Research Fellowship Award Research Articles

Ebola virus-induced eye sequelae: a murine model for evaluating glycoprotein-targeting therapeutics

Ebola virus disease (EVD) survivors experience ocular sequelae including retinal lesions, cataracts, and vision loss. While monoclonal antibodies targeting the Ebola virus glycoprotein (EBOV-GP) have shown promise in improving prognosis, their effectiveness in mitigating ocular sequelae remains uncertain. We developed and characterized a BSL-2-compatible immunocompetent mouse model to evaluate therapeutics targeting EBOV-GP by inoculating neonatal mice with vesicular stomatitis virus expressing EBOV-GP (VSV-EBOV). To examine the impact of anti-EBOV-GP antibody treatment on acute retinitis and ocular sequelae, VSV-EBOV-infected mice were treated with polyclonal antibodies or monoclonal antibody preparations with antibody-dependent cellular cytotoxicity (ADCC-mAb) or neutralizing activity (NEUT-mAb). Treatment with all anti-EBOV-GP antibodies tested dramatically reduced viremia and improved survival. Further, all treatments reduced the incidence of cataracts. However, NEUT-mAb alone or in combination with ADCC-mAb reduced viral load in the eyes, downregulated the ocular immune and inflammatory responses, and minimized retinal damage more effectively. Anti-EBOV-GP antibodies can improve survival among EVD patients, but improved therapeutics are needed to reduce life altering sequelae. This animal model offers a new platform to examine the acute and long-term effect of the virus in the eye and the relative impact of therapeutic candidates targeting EBOV-GP. Results indicate that even antibodies that improve systemic viral clearance and survival can differ in their capacity to reduce acute ocular inflammation, and long-term retinal pathology and corneal degeneration. This study was partly supported by Postgraduate Research Fellowship Awards from ORISE through an interagency agreement between the US DOE and the US FDA.

Deletion of Kcnj16 altered transcriptomic and metabolomic profiles of Dahl salt-sensitive rats

The essential role of inwardly rectifying K+ Channel Kir5.1 (encoded by the Kcnj16 gene) in renal salt handling and blood pressure control has been demonstrated in both human and animal models. However, the underlying mechanisms are still not fully understood. Here, we aimed to integrate transcriptomics and metabolomics to profile the comprehensive changes of genes and metabolites under the deletion of Kcnj16 in the Dahl salt-sensitive (SS) rat background to identify novel mechanisms. For the transcriptomics profiling, we performed RNA-sequencing on kidney cortex tissue of Kcnj16 knockout (KO) and wild-type (WT) SS rats (N=5 per group). For the metabolomics profiling, we performed untargeted metabolomics of kidney cortex tissue, plasma, and urine of KO (N=6) and WT (N=5) rats. Transcriptomics analysis revealed over 6,000 differentially expressed genes in KO rats compared to WT rats, with renin Ren (the gene encoding renin) being the most upregulated gene. Consistent with the phenotype observed in the KO rats, ingenuity pathway analysis (IPA) based on the transcriptomic profile predicted reduced blood pressure and kidney damage and increased ion transport. Canonical pathway analysis suggested activation of metabolic-related pathways [e.g., xenobiotic metabolism, tricarboxylic acid (TCA) cycle, phenylalanine degradation, etc.] and suppression of immune responses-related pathways [e.g., pathogen-induced cytokine storm signaling pathway, macrophage classical activation signaling pathway, nuclear factor of activated T cells (NFAT) pathway, etc.] in KO rats. Untargeted metabolomic analysis suggested distinct metabolic profiles of plasma, urine, and kidneys between WT and KO rats, with 219, 790, and 31 differentially expressed metabolites identified in plasma, urine, and kidney in KO rats compared to WT rats. Canonical pathway analysis identified spermine and spermidine degradation pathways in both plasma and urine and taurine biosynthesis pathways in both urine and kidney. Integration analysis based on transcriptomic and metabolomic profiles suggested an altered TCA cycle, amino acid, and reactive oxygen species metabolism. In conclusion, besides significantly altered ion transport, our transcriptomics and metabolomics data suggest significant suppressed immune responses-related and altered metabolic-related pathways in SS rats lacking Kir5.1, which provides new insights into the underlying mechanisms of the regulation of blood pressure and renal function by Kir5.1. This work is supported by National Institutes of Health Grants R35 HL135749 and R01 DK135644 (to A.S.), Department of Veteran Affairs grant I01 BX004024 (to A.S.), the American Physiological Society Postdoctoral Fellowship (to B.X.) and the American Society of Nephrology Dimitrios G. Oreopoulos Research Fellowship Award (to L.V.D.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Beneficial Effects of Mild Hyperuricemia in Salt-Sensitive Hypertension

