Publications by authors named "Laura M Saba"

44 Publications

Variants in mycophenolate and CMV antiviral drug pharmacokinetic and pharmacodynamic genes and leukopenia in heart transplant recipients.

J Heart Lung Transplant 2021 Jun 12. Epub 2021 Jun 12.

Department of Pharmaceutical Sciences, University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, Colorado, USA. Electronic address:

Background: The objective was to assess the relationship between single nucleotide polymorphisms in mycophenolate and cytomegalovirus antiviral drug pharmacokinetic and pharmacodynamic genes and drug-induced leukopenia in adult heart transplant recipients.

Methods: This retrospective analysis included n = 148 patients receiving mycophenolate and a cytomegalovirus antiviral drug. In total, 81 single nucleotide polymorphisms in 21 pharmacokinetic and 23 pharmacodynamic genes were selected for investigation. The primary and secondary outcomes were mycophenolate and/or cytomegalovirus antiviral drug-induced leukopenia, defined as a white blood cell count <3.0 × 10/L, in the first six and 12 months post-heart transplant, respectively.

Results: Mycophenolate and/or cytomegalovirus antiviral drug-induced leukopenia occurred in 20.3% of patients. HNF1A rs1169288 A>C (p.I27L) was associated with drug-induced leukopenia (unadjusted p = 0.002; false discovery rate <20%) in the first six months post-transplant. After adjusting for covariates, HNF1A rs1169288 variant C allele carriers had significantly higher odds of leukopenia compared to A/A homozygotes (odds ratio 6.19; 95% CI 1.97-19.43; p = 0.002). Single nucleotide polymorphisms in HNF1A, SLC13A1, and MBOAT1 were suggestively associated (p < 0.05) with the secondary outcome but were not significant after adjusting for multiple comparisons.

Conclusion: Our data suggest genetic variation may play a role in the development of leukopenia in patients receiving mycophenolate and cytomegalovirus antiviral drugs after heart transplantation. Following replication, pharmacogenetic markers, such as HNF1A rs1169288, could help identify patients at higher risk of drug-induced leukopenia, allowing for more personalized immunosuppressant therapy and cytomegalovirus prophylaxis following heart transplantation.
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http://dx.doi.org/10.1016/j.healun.2021.05.020DOI Listing
June 2021

Trisomy 21 results in modest impacts on mitochondrial function and central carbon metabolism.

Free Radic Biol Med 2021 Jun 12;172:201-212. Epub 2021 Jun 12.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, USA; Linda Crnic Institute for Down Syndrome, School of Medicine, University of Colorado, Aurora, CO, USA. Electronic address:

Down syndrome (DS) is the most common genetic cause of intellectual disability. Mechanistically, oxidative stress and mitochondrial dysfunction are reported to be etiological factors for many of the DS-related comorbidities and have previously been reported in a number of in vitro and in vivo models of DS. The purpose of this study was to test for the presence of mitochondrial dysfunction in fibroblast cells obtained via skin biopsy from individuals with DS, and to assess the impact of trisomy 21 on central carbon metabolism. Using extracellular flux assays in matched dermal fibroblasts from euploid and DS individuals, we found that basal mitochondrial dysfunction is quite mild. Stressing the cells with a cocktail of mitochondrial stressors revealed a significant mitochondrial deficit in DS cells compared to euploid controls. Evaluation of extracellular acidification rate did not reveal a baseline abnormality in glycolysis; however, metabolomic assessments utilizing isotopically labeled glucose and glutamine revealed altered central carbon metabolism in DS cells. Specifically, we observed greater glucose dependency, uptake and flux into the oxidative phase of the pentose phosphate pathway in DS fibroblasts. Furthermore, using induced pluripotent stem cells (iPSC) we found that mitochondrial function in DS iPSCs was similar to the previously published studies employing fetal cells. Together, these data indicate that aberrant central carbon metabolism is a candidate mechanism for stress-related mitochondrial dysfunction in DS.
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http://dx.doi.org/10.1016/j.freeradbiomed.2021.06.003DOI Listing
June 2021

Association Between Variants in Calcineurin Inhibitor Pharmacokinetic and Pharmacodynamic Genes and Renal Dysfunction in Adult Heart Transplant Recipients.

Front Genet 2021 1;12:658983. Epub 2021 Apr 1.

Department of Pharmaceutical Sciences, University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, CO, United States.

: The goal of the study was to assess the relationship between single nucleotide variants (SNVs) in calcineurin inhibitor (CNI) pharmacokinetic and pharmacodynamic genes and renal dysfunction in adult heart transplant (HTx) recipients. : This retrospective analysis included = 192 patients receiving a CNI at 1-year post-HTx. Using a candidate gene approach, 93 SNVs in eight pharmacokinetic and 35 pharmacodynamic genes were chosen for investigation. The primary outcome was renal dysfunction 1-year after HTx, defined as an estimated glomerular filtration rate (eGFR) <45 ml/min/1.73m. Renal dysfunction was present in 28.6% of patients 1-year after HTx. Two SNVs [transforming growth factor beta 1 () rs4803455 C > A and phospholipase C beta 1 () rs170549 G > A] were significantly associated with renal dysfunction after accounting for a false discovery rate (FDR) of 20%. In a multiple-SNV adjusted model, variant A allele carriers of rs4803455 had lower odds of renal dysfunction compared to C/C homozygotes [odds ratio (OR) 0.28, 95% CI 0.12-0.62; = 0.002], whereas rs170549 variant A allele carriers had higher odds of the primary outcome vs. patients with the G/G genotype (OR 2.66, 95% CI 1.21-5.84, = 0.015). : Our data suggest that genetic variation in and may contribute to the occurrence of renal dysfunction in HTx recipients receiving CNIs. Pharmacogenetic markers, such as rs4803455 and rs170549, could help identify patients at increased risk of CNI-associated renal dysfunction following HTx, potentially allowing clinicians to provide more precise and personalized care to this population.
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http://dx.doi.org/10.3389/fgene.2021.658983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8047196PMC
April 2021

Aptardi predicts polyadenylation sites in sample-specific transcriptomes using high-throughput RNA sequencing and DNA sequence.

Nat Commun 2021 03 12;12(1):1652. Epub 2021 Mar 12.

Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Annotation of polyadenylation sites from short-read RNA sequencing alone is a challenging computational task. Other algorithms rooted in DNA sequence predict potential polyadenylation sites; however, in vivo expression of a particular site varies based on a myriad of conditions. Here, we introduce aptardi (alternative polyadenylation transcriptome analysis from RNA-Seq data and DNA sequence information), which leverages both DNA sequence and RNA sequencing in a machine learning paradigm to predict expressed polyadenylation sites. Specifically, as input aptardi takes DNA nucleotide sequence, genome-aligned RNA-Seq data, and an initial transcriptome. The program evaluates these initial transcripts to identify expressed polyadenylation sites in the biological sample and refines transcript 3'-ends accordingly. The average precision of the aptardi model is twice that of a standard transcriptome assembler. In particular, the recall of the aptardi model (the proportion of true polyadenylation sites detected by the algorithm) is improved by over three-fold. Also, the model-trained using the Human Brain Reference RNA commercial standard-performs well when applied to RNA-sequencing samples from different tissues and different mammalian species. Finally, aptardi's input is simple to compile and its output is easily amenable to downstream analyses such as quantitation and differential expression.
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http://dx.doi.org/10.1038/s41467-021-21894-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955126PMC
March 2021

Systems genetics analysis of the LXS recombinant inbred mouse strains:Genetic and molecular insights into acute ethanol tolerance.

PLoS One 2020 23;15(10):e0240253. Epub 2020 Oct 23.

Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO, United States of America.

We have been using the Inbred Long- and Short-Sleep mouse strains (ILS, ISS) and a recombinant inbred panel derived from them, the LXS, to investigate the genetic underpinnings of acute ethanol tolerance which is considered to be a risk factor for alcohol use disorders (AUDs). Here, we have used RNA-seq to examine the transcriptome of whole brain in 40 of the LXS strains 8 hours after a saline or ethanol "pretreatment" as in previous behavioral studies. Approximately 1/3 of the 14,184 expressed genes were significantly heritable and many were unique to the pretreatment. Several thousand cis- and trans-eQTLs were mapped; a portion of these also were unique to pretreatment. Ethanol pretreatment caused differential expression (DE) of 1,230 genes. Gene Ontology (GO) enrichment analysis suggested involvement in numerous biological processes including astrocyte differentiation, histone acetylation, mRNA splicing, and neuron projection development. Genetic correlation analysis identified hundreds of genes that were correlated to the behaviors. GO analysis indicated that these genes are involved in gene expression, chromosome organization, and protein transport, among others. The expression profiles of the DE genes and genes correlated to AFT in the ethanol pretreatment group (AFT-Et) were found to be similar to profiles of HDAC inhibitors. Hdac1, a cis-regulated gene that is located at the peak of a previously mapped QTL for AFT-Et, was correlated to 437 genes, most of which were also correlated to AFT-Et. GO analysis of these genes identified several enriched biological process terms including neuron-neuron synaptic transmission and potassium transport. In summary, the results suggest widespread genetic effects on gene expression, including effects that are pretreatment-specific. A number of candidate genes and biological functions were identified that could be mediating the behavioral responses. The most prominent of these was Hdac1 which may be regulating genes associated with glutamatergic signaling and potassium conductance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240253PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584226PMC
December 2020

Alcohol Sensitivity as an Endophenotype of Alcohol Use Disorder: Exploring Its Translational Utility between Rodents and Humans.

Brain Sci 2020 Oct 13;10(10). Epub 2020 Oct 13.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.

Alcohol use disorder (AUD) is a complex, chronic, relapsing disorder with multiple interacting genetic and environmental influences. Numerous studies have verified the influence of genetics on AUD, yet the underlying biological pathways remain unknown. One strategy to interrogate complex diseases is the use of endophenotypes, which deconstruct current diagnostic categories into component traits that may be more amenable to genetic research. In this review, we explore how an endophenotype such as sensitivity to alcohol can be used in conjunction with rodent models to provide mechanistic insights into AUD. We evaluate three alcohol sensitivity endophenotypes (stimulation, intoxication, and aversion) for their translatability across human and rodent research by examining the underlying neurobiology and its relationship to consumption and AUD. We show examples in which results gleaned from rodents are successfully integrated with information from human studies to gain insight in the genetic underpinnings of AUD and AUD-related endophenotypes. Finally, we identify areas for future translational research that could greatly expand our knowledge of the biological and molecular aspects of the transition to AUD with the broad hope of finding better ways to treat this devastating disorder.
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http://dx.doi.org/10.3390/brainsci10100725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600833PMC
October 2020

Identification of Genes Involved in the Differentiation of R7y and R7p Photoreceptor Cells in .

G3 (Bethesda) 2020 11 5;10(11):3949-3958. Epub 2020 Nov 5.

Department of Neurology, Department of Ophthalmology, Dell Medical School, University of Texas at Austin, Austin, TX 78712

The R7 and R8 photoreceptor cells of the compound eye mediate color vision. Throughout the majority of the eye, these cells occur in two principal types of ommatidia. Approximately 35% of ommatidia are of the pale type and express in R7 cells and in R8 cells. The remaining 65% are of the yellow type and express in R7 cells and in R8 cells. The specification of an R8 cell in a pale or yellow ommatidium depends on the fate of the adjacent R7 cell. However, pale and yellow R7 cells are specified by a stochastic process that requires the genes , and To identify additional genes involved in this process we performed genetic screens using a collection of 480 transposon insertion strains. We identified genes in gain of function and loss of function screens that significantly altered the percentage of expressing R7 cells (%) from wild-type. 36 strains resulted in altered % in the gain of function screen where the insertion strains were crossed to a driver line. 53 strains resulted in altered % in the heterozygous loss of function screen. 4 strains showed effects that differed between the two screens, suggesting that the effect found in the gain of function screen was either larger than, or potentially masked by, the insertion alone. Analyses of homozygotes validated many of the candidates identified. These results suggest that R7 cell fate specification is sensitive to perturbations in mRNA transcription, splicing and localization, growth inhibition, post-translational protein modification, cleavage and secretion, signaling, ubiquitin protease activity, GTPase activation, actin and cytoskeletal regulation, and Ser/Thr kinase activity, among other diverse signaling and cell biological processes.
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http://dx.doi.org/10.1534/g3.120.401370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642934PMC
November 2020

Peptide-Spectrum Match Validation with Internal Standards (P-VIS): Internally-Controlled Validation of Mass Spectrometry-Based Peptide Identifications.

J Proteome Res 2021 01 29;20(1):236-249. Epub 2020 Sep 29.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045-0508, United States States.

Liquid chromatography-tandem mass spectrometry is an increasingly powerful tool for studying proteins in the context of disease. As technological advances in instrumentation and data analysis have enabled deeper profiling of proteomes and peptidomes, the need for a rigorous, standardized approach to validate individual peptide-spectrum matches (PSMs) has emerged. To address this need, we developed a novel and broadly applicable workflow: PSM validation with internal standards (P-VIS). In this approach, the fragmentation spectrum and chromatographic retention time of a peptide within a biological sample are compared with those of a synthetic version of the putative peptide sequence match. Similarity measurements obtained for a panel of internal standard peptides are then used to calculate a prediction interval for valid matches. If the observed degree of similarity between the biological and the synthetic peptide falls within this prediction interval, then the match is considered valid. P-VIS enables systematic and objective assessment of the validity of individual PSMs, providing a measurable degree of confidence when identifying peptides by mass spectrometry.
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http://dx.doi.org/10.1021/acs.jproteome.0c00355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775876PMC
January 2021

A long non-coding RNA (Lrap) modulates brain gene expression and levels of alcohol consumption in rats.

