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The stress hormone cortisol modulates fuel metabolism, cardiovascular homoeostasis, mood, inflammation and cognition. The CORtisol NETwork (CORNET) consortium previously identified a single locus associated with morning plasma cortisol. Identifying additional genetic variants that explain more of the variance in cortisol could provide new insights into cortisol biology and provide statistical power to test the causative role of cortisol in common diseases. The CORNET consortium extended its genome-wide association meta-analysis for morning plasma cortisol from 12,597 to 25,314 subjects and from ~2.2 M to ~7 M SNPs, in 17 population-based cohorts of European ancestries. We confirmed the genetic association with SERPINA6/SERPINA1. This locus contains genes encoding corticosteroid binding globulin (CBG) and α1-antitrypsin. Expression quantitative trait loci (eQTL) analyses undertaken in the STARNET cohort of 600 individuals showed that specific genetic variants within the SERPINA6/SERPINA1 locus influence expression of SERPINA6 rather than SERPINA1 in the liver. Moreover, trans-eQTL analysis demonstrated effects on adipose tissue gene expression, suggesting that variations in CBG levels have an effect on delivery of cortisol to peripheral tissues. Two-sample Mendelian randomisation analyses provided evidence that each genetically-determined standard deviation (SD) increase in morning plasma cortisol was associated with increased odds of chronic ischaemic heart disease (0.32, 95% CI 0.06–0.59) and myocardial infarction (0.21, 95% CI 0.00–0.43) in UK Biobank and similarly in CARDIoGRAMplusC4D. These findings reveal a causative pathway for CBG in determining cortisol action in peripheral tissues and thereby contributing to the aetiology of cardiovascular disease.

Phosphofurin acidic cluster sorting protein-1 (PACS-1) is a multifunctional membrane traffic regulator that plays important roles in organ homeostasis and disease. In this study, we elucidate a novel nuclear function for PACS-1 in maintaining chromosomal integrity. PACS-1 progressively accumulates in the nucleus during cell cycle progression, where it interacts with class I histone deacetylases 2 and 3 (HDAC2 and HDAC3) to regulate chromatin dynamics by maintaining the acetylation status of histones. PACS-1 knockdown results in the proteasome-mediated degradation of HDAC2 and HDAC3, compromised chromatin maturation, as indicated by elevated levels of histones H3K9 and H4K16 acetylation, and, consequently, increased replication stress-induced DNA damage and genomic instability.

Background The impact of COVID-19 on physical and mental health and employment after hospitalisation with acute disease is not well understood. The aim of this study was to determine the effects of COVID-19-related hospitalisation on health and employment, to identify factors associated with recovery, and to describe recovery phenotypes. Methods The Post-hospitalisation COVID-19 study (PHOSP-COVID) is a multicentre, long-term follow-up study of adults (aged ≥18 years) discharged from hospital in the UK with a clinical diagnosis of COVID-19, involving an assessment between 2 and 7 months after discharge, including detailed recording of symptoms, and physiological and biochemical testing. Multivariable logistic regression was done for the primary outcome of patient-perceived recovery, with age, sex, ethnicity, body-mass index, comorbidities, and severity of acute illness as covariates. A post-hoc cluster analysis of outcomes for breathlessness, fatigue, mental health, cognitive impairment, and physical performance was done using the clustering large applications k-medoids approach. The study is registered on the ISRCTN Registry (ISRCTN10980107). Findings We report findings for 1077 patients discharged from hospital between March 5 and Nov 30, 2020, who underwent assessment at a median of 5·9 months (IQR 4·9–6·5) after discharge. Participants had a mean age of 58 years (SD 13); 384 (36%) were female, 710 (69%) were of white ethnicity, 288 (27%) had received mechanical ventilation, and 540 (50%) had at least two comorbidities. At follow-up, only 239 (29%) of 830 participants felt fully recovered, 158 (20%) of 806 had a new disability (assessed by the Washington Group Short Set on Functioning), and 124 (19%) of 641 experienced a health-related change in occupation. Factors associated with not recovering were female sex, middle age (40–59 years), two or more comorbidities, and more severe acute illness. The magnitude of the persistent health burden was substantial but only weakly associated with the severity of acute illness. Four clusters were identified with different severities of mental and physical health impairment (n=767): very severe (131 patients, 17%), severe (159, 21%), moderate along with cognitive impairment (127, 17%), and mild (350, 46%). Of the outcomes used in the cluster analysis, all were closely related except for cognitive impairment. Three (3%) of 113 patients in the very severe cluster, nine (7%) of 129 in the severe cluster, 36 (36%) of 99 in the moderate cluster, and 114 (43%) of 267 in the mild cluster reported feeling fully recovered. Persistently elevated serum C-reactive protein was positively associated with cluster severity. Interpretation We identified factors related to not recovering after hospital admission with COVID-19 at 6 months after discharge (eg, female sex, middle age, two or more comorbidities, and more acute severe illness), and four different recovery phenotypes. The severity of physical and mental health impairments were closely related, whereas cognitive health impairments were independent. In clinical care, a proactive approach is needed across the acute severity spectrum, with interdisciplinary working, wide access to COVID-19 holistic clinical services, and the potential to stratify care. Funding UK Research and Innovation and National Institute for Health Research.

