bluuebunny/crossref_metadata_2025_split huggingface dataset:
Integration of Drug Perturbed Dose Dependent Response Data in Predictive Computational Models of Chemotherapeutic Response Using Multiple‐Kernel‐Learning
Authors: Mannheimer Joshua David , Prasad Ashok , Gustafson Daniel 
Published: April 2020

Recently there has been a great deal of work to understand the dynamics of genome expression under small molecule perturbation. This has been demonstrated by the recent expansion of the Connectivity Map (CMAP) (
www.broadinstitute.org/connectivity‐map‐cmap) with roughly 5000 small molecule signatures. While this has proven to be invaluable resource it is still limited due to many drugs being constrained to a handful of cancer cell genomes, because of this, it is difficult to leverage these datasets to make an accurate and robust predictive model. Outside CMAP there is limited data that systematically characterize small molecule induced genomic changes, however, recently Monks et al. (Cancer Res 2018 DEC 15) has made available data in the NCI60 cell line panel for treated and untreated cell lines at 2, 6, and 24hr and two different dosing concentrations for 15 anti‐cancer agents. In our work we explore how each dataset can be optimally leveraged to build a predictive model of cell IC50 within each of the 15 drugs using a multiple kernel learning approach. With this method we can reach correlations as high as 0.88 (p ~1e−6) between predicted and measured IC50 with an average correlation of 0.57 on an independent test set. Additionally, perturbation with higher drug dose yields better individual models with average correlation around 0.451 compared to non‐treated data of 0.391 suggesting drug induced genomic changes are an important predictor of drug performance. Furthermore, we present a novel method that derives signatures present in perturbed data that can be captured in basal expression data. This approach to selecting features yields model performance that is equal or better than a signature derived basally alone. Our work demonstrates the importance of both basal and dynamic gene expression in modeling and understanding of drug response. We hope this work motivates further experiments to characterize drug induced gene dynamics for both modeling applications and understanding the underlying genomic influences in drug response.Support or Funding InformationShipley Chair in Comparative Oncology
Phorbol 12‐myristate 13‐acetate Dedifferentiates Human Cardiac Myofibroblasts to Fibroblasts
Authors: Luu Vy Tran , Phan Sang , Jin Zhuqiu 
Published: April 2020

Fibroblast plays an important role in keeping heart shape and elasticity as well as maintaining normal cardiac function. The differentiation of fibroblast to myofibroblast is generally considered as an irreversible conversion which represents a critical step in pathogenesis of fibrosis. Transforming growth factor (TGF)‐β1 is a major cytokine that mediates the conversion of fibroblast to myofibroblast, defined by the overly expression of α‐smooth muscle actin (α‐SMA)‐ a biomarker of myofibroblast with little to no appearance of fibroblast specific protein 1 (FSP‐1) – a biomarker of fibroblast. The cellular and molecular mechanisms of myofibroblast differentiation from fibroblasts have been studied extensively. Reversal of myofibroblast differentiation to fibroblasts remains unclear and incompletely understood. Phorbol 12‐myristate 13 acetate (PMA) is involved in multiple cellular functions such as cell growth, differentiation, programmed cell death via protein kinase C (PKC) signaling pathways. To investigate whether PMA dedifferentiates the formed myofibroblasts, NIH 3T3 fibroblasts and human cardiac fibroblast (HCF), cultured in DMEM and fibroblast medium (FM)‐2 respectively, were induced to convert into myofibroblasts in the presence of 2 ng/ml of TGF‐β1 for 24‐ or 48‐hour incubations. Expression of α‐SMA and FSP‐1 in both cell lines was detected by using western blotting and immunofluorescence. Collagen gel contraction induced by cardiac fibroblasts was determined as well. After incubation with TGF‐β1, morphology changes in the shape and the size of NIH 3T3 and HCF cells were observed by the presence of large nuclei and cytoplasm. The levels of expression of α‐SMA were significantly increased whereas expression of FSP‐1 was reduced after 48‐hour incubation with TGF‐β1. NIH 3T3 and HCF cells were then treated with 50 ng/ml of DMSO which used as control groups and with various PMA concentrations (10 ng/ml, 50 ng/ml and 100 ng/ml) for additional 24‐ and 48‐ hour incubations. Chemiluminescence detection of Western blot confirmed the reduction in expression of α‐SMA. The shape and the size of the both cell lines were recovered under the presence of PMA. The results indicated that the reduction in expression of α‐SMA was directly proportional to the concentration of PMA. As PMA concentration increased, the expression of α‐SMA remarkably decreased. PMA also reduced TGF‐β1‐induced collagen gel contraction. These data unambiguously elucidate the reversal of myofibroblast differentiation induced by PMA. Under basic condition, TGF‐β1 induces myofibroblast conversion from fibroblast, and myofibroblast de‐differentiates back into fibroblast in the presence of PMA. Although the mechanism remains to be identified, the novel findings of this study shed light on future development of novel agents to treat fibrotic diseases.Support or Funding InformationThis project was supported by the Seed Grant from California Northstate University, College of Pharmacy.
Optogenetic stimulation of respiratory related neurons modulates swallow and swallow‐breathing coordination
Authors: Huff Alyssa D. , Karlen-Amarante Marlusa , Baertsch Nathan Andrew , Pitts Teresa , Ramirez Jan Marino 
Published: April 2020