Hyperuricemia (HrU) or increased plasma uric acid (UA) is associated with worsening hypertension (HTN) and kidney damage. Paradoxically, UA is also known to be an endogenous antioxidant. Some studies suggest that only HrU with crystals, but not asymptomatic HrU promotes the progression of these diseases. We hypothesized that mild asymptomatic HrU is beneficial in controlling the progression of salt-sensitive (SS) HTN. Due to sex differences in urate handling and SS HTN, we used both male and female Dahl SS rats in this study. Radiotelemetry was used to measure blood pressure and various approaches, including RNA-Seq, were employed to assess kidney injury and identify potential molecular mechanisms. Mild HrU was induced using 2% oxonic acid in the diet, a uricase inhibitor that prevents the breakdown of UA further into more soluble allantoin. Groups: 1-2) male and female 4% NaCl high salt (HS) diet; 3-4) male and female HS + 2% oxonic acid (HS/oxo) diet. After 3 weeks, in response to oxonic acid supplementation, both sexes showed a significant increase of UA in plasma compared to their respective HS-only controls (Males: 0.63 ±0.07 vs. 2.17 ±0.34; Females 0.78 ±0.15 vs. 2.04 ±0.35 mg/dl, HS vs. HS/oxo). Interestingly, only male HS/oxo rats showed a significant increase in uricosuria (Males: 0.23 ±0.03 vs. 0.45 ±0.06; Femaels: 0.26 ±0.06 vs. 0.26 ±0.001 UA/Cre, HS vs. HS/oxo). Moreover, the mild HrU was associated with a significant attenuation of the progression and magnitude of the mean arterial pressure in male but not female rats (Males: 157 ±3 vs. 136 ±3; Females: 155 ±6 vs. 154 ±5 mmHg, HS vs. HS/oxo). Xanthine oxidase (XO) is one of the enzymes that produce UA, and its activity has been shown to affect HTN as well. Therefore, we examined the level of XO activity in the plasma after the treatment. While there was no difference in the activity based on the diet within each sex, females had significantly lower levels of XO activity compared to males in each of the diets. To further investigate the beneficial phenotype seen in male rats, we evaluate changes in the progression of renal pathology. The HS/oxo group compared to the HS group had a lower kidney weight/body weight ratio and lower protein cast accumulation, indicating lower kidney damage. RNA-Seq analysis showed that the attenuated HTN phenotype was associated with an increased expression in Mas1 (MAS receptor), Klk-1 (Kallikrein-1), and Pcsk6 (PCSK6 enzyme) which can all lead to the activation of different vasodilatory pathways. Our study showed that in male but not female Dahl SS rats, asymptomatic mild HrU accompanied by hyperuricosuria ameliorates the progression of SS HTN and protects kidneys from further damage. This work is supported by the American Society of Nephrology Ben J. Lipps Research Fellowship Program (Dimitrios G. Oreopoulos Research Fellowship Award to L.V.D.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The mammalian sperm factor phospholipase C zeta is critical for early embryo division and pregnancy in humans and mice.

The mammalian sperm factor phospholipase C zeta is critical for early embryo division and pregnancy in humans and mice.

Undergraduate Research Learning in the Liberal Arts: A Case Study of Transformative Student-Faculty Collaborations

Undergraduate Research Learning in the Liberal Arts: A Case Study of Transformative Student-Faculty Collaborations

1148-P: Glycoprotein Acetyls (GlycA) Associate with Intraglomerular Hemodynamic Dysfunction, Albuminuria, Central Adiposity, and Insulin Resistance (IR) in Youth with Type 1 Diabetes (T1D)

