Translational Research Center Research Articles

Dopamine D1 Agonists, Working Memory, and the Prefrontal Cortex: Effect of Receptor Functional Selectivity/Signaling Bias

Dopamine D1 agonists produce an ‘inverted‐U’ dose‐response on working memory‐related behavioral tasks (i.e., low doses improve but high doses have no effect or tend to impair performance). This dose‐dependency has been observed at the single neuron level in the prefrontal cortex, where the cellular basis of working memory is represented. Few studies, however, have examined this process at the neuron population level and its signaling mechanism(s) is still unclear. In the current study, two D1 agonists, 2‐methyldihydrexidine (2MDHX) and CY208,243 (CY208), were examined and compared in rats performing a spatial working memory‐related T‐maze task. 2MDHX is a full agonist at adenylate cyclase and a super‐agonist at β‐arrestin recruitment, whereas CY208 has relatively high intrinsic activity at adenylate cyclase, but much lower at β‐arrestin recruitment. Both compounds had inverted U‐dose response curves at both the behavioral and prefrontal neuron population level, albeit with distinct differences. At behavioral level, they both caused similar improvement with high potency (10 nmol/kg). Conversely, the right‐hand (descending) part of the inverted U dose‐response curve had a great slope for 2MDHX than for CY208, suggesting an extended effective range for a compound with lower β‐arrestin activity. At the neuron population level, both drugs affected the percentage, uniformity, and ensemble strength of sensitivity in a dose‐dependent manner, but 2MDHX influenced neuron activity more before the choice in the T‐maze task whereas CY208 influenced it more after the choice. As the off‐target effects were not likely engaged at the very low doses (i.e., nmol/kg range) utilized in the current study, the differences between 2MDHX and CY208 most likely are related to their specific D1 functional selectivity profiles. The ‘inverted‐U’ dose‐response of D1 agonists on cognitive performance has been observed in many of animal models and confirmed in humans. Although the cognitive improvement at the optimal dose is impressive, the biphasic effect complicates potential clinical use of D1 drugs. Our data indicate that differential signaling may play a role in D1‐related dose‐dependent regulation of cognition, and suggest that D1‐related dose‐dependent regulation of working memory could be modified by functionally selective/biased ligands. This may help guide the discovery of novel D1 compounds that may yield a more monotonic response and therefore translate to the clinic more readily.Support or Funding InformationBrain & Behavior Research Foundation Young Investigator Grant # 19469 to YY, the 2017 Children’s Miracle Network Research Grant #10 to YY and RBM, the Penn State Translational Brain Research Center, and R01 NS105471 to RBM. Conflict of interest: Dr. Mailman is an inventor on patents related to D1 ligand discovery, and this conflict is disclosed and managed by the Penn State College of Medicine.

Mapping the Minimal Promoter Sequence for Human Prolidase (Peptidase D; PEPD‐ gene)

Collagen, the most abundant protein in humans, is the primary component of the extracellular matrix. Collagen is also a critical reservoir of proline that, along with hydroxyproline, comprise one‐fourth of the amino acid composition of collagen. During collagen turnover, sequential action of matrix metalloproteinases and peptidases convert collagen into dipeptides. In the subsequent final rate‐limiting step, prolidase (PEPD) hydrolyzes dipeptides containing C‐terminal proline or hydroxyproline into the constituent amino acids, which are then recycled towards collagen synthesis. Due to its critical role in collagen turnover and protein metabolism, prolidase is a key player in several physiological and pathological processes, including wound healing, cell proliferation, inflammation, and carcinogenesis. Consequently, alterations in prolidase expression can profoundly affect normal physiological processes and cause pathological changes. The goal of this study is to narrow down such knowledge gaps, especially, in our understanding of the regulation of PEPD transcription. First, towards determining a functionally active promoter region of PEPD (PEPDp), we cloned a 1587 base pair (bp) DNA region—spanning 1537 bp upstream, and 50 bp downstream, of the transcription start site (TSS)—of PEPD gene into a firefly luciferase reporter plasmid. We confirmed that the PEPDp is functionally active by demonstrating its ability to robustly drive the luciferase expression in the HEK293T cell line. Second, we sought to determine the minimal region of PEPDp that is sufficient and necessary to drive gene expression. We subcloned two progressively shorter deletion fragments of PEPDp—537 bp and 387 bp long into the firefly luciferase reporter system and then compared their promoter activity with that of the full‐length PEPDp. Our findings thus indicate that the lack of any cancellation in the promoter activity of the PEPDp deletion fragments points out that the functionally active region of PEPDp spans ~350 bp upstream and 50 bp downstream of the transcriptional start site (TSS), thereby advancing our knowledge of the molecular mechanisms regulating prolidase expression.Support or Funding InformationThis work is partly supported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni‐aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).

Control of Initiation and Aggressiveness of DMBA‐Induced Breast Cancer in Rats by Dietary Consumption of Hinute‐AM.