Genes Brain Behav 2021 Feb 19;20(2):e12698. Epub 2020 Oct 19.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy & Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

LncRNAs are important regulators of quantitative and qualitative features of the transcriptome. We have used QTL and other statistical analyses to identify a gene coexpression module associated with alcohol consumption. The "hub gene" of this module, Lrap (Long non-coding RNA for alcohol preference), was an unannotated transcript resembling a lncRNA. We used partial correlation analyses to establish that Lrap is a major contributor to the integrity of the coexpression module. Using CRISPR/Cas9 technology, we disrupted an exon of Lrap in Wistar rats. Measures of alcohol consumption in wild type, heterozygous and knockout rats showed that disruption of Lrap produced increases in alcohol consumption/alcohol preference. The disruption of Lrap also produced changes in expression of over 700 other transcripts. Furthermore, it became apparent that Lrap may have a function in alternative splicing of the affected transcripts. The GO category of "Response to Ethanol" emerged as one of the top candidates in an enrichment analysis of the differentially expressed transcripts. We validate the role of Lrap as a mediator of alcohol consumption by rats, and also implicate Lrap as a modifier of the expression and splicing of a large number of brain transcripts. A defined subset of these transcripts significantly impacts alcohol consumption by rats (and possibly humans). Our work shows the pleiotropic nature of non-coding elements of the genome, the power of network analysis in identifying the critical elements influencing phenotypes, and the fact that not all changes produced by genetic editing are critical for the concomitant changes in phenotype.
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http://dx.doi.org/10.1111/gbb.12698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900948PMC
February 2021

Relationship between nocturnal blood pressure patterns and end organ damage and diastolic dysfunction in heart transplant recipients.

Clin Transplant 2020 05 13;34(5):e13842. Epub 2020 Mar 13.

Department of Pharmaceutical Sciences, University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, CO, USA.

Background: We assessed the relationship between circadian blood pressure (BP) patterns and clinical outcomes in a contemporary cohort of adult heart transplant recipients.

Methods: This retrospective, cross-sectional study included adult heart transplant recipients at least 6 months post-transplant. Ambulatory BP measurements were recorded over 24 hours. Nondippers were defined as a decline in average nighttime BP ≤ 10% compared with daytime. Primary outcomes were the presence of end organ damage, that is, microalbuminuria, chronic kidney disease, and/or left ventricular hypertrophy. Secondary outcomes were measures of diastolic dysfunction (ie, mitral valve deceleration time, e/e', E/A, and isovolumetric relaxation time), microalbumin/creatinine ratio, eGFR, interventricular septal thickness, and left ventricular posterior wall thickness.

Results: Of 30 patients, 53.3% (n = 16) were systolic nondippers and 40% (n = 12) were diastolic nondippers. Diastolic nondippers had three times higher urine microalbumin/creatinine ratios than diastolic dippers (P = .03). Systolic nondippers had 16.3% lower mitral valve deceleration time (P = .05) than systolic dippers, while diastolic nondippers had 20.4% higher e/e' (P = .05) than diastolic dippers. There were no significant relationships between BP dipping status and any of the primary outcomes.

Conclusions: These data suggest that systolic and diastolic nondipping BP patterns are associated with subclinical kidney damage and diastolic dysfunction in heart transplant recipients.
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http://dx.doi.org/10.1111/ctr.13842DOI Listing
May 2020

Transcriptome analysis of rat dorsal hippocampal CA1 after an early life seizure induced by kainic acid.

Epilepsy Res 2020 03 30;161:106283. Epub 2020 Jan 30.

Department of Pediatrics, University of Colorado, School of Medicine, 80045, United States; Department of Neurology, University of Colorado, School of Medicine, 80045, United States; Department of Pharmacology, University of Colorado, School of Medicine, 80045, United States; Department of Otolaryngology, University of Colorado, School of Medicine, 80045, United States; Neuroscience Graduate Program, University of Colorado, School of Medicine, 80045, United States. Electronic address:

Seizures that occur during early development are associated with adverse neurodevelopmental outcomes. Causation and mechanisms are currently under investigation. Induction of an early life seizure by kainic acid (KA) in immature rats on post-natal day (P) 7 results in behavioral changes in the adult rat that reflect social and intellectual deficits without overt cellular damage. Our previous work also demonstrated increased expression of CA1 hippocampal long-term potentiation (LTP) and reduced desensitization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type ionotropic glutamate receptors (AMPA-R) one week following a kainic acid induced seizure (KA-ELS). Here we used RNA sequencing (RNAseq) of mRNA from dorsal hippocampal CA1 to probe changes in mRNA levels one week following KA-ELS as a means to investigate the mechanisms for these functional changes. Ingenuity pathway analysis (IPA) confirmed our previous results by predicting an up-regulation of the synaptic LTP pathway. Differential gene expression results revealed significant differences in 7 gene isoforms. Additional assessments included AMPA-R splice variants and adenosine deaminase acting on RNA 2 (ADAR2) editing sites as a means to determine the mechanism for reduced AMPA-R desensitization. Splice variant analysis demonstrated that KA-ELS result in a small, but significant decrease in the "flop" isoform of Gria3, and editing site analysis revealed significant changes in the editing of a kainate receptor subunit, Grik2, and a serotonin receptor, Htr2c. While these specific changes may not account for altered AMPA-R desensitization, the differences indicate that KA-ELS alters gene expression in the hippocampal CA1 one week after the insult.
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http://dx.doi.org/10.1016/j.eplepsyres.2020.106283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096153PMC
March 2020

The Role of A Priori-Identified Addiction and Smoking Gene Sets in Smoking Behaviors.

Nicotine Tob Res 2020 07;22(8):1310-1315

Institute for Behavioral Genetics, University of Colorado, Boulder, CO.

Introduction: Smoking is a leading cause of death, and genetic variation contributes to smoking behaviors. Identifying genes and sets of genes that contribute to risk for addiction is necessary to prioritize targets for functional characterization and for personalized medicine.

Methods: We performed a gene set-based association and heritable enrichment study of two addiction-related gene sets, those on the Smokescreen Genotyping Array and the nicotinic acetylcholine receptors, using the largest available GWAS summary statistics. We assessed smoking initiation, cigarettes per day, smoking cessation, and age of smoking initiation.

Results: Individual genes within each gene set were significantly associated with smoking behaviors. Both sets of genes were significantly associated with cigarettes per day, smoking initiation, and smoking cessation. Age of initiation was only associated with the Smokescreen gene set. Although both sets of genes were enriched for trait heritability, each accounts for only a small proportion of the single nucleotide polymorphism-based heritability (2%-12%).