GlycA is a biomarker of systemic inflammation and cardiovascular disease, yet little is known about this biomarker in T1D. We examined the associations among GlycA, central adiposity, IR, and early kidney injury in adolescents with T1D. Glomerular filtration rate (GFR) and renal plasma flow (RPF) by iohexol and p-aminohippurate clearance, respectively, urine albumin-to-creatinine ratio (UACR), central adiposity by dual-energy x-ray absorptiometry (DXA), and estimated insulin sensitivity (eIS) by the CACTI equation were assessed in youth with T1D (n=50, 16.0±3.0 years, 50% female, HbA1c 8.7±1.3%, T1D duration 5.7±2.6 years, UACR 6 [5-14] mg/g, GFR 189±40 mL/min). Intraglomerular pressure (PGLO) was estimated by the Gomez equations. Concentrations of GlycA were quantified by targeted nuclear magnetic resonance spectroscopy. Correlation and multivariable linear regression analyses were performed. GlycA was higher in girls vs. boys (1.05±0.26 vs. 0.84±0.15 mmol/L, p=0.001), and in participants who were overweight or obese vs. normal weight (1.12±0.23 vs. 0.87±0.20, p=0.0004). GlycA correlated positively with estimated PGLO (r=0.52, p=0.001), UACR (r=0.53, p<0.0001) and trunk mass (r=0.45, p=0.001) and negatively with eIS (r=-0.36, p=0.01). All relationships remained significant after adjustment for age, sex, and HbA1c. In youth with T1D, GlycA, a biomarker of inflammation, was found to be higher in girls with T1D and those of overweight/obese habitus, compared to boys with T1D. Additionally, GlycA associated with parameters of early kidney dysfunction, central adiposity, and IR. Disclosure T.Reinicke: None. S.Gross: None. D.Cherney: Other Relationship; Boehringer Ingelheim-Lilly, Merck, AstraZeneca, Sanofi, Mitsubishi-Tanabe, Abbvie, Janssen, Bayer, Prometic, BMS, Maze, Gilead, CSL-Behring, Otsuka, Novartis, Youngene, Lexicon and Novo-Nordisk, Research Support; Boehringer Ingelheim-Lilly, Merck, Janssen, Sanofi, AstraZeneca, CSL-Behring and Novo-Nordisk. T.B.Vigers: None. L.Pyle: None. L.Driscoll: None. F.H.Mahmud: None. A.Dart: None. M.E.Pavkov: None. R.G.Nelson: None. P.Bjornstad: Advisory Panel; AstraZeneca, Novo Nordisk, Lilly, Horizon Therapeutics plc, Boehringer Ingelheim (Canada) Ltd., LG Chem, Consultant; Bayer Inc., Bristol-Myers Squibb Company. A.Caldwell-mcgee: None. C.Birznieks: None. D.Carrasco: None. M.Marcovecchio: None. K.J.Nadeau: None. K.L.Tommerdahl: None. M.Pauley: None. J.K.Snell-bergeon: None. Funding Junior Research Fellowship Award

ILC3s select microbiota-specific T regcells to establish tolerance in the gut

Abstract Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood, and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory and malignant diseases. Here we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that respond to the microbiota and express the transcription factor RORγt. We profiled all RORγt +immune cells at single-cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of T regulatory (T reg) cells and lymphoid tissue inducer-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s are distinct from but having interconverting potential with extrathymic AIRE-expressing cells, abundantly express major histocompatibility complex class II (MHCII), and are necessary and sufficient to promote microbiota-specific RORγt +T regcells and prevent their expansion as inflammatory T helper 17 (T H17) cells. This occurs through ILC3-mediated antigen presentation, αV integrin and competition for interleukin-2. Finally, single-cell analyses suggest that interactions between ILC3s and RORγt +T regcells are impaired in inflammatory bowel disease. Our results define a paradigm whereby ILC3s select for antigen-specific RORγt +Treg cells, and against T H17 cells, to establish immune tolerance to the microbiota and intestinal health. Crohn’s and Colitis Foundation Research Fellowship Award, 935259

Cell scientists to watch – Jordan Beach and Patrick Oakes

ABSTRACT Jordan Beach received his Bachelor's degree from Mount Union University, Alliance, Ohio, before joining the laboratory of Thomas Egelhoff at Case Western Reserve University and Cleveland Clinic Foundation, Cleveland, Ohio for his PhD. He moved to the National Heart Lung and Blood Institute at the National Institutes of Health in Bethesda, Maryland for his postdoctoral training with John Hammer to use high-resolution imaging to explore myosin II assembly dynamics with the aid of a Lenfant Biomedical Research Fellowship and K22 Career Transition Award. In 2017, Jordan became an Assistant Professor at the Department of Cell and Molecular Physiology at Loyola University Chicago, USA. Patrick Oakes received his Bachelor's degree from Boston College, and his PhD in Physics from Brown University in the laboratory of Jay Tang. He did his postdoctoral work with Margaret Gardel at the Institute for Biophysical Dynamics and the James Franck Institute at the University of Chicago studying how cells sense and generate forces. In 2016 he started his own lab as an Assistant Professor of Physics at the University of Rochester, and in 2019 moved to the Department of Cell and Molecular Physiology at Loyola University Chicago, USA. In 2022 he was promoted to Associate Professor.