Swallow and breathing are tightly coordinated behaviors in which disruption of this coordination leads to aspiration pneumonia, the leading cause of death in many adult and pediatric diseases. Little is known about the neural coordination of these two behaviors. Previous work in the respiratory column has shown activity in swallow‐related and breathing‐related neurons, as well as neurons active during both swallow and breathing, suggesting coordination at a central network level. Evidence in the intermediate reticular nucleus (postinspiratory Complex, PiCo) suggest involvement in swallow behavior but not swallow‐breathing coordination. We hypothesize that swallow predominately occurs during the expiratory phase of breathing due to inspiratory related neurons suppressing swallow activity. Thus, inhibition of excitatory neurons within the Pre‐Bötzinger complex will increase swallow excitability, while exciting glutamatergic neurons within this network will have the opposite effect. To evaluate swallow circuitry and its coordination with respiratory related networks we use optogenetic approaches to manipulate the activity of inhibitory (Vgat‐Cre) and excitatory (Dbx1‐ERT2Cre, Vglut2‐Cre) neuron populations within the ventral respiratory centers as well as modulation of PiCo (ChAT‐Cre) in the presence of light stimulation. Optogenetic activation (blue laser, 447nm) of Dbx1 cells was performed in adult Dbx1‐ERT2Cre/Ai32(ChR2) mice after exposing the ventral surface of the brainstem in vivo, and placing bilateral glass fiber optics over the ventral surface in the region of the Pre‐Bötzinger complex. Swallow activity was measured by monopolar suction electrodes of the hypoglossal (XII) and vagus (X) nerves as well as cervical sympathetic nerve (cSNA). Phases of breathing was measured via monopolar electromyogram (EMG) of the diaphragm and swallow was stimulated orally by injection of 0.1cc of 2.5% citric acid water. Our preliminary data suggest that stimulating Dbx1 cells decreased the frequency and duration of swallows. Interestingly, we observed in the presence of light stimulation multiple swallow attempts (oral phase without the pharyngeal phase) resulting in hyoid elevation but failure to induce pharyngeal phase. The average swallow in the absence of light stimulation had a duration of 1025ms whereas swallow during light stimulation was 809ms. We believe that activation of Dbx1 neurons in the Pre‐Bötzinger complex suppresses swallow activity and decouples oral and pharyngeal phases of swallow.Support or Funding InformationNIH: P01 HL090554, R01 HL144801 and R01 HL126523
Recognition Motif of Protein Kinase C and Protein Phosphatase 2A
Authors: Jones Alexander Curtis , Newton Alexandra C. 
Published: April 2020

Protein phosphorylation plays a central role in transducing intracellular signaling and the balance between phosphorylation and dephosphorylation must be exquisitely controlled to maintain cellular homeostasis. Two central effectors of protein phosphorylation, protein kinase C (PKC) and protein phosphatase 2A (PP2A), are themselves regulated by protein phosphorylation but the mechanism of their interaction is not known. Here we identify a putative binding motif on the PKC kinase domain that is predicted to recruit PP2A via its B56 regulatory subunit. These two enzymes regulate the phosphorylation state of each other, with PP2A opposing constitutive phosphates on PKC required for structural integrity, and PKC proposed to phosphorylate B56, controlling its interaction with PP2A substrates. While many kinase consensus phosphorylation sequences are well‐characterized, emerging research into phosphatase‐recognition of substrates via short linear motifs (SLiMs) has uncovered a PP2A‐B56 SLiM comprised of L/F‐x‐x‐I/V‐x‐E. Sequence analysis of PKC reveals a potential PP2A‐B56 binding site that is conserved in conventional PKC isozymes (α, β, and γ); indeed the Glu is required for binding PP2A‐B56 and is present in a majority of the PKC family. This sequence is located on the C‐lobe of the kinase in a region involved in substrate binding. This study characterizes the effect of mutation on this motif on PP2A‐B56 binding and function, and on the phosphorylation state of PKC. Attesting to the importance of this motif, several cancer‐associated mutations have been identified in the motif, including PKCα‐E552Q and PKCβ‐E555K. Our study demonstrates that phosphatase SLiMs are able to inform new kinase/phosphatase relationships and may be a valuable predictive tool for studying signaling cascades in disease.Support or Funding InformationThis work was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752), National Science Foundation Graduate Research Fellowship Program, and NIH R35 GM122523.
Creating Educator Scholar Leaders – Effectiveness of Robust Curriculum Development of a New PhD Program in Anatomy Education.
Authors: Terrell Mark , Kulesza Randy 
Published: April 2020