We reported earlier that the consumption of soy protein controlled not only the proliferation of dimethylbenz [a] anthracene (DMBA)‐induced breast cancer in rats but also its aggressiveness. Soy protein is hydrolyzed in the gut to small peptides which are easily absorbed and are involved in various cellular functions. Fuji Oil Company, Osaka, Japan, has commercially marketed a soy protein hydrolysate, termed Hinute‐AM, composed of primarily di‐ and tripeptides, to be used as a health food, and it has anti‐inflammatory property. The purpose of the present study was to investigate whether dietary intake of Hinute‐AM can delay the development of DMBA‐induced breast cancer and its aggressiveness in rats. For this study, we obtained 22‐days old female Sprague Dawley rats from Charles River (Wilmington, MA). Animals were divided into 5 groups, each containing 5 animals. Group 1 received standard AIN‐76A diet (Envigo – Teklad Diets, Madison, WI) containing 20% casein and 0% Hinute‐AM; Group 2 received the same diet containing 15% casein + 5% Hinute‐AM; Group 3 received the same diet, but containing 10% casein + 10% Hinute‐AM; Group 4 received the same diet but containing 5% casein + 15% Hinute‐AM; and Group 5 received the same diet but containing 0% casein + 20% Hinute‐AM. For induction of mammary tumors, all groups received a single intragastric administration of DMBA (Sigma Chemical Co., St. Louis) dissolved in sesame oil (80 mg/kg b.wt.) at 50 days of age. At 120 days post DMBA injection, the animals were euthanized by CO2 asphyxiation. All tumors were weighed and measured for volume, and a section of tumor was subjected to histological grading by a certified pathologist. Our preliminary data revealed that replacement of casein by Hinute‐AM as a source of dietary protein caused a dose‐dependent delay in the development of breast tumor, decrease in breast cancer incidence, a decrease in tumor volume, and tumor grade.Support or Funding InformationThis work was supported by grants from Fuji Oil Company, Osaka, Japan and by the Meharry Translational Research Center grant NIH 5U54MD007593

HIV‐1 Capsid Binding Host Factor CPSF6 is Post Transcriptionally Regulated by the Cellular MicroRNA miR‐125b

The cleavage and polyadenylation specificity factor 6 (CPSF6) is a cellular protein involved in RNA cleavage and polyadenylation. Emerging evidence also suggest that CPSF6 plays a key role during HIV‐1 infection by guiding integration of the viral DNA into gene‐dense regions of the host genome. Since viral DNA integration is a critical step of HIV‐1 infection, the role of CPSF6 in the virus lifecycle is being intensely investigated. Surprisingly, the cellular mechanisms that regulate CPSF6 expression are largely unknown. In this study, we report a post‐transcriptional mechanism of regulation of CPSF6. Our initial bioinformatics analysis revealed that the 3′ untranslated region (3′UTR) of Cpsf6 contains a binding site for the cellular miRNA miR‐125b that is strikingly conserved across different mammalian species. Since miRNAs negatively regulate protein expression, we carried out knock‐down and over‐expression studies of miR‐125b. Results from these experiments revealed that miR‐125b expression is negatively associated with CPSF6 protein levels. Interestingly, HIV‐1 infection resulted in the down‐regulation of miR‐125b concurrent with induction of CPSF6. To probe that CPSF6 expression is post‐transcriptionally regulated by miR‐125b, we cloned the 3′UTR regions of Cpsf6 mRNA into a luciferase reporter. Results from luciferase assay provide evidence that miR‐125b expression negatively regulates Cpsf6 3′UTR activity. Accordingly, mutations in the miR‐125b seed sequences abrogated the regulatory effect of the miRNA on Cpsf6 3′UTR. Pull‐down studies demonstrated that miR‐125b physically interacts with the Cpsf6 mRNA. Continuing studies probe the necessity of productive HIV‐1 infection for negatively regulating miR‐125b expression as well as to elucidate the pathway through which HIV‐1 infection can knockdown miR‐125b expression. Collectively, these findings establish a post‐transcriptional mechanism of CPSF6 expression and describe a novel function of miR‐125b in virus‐host interaction.Support or Funding InformationThis work is partly supported by grants DA024558, DA30896, DA033892 and DA021471 from NIDA/NIH to CD. We also acknowledge the RCMI Grant G12MD007586, the Vanderbilt CTSA grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 grant MD007593 from NIMHD/NIH, and Tennessee CFAR grant (P30 AI110527).

Studying KLF6 and PEPD‐gene Promoter by Chromatin Immunoprecipitation Assay

Prolidase (PEPD) is a ubiquitously expressed cytosolic dipeptidase that has the unique ability to cleave proline or hydroxyproline on the C‐terminus of imidodipeptides and imidotripeptides. The reaction is vital for collagen degradation. Since collagen is the most abundant protein in the body, our objective is to get a better understanding of the enzymes involved in its turnover is crucial. In silico analysis on prolidase promoter showed that putative transcription factor of interest is KLF6. To test the hypothesis that the expression of PEPD gene may be regulated by the binding of the transcription factor KLF6 to candidate binding sites we used chromatin immunoprecipitation (ChIP) assay. HEK293T cells were transfected with FLAG‐hKLF6 plasmid and the cellular lysates were obtained to measure the KLF6 protein expression by immunoblotting. After that, purified DNA from the ChIP assays were used in end‐point PCR with primers targeting candidate KLF6‐binding sites in PEPD promoter. The primers used were expected to yield a 615 bp amplicon. PCR products were resolved on 1% agarose/TAE gel. The second round of PCR was performed in which endpoint PCR products from the first round of PCR were used as a template for nested PCR with primers designed to amplify 164 bp. Nevertheless, our results showed no detectable PEPD promoter‐specific signals in the PCR analysis of the ChIP products. However, due to the inherent caveats in the experimental design, choice of reagents, and analysis method, it would be premature to conclude at this time point that PEPD expression is not regulated by KLF6. Accordingly, the design of future experiments will effectively address the limitations of this current work and will help reveal the identity of transcription factors regulating PEPD expression.Support or Funding InformationSupported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni‐Aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).