Climate Change and Uncertainty: An Asset Pricing Perspective

Climate change and uncertainty about its potential consequences has become a central concern for economists, investors, and policymakers alike. I use a stochastic, dynamic general equilibrium model where final output is produced using a mix of cheap, dirty inputs and expensive, clean inputs and preferences incorporate aversion to climate model misspecification to analyze the implications of climate change and climate model uncertainty on economic and financial market outcomes. I find that climate change leads to increased clean input production and reduced emissions and that there is a negative price of climate risk that is significantly amplified and increasing in magnitude as climate change increases due to aversion to climate model uncertainty. Existing empirical estimates are consistent with the model implications, highlighting the potentially significant influence of climate model uncertainty on macroeconomic and asset pricing outcomes. This paper was accepted by Elke Weber, Special Section of Management Science on Business and Climate Change. Funding: This work was supported by the Energy Policy Institute at Chicago [Dissertation Support Fellowship], the Fama-Miller Center [Liew Fama-Miller Fellowship], the National Science Foundation [Graduate Research Fellowship Award D], and the University of Chicago [Harper (Provost) Dissertation Fellowship]. Supplemental Material: The data files and online appendices are available at https://doi.org/10.1287/mnsc.2022.4635 .

Family-integrated diabetes education for individuals with diabetes in South-west Nigeria.

This study aimed to determine the effects of family-integrated diabetes education on diabetes knowthe ledge of patients and family members, as well as its impact on patients' glycosylated haemoglobin (A1C). The design was a two-group Pretest Posttest quasi-experimental. The study took place at the diabetes clinics of two tertiary hospitals in southwestern Nigeria. People Living with Diabetes (PLWD) and family members aged 18 years and over and without cognitive impairment were placed, as clusters, into either a control group (CG) or an intervention group (IG) The CG comprised 88 patients and 88 family members while IG comprised 82 patients and 82 family members. Of these, 78 and 74 patients completed the study in CG and IG, respectively. PLWD in IG along with their family members were given an educational intervention on diabetes management and collaborative support with an information booklet provided. This was followed by three (3) complimentary Short Messaging Service (SMS). A1C and diabetes knowledge. Over half (52.4%) and about a fifth (18.2%) of family members and patients, respectively, had never had diabetes education. There was a statistically significant increase in the knowledge of patients and family members in IG. Unlike CG, the A1C of patients in IG improved significantly at three and six-month post-intervention, (p<0.01). Regression showed an independent effect of family members' knowledge on IG's A1C. Improved family members' diabetes knowledge positively impacted patients' glucose level. There is a need to integrate family members into diabetes care better. African Doctoral Dissertation Research Fellowship (ADDRF) award offered by the Africa Population and Health Research Center (APHRC) in partnership with the International Development Research Centre.

Updated International Society of Geriatric Oncology COVID-19 working group recommendations on COVID-19 vaccination among older adults with cancer

Updated International Society of Geriatric Oncology COVID-19 working group recommendations on COVID-19 vaccination among older adults with cancer

A rare case of recurrent group A streptococcal vulvovaginitis in a premenopausal woman.

A rare case of recurrent group A streptococcal vulvovaginitis in a premenopausal woman.

Long-Term Ambient Air Pollution and Childhood Eczema in the United States.

Long-Term Ambient Air Pollution and Childhood Eczema in the United States.

P059 Acceptability of remote consulting during COVID-19 among patients with two common long-term musculoskeletal conditions: findings from three qualitative studies and recommendations for practice