INTRODUCTIONThere is a current deficit in the number of high quality anatomy educators. This shortage has developed as well‐experienced anatomists move into administrative roles or retire and health profession schools expand course offerings and open new campuses. Curricular hour reductions, new technological innovations, and a focus on improved board performances forces anatomists to accomplish more with less. Although many anatomists make education a keystone in their careers, few PhD programs in anatomy provide formalized training in educational practice. AIM. To evaluate the effectiveness of a new PhD program in Anatomy Education designed to graduate educational‐scholar‐leaders as educational specialists.METHODSApplying Kern’s six step approach to curriculum development in medical education, we successfully merged the strengths existing educational programs at LECOM to create a new PhD program in Anatomy Education.RESULTSA new curriculum was planned, implemented, and evaluated. Program strengths include emphasis on the practice of teaching skills, educational research/scholarship, leadership, while developing robust knowledge in the anatomical sciences. Evidence of effectiveness of the innovative and unique curriculum includes, the number of applications, successful obtainment of accreditation, credit hours devoted to educational training, current evidence‐based dissertation research projects in anatomy education, institutional support of the program, and current PhD student evaluations.CONCLUSIONThis program has a strong commitment to producing a unique breed of new anatomists as education specialists who have the knowledge, skills and behaviors needed to lead health professions education development and research, thereby enhancing academic programs, improving health care outcomes, and strengthening the anatomical profession.
Regulation of GLS1 Expression by Scleraxis in Cardiac Fibroblasts
Authors: Chattopadhyaya Sikta , Nagalingam Raghu , Narhan Pavit , Czubryt Michael 
Published: April 2020

Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix, including fibrillar collagens, proteoglycans, and fibronectin. This process is energy‐intensive for cardiac fibroblasts, however it remains unclear how these energy metabolic requirements are met. Liver fibrosis, and in particular collagen synthesis, has been shown to be dependent on glutaminolysis, in which glutamine is converted to glutamate by the enzyme glutaminase (GLS) with further conversion to a‐ketoglutarate to fuel oxidative metabolism via the tricarboxylic acid cycle, but a role for glutaminolysis in cardiac fibrosis remains to be demonstrated. We previously showed that scleraxis (Scx) is a transcription factor that is critically required for the conversion of cardiac fibroblasts to myofibroblasts, but its potential role in glutaminolysis in these cells is unknown. We report here that freshly‐isolated rat cardiac fibroblasts (P0 – unpassaged) passaged twice to become myofibroblasts (P2) show concomitant up‐regulation of Scx by 5‐fold and GLS1 by 4‐fold in P2 versus P0. Scx over‐expression induced a 20‐fold increase in GLS1 expression. To determine whether GLS1 expression is dependent on Scx, we examined GLS1 expression in activated mouse cardiac fibroblasts (P1) isolated from wild type or Scx knockout mice. Scx knockout attenuated GLS1 expression by almost 90%. TGFb is a potent inducer of cardiac fibrosis that both dramatically up‐regulates Scx expression, and requires Scx for many of its downstream effects. TGFb treatment of wild type mouse P1 activated fibroblasts doubled the expression of GLS1, but had no effect in Scx KO cells. In silico analysis revealed four putative Scx binding sites in the GLS1 promoter. While TGFb significantly up‐regulated expression of the myofibroblast marker periostin in cardiac fibroblasts, both scleraxis knockout and the glutaminase inhibitor CB‐839 attenuated this induction, indicating a failure to convert to myofibroblasts. These findings suggest that Scx regulates GLS1 expression to facilitate increased energy metabolism during cardiac fibroblast to myofibroblast conversion.Support or Funding InformationCanadian Institutes of Health Research (PJT‐162422).
Coupling Electrophysiology with Single Cell RNA Sequencing to Uncover Transcriptional Markers of Chemosensitive Serotonergic Neurons
Authors: Mouradian Gary Charles , Duffy Erin , Liu Pegnyuan , Hodges Matt 
Published: April 2020

