bluuebunny/crossref_metadata_2025_split huggingface dataset:
OR35-03 Uncovering Causative Mutations in Polycystic Ovary Syndrome
Authors: Srivastava Jaya , Ovcharenko Ivan 
Published: October 2023

Abstract
Disclosure: J. Srivastava: None. I. Ovcharenko: None.
PCOS is a complex disorder manifesting as reproductive and metabolic abnormalities in women. The undefined etiology stems from our incomplete understanding of genetic aberrations contributing to the diseased phenotype. This has resulted in speculations surrounding the cause-consequence relationship between phenotypic features of PCOS and limited our knowledge of comprehensive genetic signatures of PCOS. Pleiotropic effects are salient features of genetic regulation in mammalian genomes. The contribution of a set of regulatory elements (REs) to upregulation of a single gene, or the genetic control of multigene loci by a single RE, give rise to heterogenous phenotypes. Enhancers in particular dictate the spatio-temporal scale of gene expression by controlling transcription factor (TF) binding events. The complex traits of PCOS are suggestive of pleiotropy originating from genetic and epigenetic changes that arise from nucleotide variations in REs of the genome. Genome Wide Association Studies (GWAS) have identified ∼100 risk variants associated with PCOS and implicated the involvement of several genes such as FSHB/R, THADA, DENND1A, etc. However, mechanisms by which they contribute to pathophysiology are unknown. Additionally, twin studies have implicated the heritability of PCOS to be ∼80% and yet, the heritability from GWAS variants accounts for less than 10%. Experimental data may account for the ‘missing heritability problem’ specifically through rare variants that are missed in GWAS. To address this issue, we developed a Deep Learning model of gene regulation in PCOS identifying hundreds of active REs orchestrating the activity of PCOS genes. We also established a foundation for quantifying the impact of single-nucleotide noncoding mutations on TF binding and RE activity. By combining thousands of experimental assays associated with a large number of biological events such as tissue-specific epigenetic maps of the human genome, multi-tissue profiling of TF binding and gene expression using data from high throughput experiments we devised a strategy to accurately identify functionally map causative mutations in the GWAS loci of PCOS genes. MPRA experimental data validates a large fraction of our predicted causative variants thus suggesting that our studies have a potential of a leap forward accurate dissection of genomic and epigenetic mechanisms of PCOS. Consolidated results of this analysis and experimental validation of our predictions will lead to detailing the regulatory mechanisms of PCOS.
Presentation Date: Sunday, June 18, 2023
FRI162 Ligand-dependent Photocrosslinking of Mineralocorticoid Receptors Using Site-directed Incorporation of Unnatural Amino Acids
Authors: Almeida-Prieto Brian , Gonzalez-Hernandez Alberto J , Giraldez Teresa , Alvarez de la Rosa Diego 
Published: October 2023

Abstract
Disclosure: B. Almeida-Prieto: None. A.J. Gonzalez-Hernandez: None. T. Giraldez: None. D. Alvarez de la Rosa: None.
Steroid receptors dimerize prior DNA binding as a key step for translational activation of target genes. It appears that several domains of the receptors play a role in the process. In addition, current evidence suggests that the active conformation of steroid receptors may not always be a dimer, but involves different quaternary structures, characteristic for each receptor. Recent data obtained by our group suggests that agonist-bound mineralocorticoid receptors (MR) adopt a tetrameric form in the nucleoplasm, forming higher order oligomers when bound to hormone response elements in the chromatin. The formation of such complexes would likely requires more than one oligomerization interface. To generate a tool for investigating inter-subunit contacts, we incorporated photoactivatable crosslinkers into specific sites in MR using a genetically-encoded unnatural amino acid (p-Benzoyl-L-phenylalanine, BzF). We created a library of 26 amber stop codon mutants substituting conserved A, F, W and Y residues distributed along the N-terminal domain (NTD), DNA-binding domain (DBD) and ligand-binding domain (LBD) of the mouse MR coding sequence. Site-directed incorporation of BzF into MR was performed by co-expressing an orthogonal pair of BzF-specific aminoacyl-tRNA synthetase and an amber-suppressor tRNA with the mutant receptors. Characterization of the library showed that 18 mutants produced functional receptors after BzF incorporation. Using an in silico approach we identified position W914 in helix 10 of the LBD as a potential candidate for photocrosslinking. Treatment of cells expressing W914BzF with UV light selectively increased aldosterone-bound, but not cortisol-bound, MR transcriptional activity by approximately 50%. Increased activity was still apparent after hormone washout, suggesting that BzF-induced photocrosslinking at position 914 contributes to stabilizing an active conformation and identifying H10 as a putative region for ligand-specific oligomerization interface. Our results support the strong potential of this methodology for studying this new organization model proposed for MR and other steroid receptors, along with the characterization of functional protein-protein interactions.