ObjectiveGenome-wide association studies (GWAS) have identified several risk loci for gallstone disease. As with most polygenic traits, it is likely many genetic determinants are undiscovered. The aim of this study was to identify novel genetic variants that represent new targets for gallstone research and treatment.DesignWe performed a GWAS of 28,627 gallstone cases and 348,373 controls in the UK Biobank and a GWA meta-analysis (43,639 cases and 506,798 controls) with the FinnGen cohort. We assessed pathway enrichment using gene-based then gene-set analysis and tissue expression of implicated genes in Genotype-Tissue Expression project data. We constructed a polygenic risk score (PRS) and evaluated phenotypic traits associated with the score.ResultsSeventy-five risk loci were identified (P−8) of which forty-six were novel. Pathway enrichment revealed associations with lipid homeostasis, glucuronidation, phospholipid metabolism and gastrointestinal motility. ANO1 and TMEM147, both in novel loci, are strongly expressed in the gallbladder and gastrointestinal tract. Both regulate gastrointestinal motility. The gallstone risk allele rs7599-A leads to suppression of hepatic TMEM147 expression suggesting the protein protects against gallstone formation. Individuals in the highest decile of the PRS demonstrated a 6-fold increased risk of gallstones compared to the lowest risk category. The PRS was strongly associated with increased body mass index, serum liver enzyme and C-reactive protein concentrations and decreased lipoprotein cholesterol concentrations.ConclusionThis GWAS demonstrates the polygenic nature of gallstone risk and identifies 46 novel susceptibility loci. For the first time, we implicate genes influencing gastrointestinal motility in the pathogenesis of gallstones.Summary BoxWhat is already known on this subject?Genome-wide association studies (GWAS) have identified 29 genetic variants within independent loci which increase the risk of gallstone disease.Most of these variants lie within or near to genes that regulate lipid or bile acid metabolism. Gallstones are known to have a significant genetic component with 25-50% of gallstone disease due to genetic risk factors.Much of this risk is not accounted for by the known gallstone-susceptibility loci.What are the new findings?We performed a GWAS in the UK Biobank (28,627 gallstone cases, 348,373 controls) and a GWA meta-analysis (43,639 cases and 506,798 controls) with the FinnGen cohort.We identified a total of 75 gallstone-susceptibility loci with 46 of these being new and the remaining 29 being those already identified.We annotated the variants based on their position within or near to genes and assessed pathway enrichment through gene-set analysis.We identify two novel gallstone-susceptibility loci in which the lead variants lie within genes governing gastrointestinal motility which highly expressed in gallbladder (ANO1 and TMEM147).We demonstrate further loci involved in primary cilia function.We report significant association of a polygenic risk score with gallstones using independent subsets of the study population.How might it impact on clinical practice in the foreseeable future?The genes and pathways identified represent novel targets for development of medication targeting primary or secondary prevention of gallstones. This may be of particular benefit to those unable to undergo cholecystectomy.The individual variants or polygenic risk score identified in this GWAS could form the basis for identification of individuals with high risk of gallstones to support screening or treatment of gallstone disease. This screening may be of particular benefit in populations with elevated risk of gallstones such as haemolytic disease or bariatric surgery.

Genome-wide association studies (GWAS) have identified several risk loci for nonalcoholic fatty liver disease (NAFLD). Previous studies have largely relied on small sample sizes and have assessed quantitative traits. We performed a case-control GWAS in the UK Biobank using recorded diagnosis of NAFLD based on diagnostic codes recommended in recent consensus guidelines. We performed a GWAS of 4,761 cases of NAFLD and 373,227 healthy controls without evidence of NAFLD. Sensitivity analyses were performed excluding other co-existing hepatic pathology, adjusting for body mass index (BMI) and adjusting for alcohol intake. A total of 9,723,654 variants were assessed by logistic regression adjusted for age, sex, genetic principal components, and genotyping batch. We performed a GWAS meta-analysis using available summary association statistics. Six risk loci were identified (P < 5*10-8 ) (apolipoprotein E [APOE], patatin-like phospholipase domain containing 3 [PNPLA3, transmembrane 6 superfamily member 2 [TM6SF2], glucokinase regulator [GCKR], mitochondrial amidoxime reducing component 1 [MARC1], and tribbles pseudokinase 1 [TRIB1]). All loci retained significance in sensitivity analyses without co-existent hepatic pathology and after adjustment for BMI. PNPLA3 and TM6SF2 remained significant after adjustment for alcohol (alcohol intake was known in only 158,388 individuals), with others demonstrating consistent direction and magnitude of effect. All six loci were significant on meta-analysis. Rs429358 (P = 2.17*10-11 ) is a missense variant within the APOE gene determining ϵ4 versus ϵ2/ϵ3 alleles. The ϵ4 allele of APOE offered protection against NAFLD (odds ratio for heterozygotes 0.84 [95% confidence interval 0.78-0.90] and homozygotes 0.64 [0.50-0.79]). Conclusion: This GWAS replicates six known NAFLD-susceptibility loci and confirms that the ϵ4 allele of APOE is associated with protection against NAFLD. The results are consistent with published GWAS using histological and radiological measures of NAFLD, confirming that NAFLD identified through diagnostic codes from consensus guidelines is a valid alternative to more invasive and costly approaches.

Background & AimsGenome-wide association studies (GWAS) have identified several risk loci for non-alcoholic fatty liver disease (NAFLD). Previous studies have largely relied on small sample sizes and have assessed quantitative traits. We performed a case-control GWAS in the UK Biobank using recorded diagnosis of NAFLD based on diagnostic codes recommended in recent consensus guidelines.Approach & ResultsWe performed a GWAS of 4,761 cases of NAFLD and 373,227 healthy controls without evidence of NAFLD. Sensitivity analyses were performed excluding other co-existing hepatic pathology, adjusting for BMI and adjusting for alcohol intake. 9,723,654 variants were assessed by logistic regression adjusted for age, sex, genetic principal components and genotyping batch. We performed a GWA meta-analysis using available summary association statistics from two previously published case-control GWAS of NAFLD. Six risk loci were identified (PConclusionsThis GWAS demonstrates that the ∈4 allele of APOE is strongly associated with protection against NAFLD.

A new time-domain impedance probe is presented in this paper. The new instrument is able to make measurements of absolute electron density and electron-neutral collision frequency in the ionosphere at temporal and spatial resolutions not previously attained. A single measurement is made in $100~\mu \text{s}$ , which yields an instantaneous spatial resolution of 0.1 m for sounding rocket experiments. A prototype of this instrument was integrated into the payload of a NASA Undergraduate Student Instrument Program sounding rocket launched out of Wallops Island on March 1, 2016. Here, we describe the instrument, and present the data obtained from the sounding rocket experiment. A 6-V amplitude Gaussian derivative excitation was applied to a dipole probe structure, and the current through the probe terminals measured with a balanced active bridge circuit. The time-domain current response was sampled at 5 MS/s, at 12-bit resolution. In the course of the flight, the instrument measured a highly nonlinear response of the plasma because of the large input voltage signal applied. The linear theory cannot explain this response, which obscured interpretation of the data. As a result, we used time- and frequency-domain trend analysis to obtain the variation of electron density over the upleg and downleg of the rocket trajectory. The obtained time and fast Fourier transform trends showed enhanced electron densities in the F layer, which confirmed that the instrument was able to measure the density variations during a significant portion of the flight.