An epigenome-wide association study of posttraumatic stress disorder in US veterans implicates several new DNA methylation loci

BackgroundPrevious studies using candidate gene and genome-wide approaches have identified epigenetic changes in DNA methylation (DNAm) associated with posttraumatic stress disorder (PTSD).MethodsIn this study, we performed an EWAS of PTSD in a cohort of Veterans (n = 378 lifetime PTSD cases and 135 controls) from the Translational Research Center for TBI and Stress Disorders (TRACTS) cohort assessed using the Illumina EPIC Methylation BeadChip which assesses DNAm at more than 850,000 sites throughout the genome. Our model included covariates for ancestry, cell heterogeneity, sex, age, and a smoking score based on DNAm at 39 smoking-associated CpGs. We also examined in EPIC-based DNAm data generated from pre-frontal cortex (PFC) tissue from the National PTSD Brain Bank (n = 72).ResultsThe analysis of blood samples yielded one genome-wide significant association with PTSD at cg19534438 in the gene G0S2 (p = 1.19 × 10-7, padj = 0.048). This association was replicated in an independent PGC-PTSD-EWAS consortium meta-analysis of military cohorts (p = 0.0024). We also observed association with the smoking-related locus cg05575921 in AHRR despite inclusion of a methylation-based smoking score covariate (p = 9.16 × 10-6), which replicates a previously observed PGC-PTSD-EWAS association (Smith et al. 2019), and yields evidence consistent with a smoking-independent effect. The top 100 EWAS loci were then examined in the PFC data. One of the blood-based PTSD loci, cg04130728 in CHST11, which was in the top 10 loci in blood, but which was not genome-wide significant, was significantly associated with PTSD in brain tissue (in blood p = 1.19 × 10-5, padj = 0.60, in brain, p = 0.00032 with the same direction of effect). Gene set enrichment analysis of the top 500 EWAS loci yielded several significant overlapping GO terms involved in pathogen response, including “Response to lipopolysaccharide” (p = 6.97 × 10-6, padj = 0.042).ConclusionsThe cross replication observed in independent cohorts is evidence that DNA methylation in peripheral tissue can yield consistent and replicable PTSD associations, and our results also suggest that that some PTSD associations observed in peripheral tissue may mirror associations in the brain.

Lessons Learned in Evaluating the Infrastructure of a Centre for Translational Research (CTR).

This paper shares lessons learned while evaluating the implementation of a Clinical and Translational Research Center (CTR). To meet its overarching goals the CTR consists of numerous research support units (e.g., biostats, community engagement, professional development, etc.) that are intended to work together collaboratively. It is then argued that an evaluation approach grounded in system thinking was the best fit to evaluate this key CTR design feature. The rationale for selecting Systems Evaluation Theory (SET) as the evaluation framework best suited to evaluate the CTR infrastructure is then presented. The application of SET and the lessons learned are then shared. The paper concludes that there are many similarly structured programs worldwide to which the lessons learned can be applied and upfront investments in using a system approach are rewarded by providing meaningful and useful evaluation recommendations for system change.

Novel Compounds Identified by Structure-Based Prion Disease Drug Discovery Using <i>In Silico</i> Screening Delay the Progression of an Illness

The accumulation of abnormal prion protein (PrPSc) produced by the structure conversion of PrP (PrPC) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, the intramolecular chemical bridging in the most fragile pocket of PrP, known as the “Hot Spot”, can stabilize the structure of PrPC and inhibit the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that can bind to the hot spot. Several candidates were identified as anti-prion drugs with strong binding to recombinant PrP in vitro that significantly reduced the levels of PrPSc. These compounds were able to decrease the number of aggresomes in persistently prion-infected cells. Remarkably, treatment with the predominant candidate compounds significantly prolonged the survival period of prion-infected mice and dominantly suppressed disease-specific pathological damage, such as vacuole degeneration, PrPSc accumulation, microgliosis and astrogliosis in the brain, suggesting their possible clinical use. Fragment molecular orbital (FMO) analysis indicated that the high affinity of the candidates to the protein is largely dependent on non-polar interactions, such as van der Waals interactions. Our results suggest that in silico drug discovery using NUDE/DEGIMA may be useful to identify candidate compounds that effectively stabilize the protein. Funding Statement: This work was supported in part by the a grant from a Grant-in-Aid of the Research Committee of Prion Disease and Slow Virus Infection from the Ministry of Health, Labour and Welfare of Japan (N.N.); a Grant-in-Aid of the Research Committee of Molecular Pathogenesis and Therapies for Prion Disease and Slow Virus Infection, the Practical Research Project for Rare and Intractable Disease from the Japan Agency for Medical Research and Development, AMED (D.I.); a grant from the Takeda Science Foundation (N.N. and D.I.); a grant from the Japan Intractable Disease Research Foundation (D.I.); a Grant-in-Aid from the Tokyo Biochemical Research Foundation (D.I.); a grant provided by the YOKOYAMA Foundation for Clinical Pharmacology (Grant No. YRY1502) (D.I.); the grant provided by the Ichiro Kanehara Foundation (D.I.); a grant provided by the Mochida Memorial Foundation for Medical and Pharmaceutical Research (D.I.); a grant provided by the Waksman Foundation of Japan Inc. (D.I.); and a grant provided by the Center for Clinical and Translational Research of Kyushu University Hospital (D.I.). Declaration of Interests: The authors state that they have no conflicts of interest. Ethics Approval Statement: Animal experiments were approved by the Animal Care and Use Committee of the Nagasaki University. All animals were treated and cared for in accordance with the Guidelines for Animal Experimentation of Nagasaki University.