Presentation Date: Friday, June 16, 2023
OR11-02 Structural Determinants of Pure Antiestrogenic Activity
Authors: Vallet Amandine , Diennet Marine , Thiombane Khady , Vivet Anaïs , Weber Sandra , El Ezzy Mohamed , Shaikh Faraz , Poupart Julien , Mendoza-Sanchez Rodrigo , Alexandra Schuetz Doris , Marinier Anne , Greene Geoffrey L , William Fanning Sean , Nathalie Mader Sylvie 
Published: October 2023

Abstract
Disclosure: A. Vallet: None. M. Diennet: None. K. Thiombane: None. A. Vivet: None. S. Weber: None. M. El Ezzy: None. F. Shaikh: None. J. Poupart: None. R. Mendoza-Sanchez: None. D. Schuetz: None. A. Marinier: None. G.L. Greene: None. S.W. Fanning: None. S.N. Mader: None.
Inhibitory activities of antiestrogens on estrogen receptor alpha (ERα) range from mixed antagonism/agonism (selective ER modulators; SERMs) to complete antiestrogenicity associated with accelerated ERα turnover (selective ER degraders; SERDs). Using a panel of SERMs, SERDs and PROTACs, we show that antiestrogens induce variable degrees of SUMOylation of ERα and that efficient induction of SUMOylation, rather than increased suppression of coactivator recruitment or accelerated ERα degradation, correlates with suppression of ERα basal and induced transcriptional activity in reporter assays. Antiestrogens did not induce SUMOylation of ERβ and did not suppress its basal transcriptional activity in the same assays. Chimeras between the two receptors identified ERα-specific residues required for induction of SUMOylation by antiestrogens and revealed differences according to the antiestrogen structure. In addition, induction of ERα SUMOylation by antiestrogens depended on the hydrophobicity of N-terminal residues of ligand binding domain (LBD) helix H12. L536 mutations, including those occurring in endocrine therapy-resistant breast cancer, abolished both induction of ERα SUMOylation and pure antiestrogenicity. Structures of the L536S human ERα LBD bound to fulvestrant analogs and molecular dynamics simulations revealed dynamic side chain interactions with ERα H12 in the coactivator-binding groove, and predict the impact of SUMOylation-suppressing L536 mutations on these interactions. Together, our results show that antiestrogen-induced ERα SUMOylation is associated with complete suppression of its transcriptional activity and provide insights into the structural determinants of pure antiestrogenicity.
Presentation Date: Friday, June 16, 2023
SAT020 Is Testosterone The Male Sex Hormone Responsible For Increased Male Mortality In Melanoma? In-vitro Studies On Two Human Melanoma (BLM, 1205Lu) Cell Models
Authors: Ramaraj Pandurangan 
Published: October 2023

Abstract
Disclosure: P. Ramaraj: None.