The analysis of the effects of autozygosity, measured as the change of the mean value of a trait among offspring of genetic relatives, reveals the existence of directional dominance or overdominance. In this study we detect evidence of the effect of autozygosity in 4 out of 13 cardiometabolic disease-associated traits using data from more than 10,000 sub-Saharan African individuals recruited from Ghana, Burkina Faso, Kenya and South Africa. The effect of autozygosity on these phenotypes is found to be sex-related, with inbreeding having a significant decreasing effect in men but a significant increasing effect in women for several traits (body mass index, subcutaneous adipose tissue, low-density lipoproteins and total cholesterol levels). Overall, the effect of inbreeding depression is more intense in men. Differential effects of inbreeding depression are also observed between study sites with different night-light intensity used as proxy for urban development. These results suggest a directional dominant genetic component mediated by environmental interactions and sex-specific differences in genetic architecture for these traits in the Africa Wits-INDEPTH partnership for Genomic Studies (AWI-Gen) cohort., The prevalence of cardiometabolic diseases (CMDs) is increasing rapidly across Africa. Here, the authors investigate autozygosity in CMD-associated traits in over 10,000 sub-Saharan African individuals, showing these traits are influenced by sex-specific inbreeding depression and environmental interactions.

To efficiently transform genetic associations into drug targets requires evidence that a particular gene, and its encoded protein, contribute causally to a disease. To achieve this, we employ a three-step proteome-by-phenome Mendelian Randomization (MR) approach. In step one, 154 protein quantitative trait loci (pQTLs) were identified and independently replicated. From these pQTLs, 64 replicated locally-acting variants were used as instrumental variables for proteome-by-phenome MR across 846 traits (step two). When its assumptions are met, proteome-by-phenome MR, is equivalent to simultaneously running many randomized controlled trials. Step 2 yielded 38 proteins that significantly predicted variation in traits and diseases in 509 instances. Step 3 revealed that amongst the 271 instances from GeneAtlas (UK Biobank), 77 showed little evidence of pleiotropy (HEIDI), and 92 evidence of colocalization (eCAVIAR). Results were wide ranging: including, for example, new evidence for a causal role of tyrosine-protein phosphatase non-receptor type substrate 1 (SHPS1; SIRPA) in schizophrenia, and a new finding that intestinal fatty acid binding protein (FABP2) abundance contributes to the pathogenesis of cardiovascular disease. We also demonstrated confirmatory evidence for the causal role of four further proteins (FGF5, IL6R, LPL, LTA) in cardiovascular disease risk., Author summary The targets of most medications prescribed today are proteins. For many common diseases our understanding of the underlying causes is often incomplete, and our ability to predict whether new drugs will be effective is remarkably poor. Attempts to use genetics to identify drug targets have an important limitation: standard study designs link disease risk to DNA but do not explain how the genotype leads to disease. In our study, we made robust statistical links between DNA variants and blood levels of 249 proteins, in two separate groups of Europeans. We then used this information to predict protein levels in large genetic studies. In many cases, this second step gives us evidence that high or low levels of a given protein play a role in causing a given disease. Among dozens of high-confidence links, we found new evidence for a causal role of a protein called SHPS1 in schizophrenia, and of another protein (FABP2) in heart disease. Our method takes advantage of information from large numbers of existing genetic studies to prioritize specific proteins as drug targets.

Publisher's version (útgefin grein)., In many species, the offspring of related parents suffer reduced reproductive success, a phenomenon known as inbreeding depression. In humans, the importance of this effect has remained unclear, partly because reproduction between close relatives is both rare and frequently associated with confounding social factors. Here, using genomic inbreeding coefficients (FROH) for >1.4 million individuals, we show that FROH is significantly associated (p < 0.0005) with apparently deleterious changes in 32 out of 100 traits analysed. These changes are associated with runs of homozygosity (ROH), but not with common variant homozygosity, suggesting that genetic variants associated with inbreeding depression are predominantly rare. The effect on fertility is striking: FROH equivalent to the offspring of first cousins is associated with a 55% decrease [95% CI 44–66%] in the odds of having children. Finally, the effects of FROH are confirmed within full-sibling pairs, where the variation in FROH is independent of all environmental confounding., This paper is the work of the ROHgen consortium. We thank the Sigma T2D Consortium, whose members are detailed in Supplementary Note 3. We thank the UK Biobank Resource, approved under application 19655; we acknowledge funding from the UK Medical Research Council Human Genetics Unit and MRC Doctoral Training Programme in Precision Medicine. We also thank Neil Robertson, Wellcome Trust Centre for Human Genetics, Oxford, for use of his author details management software, Authorial. Finally, we thank all the participants, researchers and funders of ROHgen cohorts. Cohort-specific acknowledgements are in Supplementary Data 2; personal acknowledgements and disclosures are in Supplementary Note 2. We thank Rachel Edwards for administrative assistance.

Surgical treatments for nasal airway obstruction (NAO) are commonly offered as part of otolaryngology practice. Anatomic causes include septal deviation, inferior turbinate hypertrophy, and nasal valve collapse (NVC). This study was performed to determine the prevalence of anatomic contributors to NAO. A total of 1,906 patients with sinonasal complaints were surveyed by 50 otolaryngologists in varying U.S. geographic regions. Patients were first evaluated using the Nasal Obstruction Symptom Evaluation (NOSE) instrument to assess the NAO symptoms and their severity. Physicians then examined patients for the presence of the three anatomic contributors. Presence of septal deviation and turbinate hypertrophy was assessed through an internal nasal exam with direct or endoscopic visualization based on the physician's standard methodology for diagnosis. Presence of NVC was determined by the modified Cottle maneuver. Among all patients surveyed, prevalence was 67% for NVC, 76% for septal deviation, and 72% for inferior turbinate hypertrophy. We found that 64% of the patients (n = 1,211) had severe/extreme NOSE scores (≥55), representing the most likely nasal obstruction candidates for intervention. In these patients, the prevalence of NVC, septal deviation, and inferior turbinate hypertrophy was 73, 80, and 77%, respectively. Eighty-two percent of the 236 patients with severe/extreme NOSE scores who reported prior septoplasty and/or inferior turbinate reduction had NVC. Our study revealed a comparable prevalence of all three anatomic contributors across all patients and the subset with severe/extreme NOSE scores, highlighting the importance of evaluating the lateral nasal wall as a component of NAO treatment strategy.

Teachers working with refugee families who are culturally and linguistically diverse (CLD) and receiving special education services often rely on cultural liaisons to provide interpreter and translator services during Individualized Educational Program (IEP) and Individualized Family Service Plan (IFSP) meetings. The purpose of this qualitative study was to interview nine cultural liaisons, who live in a refugee resettlement community, regarding their experiences and advice for teachers when conducting IEP and IFSP meetings with refugee families. Semistructured interviews resulted in themes or suggestions for teachers on how to prepare for the meetings, conduct the meetings, and communicate with families through the cultural liaison in addition to an appeal for teachers to learn about the cultures and consistently demonstrate respect. Implications for future research and teacher preparation are discussed.