Abstract P3-03-08: Identification of drugs that induce the death or suppress the growth of p53-mutant breast cancer

Abstract Background: P53 is the most commonly mutated protein in cancer with more than 40% of all cancer patients harboring a p53 mutation. Up to 85% of triple-negative breast cancers (TNBCs), aggressive breast cancers characterized by the absence of ER, PR, and Her2 receptors, have p53 mutations. P53 is a tumor suppressor that helps balance cellular growth and death after a genomic insult. Due to the rate of p53 mutation in cancer, the targeting of p53 in a clinical setting is an attractive option. However, currently there are no available p53-targeted drugs. We hypothesized that molecular pathways exist, that when inhibited, induce death or suppress growth of p53-mutant breast cancer cells, without affecting p53 wild-type breast cells. To investigate this hypothesis, we conducted a high-throughput drug screen to identify drugs that induce death or suppress growth of p53-mutant TNBCs. Methods: A library of 453 drugs with diverse and known mechanisms of action was used to perform high-throughput drug screening through collaboration with Texas A&M’s Center for Translational Cancer Research. An ATP luminescence assay was used to test eight TNBC cell lines with distinct p53 mutations in this screen. We then tested a subset of these drugs, identified to have an effect on p53-mutant TNBC, on the growth of five p53 wild-type breast cancer cell lines. Cell death assays to differentiate between growth suppression and death-inductive phenotypes are currently underway. Results: 145 drugs were identified that induce death or decrease growth by a minimum of 50% in p53-mutant TNBC cell lines. Of these drugs, 16 drugs were found to differentially suppress the growth of p53-mutant breast cancer cells, as compared to p53-wild type breast cancer cells. Further analysis of these drugs identified proteasome, apoptosis, and iron-dependent cell death pathways as potential p53-mutant specific targets in TNBCs. Conclusion: A screen of molecularly annotated drugs was performed in p53-mutant and p53-wild type breast cancer cells to identify drugs that preferentially kill or suppress the growth of p53-mutant breast cancers. These studies identified 145 drugs that were able to induce death or decrease growth by a minimum of 50% in p53-mutant TNBC cell lines. Testing these drugs in p53 wild-type breast cancer cell lines demonstrated that several of the identified drugs suppress growth or induce death preferentially in p53-mutant, as compared to p53 wild-type, breast cancers. These drugs affect specific cellular pathways such as proteasomal degradation, ferroptosis, and apoptosis pathways, which appear to be critical for the growth and survival of p53-mutant breast cancers. Further work will elucidate the interplay between p53-mutation and the identified cellular pathways. These studies will provide the basic science foundation for the development of new drugs for the treatment of p53-mutant breast cancer. This work was supported by a generous gift from the John Charles Cain Family. Citation Format: William Miska Tahaney, Reid Powell, Nghi Nguyen, Lakshmi Reddy Bollu, Jing Qian, Abhijit Mazumdar, Clifford Stephan, Peter JA Davies, Powel H Brown. Identification of drugs that induce the death or suppress the growth of p53-mutant breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-08.

Cell death and survival due to cytotoxic exposure modelled as a two-state Ising system.

Cancer chemotherapy agents are assessed for their therapeutic utility primarily by their ability to cause apoptosis of cancer cells and their potency is given by an IC50 value. Chemotherapy uses both target-specific and systemic-action drugs and drug combinations to treat cancer. It is important to judiciously choose a drug type, its dosage and schedule for optimized drug selection and administration. Consequently, the precise mathematical formulation of cancer cells' response to chemotherapy may assist in the selection process. In this paper, we propose a mathematical description of the cancer cell response to chemotherapeutic agent exposure based on a time-tested physical model of two-state multiple-component systems near criticality. We describe the Ising model methodology and apply it to a diverse panel of cytotoxic drugs administered against numerous cancer cell lines in a dose–response manner. The analysed dataset was generated by the Netherlands Translational Research Center B.V. (Oncolines). This approach allows for an accurate and consistent analysis of cytotoxic agents' effects on cancer cell lines and reveals the presence or absence of the bystander effect through the interaction constant. By calculating the susceptibility function, we see the value of IC50 coinciding with the peak of this measure of the system's sensitivity to external perturbations.