Abstract Background/Aims The COVID-19 pandemic led to the widespread adoption of remote consultations. Whilst remote consultations offer many potential advantages to patients and healthcare services, they are unlikely to be suitable for all. Guidance encourages clinicians to consider patient preferences when choosing face-to-face vs remote consultations. However, little is known about acceptability of, and preferences for remote consultations, particularly amongst patients with musculoskeletal conditions. This study aimed to explore the acceptability of, and preferences for, remote consultations among patients with osteoporosis and rheumatoid arthritis. Methods Data for this study derived from three UK qualitative studies: iFraP (improving fracture prevention study), Blast Off (BO; Bisphosphonate aLternAtive regimenS for the prevenTion of Osteoporotic Fragility Fractures), and ERA (Exploring people with Rheumatoid Arthritis’ experience of the pandemic). Each study explored patient experiences of accessing and receiving healthcare during the pandemic year. Transcripts from each data set relating to remote consulting were extracted. A minimum of two study team members worked independently, following a consistent approach, to conduct a rapid deductive analysis using the Theoretical Framework of Acceptability (TFA). The TFA consists of 7 constructs to understand acceptability of, in this context, remote consultations, including: affective attitudes; intervention coherence; perceived effectiveness; burden; self-efficacy; opportunity-costs; and ethicality. Following coding, the findings of all three studies were pooled. Analysis was facilitated by group meetings to discuss interpretations. Results Findings from 1 focus group and 64 interviews with 35 people, who had mostly experienced telephone consultations, were included the analysis. Participants’ emotional attitudes to remote consultations, views on fairness (ethicality) and sense making (intervention coherence) varied according to their specific needs for the consultation and values, relative to the pandemic context; participants perceived remote consultations as making more sense and being ‘fairer’ earlier in the pandemic. Some participants valued the reduced burden associated with remote consultations, while others highly valued, and did not want to give up, non-verbal communication or physical examination associated with face-to-face consults (opportunity costs); although perceived need for physical examination in participants with RA was associated with strong preference for face-to-face consultations, asymptomatic participants with RA and osteoporosis also expressed similar strong preferences. Some participants described low confidence (self-efficacy) in being able to communicate in remote consultations and others perceived remote consultations as ineffective, in part due to suboptimal communication. Conclusion Acceptability of, and preferences for remote consultation appear to be influenced by a range of societal, healthcare provider and personal factors and in this study, were not fixed, or condition-dependent. Remote care by default has the potential to exacerbate health inequalities and needs nuanced implementation. The findings have supported the development of patient-centred recommendations for practice that should be considered alongside clinician-focused recommendations when deciding whether remote consultations are appropriate. Disclosure Z. Paskins: Grants/research support; NIHR, Clinician Scientist Award (CS-2018-18-ST2-010)/NIHR Academy. L. Bullock: None. F. Manning: Grants/research support; part funded NIHR Clinical Research Network Scholar Programme. S. Bishop: None. P. Campbell: None. E. Cottrell: None. C. Jinks: Grants/research support; part funded by the NIHR Applied Research Collaboration (ARC) West Midlands. M. Narayanasamy: None. I. Scott: Grants/research support; funded by an NIHR Advanced Research Fellowship Award (NIHR300826). O. Sahota: None. S. Ryan: None.

Oocyte activation deficiency and assisted oocyte activation: mechanisms, obstacles and prospects for clinical application.

BACKGROUNDOocyte activation deficiency (OAD) is attributed to the majority of cases underlying failure of ICSI cycles, the standard treatment for male factor infertility. Oocyte activation encompasses a series of concerted events, triggered by sperm-specific phospholipase C zeta (PLCζ), which elicits increases in free cytoplasmic calcium (Ca2+) in spatially and temporally specific oscillations. Defects in this specific pattern of Ca2+ release are directly attributable to most cases of OAD. Ca2+ release can be clinically mediated via assisted oocyte activation (AOA), a combination of mechanical, electrical and/or chemical stimuli which artificially promote an increase in the levels of intra-cytoplasmic Ca2+. However, concerns regarding safety and efficacy underlie potential risks that must be addressed before such methods can be safely widely used.OBJECTIVE AND RATIONALERecent advances in current AOA techniques warrant a review of the safety and efficacy of these practices, to determine the extent to which AOA may be implemented in the clinic. Importantly, the primary challenges to obtaining data on the safety and efficacy of AOA must be determined. Such questions require urgent attention before widespread clinical utilization of such protocols can be advocated.SEARCH METHODSA literature review was performed using databases including PubMed, Web of Science, Medline, etc. using AOA, OAD, calcium ionophores, ICSI, PLCζ, oocyte activation, failed fertilization and fertilization failure as keywords. Relevant articles published until June 2019 were analysed and included in the review, with an emphasis on studies assessing large-scale efficacy and safety.OUTCOMESContradictory studies on the safety and efficacy of AOA do not yet allow for the establishment of AOA as standard practice in the clinic. Heterogeneity in study methodology, inconsistent sample inclusion criteria, non-standardized outcome assessments, restricted sample size and animal model limitations render AOA strictly experimental. The main scientific concern impeding AOA utilization in the clinic is the non-physiological method of Ca2+ release mediated by most AOA agents, coupled with a lack of holistic understanding regarding the physiological mechanism(s) underlying Ca2+ release at oocyte activation.LIMITATIONS, REASONS FOR CAUTIONThe number of studies with clinical relevance using AOA remains significantly low. A much wider range of studies examining outcomes using multiple AOA agents are required.WIDER IMPLICATIONSIn addition to addressing the five main challenges of studies assessing AOA safety and efficacy, more standardized, large-scale, multi-centre studies of AOA, as well as long-term follow-up studies of children born from AOA, would provide evidence for establishing AOA as a treatment for infertility. The delivery of an activating agent that can more accurately recapitulate physiological fertilization, such as recombinant PLCζ, is a promising prospect for the future of AOA. Further to PLCζ, many other avenues of physiological oocyte activation also require urgent investigation to assess other potential physiological avenues of AOA.STUDY FUNDING/COMPETING INTERESTSD.G. was supported by Stanford University’s Bing Overseas Study Program. J.K. was supported by a Healthcare Research Fellowship Award (HF-14-16) made by Health and Care Research Wales (HCRW), alongside a National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST). The authors have no competing interests to declare.