Ovarian cancer (OC) is the most deadly gynecological cancer and unlike most other neoplasms, survival rates for OC have not significantly improved in recent decades. We show that RAD6, an ubiquitin-conjugating enzyme, is significantly overexpressed in ovarian tumors and its expression increases in response to carboplatin chemotherapy. RAD6 expression correlated strongly with acquired chemoresistance and malignant behavior of OC cells, expression of stem cell genes and poor prognosis of OC patients, suggesting an important role for RAD6 in ovarian tumor progression. Upregulated RAD6 enhances DNA damage tolerance and repair efficiency of OC cells and promotes their survival. Increased RAD6 levels cause histone 2B ubiquitination-mediated epigenetic changes that stimulate transcription of stem cell genes, including ALDH1A1 and SOX2, leading to a cancer stem cell phenotype, which is implicated in disease recurrence and metastasis. Downregulation of RAD6 or its inhibition using a small molecule inhibitor attenuated DNA repair signaling and expression of cancer stem cells markers and sensitized chemoresistant OC cells to carboplatin. Together, these results suggest that RAD6 could be a therapeutic target to prevent and treat acquired chemoresistance and disease recurrence in OC and enhance the efficacy of standard chemotherapy.

Angiomyolipomas are benign tumors composed of blood vessels, adipose tissue, and smooth muscle. Usually, these lesions are located in the kidney and are found in association with tuberous sclerosis. The current case describes the rare presence of an angiomyolipoma within the nasal vestibule, leading to nasal obstruction. Surgical resection of the lesion was curative, and the patient’s nasal congestion improved postoperatively. Consistent with previous findings in the literature, the patient’s angiomyolipoma had not recurred at subsequent postoperative visits.

Significance Modern genetic analysis has revealed genetic differentiation across the south of Britain and Ireland. This structure demonstrates the impact of hegemonies and migrations from the histories of Britain and Ireland. How this structure compares to the north of Britain, Scotland, and its surrounding Isles is less clear. We present genomic analysis of 2,544 British and Irish, including previously unstudied Scottish, Shetlandic and Manx individuals. We demonstrate widespread structure across Scotland that echoes past kingdoms, and quantify the considerable structure that is found on its surrounding isles. Furthermore, we show the extent of Norse Viking ancestry across northern Britain and estimate a region of origin for ancient Gaelic Icelanders., Britain and Ireland are known to show population genetic structure; however, large swathes of Scotland, in particular, have yet to be described. Delineating the structure and ancestry of these populations will allow variant discovery efforts to focus efficiently on areas not represented in existing cohorts. Thus, we assembled genotype data for 2,554 individuals from across the entire archipelago with geographically restricted ancestry, and performed population structure analyses and comparisons to ancient DNA. Extensive geographic structuring is revealed, from broad scales such as a NE to SW divide in mainland Scotland, through to the finest scale observed to date: across 3 km in the Northern Isles. Many genetic boundaries are consistent with Dark Age kingdoms of Gaels, Picts, Britons, and Norse. Populations in the Hebrides, the Highlands, Argyll, Donegal, and the Isle of Man show characteristics of isolation. We document a pole of Norwegian ancestry in the north of the archipelago (reaching 23 to 28% in Shetland) which complements previously described poles of Germanic ancestry in the east, and “Celtic” to the west. This modern genetic structure suggests a northwestern British or Irish source population for the ancient Gaels that contributed to the founding of Iceland. As rarer variants, often with larger effect sizes, become the focus of complex trait genetics, more diverse rural cohorts may be required to optimize discoveries in British and Irish populations and their considerable global diaspora.

Target identification remains a crucial challenge in drug development. To enable unbiased detection of proteins and pathways that have a causal role in disease pathogenesis or progression, we propose proteome-by-phenome Mendelian Randomisation (P2MR). We first detected genetic variants associated with plasma concentration of 249 proteins. We then used 64 replicated variants in two-sample Mendelian Randomisation to quantify evidence of a causal role for each protein across 846 phenotypes: this yielded 509 robust protein-outcome links. P2MR provides substantial promise for drug target prioritisation. We provide confirmatory evidence for a causal role for the proteins encoded at multiple cardiovascular disease risk loci (FGF5, IL6R, LPL, LTA), and discovered that intestinal fatty acid binding protein (FABP2) contributes to disease pathogenesis. Additionally, we find and replicate evidence for a causal role of tyrosine-protein phosphatase non-receptor type substrate 1 (SHPS1; SIRPA) in schizophrenia. Our results provide specific prediction of the effects of changes of plasma protein concentration on complex phenotypes in humans.

Sinonasal papillomas, also known as Schneiderian papillomas, are benign but infiltrative epithelial neoplasms that arise from ectodermally derived pseudostratified ciliated (or Schneiderian) epithelium that lines the sinonasal tract and nasal cavity. We describe a rare case of a sinonasal papilloma confined to the lacrimal duct and sac in a 45-year-old man who presented with 10 months of right-sided epiphora and swelling of his medial right lower eyelid. He was found to have a 10 × 10 mm area of distension near the right lacrimal sac and underwent an external dacryocystorhinostomy. A computed tomography scan revealed an expansile mass in the right nasolacrimal duct area. Right endoscopic sinus surgery and endoscopic dacryocystorhinostomy were performed. This case illustrates the importance of including sinonasal papilloma, a benign but invasive neoplasm, in the differential diagnosis of nasolacrimal duct obstruction.

The normal female reproductive hormone estrogen has been linked with increased risk of breast and many other forms of cancer. This is largely due to metabolic conversion of estrogens into highly reactive catechol estrogen quinones which can interact with DNA and cause a variety of DNA adducts and lesions. Detection and analysis of these adducts and their associated cellular responses involve complex chemical, enzymatic, and LC-MS based methods, which are both laborious and require specialized expertise and instrumentation. Herein, we show that using a biotin-labeled estradiol allows immunodetection of estrogen-induced DNA adducts by slot blot and single-cell molecular combing and proximity ligation assays. The biotinylated and unlabeled estradiols induced similar levels of DNA single and double strand breaks as measured by comet assays. Using biotinylated estrogen, we further show that estrogens are able to activate the Fanconi anemia-BRCA tumor suppressor pathway and cause DNA strand breaks and oxidatively modified DNA bases as well as gross chromosomal aberrations. Utilization of biotin-labeled estrogens could be a powerful tool to detect estrogen adducts and associated DNA damage, and to track estrogen adduct-induced cellular responses and carcinogenic mechanisms in cultured cells. The techniques presented here allow simple and rapid detection and quantitation of estrogen adducts by slot blot as well as direct visualization on the DNA strand and could pave the way for developing new treatments to protect the genome from the effects of reactive estrogen metabolites. © 2016 Wiley Periodicals, Inc.