Central respiratory chemoreceptors are specialized neurons with intrinsic sensitivity to hypercapnia and/or acidosis that couple breathing and pH/CO2 levels. Previous data support the hypothesis that a sub‐population of brainstem serotonin (5‐HT) neurons serve as central respiratory chemoreceptors, where they become sensitive to pH/CO2 on or after post‐natal day 12 (P12) in rats. However, to date it remains unclear which 5‐HT neurons develop chemosensitivity and what molecular markers may identify this unique sub‐population of 5‐HT neurons. To address these questions, we employed a “patch‐to‐seq” technique to first distinguish 5‐HT neurons as chemosensitive (or insensitive) using electrophysiologic recording, and then to isolate each neurons’ RNA for single cell RNA Sequencing to identify potential cellular sensing mechanisms and/or unique transcriptional markers in each phenotypically‐identified 5‐HT neuron population. Acute brainstem slices (200 μm) were made from young (P18–23) transgenic rats which express eGFP in all 5‐HT neurons (SSeGFP), and action potential firing rates measured via a cell‐attached patch configuration in eGFP+ 5‐HT neurons during superfusion with artificial CSF (aCSF) containing inhibitors for synaptic blockade (10 mM CNQX, 50 mM D‐AP5, 20 mM Gabazine) bubbled with either 5% CO2 (bal. O2; pH= 7.36; 5 min) or 15% CO2 (bal. O2; pH= 7.10). Cellular phenotypes were assessed by calculating a Chemosensitivity Index (C.I.). pH‐sensitive eGFP+ neurons (n=7) had an average C.I. of 177.0±14.6 whereas pH‐insensitive eGFP+ neurons (n=9) had an average C.I. of 98.0 ± 2.9 (P < 0.0001). Cellular contents of each recorded cell were collected and subjected to single cell RNA Sequencing (scRNA‐Seq), from which an average of 19.3M reads were generated with a quality score of ~33.7 and ~83% mapping rate per sample (32,883 known genes quantified). Preliminary analyses indicate very few differentially expressed genes (q < 0.05) between pH‐sensitive and ‐ insensitive 5‐HT neuron populations, and of those differentially expressed no gene obviously contributes to pH regulation or sensing. In addition, unbiased hierarchical clustering and principal component analyses failed to clearly separate samples by phenotype. However, a supervised analysis using Support Vector Machine learning and Recursive Feature Elimination (SVM‐RFE) identified several genes in which expression levels significantly predict the predefined phenotype, but the biological/functional significance of these genes in CO2/pH sensing remains unclear and instead these genes may provide a molecular signature to distinguish pH/CO2 sensitive vs insensitive 5‐HT neurons. We conclude from these preliminary data that pH/CO2 sensitivity in 5‐HT neurons is not a transcriptionally regulated phenotype.Support or Funding InformationParker B. Francis FoundationNHLBI ‐ R01 HL122358
Endothelial Mineralocorticoid Receptor Deletion Abrogates Leptin‐Induced Endothelial Dysfunction in Pregnant Mice
Authors: Faulkner Jessica L. , Wright Derrian , Kennard Simone , Antonova Galina , Jaffe Iris , Belin de Chantemèle Eric J. 
Published: April 2020

