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Over 40 years of controversy surround the nuclear localization of actin. Now, it is well understood that actin localization to the nucleus is highly regulated, suggesting actin has critical nuclear functions. Studies have demonstrated that nuclear actin has roles in regulating transcription, chromatin remodeling, nuclear organization, and DNA damage repair. However, much remains unknown about nuclear actin, including its structure in the nucleus and its functions during development. Here we present our novel finding that Drosophila oogenesis, or follicle development, is a model for studying the structure, regulation, and function of nuclear actin. Follicles are composed of roughly 1000 somatic follicle cells and 16 germline cells, including 15 nurse or support cells and a single oocyte, and progress through a series of 14 morphological stages, from the germanium to Stage 14. During Stages 5–9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Known regulators of actin nucleocytoplasmic transport, Cofilin and Profilin, also localize to the nuclei during follicle development. Further, germline expression of GFP‐tagged Actin results in nuclear actin rod formation in nurse cells during Stages 5–9. These nuclear actin rods are static, label with both phalloidin and Cofilin. These findings suggest that nuclear actin is tightly regulated during follicle development. As we have previously determined that Fascin, an actin bundling protein, localizes to nurse cell nuclei, we postulated that Fascin may be a novel regulator of nuclear actin. Overexpression of Fascin enhances the frequency and length of GFP‐Actin rods, and Fascin colocalizes with these rods. Additionally, the loss of Fascin reduces, while the overexpression of Fascin increases, the frequency of nurse cells with high levels of endogenous nuclear actin. However, genetic manipulation of Fascin does not alter the overall nuclear level of actin within the ovary. Thus, these data suggest Fascin regulates the ability of specific cells to accumulate nuclear actin. The loss of Fascin reduces nuclear Cofilin suggesting that Fascin positively regulates nuclear actin through Cofilin, a key factor in the nuclear import of actin. Indeed, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. In conclusion, we have established Drosophila oogenesis as a new system to uncover the physiologically important functions and regulation of nuclear actin. Further, we have identified Fascin as a novel regulator of nuclear actin. The nuclear localization of Fascin and Actin is highly conserved among systems, indicating these findings are applicable far beyond Drosophila follicle development. Support or Funding Information This project is supported by grants by the National Science Foundation MCB‐1158527, National Institutes of Health R01GM116885 and Carver Trust Medical Research Initiative Grant. D.J.K. is partially supported by NINDS T32NS045549.

Steatotic hepatocytes accumulate profound numbers of large neutral lipid‐storage organelles called lipid droplets (LDs). The process by which the formation or utilization of these organelles are perturbed during NAFLD and NASH is poorly defined. EtOH consumption can also exacerbate these conditions markedly by yet unknown mechanisms. Thus, there is a critical need to understand lipolytic mechanisms for LD catabolism in hepatocytes. In adipocytes, catecholamines induce lipolysis through a β‐adrenergic/cAMP pathway that activates cytosolic lipases ATGL and HSL to catabolize LD triglycerides. Importantly, β‐adrenergic induction of LD catabolism has not been demonstrated in hepatocytes. Thus, the GOAL of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP‐mediated lipolysis in response to β‐adrenergic (β‐AR) stimulation. First, treatment of primary rat hepatocytes or Hep3B human hepatoma cells with the β‐AR agonist isoproterenol caused a 20–40% reduction in total cellular LD content within 24 hours. β‐AR induced LD loss was mimicked by the cAMP agonist forskolin/IBMX and was abrogated by the cAMP‐dependent protein kinase (PKA) inhibitor H89. In correlation, PKA activation following β‐AR stimulation was confirmed by FRET biosensors and biochemical analysis. β‐AR stimulation directly activated the lipolytic pathway, as evidenced by cAMP/PKA‐mediated phosphorylation of hormone sensitive lipase (HSL) and adipose triglyceide lipase (ATGL). In addition, pharmacological inhibitors against ATGL or HSL each blocked β‐AR induced LD loss, suggesting that lipase activation is critical for this process in stimulated cells. To examine whether β‐AR/cAMP stimulation triggered lipase recruitment directly to the LD, live‐cell imaging of Hep3B cells expressing either ATGL‐EGFP or HSL‐sfGFP showed that within 60 min of stimulation, each lipase was significantly displaced from the cytosol and recruited to the LD surface. This recruitment was blocked by the PKA inhibitor H89. Finally, to test if EtOH exposure attenuates the activation of lipolysis in hepatocytes, we isolated primary hepatocytes from rats fed a 6‐week ethanol‐containing Lieber‐DeCarli diet. Compared with controls, ethanol‐exposed hepatocytes showed a drastic inhibition in lipase activation and LD breakdown following β‐AR/cAMP stimulation. Remarkably, in ethanol‐metabolizing VA13 hepatocytes, ethanol insult completely prevented isoproterenol‐induced translocation of ATGL‐EGFP to the LD. In summary , these results show that β‐adrenergic stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic ethanol insult leading to fatty liver. Manipulation of this regulatory cascade could be useful in developing new therapies to attenuate fatty liver disease. Support or Funding Information This study was generously supported by funding from NIAAA 5R01AA020735 (MAM and CAC) and NIDDK 5R37DK044650 (MAM), T32DK007352‐36 (MBS), and P30DK084567 (Mayo Clinic Center for Cell Signaling in Gastroenterology).