Conclusions: These two gene sets are associated with smoking behaviors, but collectively account for a limited amount of the genetic and phenotypic variation of these complex traits, consistent with high polygenicity.

Implications: We evaluated evidence for the association and heritable contribution of expert-curated and bioinformatically identified sets of genes related to smoking. Although they impact smoking behaviors, these specifically targeted genes do not account for much of the heritability in smoking and will be of limited use for predictive purposes. Advanced genome-wide approaches and integration of other 'omics data will be needed to fully account for the genetic variation in smoking phenotypes.
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http://dx.doi.org/10.1093/ntr/ntaa006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749195PMC
July 2020

Toxic consequences and oxidative protein carbonylation from chloropicrin exposure in human corneal epithelial cells.

Toxicol Lett 2020 Apr 26;322:1-11. Epub 2019 Dec 26.

Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, 80045, USA; Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan, 48824, USA. Electronic address:

Chloropicrin (CP), a warfare agent now majorly used as a soil pesticide, is a strong irritating and lacrimating compound with devastating toxic effects. To elucidate the mechanism of its ocular toxicity, toxic effects of CP (0-100 μM) were studied in primary human corneal epithelial (HCE) cells. CP exposure resulted in reduced HCE cell viability and increased apoptotic cell death with an up-regulation of cleaved caspase-3 and poly ADP ribose polymerase indicating their contribution in CP-induced apoptotic cell death. Following CP exposure, cells exhibited increased expression of heme oxygenase-1, and phosphorylation of H2A.X and p53 as well as 4-hydroxynonenal adduct formation, suggesting oxidative stress, DNA damage and lipid peroxidation. CP also caused increases in mitogen activated protein kinase-c-Jun N-terminal kinase and inflammatory mediator cyclooxygenase-2. Proteomic analysis revealed an increase in the carbonylation of 179 proteins and enrichment of pathways (including proteasome pathway and catabolic process) in HCE cells following CP exposure. CP-induced oxidative stress and lipid peroxidation can enhance protein carbonylation, prompting alterations in corneal epithelial proteins as well as perturbing signaling pathways resulting in toxic effects. Pathways and major processes identified following CP exposure could be lead-hit targets for further biochemical and molecular characterization as well as therapeutic intervention.
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http://dx.doi.org/10.1016/j.toxlet.2019.12.023DOI Listing
April 2020

Investigating RNA expression profiles altered by nicotinamide mononucleotide therapy in a chronic model of alcoholic liver disease.

Hum Genomics 2019 12 10;13(1):65. Epub 2019 Dec 10.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA.

Background: Chronic alcohol consumption is a significant cause of liver disease worldwide. Several biochemical mechanisms have been linked to the initiation and progression of alcoholic liver disease (ALD) such as oxidative stress, inflammation, and metabolic dysregulation, including the disruption of NAD/NADH. Indeed, an ethanol-mediated reduction in hepatic NAD levels is thought to be one factor underlying ethanol-induced steatosis, oxidative stress, steatohepatitis, insulin resistance, and inhibition of gluconeogenesis. Therefore, we applied a NAD boosting supplement to investigate alterations in the pathogenesis of early-stage ALD.

Methods: To examine the impact of NAD therapy on the early stages of ALD, we utilized nicotinamide mononucleotide (NMN) at 500 mg/kg intraperitoneal injection every other day, for the duration of a Lieber-DeCarli 6-week chronic ethanol model in mice. Numerous strategies were employed to characterize the effect of NMN therapy, including the integration of RNA-seq, immunoblotting, and metabolomics analysis.

Results: Our findings reveal that NMN therapy increased hepatic NAD levels, prevented an ethanol-induced increase in plasma ALT and AST, and changed the expression of 25% of the genes that were modulated by ethanol metabolism. These genes were associated with a number of pathways including the MAPK pathway. Interestingly, our analysis revealed that NMN treatment normalized Erk1/2 signaling and prevented an induction of Atf3 overexpression.

Conclusions: These findings reveal previously unreported mechanisms by which NMN supplementation alters hepatic gene expression and protein pathways to impact ethanol hepatotoxicity in an early-stage murine model of ALD. Overall, our data suggest further research is needed to fully characterize treatment paradigms and biochemical implications of NAD-based interventions.
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http://dx.doi.org/10.1186/s40246-019-0251-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902345PMC
December 2019

Development of a tissue augmented Bayesian model for expression quantitative trait loci analysis.

Math Biosci Eng 2019 09;17(1):122-143

Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado Denver Anschutz Medical Campus, Mail Stop B119, 13001 E. 17th Place, Aurora, 80045, USA.

Expression quantitative trait loci (eQTL) analyses detect genetic variants (SNPs) associated with RNA expression levels of genes. The conventional eQTL analysis is to perform individual tests for each gene-SNP pair using simple linear regression and to perform the test on each tissue separately ignoring the extensive information known about RNA expression in other tissue(s). Although Bayesian models have been recently developed to improve eQTL prediction on multiple tissues, they are often based on uninformative priors or treat all tissues equally. In this study, we develop a novel tissue augmented Bayesian model for eQTL analysis (TA-eQTL), which takes prior eQTL information from a different tissue into account to better predict eQTL for another tissue. We demonstrate that our modified Bayesian model has comparable performance to several existing methods in terms of sensitivity and specificity using allele-specific expression (ASE) as the gold standard. Furthermore, the tissue augmented Bayesian model improves the power and accuracy for local-eQTL prediction especially when the sample size is small. In summary, TA-eQTL's performance is comparable to existing methods but has additional flexibility to evaluate data from different platforms, can focus prediction on one tissue using only summary statistics from the secondary tissue(s), and provides a closed form solution for estimation.
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http://dx.doi.org/10.3934/mbe.2020007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384761PMC
September 2019

Cholestatic liver disease results increased production of reactive aldehydes and an atypical periportal hepatic antioxidant response.

Free Radic Biol Med 2019 11 1;143:101-114. Epub 2019 Aug 1.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, United States.