Camptothecin (CPT) and its analogues are chemotherapeutic agents that covalently and reversibly link DNA Topoisomerase I to its nicked DNA intermediate eliciting the formation of DNA double strand breaks (DSB) during replication. The repair of these DSB involves multiple DNA damage response and repair proteins. Here we demonstrate that CPT-induced DNA damage promotes functional interactions between BRCA2, FANCD2, Rad18, and Rad51 to repair the replication-associated DSB through homologous recombination (HR). Loss of any of these proteins leads to equal disruption of HR repair, causes chromosomal aberrations and sensitizes cells to CPT. Rad18 appears to function upstream in this repair pathway as its downregulation prevents activation of FANCD2, diminishes BRCA2 and Rad51 protein levels, formation of nuclear foci of all three proteins and recovery of stalled or collapsed replication forks in response to CPT. Taken together this work further elucidates the complex interplay of DNA repair proteins in the repair of replication-associated DSB.

Ovarian cancer is the fifth most deadly cancer in women in the United States and despite advances in surgical and chemotherapeutic treatments survival rates have not significantly improved in decades. The poor prognosis for ovarian cancer patients is largely due to the extremely high (80%) recurrence rate of ovarian cancer and because the recurrent tumors are often resistant to the widely utilized platinum-based chemotherapeutic drugs. In this study, expression of Rad6, an E2 ubiquitin-conjugating enzyme, was found to strongly correlate with ovarian cancer progression. Furthermore, in ovarian cancer cells Rad6 was found to stabilize β-catenin promoting stem cell-related characteristics, including expression of stem cell markers and anchorage-independent growth. Cancer stem cells can promote chemoresistance, tumor recurrence and metastasis, all of which are limiting factors in treating ovarian cancer. Thus it is significant that Rad6 overexpression led to increased resistance to the chemotherapeutic drug carboplatin and correlated with tumor cell invasion. These findings show the importance of Rad6 in ovarian cancer and emphasize the need for further studies of Rad6 as a potential target for the treatment of ovarian cancer.

In hypoxia, mitochondria-generated reactive oxygen species not only stimulate accumulation of the transcriptional regulator of hypoxic gene expression, hypoxia inducible factor-1 (Hif-1), but also cause oxidative base modifications in hypoxic response elements (HREs) of hypoxia-inducible genes. When the hypoxia-induced base modifications are suppressed, Hif-1 fails to associate with the HRE of the VEGF promoter, and VEGF mRNA accumulation is blunted. The mechanism linking base modifications to transcription is unknown. Here we determined whether recruitment of base excision DNA repair (BER) enzymes in response to hypoxia-induced promoter modifications was required for transcription complex assembly and VEGF mRNA expression. Using chromatin immunoprecipitation analyses in pulmonary artery endothelial cells, we found that hypoxia-mediated formation of the base oxidation product 8-oxoguanine (8-oxoG) in VEGF HREs was temporally associated with binding of Hif-1α and the BER enzymes 8-oxoguanine glycosylase 1 (Ogg1) and redox effector factor-1 (Ref-1)/apurinic/apyrimidinic endonuclease 1 (Ape1) and introduction of DNA strand breaks. Hif-1α colocalized with HRE sequences harboring Ref-1/Ape1, but not Ogg1. Inhibition of BER by small interfering RNA-mediated reduction in Ogg1 augmented hypoxia-induced 8-oxoG accumulation and attenuated Hif-1α and Ref-1/Ape1 binding to VEGF HRE sequences and blunted VEGF mRNA expression. Chromatin immunoprecipitation-sequence analysis of 8-oxoG distribution in hypoxic pulmonary artery endothelial cells showed that most of the oxidized base was localized to promoters with virtually no overlap between normoxic and hypoxic data sets. Transcription of genes whose promoters lost 8-oxoG during hypoxia was reduced, while those gaining 8-oxoG was elevated. Collectively, these findings suggest that the BER pathway links hypoxia-induced introduction of oxidative DNA modifications in promoters of hypoxia-inducible genes to transcriptional activation.

PACS-1 is a protein involved in trafficking, especially transport of proteins like furin and Mannose-6-phosphate into the trans-golgi network. Intriguingly, our studies identified dynamic translocation of PACS-1 during cell division and its association with chromatin in tumor cells. Downregulation of PACS-1 in cancer cell lines attenuated cell growth, induced replication stress mediated DNA damage response (DDR), such as ATM/ATR-mediated checkpoint activation, and formation RPA, FANCD2 and pH2AX foci. Consistent with these observations, live-cell imaging revealed increased nuclear translocation of PACS-1 during S-phase and until the complete cell division. These results point to a novel role for PACS-1 during normal replication or in the suppression of oncogenic replication in tumor cells. However, a direct role for PACS-1 in cancer development or progression is not clear. Analysis of the cancer genome atlas (TCGA) data revealed occurrence of PACS-1 amplifications, mutations and deletions in cancers of different tissues. PACS-1 depletion leads to proteasome-mediated degradation of HDAC2 and HDAC3, compromised chromatin maturation, leads to replication stress and genomic instability. Intriguingly, stable overexpression of PACS-1 in MDA-MB-231 cells enhanced malignant behavior of tumor cells in their ability to form soft agar colonies and matrigel invasion compared to the cells that are expressing vector control. PACS-1 downregulation promotes replication stress in tumor cells suggesting that its upregulation in tumors may be important for replication stress recovery in cancer cells and for an efficient response to chemotherapy induced DNA damage. Furthermore, Prognoscan analysis indicated that PACS-1 expression levels correlates with poor survival in multiple studies, including breast, ovarian and lung cancers, suggesting a role for PACS-1 in tumorigenesis and a biomarker for poor prognosis. Citation Format: Chinnadurai Mani, Kaushlendra Tripathi, David W. Clark, Gary Thomas, Komaraiah Palle. A novel role for PACS-1 in suppression of oncogenic replication stress in cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2380.