Background Therapy for celiac disease (CD) mainly relies on following a gluten‐free diet (GFD); however, a serum marker for gluten intake has yet to be established. Aims To evaluate the utility of alkylresorcinol concentrations for detecting gluten intake in studies of human and mouse. Methods Alkylresorcinol concentrations were compared among treated CD patients (n=34), untreated CD patients (n=36), and controls (n=33). Furthermore, 7 additional CD patients whose serum samples were available at diagnosis and after GFD were evaluated. In mice studies, alkylresorcinol concentrations were compared in the serum of 5 mice fed a regular chow and 10 mice fed lifelong with a gluten‐free chow. In addition, the effect of adding gluten on changes of alkylresorcinol concentrations was also evaluated. Results Total alkylresorcinol concentrations were significantly lower in treated CD patients (median [IQR], 3 (2–8) nmol/L), compared to untreated CD patients (median [IQR], 32 [11–74] nmol/L; P<.0001) or healthy controls (median [IQR], 54 [23–112] nmol/L; P<.0001). Moreover, alkylresorcinol concentrations in CD patients significantly decreased after introduction of a GFD (median, 34 nmol/L at diagnosis vs. 5 nmol.L after GFD, p=0.02). In the mice, median (IQR) total alkylresorcinol concentrations in serum samples of mice fed lifelong with a gluten‐free chow was 1.8 (1.6–2.3) nmol/L, which was further significantly increased to 16 (11–22) nmol/L after 8 days of feeding with the gluten free chow that had gluten added to it. (p=.008). Conclusion Serum alkylresorcinol concentrations could be a useful marker for dietary gluten in CD. Support or Funding Information No funding to report.

Background The ability to maintain adequate cerebral blood flow and oxygenation determines tolerance to central hypovolemia. It is unclear whether exposure to acute hypoxia changes cerebral blood flow during central hypovolemia. We hypothesized that cerebral blood velocity during lower body negative pressure would be lower during hypoxic versus normoxic conditions. Methods Ten healthy subjects (35 ± 3 y, BMI 25 ± 1 kg/m 2 ) were exposed to stepwise lower body negative pressure (LBNP, 5 minutes at 0, −15, −30, and −45 mmHg) during both normoxia and hypoxia (F i O 2 = 0.12–0.15 O 2 ). Normoxia and hypoxia protocols were randomized and blinded. Mean arterial blood pressure (MAP, brachial arterial catheter), middle cerebral artery blood velocity (MCAv, transcranial doppler) and end tidal CO 2 (ET CO2 , mass spectrometer) were continuously recorded. We calculated cardiac output (CO) and total peripheral resistance (TPR) from the arterial pressure waveform. Cerebral vascular conductance (CVC) was calculated as MCAv/MAP. The cumulative stress index (CSI) was calculated as the cumulative product of LBNP pressure and time at each LBNP stage. Results During the hypoxia protocol, 7 participants developed presyncopal symptoms and the protocol was terminated early. Three of these 7 did not complete the normoxia protocol due to presyncopal symptoms as well. Subsequently, CSI was lower during hypoxia (278 ± 63 vs. 513 ± 47 mmHg·min −1 , p < 0.01). MAP (p < 0.01), TPR (p < 0.01), MCAv (p = 0.06) and ET CO2 (p = 0.01) were lower during hypoxia than normoxia – . CO was higher (p = 0.03) during