Obesity is a risk factor for endothelial dysfunction and hypertension in pregnancy, both of which are hallmarks of preeclampsia. Our lab has demonstrated that obesity‐associated endothelial dysfunction and hypertension in female mouse models is mediated via leptin‐induced aldosterone production and subsequent mineralocorticoid receptor (MR) activation. We also showed that progesterone increases endothelial MR (ECMR) expression and that high progesterone levels in pregnant mice are associated with highly elevated ECMR expression, potentially rendering pregnant female mice susceptible to leptin‐induced endothelial dysfunction in obese pregnancy. Therefore, we hypothesized that ECMR deletion would prevent leptin‐induced endothelial dysfunction in pregnant mice in late gestation. Endothelial function was experimentally measured by wire myography concentration response curves to acetylcholine in mouse aorta. Chronic leptin infusion (0.9mg/kg/day, s.c. osmotic minipump) throughout late‐gestation (gestational days 11–18) induced endothelial impairment in wild‐type pregnant mice that was impaired both compared to sham‐treated pregnant (2‐way ANOVA with repeated measures, *P<0.05) and non‐pregnant leptin‐infused females (*P<0.05). However, leptin infusion in pregnant ECMR−/− mice did not induce similar endothelial dysfunction (*P<0.05). Relaxation responses to acetylcholine in the presence of LNAME indicated that endothelial dysfunction in pregnant leptin‐infused mice was attributable to reduction in nitric oxide activity. In addition, no changes in endothelial‐independent sodium nitroprusside responses were observed among groups. Placental dysfunction is a characteristic of preeclampsia that precludes the development of endothelial dysfunction and hypertension. In homogenized placentas, mRNA expression of hypoxia‐inducible factor 1α (HIF1α), a marker of ischemia, was increased by leptin infusion in wild‐type (1.9±0.2‐fold change from sham wild‐type, P=0.08), but not in ECMR−/− (1.2±0.2‐fold change from sham ECMR−/−) pregnant mice. These changes in placental function were associated with adverse fetal effects in leptin‐infused wild‐type mice as indicated by decreased pup weight (0.86±0.04g sham wild‐type vs 0.52±0.11g wild‐type +leptin, *P<0.05). However, ECMR deletion rescued pup weight in leptin‐infused pregnant mice (0.79±0.08g, sham ECMR−/− vs 0.86±0.11g, ECMR−/− +leptin). Collectively, these data indicate that high leptin levels in obese pregnancies may predispose to placental dysfunction and endothelial dysfunction, which are characteristics of preeclampsia.Support or Funding Information1R01HL130301‐01, 19EIA34760167, Adrenal Center pilot grant, 1K99HL146948‐01A1
A Brain Tissue Metabolomic Signature Discloses Alzheimer’s Disease Post‐Mortem
Authors: Jasbi Paniz , Shi Xiaojian , Turner Cassidy , Jentarra Garilyn , Gu Haiwei 
Published: April 2020

Alzheimer’s disease (AD) is the most common cause of dementia, accounting for an estimated 60 to 80% of cases, and is the sixth‐leading cause of death in the United States. A major challenge persists in the post‐mortem diagnosis of AD patients from those suffering mild cognitive impairment (MCI) and control patients with high pathology (HPC). Mass spectrometry (MS)‐based metabolomics has shown significant potential in disease diagnosis over the past two decades. In this study, we present a combination of gas chromatography‐MS (GC‐MS) for global metabolic profiling and analysis of long‐ and short‐chain fatty acids, in addition to liquid chromatography‐tandem MS (LC‐MS/MS) for the detection of targeted aqueous metabolites and lipids. Using this approach, 2084 metabolites and features were reliably detected and monitored in 48 tissue samples harvested from the superior frontal gyrus of male and female subjects post‐mortem. Samples were taken from four groups: 12 normal control (NC) patients, 12 subjects characterized as HPC, 12 with sub‐clinical MCI, and 12 diagnosed with AD. Multivariate significance testing informed the construction and cross‐validation (p < 0.05) of partial least squares‐discriminant analysis (PLS‐DA) models defined by a 9‐metabolite panel of potential diagnostic biomarkers. Receiver operating characteristic (ROC) analysis showed high predictive accuracy of the resulting PLS‐DA models for discrimination of NC (96.6%), HPC (91.7%), MCI (93.9%) and AD (95.1%). Pathway analysis revealed significant disturbances in lysine degradation (p = 0.007), fatty acid metabolism (p = 0.009), and the degradation of branched‐chain amino acids (p = 0.025). Over representation analysis of lipid signatures revealed significant alterations in glycerophospholipid (FDR q = 8.3E‐07) and choline metabolism (FDR q = 0.006). Network analysis showed significant enrichment in 11 enzymes, mostly of the mitochondria. The current results allow for improved clinical staging post‐mortem, expand basic knowledge of the metabolome related to AD pathogenesis, and reveal pathways that can be targeted therapeutically in future studies. This study also provides a promising basis for the development of larger multi‐site projects to validate these candidate markers in readily available biospecimens such as blood to enable the effective screening, rapid diagnosis, accurate surveillance, and therapeutic monitoring of AD across population groups.Support or Funding InformationSupport from the College of Health Solutions at Arizona State University and the National Institutes of Health is gratefully acknowledged.