Elevated estrogens exert their carcinogenic effects by at least three different mechanisms; they promote cell proliferation by transcriptional regulation of estrogen responsive genes, cause generation of reactive oxygen species and other free radicals, and directly react with DNA and form potentially mutagenic adducts. However, unbalanced estrogen metabolism has been linked to development of several malignant diseases including breast, endometrial and ovarian cancer and has been identified as a risk factor for these cancers. The metabolic breakdown of estogens is regulated by a series of reactions mediated by hepatic and peripheral enzymes that balances the induction and reduction of cellular stress. An imbalance in this enzymes and processes can lead to highly reactive catechol estrogen metabolites that can react with DNA, form carcinogenic DAN-adducts and lesions. Although several studies established a connection between estrogen-induced DNA damage and carcinogenesis, the underlying molecular mechanisms have been difficult to study because of the technical challenges in detecting and analyzing the variety of different DNA lesions that are formed by estrogen compounds. Moreover, detection and analysis of these adducts are important to directly monitor estrogen metabolites induced cellular responses in the cells. Towards this, we developed a novel method using biotinylated-estrogens that allows immunodetection of estrogen-induced DNA adducts by Slot-blot and single-cell molecular combing and proximity ligation assays. Using these modified estrogens we first time quantitatively detected these adducts on DNA by immune Slot-blot techniques and on DNA fibers. Furthermore, similar to other environmental carcinogens estrogens activates replication associated DNA damage responses and induces chromosomal instability. Hence, first time our studies demonstrate that biotin-labeled estrogens could be a powerful tool to detect estrogen adducts and to probe associated DNA damage responses and cellular responses. Citation Format: Kaushlendra Tripathi, Chinnadurai Mani, David W. Clark, Komaraiah Palle. BIOTINYLATED ESTROGENS A NOVEL TOOL FOR EARLY DETECTION OF ADDUCT IN OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-015.

We use a multi-stage genome-wide association of 1 million parental lifespans of genotyped subjects and data on mortality risk factors to validate previously unreplicated findings near CDKN2B-AS1, ATXN2/BRAP, FURIN/FES, ZW10, PSORS1C3, and 13q21.31, and identify and replicate novel findings near GADD45G, KCNK3, LDLR, POM121C, ZC3HC1, and ABO. We also validate previous findings near 5q33.3/EBF1 and FOXO3, whilst finding contradictory evidence at other loci. Gene set and tissue-specific analyses show that expression in foetal brain cells and adult dorsolateral prefrontal cortex is enriched for lifespan variation, as are gene pathways involving lipid proteins and homeostasis, vesicle-mediated transport, and synaptic function. Individual genetic variants that increase dementia, cardiovascular disease, and lung cancer –but not other cancers-explain the most variance, possibly reflecting modern susceptibilities, whilst cancer may act through many rare variants, or the environment. Resultant polygenic scores predict a mean lifespan difference of around five years of life across the deciles.

Inconsistencies in the nomenclature of structures of the frontal sinus have impeded the development of a validated "reference standard" classification system that surgeons can reliably agree upon. The International Frontal Sinus Anatomy Classification (IFAC) system was developed as a consensus document, based on expert opinion, attempting to address this issue. The purposes of this study are to: establish the reliability of the IFAC as a tool for classifying cells in the frontal recess among an international group of rhinologists; and improve communication and teaching of frontal endoscopic sinus surgery (ESS).Forty-two computed tomography (CT) scans, each with a marked frontal cell, were reviewed by 15 international fellowship-trained rhinologists. Each marked cell was classified into 1 of 7 categories described in the IFAC, on 2 occasions separated by 2 weeks. Inter- and intrarater reliability were evaluated using Light's kappa (κ), the interclass correlation coefficient (ICC), and simple proportion of agreement.Interrater reliability showed pairwise κ values ranging from 0.7248 to 1.0, with a mean of 0.9162 (SD, 0.0537). The ICC was 0.98. Intrarater reliability showed κ values ranging from 0.8613 to 1.0, with a mean of 0.9407 (SD, 0.0376). The within-rater ICC was 0.98.Among a diverse sample of rhinologists (raters), there was substantial to almost perfect agreement between raters, and among individual raters at different timepoints. The IFAC is a reliable tool for classification of cells in the frontal sinus. Further outcome studies are still needed to determine the validity of the IFAC.

Humans vary substantially in their willingness to take risks. In a combined sample of over one million individuals, we conducted genome-wide association studies (GWAS) of general risk tolerance, adventurousness, and risky behaviors in the driving, drinking, smoking, and sexual domains. We identified 611 approximately independent genetic loci associated with at least one of our phenotypes, including 124 with general risk tolerance. We report evidence of substantial shared genetic influences across general risk tolerance and risky behaviors: 72 of the 124 general risk tolerance loci contain a lead SNP for at least one of our other GWAS, and general risk tolerance is moderately to strongly genetically correlated (to 0.50) with a range of risky behaviors. Bioinformatics analyses imply that genes near general-risk-tolerance-associated SNPs are highly expressed in brain tissues and point to a role for glutamatergic and GABAergic neurotransmission. We find no evidence of enrichment for genes previously hypothesized to relate to risk tolerance.

Mortality in ovarian cancer is predominantly due to acquired chemoresistance and tumor recurrence. UBIQUITIN CONJUGATING ENZYME E2 or RAD6 expression increases in cell lines and patient tumors in response to platinum-based chemotherapy and promotes both activation of DNA damage response pathways and expression of stemness genes and a stem cell-like phenotype driving ovarian cancer chemoresistance.

Preview As many as 30% of patients with long-standing diabetes mellitus may have symptoms of gastroparesis, and others are found to have the condition despite being asymptomatic. When should diabetic gastroparesis be suspected, and which are the best tests for detecting it? What factors can exacerbate it? Drs Clark and Nowak address these questions and discuss the therapeutic value of dietary modification, pro- kinetic agents, and surgical intervention.

Inverted papilloma of the nasal cavity is a benign neoplasm, although it can be locally invasive and has the potential for malignant degeneration. Inverted papilloma of the temporal bone is extremely rare. We describe a case of a 44-year-old woman who was treated for nasal inverted papilloma and was later found to have inverted papilloma of her temporal bone. The patient required several procedures to remove the inverted papilloma from the nasal cavity and temporal bone, and she is currently free of recurrence.

Fanconi anemia (FA) is a rare genome instability syndrome with progressive bone marrow failure and cancer susceptibility. FANCJ is one of 17 genes mutated in FA-patients, comprises a DNA helicase that is vital for properly maintaining genomic stability and is known to function in the FA-BRCA DNA repair pathway. While exact role(s) of FANCJ in this repair process is yet to be determined, it is known to interact with primary effector FANCD2. However, FANCJ is not required for FANCD2 activation but is important for its ability to fully respond to DNA damage. In this report, we determined that transient depletion of FANCJ adversely affects stability of FANCD2 and its co-regulator FANCI in multiple cell lines. Loss of FANCJ does not significantly alter cell cycle progression or FANCD2 transcription. However, in the absence of FANCJ, the majority of FANCD2 is degraded by both the proteasome and Caspase-3 dependent mechanism. FANCJ is capable of complexing with and stabilizing FANCD2 even in the absence of a functional helicase domain. Furthermore, our data demonstrate that FANCJ is important for FANCD2 stability and proper activation of DNA damage responses to replication blocks induced by hydroxyurea.