hypoxia than normoxia, while CVC (p = 0.35) was similar. Conclusion The attenuated rise in TPR during the hypoxia protocol was associated with a reduced MAP observed during hypoxia. These findings indicate that lower MCAv and LBNP tolerance during hypoxia are due to reductions in cerebral perfusion pressure (MAP). The similar CVC during both conditions renders it unlikely that significant changes in cerebrovascular resistance contribute to the differences between normoxia and hypoxia in LBNP tolerance and MCAv. Support or Funding Information Funding: W81XWH‐13‐2‐0038 Physiological responses to LBNP with hypoxia and normoxia Baseline LBNP−15mmHg LBNP−30mmHg LBNP−45mmHg MAP, mmHg Normoxia 103 ± 3 104 ± 3 101 ± 3 98 ± 41 Hypoxia 100 ± 3 94 ± 4 93 ± 4 83 ± 4 CO, 1/min Normoxia 4.6 ± 0.3 4.5 ± 0.3 4.1 ± 0.3 3.7 ± 0.31 Hypoxia 5.4 ± 0.3 4.8 ± 0.3 4.1 ± 0.3 3.6 ± 0.3 TPR, mmHg·min·mL −1 Normoxia 22.6 ± 1.3 23.7 ± 1.3 25.0 ± 1.3 26.6 ± 1.41 Hypoxia 18.5 ± 1.3 19.8 ± 1.3 22.7 ± 1.4 23.7 ± 1.6 MCAv, cm·s −1 Normoxia 65 ± 4 64 ± 4 61 ± 4 55 ± 5 Hypoxia 58 ± 4 59 ± 4 57 ± 5 54 ± 6 CVC, cm·s −1 ·mmHg −1 Normoxia 0.67 ± 0.05 0.65 ± 0.05 0.63 ± 0.05 0.58 ± 0.05 Hypoxia 0.61 ± 0.05 0.64 ± 0.05 0.63 ± 0.05 0.64 ± 0.06 ET CO2 , mmHg Normoxia 38 ± 1 38 ± 1 38 ± 1 36 ± 1 Hypoxia 37 ± 1 36 ± 1 35 ± 1 34 ± 2 Different vs. normoxia p<0.06, } indicates a main effect.

Background Hypoxia increases susceptibility to syncope during orthostatic stress. Furthermore, cardiac baroreflex sensitivity (cBRS) is attenuated during lower body negative pressure (LBNP). Recent studies suggest that there is cross‐talk between baroreceptors and carotid body chemoreceptors. With this in mind, hypoxia may further blunt cardiac baroreflex sensitivity (cBRS) during central hypovolemia in individuals susceptible to hypoxic syncope. Therefore, we hypothesized that participants susceptible to hypoxic syncope (“fainters”) during progressive central hypovolemia would have lower spontaneous cBRS during LBNP when compared to “nonfainters”. Methods Ten healthy male participants (35 ± 3 yr, BMI 25 ± 1 kg/m 2 ) were exposed to stepwise lower body negative pressure (LBNP, 5 minutes at 0, −15, −30, −45 mmHg) during both normoxia and hypoxia (FiO 2 = 0.12 – 0.15; S p O 2 ~85%). LBNP protocols were terminated early if participants developed presyncopal symptoms. Data from uncompleted stages were excluded. Heart rate (HR, 3 lead electrocardiogram) and mean arterial blood pressure (MAP, brachial arterial catheter) were continuously recorded. Spectral cBRS was calculated from the heart rate and blood pressure signals. Following experimentation, participants were stratified as fainters (developed presyncopal symptoms during hypoxic conditions, n=7) and nonfainters (n=3). Results MAP was lower in fainters than in nonfainters during hypoxia (interaction with LBNP p<0.05) while HR (p=0.38) and cBRS (p=0.43) were similar – . Hypoxia had a main effect vs normoxia on MAP, HR and cBRS in fainters (all p<0.02). In nonfainters hypoxia had a main effect vs normoxia on HR and cBRS (all p<0.03) and an interaction effect with LBNP on MAP (p<0.01). Conclusion Spontaneous cardiac baroreflex sensitivity is not attenuated in participants susceptible to hypoxic syncope during lower body negative pressure. This