The Impact of Obesity On Insulin Sensitivity and Secretion During Pubertal Progression: A Longitudinal Study.

Physiologic changes in glucose metabolism are well-described to occur during puberty. However, there are important gaps in understanding the interaction between obesity and the normal physiologic changes during puberty, as well as how these changes could contribute to the increased risk of comorbidities, such as type 2 diabetes and dyslipidemia, in youth with obesity. The objective of this study was to compare longitudinal changes in insulin sensitivity (Si) and secretion during pubertal progression in youth with obesity versus those with normal weight. Longitudinal observational study evaluating youth from early puberty (Tanner [T]2-T3) until puberty completion (T5). Pediatric academic hospital Clinical Translational Research Center. Pubertal youth with normal weight (n = 47; 22 female, 25 male) and obesity (n = 37; 23 female, 14 male). Si, insulin response (acute insulin response to glucose, AIRg) and disposition index (DI) by intravenous glucose tolerance test at baseline (T2-T3), T4, and T5. Youth with obesity had significantly lower Si and higher AIRg at each time point (P < 0.001), but DI was similar between the groups. There were no group differences in trajectory of Si, AIRg or DI over time. Leptin, insulin-like growth factor-1, and obesity were most strongly associated with Si and AIRg at all time points. Obesity significantly impacts Si during puberty, even at the earliest stages. However, in general, obese youth have adequate β-cell compensation for the significantly reduced Si of puberty. Future studies are needed to better predict the subset of youth who fail to maintain β-cell compensation during puberty.

Primary Care Contact for Mental Illness and Self-Harm Before, During and After the Peak of the COVID-19 Pandemic in the UK: Cohort Study of 13 Million Individuals

Background: The Covid-19 pandemic has adversely affected population mental health.Methods: Using electronic health records from 1714 UK general practices registered with the Clinical Practice Research Datalink we examined incidence and event rates of depression and anxiety disorders, self-harm, prescriptions for antidepressants and benzodiazepines and GP referrals to mental health services per 100,000 person-months, before, during and after the peak of the Covid-19 emergency. Analyses were stratified by gender, age group and practice-level Index of Multiple Deprivation quintile.Findings: In April 2020, primary care-recorded incident depression reduced by 43·6% (95% CI 39·0% to 47·9%), anxiety disorders by 48·2% (CI 44·6% to 51·5%) and antidepressant prescribing by 36·2% (CI 33·7% to 38·6%) compared to expected rates based on prior trends. Reductions in first diagnoses of depression and anxiety disorders were particularly stark for working-age adults and patients registered at practices in more deprived areas. Self-harm incidence was 38·5% (CI 35·7% to 41·3%) lower than expected in April 2020. Total self-harm contacts fell by 28·2% (CI 25·5% to 30·8%). Rates of both incident and any self-harm remained around thirty percent lower than expected up to June 2020. Interpretation: Our findings reveal a stark treatment gap that was greater for first diagnoses of depression and anxiety disorders in working age adults, for practice populations in deprived areas, and for self-harm. Consequences could include more patients subsequently presenting with greater acuity and severity of mental illness and rising rates of non-fatal self-harm and suicide. Funding: This work was funded by the National Institute for Health Research (NIHR) Greater Manchester Patient Safety Translational Research Centre, UK Research and Innovation/Medical Research Council COVID-19 Rapid Response Initiative funding and by a University of Manchester Presidential Fellowship (SS). CC-G received funding from the NIHR Applied Research Collaboration West Midlands. Declaration of Interests: All authors have completed the Unified Competing Interest form (available on request from the corresponding author) and declare: no support from any organisation for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years, no other relationships or activities that could appear to have influenced the submitted work.Ethics Approval Statement: This study is based on data from the Clinical Practice Research Datalink obtained under license from the UK Medicines and Healthcare products Regulatory Agency. The study was approved by the Independent Scientific Advisory Committee for Clinical Practice Research Datalink research (protocol number 20_094R1).

Evaluating the impact of individual alcohol licensing decisions on local health and crime: a natural experiment with synthetic controls