Cholangiopathies such as primary sclerosing cholangitis (PSC) are chronic liver diseases characterized by increased cholestasis, biliary inflammation and oxidative stress. The objective of this study was to elucidate the impact of cholestatic injury on oxidative stress-related factors. Using hepatic tissue and whole cell liver extracts (LE) isolated from 11-week old C57BL/6J (WT) and Mdr2 mice, inflammation and oxidative stress was assessed. Concurrently, specific targets of carbonylation were assessed in LE prepared from murine groups as well as from normal and human patients with end-stage PSC. Identified carbonylated proteins were further evaluated using bioinformatics analyses. Picrosirius red staining revealed extensive fibrosis in Mdr2 liver, and fibrosis colocalized with increased periportal inflammatory cells and both acrolein and 4-HNE staining. Western blot analysis revealed elevated periportal expression of antioxidant proteins Cbr3, GSTμ, Prdx5, TrxR1 and HO-1 but not GCLC, GSTπ or catalase in the Mdr2 group when compared to WT. From immunohistochemical analysis, increased periportal reactive aldehyde production colocalized with elevated staining of Cbr3, GSTμ and TrxR1 but surprisingly not with Nrf2. Mass spectrometric analysis revealed an increase in carbonylated proteins in the Mdr2 and PSC groups compared to respective controls. Gene ontology and KEGG pathway analysis of carbonylated proteins revealed a propensity for increased carbonylation of proteins broadly involved in metabolic processes as well more specifically in Rab-mediated signal transduction, lysosomes and the large ribosomal subunit in human PSC. Western blot analysis of Rab-GTPase expression revealed no significant differences in Mdr2 mice when compared to WT livers. In contrast, PSC tissue exhibited decreased levels of Rabs 4, 5 and increased abundance of Rabs 6 and 9a protein. Results herein reveal that cholestasis induces stage-dependent increases in periportal oxidative stress responses and protein carbonylation, potentially contributing to pathogenesis in Mdr2. Furthermore, during early stage cholestasis, there is cell-specific upregulation of some but not all, antioxidant proteins.
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http://dx.doi.org/10.1016/j.freeradbiomed.2019.07.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6848778PMC
November 2019

Networking in Biology: The Hybrid Rat Diversity Panel.

Methods Mol Biol 2019 ;2018:213-231

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Aurora, CO, USA.

One of the most fruitful resources for systems genetic studies of nonhuman mammals is a panel of inbred strains that exhibits significant genetic diversity between strains but genetic stability (isogenicity) within strains. These characteristics allow for fine mapping of complex phenotypes (QTLs) and provide statistical power to identify loci which contribute nominally to the phenotype. This type of resource also allows the planning and performance of investigations using the same genetic backgrounds over several generations of the test animals. Often, rats are preferred over mice for physiologic and behavioral studies because of their larger size and more distinguishable anatomy (particularly for their central nervous system). The Hybrid Rat Diversity Panel (HRDP) is a panel of inbred rat strains, which combines two recombinant inbred panels (the HXB/BXH, 30 strains; the LEXF/FXLE, 34 strains and 35 more strains of inbred rats which were selected for genetic diversity, based on their fully sequenced genomes and/or thorough genotyping). The genetic diversity and statistical power of this panel for mapping studies rivals or surpasses currently available panels in mouse. The genetic stability of this panel makes it particularly suitable for collection of high-throughput omics data as relevant technology becomes available for engaging in truly integrative systems biology. The PhenoGen website ( http://phenogen.org ) is the repository for the initial transcriptome data, making the raw data, the processed data, and the analysis results, e.g., organ-specific protein coding and noncoding transcripts, isoform analysis, expression quantitative trait loci, and co-expression networks, available to the research public. The data sets and tools being developed will complement current efforts to analyze the human transcriptome and its genetic controls (the Genotype-Tissue Expression Project (GTEx)) and allow for dissection of genetic networks that predispose to particular phenotypes and gene-by-environment interactions that are difficult or even impossible to study in humans. The HRDP is an essential population for exploring truly integrative systems genetics.
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http://dx.doi.org/10.1007/978-1-4939-9581-3_10DOI Listing
November 2019

Effects of Chronic Proton-Pump Inhibitor Use on Kidney Function in Older Adults.

Sr Care Pharm 2019 May;34(5):325-333

Proton-pump inhibitors (PPIs) have been associated with adverse renal outcomes in older adults; however, there are little data regarding the magnitude of the change in renal function in this population. The objective of this study was to quantify the change in kidney function associated with chronic PPI therapy at two years in older adults using estimated glomerular filtration rate (eGFR).
The study was a retrospective, pre/post, observational cohort.
The study included University of Colorado Health primary care patients 60 to 89 years of age who were newly initiated on a PPI between August 1, 2012, and March 1, 2015, and remained on therapy for at least two years. The primary outcome was the change in kidney function, measured by eGFR, two years after starting PPI therapy. Secondary outcomes included change in kidney function and incidence of reduction in eGFR to < 60 mL/min/1.73 m² two years post-index date between patients with and without diabetes mellitus.
Of 877 electronic health records reviewed, 100 patients met inclusion criteria. The mean change in eGFR was -6.15 mL/min/1.73 m² (standard error of the mean = 1.03) at two years compared with baseline
(95% confidence interval -8.20 to -4.10; < 0.0001). There were no differences in the secondary outcomes based on concomitant diabetes mellitus.
Chronic PPI use was associated with a significant reduction in eGFR in ambulatory older adults at two years, beyond that expected based on increased age alone. Prescribers should be aware of the potential adverse renal effects of chronic PPI use.
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May 2019

Unsupervised discovery of phenotype-specific multi-omics networks.

Bioinformatics 2019 11;35(21):4336-4343

Department of Biostatistics and Informatics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Motivation: Complex diseases often involve a wide spectrum of phenotypic traits. Better understanding of the biological mechanisms relevant to each trait promotes understanding of the etiology of the disease and the potential for targeted and effective treatment plans. There have been many efforts towards omics data integration and network reconstruction, but limited work has examined the incorporation of relevant (quantitative) phenotypic traits.

Results: We propose a novel technique, sparse multiple canonical correlation network analysis (SmCCNet), for integrating multiple omics data types along with a quantitative phenotype of interest, and for constructing multi-omics networks that are specific to the phenotype. As a case study, we focus on miRNA-mRNA networks. Through simulations, we demonstrate that SmCCNet has better overall prediction performance compared to popular gene expression network construction and integration approaches under realistic settings. Applying SmCCNet to studies on chronic obstructive pulmonary disease (COPD) and breast cancer, we found enrichment of known relevant pathways (e.g. the Cadherin pathway for COPD and the interferon-gamma signaling pathway for breast cancer) as well as less known omics features that may be important to the diseases. Although those applications focus on miRNA-mRNA co-expression networks, SmCCNet is applicable to a variety of omics and other data types. It can also be easily generalized to incorporate multiple quantitative phenotype simultaneously. The versatility of SmCCNet suggests great potential of the approach in many areas.

Availability And Implementation: The SmCCNet algorithm is written in R, and is freely available on the web at https://cran.r-project.org/web/packages/SmCCNet/index.html.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6931269PMC
November 2019

Quantifying Competition among Mitochondrial Protein Acylation Events Induced by Ethanol Metabolism.

J Proteome Res 2019 04 31;18(4):1513-1531. Epub 2019 Jan 31.

Skaggs School of Pharmacy and Pharmaceutical Sciences , University of Colorado Anschutz Medical Campus , Aurora , Colorado 80045 , United States.