Impedance spectroscopy is a powerful technique that can be employed to determine the physical properties of different materials. In principle the technique can be implemented in the time domain using pulsed signal excitation rather than sweeping across frequencies. The advantage is particularly apparent when the measuring instrument is either traveling through the material medium rapidly, as in the case of a satellite moving through space plasma, or if the medium is for example a fluid that flows past an instrument quickly. Here we describe a Time Domain Impedance Probe (TDIP) circuit design that is used to measure the absolute density of ionospheric plasmas. A preliminary version of this instrument was flown on a sounding rocket, but here we outline the system and circuit design that is being implemented for a Low Earth Orbit (LEO) micro-satellite. The design employs a bridge architecture together with a software adaptive filter and LMS algorithm for fast calibration and data compression. We propose that the design can be generalized, and we present a System On Chip (SOC) concept based on the time domain architecture. The proposed concept appears to be well suited to ultra-fast time domain spectroscopic measurements, but does have some inherent limitations such as increased noise. We suggest that this unavoidable shortcoming can be somewhat mitigated through repetitive pulsing and averaging during the measurement process.

A new Time Domain Impedance Probe (TDIP) is presented in this paper. The new instrument is able to make measurements of absolute electron density and electron neutral collision frequency in the ionosphere at temporal and spatial resolutions not previously attained. A single measurement is made in 100 microseconds, which yields an instantaneous spatial resolution of 0.1 meters for sounding rocket experiments. A prototype of this instrument was integrated into the payload of a NASA USIP sounding rocket launched out of Wallops Island on March 1 2016. The sounding rocket launched at 8:50 am and reached an reached an altitude of 170 km, passing through the D and E and F layers of the ionosphere. The TDIP was active for 206 seconds during the flight. Here we describe the instrument, and present some time domain data obtained from the sounding rocket experiment. A 6 Volt amplitude Gaussian derivative excitation was applied to a dipole probe structure, and the current through the probe terminals measured with a balanced active bridge circuit. The time domain current response was sampled at 5 MS/s, at 12 bit resolution. In the course of the flight, the instrument measured what appeared to be a highly nonlinear response of the plasma because of the large input voltage signal applied. These are the first measurements of this type of response, to our knowledge. Post-flight laboratory calibration indicated that the instrument worked correctly through the flight. Further modeling, simulation and theoretical work needs to be performed to understand and interpret the measurements.

In the original version of this Article, the legend in the upper panel of Figure 2 incorrectly read ‘paternal imprinting’ and should have read ‘maternal imprinting’. This has been corrected in both the PDF and HTML versions of the Article.

A deep neck abscess is uncommon in the newborn period. In this case, we noted a clindamycin-sensitive methicillin-resistant Staphylococcus aureus infection characterized as a deep neck abscess in an 8-day-old boy. He was admitted to the pediatric intensive care unit with a progressively enlarging indurated mass below the mandible. Imaging confirmed the mass as a submandibular abscess. The patient received antibiotics in addition to incision and drainage, with resolution of the abscess.

The normal female reproductive hormone estrogen has been linked with increased risk of breast and many other forms of cancer. This is largely due to metabolic conversion of estrogens into highly reactive catechol estrogen quinones which can interact with DNA and cause a variety of DNA adducts and lesions. Detection and analysis of these adducts and their associated cellular responses involve complex chemical, enzymatic, and LC-MS based methods, which are both laborious and require specialized expertise and instrumentation. Herein, we show that using a biotin-labeled estradiol allows immunodetection of estrogen-induced DNA adducts by slot blot and single-cell molecular combing and proximity ligation assays. The biotinylated and unlabeled estradiols induced similar levels of DNA single and double strand breaks as measured by comet assays. Using biotinylated estrogen, we further show that estrogens are able to activate the Fanconi anemia-BRCA tumor suppressor pathway and cause DNA strand breaks and oxidatively modified DNA bases as well as gross chromosomal aberrations. Utilization of biotin-labeled estrogens could be a powerful tool to detect estrogen adducts and associated DNA damage, and to track estrogen adduct-induced cellular responses and carcinogenic mechanisms in cultured cells. The techniques presented here allow simple and rapid detection and quantitation of estrogen adducts by slot blot as well as direct visualization on the DNA strand and could pave the way for developing new treatments to protect the genome from the effects of reactive estrogen metabolites. © 2016 Wiley Periodicals, Inc.

BRCA1-Interacting Protein 1 (BRIP1), a DNA helicase and tumor suppressor also known as FANCJ, is implicated in breast and ovarian cancers as well as the genetic instability syndrome Fanconi anemia (FA) which predisposes patients to multiple neoplasms. This study investigates the role of FANCJ in repair of replication-associated DNA damage which can lead to DNA strand breaks, chromosomal instability, and tumorigenesis. FANCJ has long been known to function in repair of DNA crosslinks; however, its role in protecting cells from replication-associated DNA damage is less well known. We have shown that loss of FANCJ sensitizes cells to damage caused by compounds that block DNA replication, such as hydroxyurea (HU) and the topoisomerase I poison camptothecin (CPT). Loss of FANCJ disrupts the cell's response to replication-associated DNA damage by impairing Chk1 and Chk2 phosphorylation and stability and activation of multiple DNA repair proteins. FANCJ-deficient cells exposed to low levels of CPT failed to progress through S-phase indicating that FANCJ is important for intra-S phase DNA damage checkpoint and recovery from replication fork blocks. FANCJ depletion leads to a 90% decrease in homologous recombination repair in assays designed to measure repair of DNA double strand breaks as well as a loss of expression of repair-associated proteins and DNA repair foci in response to damage from HU and CPT. Loss of FANCJ impairs DNA replication and leads to accumulation of gross chromosomal aberrations. Importantly, some of the DNA damage response functions of FANCJ are independent of its helicase activity, as a helicase-deficient mutant is able to compensate for loss of FANCJ in activation and stability of FANCD2 and other HR proteins. Collectively our findings indicate a novel role for FANCJ as a key regulator of the DNA damage response to errors in DNA replication potentially leading to mutagenesis and tumorigenesis. Citation Format: David W. Clark, Chinnadurai Mani, Komariah Palle. BRIP1/FANCJ is vital for efficient response to and repair of replication-associated DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3380.