BackgroundLocal authorities in England can influence the local alcohol environment by contributing to the licensing process and controlling the enforcement of existing licenses. However, a gap remains in the availability of quantitative evidence of effectiveness and impact of these local interventions, including a shortage of public health evidence around individual premises. Natural experiments offer the opportunity to evaluate these interventions where formal randomisation is not possible. We aimed to assess whether it is possible to quantitatively evaluate three natural experiments of alcohol licensing decisions at small spatial scale. MethodsThree situations presenting a natural experiment were identified in different English Local Authority areas by public health or licensing practitioners: (i) the closure of a nightclub in 2013, (ii) the closure of a restaurant-nightclub following reviews in 2016, and (iii) the implementation of new local licensing guidance (LLG) in 2013/14. We obtained monthly numbers of reported incidents of emergency department admissions for alcohol-related reasons, ambulance call-outs, and various crimes at lower/middle-super-output-area level (from 2010–14 for case i, 2015–17 for case ii, and 2008–14 for case iii). Bayesian structural timeseries were used to compare trends to their counterfactuals, approximated by synthetic controls based on time series of the same outcomes in other, comparable, areas. FindingsClosure of the nightclub was associated with temporary reductions in antisocial behaviour (–18%; 95% Bayesian Credible Interval [BCI] –37 to –4); equivalent to 60 averted incidents in 4 months. Closure of the restaurant-nightclub was not associated with measurable changes in outcomes. There was some evidence that the LLG introduction was associated with a reduction in drunk and disorderly behaviour (–42%, 95% BCI –109 to 23), but this reduction equated to less than one incident per month. The unplanned end of the LLG might have contributed to an increase in domestic violence (11%, 95% BCI –10 to 35), corresponding to two additional incidents per month. InterpretationIt is possible to evaluate the impact of local alcohol policies, even at the level of individual premises, using this methodology. We provide quantitative evidence that local government actions to influence the local alcohol environment can have a positive impact on health and crime in the area but could also have unintended consequences. FundingThis work was funded by the National Institute for Health Research School for Public Health Research (NIHR SPHR), a partnership between the Universities of Sheffield, Bristol, Cambridge, Exeter, and University College London); the London School for Hygiene and Tropical Medicine; the LiLaC collaboration between the Universities of Liverpool and Lancaster and Fuse; and the Centre for Translational Research in Public Health, a collaboration between Newcastle, Durham, Northumbria, Sunderland and Teesside Universities. The views expressed are those of the authors and not necessarily those of the National Health Service, the NIHR, or the UK Department of Health.

Obituary in Memory of Professor Dr. Luc Missotten

Professor Missotten was born in Hasselt, Belgium, on the 9th of October 1931. He passed away on Thursday, 27 June 2019. He is survived by his wife Marie-Helene Verhamme, his sons Pieter and Jan, his daughters Anne and Kathleen and his grandson Wolf. Professor Missotten graduated and received his medical degree from KU Leuven, Belgium, in 1955. He obtained his PhD degree in 1965, with a thesis entitled ‘The Ultrastructure of the Retina’. He was promoted to professor in 1975 and became the chair of the Department of Ophthalmology, College of Medicine, KU Leuven in 1978. Under his leadership, the Department of Ophthalmology progressed into an international centre for basic and translational research, establishing strong connections and productive international collaborations. As a research leader and mentor, Professor Missotten established a group of clinician–scientists at the KU Leuven with an outstanding contribution to translational science. His passion for research motivated many young Belgian and international ophthalmologists to pursue a research career. His scientific career was outstanding as he was the leading author or co-author of over 240 national and international publications. Professor Missotten had an encyclopedic knowledge of basic science, engineering, optics and clinical practice of Ophthalmology, which allowed him to lead in many areas of ophthalmic research. Many generations of ophthalmologists, to whom he served as a role model and mentor, will remember him as a very kind, open-minded, honest, dedicated and committed clinician–scientist. Professor Missotten has been my mentor and friend for the last 36 years, with whom I shared my research ideas and plans. Our friendship was deep, and his loss will be unforgettable. In addition to his contributions to clinical practice and research, Professor Missotten served as the editor of international research journals and was involved in several research societies. His efforts were instrumental to unite ophthalmic research groups in Europe and to establish European Association for Vision and Eye Research (EVER) in 1997. From 1997 to 2002, he served as the secretary-general of EVER. He was also very important for the European Society of Ophthalmology (SOE). He was Secretary General from 1984 to 1996 and then became the President and elder statesman of SOE from 1996 to 1998. His efforts transformed SOE from a club to a scientific society. He was honored by the Society with both the Helmholtz Medal and the Henkes Medal. Professor Missotten received many awards to his work, including the degree of ‘Doctor Honoris Causa’ from the University of Mansoura, Egypt, in 1996 for his efforts to supervise the research of many young Egyptian ophthalmologists to obtain PhD degrees. Professor Missotten, ‘you shall be remembered forever’.

Patient-reported health-related quality of life up to three years after the treatment with permanent brachytherapy: Outcome of the large-scale, prospective longitudinal study in Japanese–Prostate Cancer Outcome Study by Permanent I-125 Seed Implantation (J-POPS)

PurposeTo evaluate long-term health-related quality of life (HRQOL) impact of I-125 permanent seed implantation (PI) in Japanese patients with localized prostate cancer. Materials and MethodsThis is a longitudinal, prospective study of 2353 patients who enrolled in the nationwide Japanese–Prostate Cancer Outcome Study by Permanent I-125 Seed Implantation (J-POPS) from July 2005 to June 2007. HRQOL was measured before and after treatment (3, 12, 24, and 36 months) using the Medical Outcomes Study 8-items Short-Form Health Survey (SF-8) and the Expanded Prostate Cancer Index Composite Japanese version questionnaire. ResultsOf the total of 1511 patients who returned anonymized HRQOL questionnaires to Translational Research Center for Medical Innovation, 1079 underwent PI alone (PI group) and 432 underwent combined PI and external beam radiation therapy (EBRT) treatment (EBRT combination therapy group). The mental component summary score of SF-8 was improved at 12, 24, and 36 months compared with the pretreatment level. Although the urinary summary score at 3 months in the PI group was significantly lower than that in the EBRT combination therapy group, which recovered thereafter and was comparable to the score of the EBRT combination therapy group. The bowel summary score in the PI group was significantly higher than that in the EBRT combination therapy group at 12, 24, and 36 months. ConclusionThis longitudinal, large-scale prospective study indicated that the general HRQOL for PI was almost maintained 3 years after treatment. These results provide important information for patients with localized prostate cancer, who may select to receive I-125 brachytherapy.