suggests that spontaneous cardiac baroreflex sensitivity does not contribute to orthostatic intolerance during hypoxia. Support or Funding Information Funding: W81XWH‐13‐2‐0038 Physiological response in fainters and nonfainters to LBNP during hypoxia and normoxia MAP, mmHg HR, beats·min −1 cBRS, ms·mmHg −1 Nonfainters Fainters Nonfainters Fainters Nonfainters Fainters Baseline Normoxia 104 ± 5 102 ± 3 62 ± 5 61 ± 3 13 ± 3 19 ± 2 Hypoxia 106 ± 6 97 ± 4 80 ± 6 72 ± 4 9 ± 3 11 ± 2 LBNP −15 mmHg Normoxia 109 ± 5 103 ± 3 68 ± 5 64 ± 3 11 ± 3 14 ± 2 Hypoxia 106 ± 6 90 ± 4 88 ± 6 72 ± 4 9 ± 3 14 ± 2 LBNP −30 mmHg Normoxia 107 ± 5 98 ± 3 79 ± 5 74 ± 3 8 ± 3 11 ± 2 Hypoxia 103 ± 6 82 ± 5 95 ± 6 79 ± 5 8 ± 3 9 ± 3 LBNP −45 mmHg Normoxia 101 ± 5 99 ± 3 92 ± 5 84 ± 4 6 ± 2 5 ± 2 Hypoxia 89 ± 6 N/A 113 ± 6 N/A 3 ± 3 N/A Different vs. nonfainters p<0.05 Different vs. normoxia p<0.05

INTRODUCTION Treatment with β‐antagonists in heart failure (HF) elicits known cardioprotective benefits. Recent work has suggested that administration of a β‐agonist can induce lung fluid clearance in healthy subjects and HF patients. However, the impact of β‐antagonists on the diffusing capacity of the lungs for carbon monoxide (DLCO, to assess lung fluid changes) remains unclear. PURPOSE To examine the relationship between β‐antagonist use and lung diffusion in HF patients. METHODS A retrospective analysis was performed on 102 HF patients seen at Mayo Clinic between 1995–2010. Patients were divided into two groups: BB=patients prescribed β‐antagonists (n=70) and NB=patients not prescribed β‐antagonists (n=32). Medical records were examined ±1 year from the time of the most recent exercise stress test for age (yrs), sex, height (cm), weight (kg), body mass index (BMI) and β‐antagonist status. Outcome variables included DLCO, left ventricular ejection fraction (LVEF), cardiac output (Q), and cardiac output corrected for body surface area (CI). Independent‐samples t‐test compared age, height, weight, and BMI between BB and NB. A Chi‐Squared test compared New York Heart Association (NYHA) classification and sex between NB and BB. Comparison of DLCO, LVEF, Q, CI, DLCO corrected for CI (DLCO/CI), and DLCO corrected for Q (DLCO/Q) between NB and BB was performed with an independent‐samples t‐test. Data are reported as mean ± standard deviation (SD) or frequency (percentage) where applicable. RESULTS There were no differences in age (75±12 vs. 76±13yrs), height (172±10 vs. 173±9cm), weight (84±20 vs. 86±18kg), or BMI (28±6 vs. 29±5kg/m 2 ) between NB vs. BB, respectively. Neither NYHA classification (χ 2 =0.159; p =0.98) or sex (χ 2 =0.066; p =0.80) were different between NB and BB groups. No differences were found for LVEF (36.8±17.3 vs. 31.6±15.6%) or DLCO (20.32±7.6 vs. 19.35±5.7ml/min/mmHg) for NB vs. BB, respectively. Compared to NB, the BB group demonstrated lower CI (0.72±0.3 vs. 0.56±0.3, p =0.006) and Q (1.47±0.6 vs. 1.15±0.6, p =0.01), while both DLCO/CI and DLCO/Q were higher in BB (31.32±14.1 vs. 43.84±29.5, p =0.005 and 15.84±7.5 vs. 21.93±16.2, p =0.01, respectively). CONCLUSIONS These data suggest that HF patients prescribed β‐antagonist therapy demonstrate better lung diffusion for a given cardiac output compared to HF patients not prescribed β‐antagonist therapy.