Bromodomain Inhibition Is Effective Against Acute Myeloid Leukemia Cells Under Hypoxia and Induces Genome-Dependent Metabolic Perturbations

Inhibition of bromodomain-containing (BRD) proteins showed anti-tumor activity against different subtypes of acute myeloid leukemia (AML; Dawson et al. Nature 2011; Zuber et al. Nature 2011; Dawson et al. Leukemia 2013; Chen et al. Cancer Cell 2014; Gröschel et al. Cell 2014; Zhao et al. Cell Reports 2016). However, epigenetic and functional changes occurring under hypoxic conditions may affect BRD protein activity and the consequences of its inhibition. Moreover, the levels of c-MYC, a main target of bromodomain inhibitors, are decreased at low oxygen concentrations mimicking the bone marrow microenvironment which harbor leukemia stem cells, the final target of successful therapies.We treated AML cell lines representative of diverse genetic background (KG1; MLL-driven: MOLM-13, NOMO-1, NPM1 and DNMT3A mutated: OCI-AML3; t(8;21): Kasumi-1; c-MYC-amplified: HL-60) for 48h with the bromodomain inhibitor GSK1215101A (250 µM or 500 µM) under hypoxic conditions (1% O2). GSK1215101A reduced cell viability in a dose-dependent manner in all the cell lines (15%-35% reduction at 250 µM and 25%-65% reduction at 500 µM) except for HL60, with Kasumi-1 being the most sensitive model. A significant increase of Annexin V+ apoptotic cells was detected in NOMO-1 and Kasumi-1 cells, while OCI-AML3, Kasumi-1, HL-60 and KG-1 showed a marked decrease in cell proliferation with reduction of the percentage of cells in S phase and arrest into G0/G1 phase.To better understand GSK1215101A effects on AML cells, we performed gene expression profiling of actively translated mRNAs isolated by polysome profiling, along with western blot analysis of OCI-AML3, Kasumi-1 (drug-sensitive) and HL-60 (drug-resistant) cell lines after 16 hour of treatment (in order to allow cell adaption to the selective pressure induced by the drug, while avoiding massive cell death). GSK1215101A enforced hypoxia-induced c-MYC downregulation in all the cell lines and induced HIF-1a upregulation specifically in drug-sensitive ones, which was confirmed by immuofluorescence.Moreover, since c-MYC is a known regulator of cancer cell metabolism, we analyzed the metabolic changes induced by GSK1215101A by Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (Metabolon, Inc). GSK1215101A treatment resulted in changes in metabolite levels that are consistent with alterations in energy metabolism and cell component biosynthesis, with marked differences between the OCI-AML3 and Kasumi-1 models. Lactate levels were significantly reduced in hypoxic Kasumi-1 cells after treatment, which may be consistent with a drug-dependent decrease in lactate dehydrogenase activity, while no differences were observed at transcriptional and translational level. Little effects were observed on OCI-AML3 lactate levels. In parallel, OCI-AML3 showed a significant increase of both the reduced (3.9-fold) and oxidized (2.6-fold) forms of glutathione, which plays an important role in antioxidant defense, redox-homeostasis and protein folding. Consistently, the translational profile of OCI-AML3 cells showed enrichment of deregulated genes involved in superoxide metabolic process and response to oxidative stress (NCF1/2, ADNP2, XBP1, HSPA1A/B).Taken together, these data indicate that inhibition of bromodomain-containing proteins is effective against AML cells under hypoxic conditions and offer insights into the drug effects on leukemic cell metabolism, which is highly dependent on the genomic backgrounds. Moreover, the results suggest potential targets for novel combination therapies interfering with metabolic pathways or HIF-1a activity.Supported by: EHA research fellowship award, ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. DisclosuresMartinelli:ARIAD/INCYTE: Consultancy; PFIZER: Consultancy; ROCHE: Consultancy; JOHNSON & JOHNSON: Consultancy; CELGENE: Consultancy; AMGEN: Consultancy.

