The 5-lipoxygenase/cyclooxygenase-2 cross-over metabolite, hemiketal E2, enhances VEGFR2 activation and promotes angiogenesis.

Consecutive oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2 yields the hemiketal eicosanoids, HKE2 and HKD2. Hemiketals stimulate angiogenesis by inducing endothelial cell tubulogenesis in culture; however, how this process is regulated has not been determined. Here, we identify vascular endothelial growth factor receptor 2 (VEGFR2) as a mediator of HKE2-induced angiogenesis in vitro and in vivo. We found that HKE2 treatment of human umbilical vein endothelial cells dose-dependently increased the phosphorylation of VEGFR2 and the downstream kinases ERK and Akt that mediated endothelial cell tubulogenesis. In vivo, HKE2 induced the growth of blood vessels into polyacetal sponges implanted in mice. HKE2-mediated effects in vitro and in vivo were blocked by the VEGFR2 inhibitor vatalanib, indicating that the pro-angiogenic effect of HKE2 was mediated by VEGFR2. HKE2 covalently bound and inhibited PTP1B, a protein tyrosine phosphatase that dephosphorylates VEGFR2, thereby providing a possible molecular mechanism for how HKE2 induced pro-angiogenic signaling. In summary, our studies indicate that biosynthetic cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways gives rise to a potent lipid autacoid that regulates endothelial cell function in vitro and in vivo. These findings suggest that common drugs targeting the arachidonic acid pathway could prove useful in antiangiogenic therapy.

Machine Learning Informs RNA-Binding Chemical Space.

Small molecule targeting of RNA has emerged as a new frontier in medicinal chemistry, but compared to the protein targeting literature our understanding of chemical matter that binds to RNA is limited. In this study, we reported Repository Of BInders to Nucleic acids (ROBIN), a new library of nucleic acid binders identified by small molecule microarray (SMM) screening. The complete results of 36 individual nucleic acid SMM screens against a library of 24 572 small molecules were reported (including a total of 1 627 072 interactions assayed). A set of 2 003 RNA-binding small molecules was identified, representing the largest fully public, experimentally derived library of its kind to date. Machine learning was used to develop highly predictive and interpretable models to characterize RNA-binding molecules. This work demonstrates that machine learning algorithms applied to experimentally derived sets of RNA binders are a powerful method to inform RNA-targeted chemical space.

The Importance of Risk Management: What is Missing in ISO Standards?

The overall aim of this article is to contribute to the further development of the area of risk analysis and risk management in the International Organization for for Standardization (ISO) standards by strengthening its scientific basis. Industrial standards, especially ISO standards, are the tools organizations use to manage their risk, through following their guidance and complying with their requirements. Organizations confirm their compliance with these standards through certification, which means that they heavily depend upon the quality of the ISO standards to enable them to effectively manage their risk. The purpose of this study is to investigate what guidance is given on key elements of risk management and how well ISO standards are aligned with state-of-the-art risk management literature. Eighteen ISO standards, all addressing risk management, were reviewed in this study with regard to risk terminology and guidance. The results of the study confirm the increasing importance of risk management for business. However, the study also shows a lack of guidance on doing risk analysis in the industrial standards examined. The ISO management system standards and guidelines are not aligned with the scientific literature on risk and are not appropriate for the management of risk arising from complex interactions and emergent behavior that is inherent in present-day sociotechnical systems.

HNRNPH1-dependent splicing of a fusion oncogene reveals a targetable RNA G-quadruplex interaction.