Resistance to current chemotherapeutic or radiation-based cancer treatment strategies is a serious concern. Cancer stem cells (CSCs) are typically able to evade treatment and establish a recurrent tumor or metastasis, and it is these that lead to the majority of cancer deaths. Therefore, a major current goal is to develop treatment strategies that eliminate the resistant CSCs as well as the bulk tumor cells in order to achieve complete disease clearance. Aldehyde dehydrogenases (ALDHs) are important for maintenance and differentiation of stem cells as well as normal development. There is expanding evidence that ALDH expression increases in response to therapy and promotes chemoresistance and survival mechanisms in CSCs. This perspective will discuss a paper by Cojoc and colleagues recently published in Cancer Research, that indicates ALDHs play a key role in resistance to radiation therapy and tumor recurrence in prostate cancer. The authors suggest that ALDHs are a potential therapeutic target for treatment prostate cancer patients to limit radiation resistance and disease recurrence. The findings are consistent with work from other cancers showing ALDHs are major contributors of CSC signaling and resistance to anti-cancer treatments. This perspective will address representative work concerning the validity of ALDH and the associated retinoic acid signaling pathway as chemotherapeutic targets for prostate as well as other cancers.

Breast cancer associated gene 2 (BCA2) has been originally identified from invasive breast cancer cells and shown to be overexpressed in over 50% of invasive breast cancers. Its expression is known to be highly associated with estrogen receptor-alpha (ER-α) status and promote cell proliferation and invasive properties. Importantly, expression of BCA2 is minimal or undetectable in most normal cells and tissues, which makes it as a valuable biomarker for ER-α positive breast cancers and a potential therapeutic target. BCA2 protein is a RING and ZINC-finger domain containing E3 ubiquitin ligase that has been shown to auto-ubiquitylate and interact with several proteins including Rab7, tetherin, ubiquitin, Ubc9 and p21, which are involved in different cellular processes. However, most of these studies have been focused on tumor progression, migration and invasive properties and almost no information on its role in carcinogenesis. Since many RING and ZINC-finger domain containing ubiquitin ligases are implicated in oncogenic signaling and DNA damage responses (DDR), in this study we examined the role of BCA2 in regulation of spontaneous and chemotherapeutics induced DDR in different breast cancer cell lines (ER-α positive versus triple negative). Interestingly, siRNA mediated down regulation of BCA2 induced spontaneous DDR, such as activation of replication checkpoint, slow cell cycle progression and double strand breaks (γH2AX foci). Exposure of BCA2 knockdown cells to DNA topoisomerase inhibitors (camptothecin and etoposide) potentiated DDR induced by these drugs. However, the molecular basis for this enhanced DDR is yet to be determined. Consistent with the previous studies, BCA2 knockdown attenuated cell proliferation, and compromised migration and invasion properties of these cells. Moreover, this novel role for BCA2 in DDR strongly suggests its status may also influence tumor response to chemo and radiation therapies and in carcinogenesis process. Further evaluation of breast cancer cell’s responses to different chemotherapeutic agents revealed distinct cellular responses based on the status of BCA2 and ER-α status. Taken together, our studies implicate a novel role for BCA2 in regulation of DDR and its influence on tumor response to chemotherapy. Additionally, we aim to present E3 ligase dependent and independent roles of BCA2 in these processes and tumor responses to different therapeutic agents. Acknowledgment: This is work is supported by Abraham Mitchell Cancer Research Scholar Endowment grant. Citation Format: Yuan-Hao Lee, Kaushlendra Tripathi, David Clark, Komaraiah Palle. A novel role for breast cancer associated protein 2 (BCA2) in regulation replication-stress mediated DNA damage responses [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-06-07.

Ovarian cancer (OC) is the most lethal gynecological cancer in women in the United States. Advances in surgery and chemotherapy have not significantly changed the overall survival rate of OC for the last few decades, which highlights the need for new therapeutic strategies. Platinum drug resistance and refractory disease pose major challenges in treating this disease and are major factors contributing to the poor survival rate of OC patients. Although most patients initially respond to platinum based chemotherapy, about 80% of cases present with recurrent disease, develop platinum resistance, and die with the advanced disease. Considering the heterogeneity, small fractions of the cells could be inherently resistant to chemotherapy and/or dormant and exhibit stem-like cell properties, contributing to the resistant phenotype and disease recurrence. Although the Cancer stem cell (CSC) theory of therapeutic resistance proposes that the proportion of CSCs correlate to enhanced chemoresistance and early disease recurrence, the specific molecular mechanisms that regulate tumor cell behavior (stemness) and integrate signaling networks with aberrant oncogenic signaling in OC cells are not known. Our analysis of clinical samples revealed upregulation of Rad6, an E2 ubiquitin conjugating enzyme, in more than 80% of ovarian tumors compared to normal ovarian tissues. Upregulation of Rad6 also correlated well with tumor progression. Further analysis of molecular pathways in OC cells revealed a strong correlation between Rad6 upregulation and increased β-catenin and hedgehog signaling, stem cell like characteristics and platinum resistance. Downregulation of Rad6 using siRNAs or inhibition of its catalytic activity by a small molecule inhibitor, attenuated carboplatin induced monoubiquitination of its target proteins such as histone 2B, PCNA and proteins of the Fanconi anemia pathway thereby sensitizing OC cells to carboplatin. Interestingly, inhibition of Rad6 alone in OC cells induced replication stress and reduced cell survival and proliferation by arresting cells in the G2/M phase. Moreover, inhibition of Rad6 in various OC cell lines reduced expression of β-catenin, Gli1 and several OC stem cell markers. Moreover, Rad6 plays an important role in the activation of the trans-lesion synthesis (TLS) pathway by monoubiquitinating PCNA and in the activation of the Fanconi Anemia (FA) DNA repair pathway. These are critical mechanisms for cells to repair DNA crosslinks induced by platinum drugs. Together with these observations, our data suggest that inhibition of Rad6 could be a viable therapeutic target for overcoming platinum resistance and disease recurrence in ovarian cancer. Citation Format: Sebastian M. Spencer, Ranganatha R. Somasagara, Kaushlendra Tripathi, David W. Clark, Hend Kothayer, Andrew D. Westwell, Rodney P. Rocconi, Komaraiah Palle. Preclinical evaluation of Rad6 inhibition to overcome platinum resistance in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3734.