Mediterranean Diet and Lifestyle Index Development for Older Adults

ObjectivesThe Mediterranean diet is associated with many health benefits, yet it is typically only the food pattern that is assessed without consideration for lifestyle attributes that accompany a Mediterranean way of life. The Mediterranean diet pyramid includes lifestyle activities at the base of the pyramid such as regular physical activity (PA), adequate rest, conviviality, biodiversity and seasonality, traditional local and eco-friendly products, and culinary activities. The purpose of this study was to design and pilot test a Mediterranean diet and lifestyle index for older adults in the U.S.MethodsThe Harvard Food Frequency Questionnaire was used to determine the alternative Mediterranean Diet Score (aMed). The short version of the Minnesota Leisure Time PA questionnaire and additional Mediterranean diet-related dietary habit and lifestyle questions were piloted in 75 older adults attending senior centers.ResultsParticipants were primarily women (80.6%) and Caucasian (68%) with an average age of 71.89+/−7.60 years. A 27-item index including the aMed food groups, dietary habits, PA, culinary activities, purchasing of local and seasonal foods, and adequate rest resulted in a reliable score (α = 0.75). Individual index factors correlated with the overall Mediterranean diet and lifestyle score.ConclusionsWhile this Mediterranean diet and lifestyle index resulted in good internal consistency; assessment of conviviality, especially for older adults in the time of Covid-19, need be re-evaluated as a lifestyle measure that can impact dietary intake and overall health.Funding SourcesEastern Michigan University Faculty Research Fellowship Award.

DEVELOPMENT OF SMALL MOLECULE EPIGENETIC THERAPEUTICS FOR INFLAMMATORY BOWEL DISEASE

Abstract Long-term persistence of chronic inflammation in inflammatory bowel disease (IBD) including ulcerative colitis (UC) and Crohn’s disease (CD) is among the major factors contributing to neoplastic transformation and the development of colitis-associated colorectal cancer. There exists a lack of efficient medications for IBD, primarily due to emergence of resistance or side effects. Antibodies against tumor necrosis factor-α (anti-TNFα) are effective therapies in the armamentarium; however, up to 40% of patients become resistant to this therapy. Thus, target-based improved therapy with higher efficacy and enhanced safety profile is urgently needed to fill these gaps. We have demonstrated that bromodomain-containing protein 4 (BRD4), an epigenetic regulator, is required for stabilization of NFkB binding on the promoters of inflammatory genes, activation of RNA polymerase II, and histone H3 Lys122 acetylation (H3K122ac) to permit high levels of inflammatory gene expressions in sentinel immune cells, epithelial cells, and fibroblasts. Our compelling data using human IBD patient samples suggest that BRD4 activation is coincident with the initiation of colonic inflammation. Inhibition of the BRD4 activation is an attractive target for the development of superior therapeutics for IBD, especially for anti-TNFα-resistant patients. We have successfully identified proprietary highly potent and specific BRD4 inhibitors (patent WO 2018/112037 A1) with direct binding modes validated by the solved co-complex crystal structures. We observed that our lead compounds exhibit low toxicity both in vitro and in vivo. Therapeutic administration of our lead inhibitors ZL0516 and ZL0590 significantly reduces mucosal inflammation in several animal models of IBD and restores tissue architecture. Our data show that inhibition of BRD4 results in a decrease of key inflammatory cytokines associated with pathological responses in IBD such as TNFα, IL-6, IL-17A, and IL-1β expression in human colonic epithelial cells and peripheral blood mononuclear cells as well as several animal models of IBD colitis. Collectively, our data suggest that BRD4 activation is critical to the initiation of human colonic inflammation during IBD. BRD4 inhibition disrupts its protein-protein interactions with acetylated histone lysine residues, thereby blocking the pathological activation of the BRD4-NFκB signaling and suppressing colonic inflammation. Thus, BRD4 represents a unique drug target, and novel BRD4 inhibitors ZL0516 and ZL0590 have been identified as promising drug candidates towards the development of small molecule epigenetic therapeutics for the treatment of IBD and its chronic sequelae. Funding support: Crohn’s &amp; Colitis Foundation Entrepreneurial Investing (EI) Initiative award, Litwin IBD Pioneers Program, and a research fellowship award from the Crohn’s &amp; Colitis Foundation of America.