Epidemiological data pointed to increased male mortality in melanoma and clinical studies pointed to better protection of menstruating females in melanoma than post-menopausal women and men of any age. Our in-vitro studies with progesterone (P) on human melanoma (BLM and 1205Lu) cell lines showed a significant decrease in cell growth along with a decrease in the secretion of proinflammatory cytokine IL-8 by these cells. These in-vitro studies correlated with the hormonal data which showed that progesterone levels in menstruating females ranged between 1000 - 1500 ng/dl, compared to post-menopausal women 20 - 100 ng/dl and men 27-90 ng/dl, suggesting low progesterone levels meant no protection in melanoma. This observation prompted us to hypothesize that lack of progesterone in males could be a cause for increased male mortality. This hypothesis also raised the question of the role of male sex hormones (AD-androstenedione and T-testosterone) in increased male mortality. In order to address these questions in-vitro experiments were planned. First, to indirectly induce endogenous IL-8 by pretreating the cells with endothelin and then adding steroid hormones (AD, T, P). Secondly, to add IL-8 directly to the cells and then add steroid hormones. Finally, to suppress endogenous IL-8 with curcumin and then add T to one set of cells and IL-8 + T to another set of cells. For the first experiment, the result was a marginal increase in cell growth (67%) in cells pretreated with endothelin followed by T-treatment compared to straight T-treated cells (60% cell growth) without endothelin pretreatment. For the second experiment, there was a slight increase in cell growth in IL-8-added cells, but it was not statistically significant. For the final experiment, the cells treated with IL-8 + T after curcumin pretreatment showed a partial restoration of cell growth (49%) compared to cells (40% cell growth) treated with plain T after curcumin treatment, suggesting T was not able to control or regulate IL-8 action like progesterone. These in-vitro experiments suggested that in males 2 reasons 1) a deficiency of progesterone and 2) inefficiency of T to control endogenous IL-8 could possibly result in increased IL-8 levels. This increased IL-8 level in males could lead to increased cell growth and metastasis in melanoma leading to death. So, a situation arising out of male reproductive endocrine physiology could probably be responsible for increased male mortality in melanoma.
Presentation: Saturday, June 17, 2023
OR22-05 Progesterone Receptor-Grb2 Interaction Is Associated With Better Outcomes In Breast Cancer
Authors: Wittayavimol Nattamolphan , Iwabuchi Erina , Pateetin Prangwan , Miki Yasuhiro , Onodera Yoshiaki , Sasano Hironobu , Boonyaratanakornkit Viroj 
Published: October 2023

Abstract
Disclosure: N. Wittayavimol: None. E. Iwabuchi: None. P. Pateetin: None. Y. Miki: None. Y. Onodera: None. H. Sasano: None. V. Boonyaratanakornkit: None.
The presence of progesterone receptor (PR) is usually associated with better clinical outcomes for several endocrine-related cancers. However, the molecular mechanism of how PR mediates growth inhibition remains unclear. In addition to its role as a nuclear transcription, PR also possesses an extranuclear function, mediated mainly through PR polyproline domain (PPD) interaction with the SH3 domains of cytoplasmic signaling molecules. Our previous study suggested a possible role of PR-PPD in mediating SH3-domain interaction. We showed that PR-PPD and SH3 domain interaction inhibited EGF-mediated signaling and decreased lung cancer cell proliferation. An essential adapter protein that helps transduce growth factor signaling is Grb2 which has an SH2 domain flanked by two SH3 domains. In this study, we hypothesized that PR interferes with cytoplasmic signaling through interaction between PR-PPD and Grb2-SH3. We used several protein-protein interaction assays in vitro and in cells to demonstrate PR-Grb2 interaction. GST-pulldown analysis showed that PR-PPD interacts specifically with SH3 domains of Grb2. No interaction was detected when key prolines in the PR-PPD to alanines. Immunofluorescence staining showed colocalization of PR and Grb2 in both the nucleus and cytoplasm of BT474 breast cancer cells. Using Bimolecular Fluorescence Complementation (BiFC) analysis, we showed that PR and Grb2 interacted in breast cancer cells, specifically through the Grb2-SH3 domain. No BiFC signal was detected when Grb2-SH3 domains were deleted. Furthermore, we used Proximity Ligation Assay (PLA) analysis to examine protein-protein interaction of a distance less than 40nm in cells. We found PR-Grb2 interaction in PR-positive breast cancer tissue sections. No PLA signal was detected in PR-negative breast cancer cells. Importantly, we examined 43 PR-positive breast cancer specimens and showed that PR-Grb2 interaction was associated with low histological stage and negatively correlated with lymph node invasion and metastasis of breast cancer. Our results provide a molecular mechanism for how PR can inhibit growth factor signaling by interfering with Grb2 signaling and a basis for developing novel therapeutic strategies for future cancer treatment.
