altering codons in mRNAs. The functions of ADARs in known substrates suggest that the enzymes serve to fine-tune and optimize many biological pathways, in ways that we are only starting to imagine. ADARs are also interesting in regard to the remarkable double-stranded structures of their substrates and how enzyme specificity is achieved with little regard to sequence. This review summarizes ongoing investigations of the enzyme family and their substrates, focusing on biological function as well as biochemical mechanism.