Does Age Influence The Effects Of Exercise On Anxiety Levels Of Children With ASD?

Anxiety is a common comorbidity among children with Autism Spectrum Disorder (ASD), with approximately 40% of youths with ASD meeting the criteria for at least one anxiety disorder (van Steensel et al., 2011). Furthermore, anxiety has been shown to be more likely in adolescents with ASD (Mayes et al., 2011; Vasa et al., 2013). Only one study has shown that exercise has potential benefits for anxiety in 13-27 year olds with mainly mild ASD, following an eight week programme (Hillier et al., 2011). Further research is need to examine the effects of exercise on anxiety in children with more severe symptoms of ASD and to establish if age is a factor on the effects of exercise on anxiety. PURPOSE: To determine whether age influences the effects of exercise on anxiety levels of children with moderate to severe ASD. METHODS: Twenty children (5-18 years) with moderate to severe ASD, were included in the study. A 16-week school-based exercise programme was implemented for 60 minutes, three days a week. Anxiety was measured using the Anxiety Scale for Children with ASD (ASC-ASD), which was given to the children’s teacher, before and at the end of the programme. A Spearman’s rank-order correlation was run to measure the relationship between age and the responsiveness of the intervention. RESULTS: There was a significant, strong negative correlation between age and the effectiveness of the intervention on total ASC-ASD scores, rs(14) = -0.77, p = 0.001, and on performance anxiety, rs(14) = -0.73, p = 0.003. There was a significant, moderate negative correlation between age and the effectiveness of the intervention on anxious arousal, rs(14) = -0.65, p = 0.012, and on uncertainty, rs(14) = -0.58, p = 0.028. There was an insignificant, weak negative correlation between age and the effectiveness of the intervention on separation anxiety. CONCLUSION: Age is associated with the effectiveness of exercise on anxiety levels of children with moderate to severe ASD. The older the age of the child, the greater improvement was seen in anxiety levels at school following the exercise programme. This research was funded by the Institute of Technology Carlow, President’s research fellowship award.

Evaluation of In Vitro Models for Pyrrolizidine Alkaloid‐Induced Hepatocellular Injury

ObjectivePyrrolizidine alkaloids (PAs) are toxic chemicals found in many herbal medicines, dietary supplements and even food sources such as honey, bread, and tea. Cases of drug‐induced liver injury due to herbal and dietary supplements are relatively common in Asia and are increasing in the United States. The literature suggests that PAs containing macrocyclic structure are more toxic than those with smaller sidechains. The goal of this project was to evaluate two PAs with macrocyclic structure (monocrotaline and retrorsine) and one PA will a smaller sidechain (retronecine) for their potential to elicit toxicity across two in vitro model systems.MethodsPA toxicity was measured by determining the concentration at which 50% of the cell population was killed (LC50). PAs were dissolved in DMSO and PA‐induced toxicity was measured after 48 hours in two in vitro models: TAMH (transforming growth factor‐α transgenic mouse hepatocytes) and THLE‐2 (transformed human liver epithelial‐2 cells). Statistical analysis of cell viability data was performed using SPSS (Probit regression) to obtain LC50 values.ResultsA decrease in cell viability was observed as PA concentration increased. Interestingly, both cell lines were relatively resistant to PA‐induced toxicity. In many cases only predicted LC50 values for each compound could be generated and are indicated by an asterisk (7.0mM*, 11mM*, and 30.mM* in TAMH for monocrotaline, retrorsine and retronecine, respectively; and 2.2mM, 3.2mM, and &gt;100mM* in THLE‐2 for monocrotaline, retrorsine and retronecine, respectively. Based on the toxicologic parameters, monocrotaline and retrorsine were more toxic (lower LC50 values) than retronecine.ConclusionsWhile PAs have been associated with hepatotoxicity, limited toxicity was seen in both the TAMH and THLE‐2 cell lines. Predicted LC50 values were higher than expected, suggesting TAMH and THLE‐2 cells are not good models for studying PA‐induced liver toxicity. Retronecine, which contains a smaller sidechain, was found to be less toxic than the larger compounds. Future work is currently underway to identify biomarkers responsible for the observed resistance to PA‐induced toxicity as potential targets for preventing hepatocellular injury.Support or Funding InformationThis work was supported by the Pacific University Research Incentive Grant Program and an ASPET Summer Undergraduate Research Fellowship (SURF) award at Pacific University.