The primary oncogenic event in ∼85% of Ewing sarcomas is a chromosomal translocation that generates a fusion oncogene encoding an aberrant transcription factor. The exact genomic breakpoints within the translocated genes, EWSR1 and FLI1, vary; however, in EWSR1, breakpoints typically occur within introns 7 or 8. We previously found that in Ewing sarcoma cells harboring EWSR1 intron 8 breakpoints, the RNA-binding protein HNRNPH1 facilitates a splicing event that excludes EWSR1 exon 8 from the EWS-FLI1 pre-mRNA to generate an in-frame mRNA. Here, we show that the processing of distinct EWS-FLI1 pre-mRNAs by HNRNPH1, but not other homologous family members, resembles alternative splicing of transcript variants of EWSR1 We demonstrate that HNRNPH1 recruitment is driven by guanine-rich sequences within EWSR1 exon 8 that have the potential to fold into RNA G-quadruplex structures. Critically, we demonstrate that an RNA mimetic of one of these G-quadruplexes modulates HNRNPH1 binding and induces a decrease in the growth of an EWSR1 exon 8 fusion-positive Ewing sarcoma cell line. Finally, we show that EWSR1 exon 8 fusion-positive cell lines are more sensitive to treatment with the pan-quadruplex binding molecule, pyridostatin (PDS), than EWSR1 exon 8 fusion-negative lines. Also, the treatment of EWSR1 exon 8 fusion-positive cells with PDS decreases EWS-FLI1 transcriptional activity, reversing the transcriptional deregulation driven by EWS-FLI1. Our findings illustrate that modulation of the alternative splicing of EWS-FLI1 pre-mRNA is a novel strategy for future therapeutics against the EWSR1 exon 8 containing fusion oncogenes present in a third of Ewing sarcoma.

Validity of Race, Ethnicity, and National Origin in Population-based Cancer Registries and Rapid Case Ascertainment Enhanced With a Spanish Surname List.

BACKGROUND: Accurate information regarding race, ethnicity, and national origins is critical for identifying disparities in the cancer burden. OBJECTIVES: To examine the use of a Spanish surname list to improve the quality of race-related information obtained from rapid case ascertainment (RCA) and to estimate the accuracy of race-related information obtained from cancer registry records collected by routine reporting. SUBJECTS: Self-reported survey responses of 3954 participants from California enrolled in the Cancer Care Outcomes Research and Surveillance Consortium. MEASURES: Sensitivity, specificity, positive predictive value, and percent agreement. We used logistic regression to identify predictors of underreporting and overreporting of a race/ethnicity. RESULTS: Use of the Spanish surname list increased the sensitivity of RCA for Latino ethnicity from 37% to 83%. Sensitivity for cancer registry records collected by routine reporting was ≥95% for whites, blacks, and Asians, and specificity was high for all groups (86%-100%). However, patterns of misclassification by race/ethnicity were found that could lead to biased cancer statistics for specific race/ethnicities. Discordance between self-reported and registry-reported race/ethnicity was more likely for women, Latinos, and Asians. CONCLUSIONS: Methods to improve race and ethnicity data, such as using Spanish surnames in RCA and instituting data collection guidelines for hospitals, are needed to ensure minorities are accurately represented in clinical and epidemiological research.

Mechanistic Analysis of Riboswitch Ligand Interactions Provides Insights into Pharmacological Control over Gene Expression.

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device.

Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect posttranslational modifications with single-molecule sensitivity. Here, we demonstrate single-molecule protein sequencing using a dynamic approach in which single peptides are probed in real time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. We annotate amino acids and identify the peptide sequence by measuring fluorescence intensity, lifetime, and binding kinetics on an integrated semiconductor chip. Our results demonstrate the kinetic principles that allow recognizers to identify multiple amino acids in an information-rich manner that enables discrimination of single amino acid substitutions and posttranslational modifications. With further development, we anticipate that this approach will offer a sensitive, scalable, and accessible platform for single-molecule proteomic studies and applications.

Chemical Modulation of Pre-mRNA Splicing in Mammalian Systems.

RNA splicing is a key component of gene expression and proteomic diversity in humans. The spliceosome assembles on and processes individual nascent pre-mRNA transcripts into distinct mature mRNAs that can code for different proteins. Splicing programs can be affected by somatic mutations and changes in response to exogenous stimuli. Importantly, alterations in splicing can be direct drivers of diseases including cancers. This Review describes recent advances and the potential for targeting and controlling pre-mRNA splicing in humans with small molecules, ranging from targeting spliceosomal proteins to direct targeting of individual RNA transcripts.