Presentation: Saturday, June 17, 2023
OR33-01 Live Cell Imaging Of Enhancer Transcriptional States
Authors: Chapus Fleur , Day Christopher , Yasar Pelin , Bregio Celyn , Palia Alyson , Scappini Erica , Tucker Charles , Rodriguez Joseph 
Published: October 2023

Abstract
Disclosure: F. Chapus: None. C. Day: None. P. Yasar: None. C. Bregio: None. A. Palia: None. E. Scappini: None. C. Tucker: None. J. Rodriguez: None.
Intra-tumoral gene expression heterogeneity of estrogen receptor positive breast cancers is a roadblock toward effective treatment due to subpopulations acquiring resistance and metastatic ability. This expression heterogeneity can be explained by the stochastic nature of transcription. Transcriptional initiation is coordinated through distal elements known as enhancers. These elements aid in the recruitment of cofactors and chromatin remodelers to promoters. During transcriptional activation, genes are transcribed during short periods of nascent RNA synthesis known as transcriptional bursts. Furthermore, estrogen responsive genes can transition between transcriptional bursting and variably long periods of inactivity lasting from minutes to days. Here, we investigated whether estrogen responsive enhancers of variably expressed genes also exhibited transcriptional states which reflect promoter states. To observe live cell transcription dynamics, we integrated MS2 stem loops into several endogenous enhancers using CRISPR. After transcription initiation, these stem loops are transcribed and subsequently bound by the RNA binding protein, MS2-GFP. This enrichment in MS2-GFP at the transcription site is visible as a bright punctate spot. We observed that the estrogen responsive TFF1 enhancer exhibited short periods of transcriptional activity and long periods of inactivity. Moreover, the TFF1 enhancer RNA (eRNA) sporadically formed dynamic, but rare and long-lived condensates. To correlate enhancer and promoter transcription we used single molecule fluorescence in situ hybridization of several eRNA. We observed that eRNA aggregates were correlated with an increase of mRNA content and occasionally localized to different nuclear bodies. Lastly, we enriched for chromatin bound RNA and observed long enhancer RNA transcripts. Taken together, our work highlights the dynamic coupling of enhancer and promoter regulation and the importance of understanding the regulators of enhancer transcriptional states in cancer.
Presentation: Sunday, June 18, 2023
THU653 Subacute Thyroiditis Following Covid19 Infection
Authors: Fanous Ereeny , Georges Karam Jocelyne 
Published: October 2023

Abstract
Disclosure: E. Fanous: None. J.G. Karam: None.
Background: Subacute thyroiditis is a self-limiting inflammatory condition of the thyroid gland which usually occurs as a post-viral reaction, leading to the destruction of thyroid follicular cells due to infiltration of lymphocytes. During the first outbreak of COVID-19 virus, subacute thyroiditis had been reported as a complication that may occur in patients without any previous thyroid disorder [1]. Clinical Case: A 62-year-old female patient presented with few weeks history of generalized weakness, fatigue, chills, malaise, low appetite, and unintentional weight loss of around 10lbs. She stated that she feels "internal tremors" and palpitations with heart rate increasing from baseline 60 bpm to 90bpm. She had one episode of near syncope at work prompting her presentation. She reports no change in bowel movements, no heat intolerance, no fever or night sweat, no shortness of breath, no visual changes or eyes problems. Five weeks prior to presentation, the patient was diagnosed with COVID-19 infection by PCR, with a mild disease pattern that consisted of upper respiratory symptoms lasting for few days, however she continued to feel fatigue and low appetite since then, attributing her symptoms to long-term sequelae of Coivd-19. Her past medical history is remarkable for obesity, hypercholesterolemia, and two prior Covid 19 infections: in 2020, treated with monoclonal antibodies, and early 2022. The physical exam was significant for slightly tender thyroid and heart rate of 94 bpm. Thyroid function tests were consistent with hyperthyroidism; TSH&lt;0.01mIU/ml, FT4=2.55ng/dl (0.58-1.64), T4=19.6 mcg/dl (4.4-9.5), T3=1.45ng/ml (0.72-1.35), with negative TSI, TPO AB, and TG AB. ESR was 44mm/hr, RAI uptake and scan showed very low uptake which is indicative for subacute thyroiditis.