Abstract 4538: Global and phosphoproteomic analysis of AML cell line response to phosphatase inhibitor treatment

Abstract In the United States, approximately 21,000 people are diagnosed with AML each year and over 10,000 AML related deaths are reported. Genomic analysis has revealed at least 11 genetic classes of AML, and more than 20 subsets can be assigned when considering the differentiation states of Leukaemic blasts. This variability has generated significant interest in the development of targeted therapies for AML. However, complex mutational patterns and a lack of pharmacological agents for most mutational events have challenged the development of effective treatments. Recent results from the Beat AML clinical trial revealed that response to drugs is associated with mutational status, including instances where drug sensitivity is specific to combinatorial mutational events. To better understand the molecular mechanisms underpinning drug response, we performed global and phosphoproteomic analyses on 4 AML cell lines that carry mutations associated with AML: K562 with a BCR-ABL fusion, MOLM14 with a FLT3 mutation, HL60 with a RAS mutation, and CMK with a JAK3 mutation. These cell lines were each treated with a different kinase inhibitor drug for which they have a known sensitivity. Cell samples were collected prior to treatment and time points of 30 min, 3h, and 16 hrs. Frozen cell pellets were prepared for analysis using the workflow for multiplexed, deep-scale, proteome and phosphoproteome analysis developed by the NCI CPTAC program. The goal of our Proteogenomic Translational Research Center is to develop drug response signatures allowing the use of baseline proteome and phosphoproteome measurements to predict patient drug response, providing a foundation for rational selection of combination therapies. Preliminary analysis of the data show a dynamic temporal response to drug treatment in both the global and phosphoproteome, Figure 1. Current efforts are focused on combining knowledge-driven and data-driven analysis approaches to identify the pathways associated with drug response. Citation Format: Paul D. Piehowski, Jason E. McDermott, Joshua R. Hansen, Samantha L. Savage, Cristina E. Tognon, Anupriya Agarwal, Jeffrey W. Tyner, Brian J. Druker, Karin D. Rodland. Global and phosphoproteomic analysis of AML cell line response to phosphatase inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4538.

Trauma-related psychiatric and behavioral conditions are uniquely associated with sustained attention dysfunction.

It is increasingly recognized that trauma victims, particularly Veterans, have co-occurring psychological and physical conditions that impact cognition, especially the domains of sustained attention and executive functioning. Although previous work has generally attempted to isolate the unique cognitive effects of common combat-related comorbidities, less work has been done to examine how these conditions co-occur, and whether unique cognitive signatures accompany certain clinical combinations. To address this gap, we examined how several deployment-related conditions were associated with performance on a well-validated measure of sustained attention (i.e., gradual onset continuous performance task [gradCPT]) and a battery of standard neuropsychological measures in 123 Veterans from the Translational Research Center for TBI and Stress Disorders. Initially, a Principal component analysis was conducted to investigate how comorbid conditions grouped together. Several sustained attention measures from the gradCPT were differentially associated with four unique combinations of trauma-related pathology. Specifically, a somatic component representing the combination of current pain, sleep disturbance, and mild traumatic brain injury was associated with a higher rate of failures of attentional engagement. On the other hand, a comorbid posttraumatic stress disorder (PTSD) and mood disorder component (moodPTSD), as well as a substance use disorder component, were associated with higher rates of inhibitory control failures. Increased attentional instability was associated with moodPTSD as well as an anxiety disorder component. In contrast, the cognitive effects of deployment-related trauma were not observed on standard neuropsychological measures. These findings suggest that unique combinations of trauma-related pathology have dissociable effects on sustained attentional control. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

The effects and preventability of 2627 patient safety incidents related to health information technology failures: a retrospective analysis of 10 years of incident reporting in England and Wales

The use of health information technology (IT) is rapidly increasing to support improvements in the delivery of care. Although health IT is delivering huge benefits, new technology can also introduce unique risks. Despite these risks, evidence on the preventability and effects of health IT failures on patients is scarce. In our study we therefore sought to evaluate the preventability and effects of health IT failures by examining patient safety incidents in England and Wales. We designed our study as a retrospective analysis of 10 years of incident reporting in England and Wales. We used text mining with the words "computer", "system", "workstation", and "network" to explore free-text incident descriptors to identify incidents related to health IT failures following a previously described approach. We then applied an n-gram model of searching to identify contiguous sequences of words and provide spatial context. We examined incident details, recorded harm, and preventability. Standard descriptive statistics were applied. Degree of harm was identified according to standardised definitions and preventability was assessed by two independent reviewers. We identified 2627 incidents related to health IT failures. 2557 (97%) of 2627 incidents were assessed for harm (70 incidents were excluded). 2106 (82%) of 2557 health IT failures caused no harm to patients, 331 (13%) caused low harm, 102 (4%) caused moderate harm, 14 (1%) caused severe harm, and four (<1%) contributed to the death of a patient. 1964 (75%) of 2627 incidents were deemed to be preventable. Health IT is fundamental to the delivery of high-quality care, yet there is a poor understanding of the effects of IT failures on patient safety and whether they can be prevented. Failures are complex and involve interlinked aspects of technology, people, and the environment. Health IT failures are undoubtedly a potential source of substantial harm, but they are likely to be under-reported. Worryingly, three-quarters of IT failures are potentially preventable. There is a need to see health IT as a fundamental tenet of patient safety, develop better methods for capturing the effects of IT failures on patients, and adopt simple measures to reduce their probability and mitigate their risk. The National Institutes of Health Research Imperial Patient Safety Translational Research Centre at Imperial College London.