Custom DNA Microarrays Reveal Diverse Binding Preferences of Proteins and Small Molecules to Thousands of G-Quadruplexes.

Single-stranded DNA (ssDNA) containing four guanine repeats can form G-quadruplex (G4) structures. While cellular proteins and small molecules can bind G4s, it has been difficult to broadly assess their DNA-binding specificity. Here, we use custom DNA microarrays to examine the binding specificities of proteins, small molecules, and antibodies across ∼15,000 potential G4 structures. Molecules used include fluorescently labeled pyridostatin (Cy5-PDS, a small molecule), BG4 (Cy5-BG4, a G4-specific antibody), and eight proteins (GST-tagged nucleolin, IGF2, CNBP, FANCJ, PIF1, BLM, DHX36, and WRN). Cy5-PDS and Cy5-BG4 selectively bind sequences known to form G4s, confirming their formation on the microarrays. Cy5-PDS binding decreased when G4 formation was inhibited using lithium or when ssDNA features on the microarray were made double-stranded. Similar conditions inhibited the binding of all other molecules except for CNBP and PIF1. We report that proteins have different G4-binding preferences suggesting unique cellular functions. Finally, competition experiments are used to assess the binding specificity of an unlabeled small molecule, revealing the structural features in the G4 required to achieve selectivity. These data demonstrate that the microarray platform can be used to assess the binding preferences of molecules to G4s on a broad scale, helping to understand the properties that govern molecular recognition.

Synthetic ligands for PreQ1 riboswitches provide structural and mechanistic insights into targeting RNA tertiary structure.

Riboswitches are naturally occurring RNA aptamers that regulate gene expression by binding to specific small molecules. Riboswitches control the expression of essential bacterial genes and are important models for RNA-small molecule recognition. Here, we report the discovery of a class of synthetic small molecules that bind to PreQ1 riboswitch aptamers. These molecules bind specifically and reversibly to the aptamers with high affinity and induce a conformational change. Furthermore, the ligands modulate riboswitch activity through transcriptional termination despite no obvious chemical similarity to the cognate ligand. X-ray crystallographic studies reveal that the ligands share a binding site with the cognate ligand but make different contacts. Finally, alteration of the chemical structure of the ligand causes changes in the mode of RNA binding and affects regulatory function. Thus, target- and structure-based approaches can be used to identify and understand the mechanism of synthetic ligands that bind to and regulate complex, folded RNAs.

Cost-effectiveness of overactive bladder treatments: from the US payer perspective.

AIM: To assess the cost-effectiveness of onabotulinumtoxinA (onabotA), implantable sacral nerve stimulation devices, percutaneous tibial nerve stimulation, anticholinergic medications and mirabegron compared with best supportive care (BSC) for management of refractory overactive bladder (OAB). METHODS: A Markov model was developed to compare the cost-effectiveness of treatment options with BSC over a 10-year time horizon. Resource utilization, discontinuation rates and costs were derived from unpublished and published sources. Quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratios were reported. RESULTS: Treatment with onabotA 100U produced the largest gain in QALYs (7.179) and lowest estimated incremental cost-effectiveness ratio ($32,680/QALY) of all assessed treatments compared with BSC. CONCLUSION: Compared with BSC, onabotA 100U was the most cost-effective treatment option for patients with refractory OAB.

Targeting Mammalian Translational Inhibition with Tetracyclines.

In this issue of Cell Chemical Biology, Mortison et al. (2018) report an in-depth mechanistic study of targets of two different tetracyclines in mammalian cells. Unbiased chemoproteomics and RNA sequence mapping help identify specific ribosomal substructures bound by tetracyclines, providing insight into the therapeutic potential for tetracyclines in many diseases.

Total Synthesis and Biological Activity of the Arachidonic Acid Metabolite Hemiketal E2.