The patient was given supportive therapy. She developed mild and transient hypothyroidism at 6 weeks follow-up with TSH of 8.59 IU/ml and FT4=0.62 ng/dl (0.89-1.76) before her thyroid tests return to normal at 9 weeks with complete resolution of her symptoms. Conclusion: Prolonged diverse symptoms following Covid19 infection are often attributed to long term Covid symptoms. Health Care providers should be aware of the possibility of subacute thyroiditis following Covid 19 infection and should obtain thyroid function tests in patients who present with malaise, decreased appetite, weight loss and palpitations in the weeks following Covid 19 infection. Reference:1. Mattar SAM, Koh SJQ, Rama Chandran S, Cherng BPZ. Subacute thyroiditis associated with COVID-19. BMJ Case Rep. doi: 10.1136/bcr-2020-237336
Presentation: Thursday, June 15, 2023
THU656 Subacute Thyroiditis In Early Pregnancy: A Rare Case Report
Authors: El Sayed Farah , Shelden Daniel 
Published: October 2023

Abstract
Disclosure: F. El Sayed: None. D. Shelden: None.
Background Subacute thyroiditis (SAT) is a self-limiting inflammatory disorder that can be caused by a viral infection and presents with neck pain, fever, and fatigue. To date, only 8 cases of SAT have been reported in pregnant women. Hyperthyroidism during pregnancy is associated with both maternal and neonatal complications. The early diagnosis and treatment of SAT during pregnancy may play a vital role in prevention of adverse fetal outcomes. Case Presentation A 37-year-old female in her 6th week was referred to the endocrinology clinic for neck pain that started two months ago following a viral sore throat. She also reported exhaustion, palpitations, and occasional dysphagia. This is her 10th pregnancy, and she reports a history of thyroid isthmic cysts with hemorrhagic degeneration drained in 2014.The patient is a former occasional smoker and denies alcohol or drug use. Family history is significant for autoimmune disease in her sibling, and type 2 diabetes mellitus in her father. On physical exam, her neck is soft, supple, with no lymphadenopathy. The thyroid gland is enlarged to approximately 40 grams and is mildly tender to palpation. Labs were significant for thyroid-stimulating hormone (TSH) of 0.01 uIU/ml (reference range 0.4-4.5 uIU/ml), serum-free triiodothyronine (FT3) of 4.7 pg/ml (reference rage 1.7-3.7 ng/dl), serum-free thyroxine (FT4) 1.6 pg/ml (reference range 0.7-1.5 pg/ml), ESR 67 mm/hr (reference range 0-18 mm/hr), CRP 52.6 mg/L(reference range &lt;8 mg/L). Thyroid ultrasound revealed enlarged heterogeneous thyroid gland , with poorly defined hypoechoic areas. Based on patients’ symptoms, physical examination, and laboratory findings she was diagnosed with SAT. After review of prior literature and coordination with the patient’s obstetrician, she was started on prednisone 20 mg with a plan to taper by 5 mg weekly with close observation of thyroid function tests. Antibody assessment of thyroid was diffusely negative. Repeat assessment of the patient’s thyroid function tests showed improvement in tsh, FT3, and FT4 to 0.04, 2.4, and 0.9 respectively. The patient reported feeling significantly improved at the 2 week mark. Discussion and conclusion SAT during pregnancy is rare but requires careful management. To date, various treatment modalities are reported in case studies but there is a lack of consensus on medication dosing given the rarity of the presentation. The pathogenesis of SAT is still unknown but is thought to be secondary to destruction of thyroid follicular cells, resulting in the release of thyroid hormone and symptoms of thyrotoxicosis. SAT diagnosis is based on the patient's medical history, symptoms, physical and laboratory findings, and exclusion of other reasons for thyroid dysfunction. Pregnant women with hyperthyroidism need to be differentiated from Graves' disease, and monitoring of thyroid hormone levels is important for fetal outcomes.
Presentation: Thursday, June 15, 2023