Mitochondrial dysfunction is one of many key factors in the etiology of alcoholic liver disease (ALD). Lysine acetylation is known to regulate numerous mitochondrial metabolic pathways, and recent reports demonstrate that alcohol-induced protein acylation negatively impacts these processes. To identify regulatory mechanisms attributed to alcohol-induced protein post-translational modifications, we employed a model of alcohol consumption within the context of wild type (WT), sirtuin 3 knockout (SIRT3 KO), and sirtuin 5 knockout (SIRT5 KO) mice to manipulate hepatic mitochondrial protein acylation. Mitochondrial fractions were examined by label-free quantitative HPLC-MS/MS to reveal competition between lysine acetylation and succinylation. A class of proteins defined as "differential acyl switching proteins" demonstrate select sensitivity to alcohol-induced protein acylation. A number of these proteins reveal saturated lysine-site occupancy, suggesting a significant level of differential stoichiometry in the setting of ethanol consumption. We hypothesize that ethanol downregulates numerous mitochondrial metabolic pathways through differential acyl switching proteins. Data are available via ProteomeXchange with identifier PXD012089.
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http://dx.doi.org/10.1021/acs.jproteome.8b00800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450748PMC
April 2019

Elevated Nrf-2 responses are insufficient to mitigate protein carbonylation in hepatospecific PTEN deletion mice.

PLoS One 2018 25;13(5):e0198139. Epub 2018 May 25.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America.

Objective: In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease (NASH) is oxidative stress, which leads to the accumulation of highly reactive electrophilic α/β unsaturated aldehydes. The objective of this study was to determine the impact of NASH on protein carbonylation and antioxidant responses in a murine model.

Methods: Liver-specific phosphatase and tensin homolog (PTEN)-deletion mice (PTENLKO) or control littermates were fed a standard chow diet for 45-55 weeks followed by analysis for liver injury, oxidative stress and inflammation.

Results: Histology and Picrosirius red-staining of collagen deposition within the extracellular matrix revealed extensive steatosis and fibrosis in the PTENLKO mice but no steatosis or fibrosis in controls. Increased steatosis and fibrosis corresponded with significant increases in inflammation. PTEN-deficient livers showed significantly increased cell-specific oxidative damage, as detected by 4-hydroxy-2-nonenal (4-HNE) and acrolein staining. Elevated staining correlated with an increase in nuclear DNA repair foci (γH2A.X) and cellular proliferation index (Ki67) within zones 1 and 3, indicating oxidative damage was zonally restricted and was associated with increased DNA damage and cell proliferation. Immunoblots showed that total levels of antioxidant response proteins induced by nuclear factor erythroid-2-like-2 (Nrf2), including GSTμ, GSTπ and CBR1/3, but not HO-1, were elevated in PTENLKO as compared to controls, and IHC showed this response also occurred only in zones 1 and 3. Furthermore, an analysis of autophagy markers revealed significant elevation of p62 and LC3II expression. Mass spectrometric (MS) analysis identified significantly more carbonylated proteins in whole cell extracts prepared from PTENLKO mice (966) as compared to controls (809). Pathway analyses of identified proteins did not uncover specific pathways that were preferentially carbonylated in PTENLKO livers but, did reveal specific strongly increased carbonylation of thioredoxin reductase and of glutathione-S-transferases (GST) M6, O1, and O2.

Conclusions: Results show that disruption of PTEN resulted in steatohepatitis, fibrosis and caused hepatic induction of the Nrf2-dependent antioxidant system at least in part due to elevation of p62. This response was both cell-type and zone specific. However, these responses were insufficient to mitigate the accumulation of products of lipid peroxidation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198139PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969769PMC
November 2018

Knockout of the Gsta4 Gene in Male Mice Leads to an Altered Pattern of Hepatic Protein Carbonylation and Enhanced Inflammation Following Chronic Consumption of an Ethanol Diet.

Alcohol Clin Exp Res 2018 07 30;42(7):1192-1205. Epub 2018 May 30.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Background: Glutathione S-transferase A4-4 (GSTA4) is a key enzyme for removal of toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE). In this study, we examined the potential role of GSTA4 on protein carbonylation and progression of alcoholic liver disease by examining the development of liver injury in male wild-type (WT) SV/J mice and SV/J mice lacking functional GSTA4 (GSTA4 mice).

Methods: Adult male WT and GSTA4 mice were fed chow (N = 10 to 12) or high-fat Lieber-DeCarli liquid diets containing up to 28% calories as ethanol (EtOH) (N = 18 to 20) for 116 days. At the end of the study, half of the EtOH-fed mice were acutely challenged with an EtOH binge (3 g/kg given intragastrically) 12 hours before sacrifice. Carbonylation of liver proteins was assessed by immunohistochemical staining for 4-HNE adduction and by comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) of purified carbonylated proteins.

Results: Chronic EtOH intake significantly increased hepatic 4-HNE adduction and protein carbonylation, including carbonylation of ribosomal proteins. EtOH intake also resulted in steatosis and increased serum alanine aminotransferase. Hepatic infiltration with B cells, T cells, and neutrophils and mRNA expression of pro-inflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)γ was modest in WT mice. However, an EtOH binge increased hepatic necrosis, hepatic cell proliferation, and expression of TNFα mRNA (p < 0.05). EtOH treatment of GSTA4 mice increased B-cell infiltration and increased mRNA expression of TNFα and IFNγ and of matrix remodeling markers MMP9, MMP13, and Col1A1 (p < 0.05). GSTA4 mice exhibited panlobular rather than periportal distribution of 4-HNE-adducted proteins and increased overall 4-HNE staining after EtOH binge. Comprehensive LC-MS of carbonylated proteins identified 1,022 proteins of which 189 were unique to the GSTA4 group.

Conclusions: These data suggest long-term adaptation to EtOH in WT mice does not occur in GSTA4 mice. Products of lipid peroxidation appear to play a role in inflammatory responses due to EtOH. And EtOH effects on B-cell infiltration and autoimmune responses may be secondary to formation of carbonyl adducts.
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http://dx.doi.org/10.1111/acer.13766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028296PMC
July 2018

Unsupervised, Statistically Based Systems Biology Approach for Unraveling the Genetics of Complex Traits: A Demonstration with Ethanol Metabolism.

Alcohol Clin Exp Res 2018 07 13;42(7):1177-1191. Epub 2018 Jun 13.

Department of Pharmaceutical Sciences , Skaggs School of Pharmacy & Pharmaceutical Sciences, University of Colorado, Aurora, Colorado.

Background: A statistical pipeline was developed and used for determining candidate genes and candidate gene coexpression networks involved in 2 alcohol (i.e., ethanol [EtOH]) metabolism phenotypes, namely alcohol clearance and acetate area under the curve in a recombinant inbred (RI) (HXB/BXH) rat panel. The approach was also used to provide an indication of how EtOH metabolism can impact the normal function of the identified networks.