835-P: Examining and Validating Social Support (SS) Related to Reproductive Health (RH) Behaviors and Seeking Preconception Counseling (PC) in Female Adolescents with Diabetes

Adolescent girls with diabetes are at risk for RH complications and unplanned pregnancies. Tight glycemic control with PC can reduce these risks. Social support from significant others has improved health outcomes in people with diabetes, and may help adolescent girls regarding RH. This secondary analysis: 1)described the amount (1=no help, to 7=a lot of help) and type of available SS (3 subscales: emotional, informational, and instrumental) perceived by adolescent females with diabetes toward RH behaviors and seeking PC; 2)examined the construct validity among total and subscale scores of a SS measure adapted for RH and diabetes; 3)examined the associations of SS with RH behaviors (e.g., birth control), seeking PC, and RH communication with healthcare providers (HCPs). Baseline data were used from 113 adolescent girls with diabetes (90% T1D; duration of diabetes of 9.3±3.9 years; average HbA1c 8.4±1.7%; mean±SD age of 16.2±1.6 years; 81% white, 20% sexually active), who participated in READY-Girls Trial testing the efficacy of a PC intervention to reduce RH complications among adolescents with diabetes. Teens completed measures on SS, RH behaviors, seeking PC, and communication with HCPs. Descriptive, correlational, and regression analyses were performed. Adolescents perceived on average moderately high levels of SS: total (5.5±1.5), emotional (5.5±1.6), instrumental (5.4±1.5), and informational (5.6±1.5); 31% had some communication with HCPs. There were significant associations between total SS and all its subscales (p&amp;lt;.05) and among all SS subscales (p&amp;lt;.05). Significant associations were found between emotional and instrumental SS with effective use of birth control (p=.047, .055). Our measure of SS appears to have strong construct validity with trends for concurrent validity. Following further psychometric analyses, this SS measure could help advance research in the area. Disclosure H. Abujaradeh: None. S.M. Sereika: None. D.J. Becker: Other Relationship; Self; Jaeb Center for Health Research, MannKind Corporation, Sanofi Research &amp; Development. A.F. Fischl: None. D. Charron-Prochownik: None. Funding Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD-044097); Children’s Hospital of Pittsburgh (M01-RR-0084); Pediatric Clinical and Translational Research Center; National Center for Research Resources (UL1-RR-024153); National Institute of Nursing Research (P30-NR-03924)

Leucine Supplementation Partially Protects Leg Lean Mass in Older Adults During Seven Days of Bed Rest (OR18-02-19)

ObjectivesOlder adults are at increased risk of being hospitalized or bedridden and experiencing a host of negative health outcomes including loss of lean body mass. We hypothesized that supplementing daily meals with a small quantity (3–4 g/meal) of leucine will partially preserve lean leg mass and function during bed rest. MethodsMen and women (67.8 ± 1.1 y) were randomized to receive isoenergentic meals supplemented with leucine (LEU, 0.06 g/kg/meal; n = 10) or an alanine control, (CON, 0.06 g/kg/meal; n = 10). Subjects were admitted to the Clinical Research Center for 7 days of bed rest followed by 7 days of rehabilitation. Muscle fiber type and cross-sectional area (CSA) as well as single fiber characteristics were assessed from biopsies of the vastus lateralis obtained prior to (Pre-BR) and after bed rest (Post-BR) and after rehabilitation (Post-RE). Body composition measured using iDEXA was also determined at the same time points. ResultsLeucine-supplementation partially protected leg lean mass during bed rest (–1035 vs. –423 ± 143 g; P = 0.008). Leg lean mass did not differ between the groups Post-RE (P = 0.16). There was no significant effect of time (P = 0.16) or treatment (P = 0.92) on muscle fiber CSA; however, CON subjects, but not LEU subjects, exhibited an increased number of smaller fibers (<2000 um) and fewer larger fibers (>6000 um) Post-BR. Additionally, CON subjects tended to have a greater decrement in fiber width (P = 0.085) that did not return to baseline following rehabilitation. ConclusionsSupplementing older adults with moderate amounts of leucine has the potential to partially negate some of the deleterious effects on muscle health during short bouts of inactivity. Funding SourcesNational Institutes of Health, The Claude D. Pepper Older Americans Independence Center, Sealy Center on Aging and Institute for Translational Sciences-Clinical Research Center.