INTRODUCTION Bronchodilator therapy via β‐agonists has been considered a controversial treatment option for patients with cardiovascular disease, including heart failure (HF), due to previous studies suggesting increased rates of mortality. A major limitation to several previous studies is the lack of similar baseline characteristics between groups; therefore, a matched control comparison is yet to be explored. PURPOSE To evaluate the relationship between bronchodilator therapy and survival in HF patients. METHODS A retrospective analysis was performed on 96 HF patients who underwent a cardiopulmonary exercise test between the years 1994–2010. Patients were divided into two groups: 48 patients on bronchodilator therapy (BDT) and 48 matched controls (CTL). Patients were matched based on age (yrs), height (cm), weight (kg), body mass index (BMI), New York Heart Association (NYHA) classification, and cardioselective β‐blocker medication during the time of their cardiopulmonary exercise test. Patients were followed for an average of ten years for an endpoint of cardiovascular mortality. An independent samples t‐test was performed comparing BDT and CTL for age (yrs), height (cm), weight (kg), and BMI, to determine comparable baseline characteristics. A Chi‐Squared test compared NYHA classification, cardioselective β‐blocker status, and sex between BDT and CTL. A log rank test was performed to determine differences in survival distributions between BDT and CTL. RESULTS There were no significant demographic differences between groups (age: 59.8 vs. 60.3yrs; height: 172.1 vs. 171.9cm; weight: 86.9 vs. 86.2kg; and BMI: 28.9 vs. 28.6kg/m 2 for BDT vs. CTL, respectively). NYHA classification (χ 2 =0.00; p =1.0), cardioselective β‐blocker status (χ 2 =0.14; p =0.71) and sex (χ 2 =0.202; p =0.65) were not different between groups. BDT patients demonstrated an event rate of 51.1% compared to 42.6% for CTL patients over the same time period. The survival distribution for BDT compared to CTL was not statistically different, χ 2 (1) = 1.49, p =0.22. CONCLUSION These data suggest that HF patients on bronchodilator therapy demonstrate no significant difference in survival compared to matched HF controls. In contrast to previous work, our data suggests bronchodilator therapy does not elicit increased mortality in these HF patients.

Objective CYP2C9 and CYP2C19 are highly polymorphic pharmacogenes that encodes enzymes that metabolize a large number of clinically relevant drugs. While the majority of variability in drug metabolism can be explained by a few common alleles, identification of gene sequence variation that alters enzyme activity and incorporation of that information into pharmacogenomic rules for clinical decision support might lead to improved therapy. Using sequence data derived from the Right Drug, Right Dose, Right Time: Using Genomic Data to Individualize Treatment (RIGHT Protocol), we sought to identify variants of unknown significance (VUSs) for CYP2C9 and CYP2C19, and to characterize the function of the encoded enzymes. Methods Targeted next‐generation sequencing for 84 pharmacogenes using the PGRN‐Seq v1.0 capture reagent was performed with DNA samples from 1013 RIGHT Protocol participants. Plasmids containing VUSs were created using site directed mutagenesis and were transiently expressed in COS‐1 cells. Initial determination of enzyme activity was performed using fluorogenic substrates. Enzyme kinetic parameters (Km and Vmax) for each of the variant allozymes were determined using liquid chromatography mass spectrometry with tolbutamide (CYP2C9) and mephenytoin (CYP2C19) as prototypic substrates. We also compared our results with in silico functional predictions by SIFT and Polyphen. Results Seven VUSs were identified for each gene, none of which had been functionally characterized. There were significant positive correlations between protein content and fluorometric enzyme activity for all variants for both CYP2C9 (P<0.05) and CYP2C19 (P<0.005). Notably, CYP2C9 V237L and CYP2C19 A173V activity was much lower than predicted based on protein content. The substrate intrinsic clearance of CYP2C9 variants P73L , S115R , V237L [6.23E‐05±1.08E‐05; 2.55E‐05±4.1E‐06; 7.28E‐05±2.4E‐05 (uL/mg protein/min)], and CYP2C19 variants V113I , A173V , R186C and R186H [2.49E‐04±2.78E‐05; 1.80E‐05±2.19E‐06; 2.33E‐05±9.2E‐06;1.45E‐04±1.2E‐05 (uL/mg protein/min)] were less than 25% of their respective WT enzyme . Conclusion Sequencing of CYP2C9 and CYP2C19 from 1013 subjects identified low frequency variants that had not been functionally characterized. In silico predictions which showed variable results, can be helpful but are not always accurate, hence functional validation is necessary for VUSs requiring protein expression and determination of kinetic parameters. Efforts to identify variation in diverse populations is resulting in a very large number of VUSs, so there is a need for the application of high throughput mutagenesis approaches and functional characterization methods to help pharmacogenomics contribute to precision medicine. Support or Funding Information NIH RO1 GM28157 and the Mayo Center for Individualized Medicine Pharmacogenomics Program.