Methods: RNA was extracted from alcohol-naïve liver tissue of 30 strains of HXB/BXH RI rats. The reconstructed transcripts were quantitated, and data were used to construct gene coexpression modules and networks. A separate group of rats, comprising the same 30 strains, were injected with EtOH (2 g/kg) for measurement of blood EtOH and acetate levels. These data were used for quantitative trait loci (QTL) analysis of the rate of EtOH disappearance and circulating acetate levels. The analysis pipeline required calculation of the module eigengene values, the correction of these values with EtOH metabolism rates and acetate levels across the rat strains, and the determination of the eigengene QTLs. For a module to be considered a candidate for determining phenotype, the module eigengene values had to have significant correlation with the strain phenotypic values and the module eigengene QTLs had to overlap the phenotypic QTLs.

Results: Of the 658 transcript coexpression modules generated from liver RNA sequencing data, a single module satisfied all criteria for being a candidate for determining the alcohol clearance trait. This module contained 2 alcohol dehydrogenase genes, including the gene whose product was previously shown to be responsible for the majority of alcohol elimination in the rat. This module was also the only module identified as a candidate for influencing circulating acetate levels. This module was also linked to the process of generation and utilization of retinoic acid as related to the autonomous immune response.

Conclusions: We propose that our analytical pipeline can successfully identify genetic regions and transcripts which predispose a particular phenotype and our analysis provides functional context for coexpression module components.
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http://dx.doi.org/10.1111/acer.13763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028286PMC
July 2018

Interindividual Regulation of the Breast Cancer Resistance Protein/ Transporter in Term Human Placentas.

Drug Metab Dispos 2018 05 31;46(5):619-627. Epub 2018 Jan 31.

Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy (K.M.B., J.E.M., X.W., L.M.A.), Environmental and Occupational Health Sciences Institute (L.M.A.), and Lipid Center (L.M.A.), Rutgers, The State University of New Jersey, Piscataway, New Jersey; Department of Pediatrics, Rutgers University Robert Wood Johnson Medical School, New Brunswick, New Jersey (F.A., P.Y.S.Y., M.M., N.M., A.M.V.); Hofstra Northwell School of Medicine, Cohen Children's Medical Center of New York, New Hyde Park, New York (B.I.W.); and Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Aurora, Colorado (L.M.S.)

The breast cancer resistance protein (BCRP/) is a maternally-facing efflux transporter that regulates the placental disposition of chemicals. Transcription factors and gene variants are important regulatory factors that influence transporter expression. In this study, we sought to identify the genetic and transcriptional mechanisms underlying the interindividual expression of BCRP mRNA and protein across 137 term placentas from uncomplicated pregnancies. Placental expression of BCRP and regulatory transcription factor mRNAs was measured using multiplex-branched DNA analysis. BCRP expression and genotypes were determined using Western blot and Fluidigm Biomark genetic analysis, respectively. Placentas were obtained from a racially and ethnically diverse population, including Caucasian (33%), African American (14%), Asian (14%), Hispanic (15%), and mixed (16%) backgrounds, as well as unknown origins (7%). Between placentas, BCRP mRNA and protein varied up to 47-fold and 14-fold, respectively. In particular, BCRP mRNA correlated significantly with known transcription factor mRNAs, including nuclear factor erythroid 2-related factor 2 and aryl hydrocarbon receptor. Somewhat surprisingly, single-nucleotide polymorphisms (SNPs) in the noncoding regions were not associated with variation in placental BCRP mRNA or protein. Instead, the coding region polymorphism (C421A/Q141K) corresponded with 40%-50% lower BCRP protein in 421C/A and 421A/A placentas compared with wild types (421C/C). Although BCRP protein and mRNA expression weakly correlated ( = 0.25, = 0.040), this relationship was absent in individuals expressing the C421A variant allele. Study results contribute to our understanding of the interindividual regulation of BCRP expression in term placentas and may help to identify infants at risk for increased fetal exposure to chemicals due to low expression of this efflux protein.
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http://dx.doi.org/10.1124/dmd.117.079228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5896368PMC
May 2018

Voluntary exposure to a toxin: the genetic influence on ethanol consumption.

Mamm Genome 2018 02 1;29(1-2):128-140. Epub 2017 Dec 1.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy & Pharmaceutical Sciences, University of Colorado, Aurora, CO, 80045, USA.

Ethyl alcohol is a toxin that, when consumed at high levels, produces organ damage and death. One way to prevent or ameliorate this damage in humans is to reduce the exposure of organs to alcohol by reducing alcohol ingestion. Both the propensity to consume large volumes of alcohol and the susceptibility of human organs to alcohol-induced damage exhibit a strong genetic influence. We have developed an integrative genetic/genomic approach to identify transcriptional networks that predispose complex traits, including propensity for alcohol consumption and propensity for alcohol-induced organ damage. In our approach, the phenotype is assessed in a panel of recombinant inbred (RI) rat strains, and quantitative trait locus (QTL) analysis is performed. Transcriptome data from tissues/organs of naïve RI rat strains are used to identify transcriptional networks using Weighted Gene Coexpression Network Analysis (WGCNA). Correlation of the first principal component of transcriptional coexpression modules with the phenotype across the rat strains, and overlap of QTLs for the phenotype and the QTLs for the coexpression modules (module eigengene QTL) provide the criteria for identification of the functionally related groups of genes that contribute to the phenotype (candidate modules). While we previously identified a brain transcriptional module whose QTL overlapped with a QTL for levels of alcohol consumption in HXB/BXH RI rat strains and 12 selected rat lines, this module did not account for all of the genetic variation in alcohol consumption. Our search for QTL overlap and correlation of coexpression modules with phenotype can, however, be applied to any organ in which the transcriptome has been measured, and this represents a holistic approach in the search for genetic contributors to complex traits. Previous work has implicated liver/brain interactions, particularly involving inflammatory/immune processes, as influencing alcohol consumption levels. We have now analyzed the liver transcriptome of the HXB/BXH RI rat panel in relation to the behavioral trait of alcohol consumption. We used RNA-Seq and microarray data to construct liver transcriptional networks, and identified a liver candidate transcriptional coexpression module that explained 24% of the genetic variance in voluntary alcohol consumption. The transcripts in this module focus attention on liver secretory products that influence inflammatory and immune signaling pathways. We propose that these liver secretory products can interact with brain mechanisms that affect alcohol consumption, and targeting these pathways provides a potential approach to reducing high levels of alcohol intake and also protecting the integrity of the liver and other organs.
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http://dx.doi.org/10.1007/s00335-017-9726-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988204PMC
February 2018

Systems genetic analysis of brown adipose tissue function.

Physiol Genomics 2018 01 10;50(1):52-66. Epub 2017 Nov 10.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus , Aurora, Colorado.