The total synthesis of hemiketal E2 (HKE2) has been accomplished using a gold(I)-mediated cycloisomerization followed by oxidation of the enol ether product to introduce a unique keto-hemiketal, the core structure of HKE2. Synthetic hemiketal E2 reproduced biosynthetically derived HKE2 in the inhibition of human platelet aggregation.

Discovery of Inhibitors of MicroRNA-21 Processing Using Small Molecule Microarrays.

The identification of small molecules that bind to and perturb the function of microRNAs is an attractive approach for the treatment for microRNA-associated pathologies. However, there are only a few small molecules known to interact directly with microRNAs. Here, we report the use of a small molecule microarray (SMM) screening approach to identify low molecular weight compounds that directly bind to a pre-miR-21 hairpin. Compounds identified using this approach exhibit good affinity for the RNA (ranging from 0.8-2.0 µM) and are not composed of a polycationic scaffold. Several of the highest affinity compounds inhibit Dicer-mediated processing, while in-line probing experiments indicate that the compounds bind to the apical loop of the hairpin, proximal to the Dicer site. This work provides evidence that small molecules can be developed to bind directly to and inhibit miR-21.

Cardiovascular and Renal Outcomes Trials-Is There a Difference?

There is a general sense that most outcomes trials in patients receiving dialysis failed to yield statistically significant benefits, in contrast to many cardiovascular (CV) trials in the general population. It is unknown whether methodologic reasons caused this discrepancy. We performed a systematic MEDLINE search for randomized trials with mortality end points of the 42 compounds most commonly used for CV indications. In total, 115 trials were selected for review. We further reviewed 9 mortality end point trials in patients receiving dialysis. The CV trials in populations not receiving dialysis enrolled from 66 to 33,357 participants with an average of 4,910; 59% of the trials showed statistically significant results. The average hazard ratio (HR) was 0.77, ranging from 0.10 to 1.65; 10 drugs had ≥5 published trials each. In the population receiving dialysis, most drugs were studied in single trials; the average number of patients was 1,500 with a range of 127 to 3,883. The average HR was 0.77 and ranged from 0.06 to 1.30. Only 22% of the trials showed statistically significant results. The limitations listed in the general population and dialysis studies were similar. In conclusion, no apparent methodologic issues were detected (other than sample size) that could justify the lower frequency of randomized trials with statistically significant results in patients receiving dialysis. The most obvious difference was the paucity of trials with each drug in the dialysis cohorts; this lowers the chances of at least 1 trial being successful.

Impact of reduced tobacco smoking on lung cancer mortality in the United States during 1975-2000.

BACKGROUND: Considerable effort has been expended on tobacco control strategies in the United States since the mid-1950s. However, we have little quantitative information on how changes in smoking behaviors have impacted lung cancer mortality. We quantified the cumulative impact of changes in smoking behaviors that started in the mid-1950s on lung cancer mortality in the United States over the period 1975-2000. METHODS: A consortium of six groups of investigators used common inputs consisting of simulated cohort-wise smoking histories for the birth cohorts of 1890 through 1970 and independent models to estimate the number of US lung cancer deaths averted during 1975-2000 as a result of changes in smoking behavior that began in the mid-1950s. We also estimated the number of deaths that could have been averted had tobacco control been completely effective in eliminating smoking after the Surgeon General's first report on Smoking and Health in 1964. RESULTS: Approximately 795,851 US lung cancer deaths were averted during the period 1975-2000: 552,574 among men and 243,277 among women. In the year 2000 alone, approximately 70,218 lung cancer deaths were averted: 44,135 among men and 26,083 among women. However, these numbers are estimated to represent approximately 32% of lung cancer deaths that could have potentially been averted during the period 1975-2000, 38% of the lung cancer deaths that could have been averted in 1991-2000, and 44% of lung cancer deaths that could have been averted in 2000. CONCLUSIONS: Our results reflect the cumulative impact of changes in smoking behavior since the 1950s. Despite a large impact of changing smoking behaviors on lung cancer deaths, lung cancer remains a major public health problem. Continued efforts at tobacco control are critical to further reduce the burden of this disease.