Shivering compromises post‐operative recovery and patient comfort. Shivering is also an impediment to targeted temperature management (TTM), now standard of care for cardiac arrest. Without an effective drug to suppress shivering (referred to here as a thermolytic) high cost and difficulty of temperature management is restricting access to TTM outside of large hospitals and for conditions such as stroke. A more efficacious thermolytic is needed for post‐operative control of shivering and to improve patient comfort during TTM. Our prior work in rats, based on strong scientific premise from the study of hibernating animals, suggests that stimulating CNS A 1 adenosine receptors (A 1 AR) by combining the A 1 AR agonist 6 N cyclohexyladenosine (CHA) with the peripherally acting adenosine receptor antagonist 8‐(p‐sulphophenyl)theophylline (8‐SPT) will provide an effective means to control shivering. This approach works in rodents however, thermolytic efficacy has not been demonstrated in a larger mammal that, like humans, depends primarily on shivering thermogenesis to maintain body temperature. To assess the thermolytic efficacy of CHA in 50–55 kg swine, a species that lacks brown adipose tissue, three pigs were anesthetized with Telazol/Xylazine, propofol, or isoflurane and cooled to a target body temperature of 32 to 34°C with a water blanket set to 4°C. Probes measured core body temperature at the tympanum, lower esophagus, rectum and bladder. Electrocardiography, pulse oximetery and arterial blood pressure were monitored. Shivering was measured by respirometry and by a qualitative visual scale. When shivering was noted, CHA was administered (bolus, 4.2mg; infusion,113μg/kg/h). 8‐SPT (bolus, 186mg), atropine (bolus, 0.5mg) or epinephrine (bolus, 100μg) was then given IV to reverse bradycardia and hypotension. At completion, animals were euthanized and tissues collected for histopathology. Results show that CHA inhibits shivering and that 8‐SPT restores blood pressure and heart rate without reversing the thermolytic effect of CHA. No histopathology was noted. In conclusion, CHA is an effective thermolytic in swine. Support or Funding Information This work was supported by Be Cool Pharmaceutics LLC and the Mayo Clinic

Tissue fibrosis represents an increasing cause of morbidity and mortality worldwide, with fibroblast‐derived myofibroblasts playing an essential pathogenic role. Myofibroblasts are characterized by their increased expression of smooth muscle actin (α‐SMA), and contribute to tissue fibrosis through their increased abundance, matrix deposition and tissue contraction. Unlike in physiological wound repair, where fibroblast activation is spontaneously reversible, the fibroblast activation associated with pathological fibrosis is perpetuated, and fibroblasts maintain their activated phenotype even after the initial injury has been resolved. Previous studies have shown that aberrant epigenetic signaling may drive sustained fibroblast activation during fibrosis and several small‐molecule epigenetic modifiers have been shown to be effective in protecting against fibrosis in various animal models, however no systematic effort has been reported to identify epigenetic small molecule regulators of myofibroblast activation. Here we screened a library of 98 well‐annotated epigenetic modifying compounds from Mayo Clinic's collaboration with Nanosyn (Santa Clara, CA) for their ability to inhibit fibroblast to myofibroblast activation. To analyze compound effects, we designed a high‐content image‐based screening method to quantify α‐SMA expression, organization, and area coverage. For each metric we calculated an independent Z‐score, and to identify which compounds most effectively blocked myofibroblast activation we created a robust, multiparametric “Deep Z Analysis” which stacked together individual Z‐scores. In the initial screen each compound was run in triplicate using three concentrations (0.5, 2.5, and 10 μM). Screen hits were defined by any compound which displayed an estimated IC50 < 2.5μM, resulting in a hit rate of 12.2%. These 12 hits were then validated for their efficacy in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis. In support of our overall screening approach, among the primary screen hits were compounds previously shown to be antifibrotic, including: bromodomain inhibitors JQ1 and CPI‐0610, DNA methyl transferase inhibitor azacitidine, and Poly (ADP‐ribose) Polymerase (PARP) inhibitor ABT‐888. While several epigenetic therapies are already approved by the FDA for cancer treatment, no epigenetic drugs for the treatment of fibrotic diseases have reached the clinic. Our approach will help identify epigenetic modifiers attractive for further detailed research, with a long term goal of defining new targets to move forward towards eventual therapies for lung fibrosis.