Brown adipose tissue (BAT) has been suggested to play an important role in lipid and glucose metabolism in rodents and possibly also in humans. In the current study, we used genetic and correlation analyses in the BXH/HXB recombinant inbred (RI) strains, derived from Brown Norway (BN) and spontaneously hypertensive rats (SHR), to identify genetic determinants of BAT function. Linkage analyses revealed a quantitative trait locus (QTL) associated with interscapular BAT mass on chromosome 4 and two closely linked QTLs associated with glucose oxidation and glucose incorporation into BAT lipids on chromosome 2. Using weighted gene coexpression network analysis (WGCNA) we identified 1,147 gene coexpression modules in the BAT from BXH/HXB rats and mapped their module eigengene QTLs. Through an unsupervised analysis, we identified modules related to BAT relative mass and function. The Coral4.1 coexpression module is associated with BAT relative mass (includes Cd36 highly connected gene), and the Darkseagreen coexpression module is associated with glucose incorporation into BAT lipids (includes Hiat1, Fmo5, and Sort1 highly connected transcripts). Because multiple statistical criteria were used to identify candidate modules, significance thresholds for individual tests were not adjusted for multiple comparisons across modules. In summary, a systems genetic analysis using genomic and quantitative transcriptomic and physiological information has produced confirmation of several known genetic factors and significant insight into novel genetic components functioning in BAT and possibly contributing to traits characteristic of the metabolic syndrome.
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http://dx.doi.org/10.1152/physiolgenomics.00091.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866413PMC
January 2018

Differential carbonylation of proteins in end-stage human fatty and nonfatty NASH.

Free Radic Biol Med 2017 12 6;113:280-290. Epub 2017 Oct 6.

Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States.

Objective: In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease is oxidative stress leading to the accumulation of highly reactive electrophilic α/β unsaturated aldehydes. The objective of this study was to determine if significant differences were evident when evaluating carbonylation in human end-stage fatty nonalcoholic steatohepatitis (fNASH) compared to end-stage nonfatty NASH (nfNASH).

Methods: Using hepatic tissue obtained from healthy humans and patients diagnosed with end stage nfNASH or fNASH, overall carbonylation was assessed by immunohistochemistry (IHC) and LC-MS/MS followed by bioinformatics.

Results: Picrosirius red staining revealed extensive fibrosis in both fNASH and nfNASH which corresponded with increased reactive aldehyde staining. Although significantly elevated when compared to normal hepatic tissue, no significant differences in overall carbonylation and fibrosis were evident when comparing fNASH with nfNASH. Examining proteins that are critical for anti-oxidant defense revealed elevated expression of thioredoxin, thioredoxin interacting protein, glutathione S-transferase p1 and mitochondrial superoxide dismutase in human NASH. As important, using immunohistochemistry, significant colocalization of the aforementioned proteins occurred in cytokeratin 7 positive cells indicating that they are part of the ductular reaction. Expression of catalase and Hsp70 decreased in both groups when compared to normal human liver. Mass spectrometric analysis revealed a total of 778 carbonylated proteins. Of these, 194 were common to all groups, 124 unique to tissue prepared from healthy individuals, 357 proteins exclusive to NASH, 124 proteins distinct to samples from patients with fNASH and 178 unique to nfNASH. Using functional enrichment analysis of hepatic carbonylated proteins revealed a propensity for increased carbonylation of proteins regulating cholesterol and Huntington's disease related pathways occurred in nfNASH. Examining fNASH, increased carbonylation was evident in proteins regulating Rho cytoskeletal pathways, nicotinic acetylcholine receptor signaling and chemokine/cytokine inflammatory pathways. Using LC-MS/MS analysis and trypsin digests, sites of carbonylation were identified on peptides isolated from vimentin, endoplasmin and serum albumin in nfNASH and fNASH respectively.

Conclusions: These results indicate that cellular factors regulating mechanisms of protein carbonylation may be different depending on pathological diagnosis of NASH. Furthermore these studies are the first to use LC-MS/MS analysis of carbonylated proteins in human NAFLD and explore possible mechanistic links with end stage cirrhosis due to fatty liver disease and the generation of reactive aldehydes.
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http://dx.doi.org/10.1016/j.freeradbiomed.2017.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704928PMC
December 2017

Zolpidem prescribing practices before and after Food and Drug Administration required product labeling changes.

SAGE Open Med 2017 5;5:2050312117707687. Epub 2017 May 5.

Department of Clinical Pharmacy, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

Background: Women have higher morning serum zolpidem concentrations than men after taking an evening dose, potentially leading to increased risk of harm. On 19 April 2013, the United States Food and Drug Administration required labeling changes for zolpidem, recommending an initial dose of no greater than 5 mg (immediate release) or 6.25 mg (controlled release) per night in women.

Objectives: The primary objective of this study was to compare prescribing practices before and after the 2013 zolpidem labeling change. A secondary objective was to evaluate serious adverse events potentially related to zolpidem.

Methods: Electronic medical records of adults receiving care through the University of Colorado Health system were accessed for study inclusion if patients were provided a first-time prescription for zolpidem either prior to or after the Food and Drug Administration labeling change. Patients were randomly chosen from eight strata based on age, gender, and date of zolpidem initiation (before/after the labeling change). Demographic and zolpidem prescribing data were collected. Low-dose zolpidem was considered 5 mg (immediate release) or 6.25 mg (controlled release) daily or less. Documentation of potentially related serious adverse events within the patients' records was also evaluated.

Results: A total of 400 patients were included in the study. The overall percentage of patients prescribed low-dose zolpidem increased from 44% to 58% after the labeling change (p = 0.0020). In a pre-specified subgroup analysis, the percentage of patients prescribed low-dose zolpidem increased in all groups, including young men (38%-50%, p = 0.23), elderly men (34%-40%, p = 0.53), and elderly women (60%-74%, p = 0.14), but the change was only significant in young women (42%-70%, p = 0.0045).

Conclusion: After Food and Drug Administration-mandated labeling changes for zolpidem in 2013, the percentage of overall patients in our health system, and specifically young women, with initial prescriptions for low-dose zolpidem significantly increased as compared to before the labeling change.
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http://dx.doi.org/10.1177/2050312117707687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423710PMC
May 2017

Model based heritability scores for high-throughput sequencing data.

BMC Bioinformatics 2017 Mar 2;18(1):143. Epub 2017 Mar 2.

Department of Biostatistics and Informatics, University of Colorado School of Public Health, Aurora, CO 80045, USA.

Background: Heritability of a phenotypic or molecular trait measures the proportion of variance that is attributable to genotypic variance. It is an important concept in breeding and genetics. Few methods are available for calculating heritability for traits derived from high-throughput sequencing.

Results: We propose several statistical models and different methods to compute and test a heritability measure for such data based on linear and generalized linear mixed effects models. We also provide methodology for hypothesis testing and interval estimation. Our analyses show that, among the methods, the negative binomial mixed model (NB-fit), compound Poisson mixed model (CP-fit), and the variance stabilizing transformed linear mixed model (VST) outperform the voom-transformed linear mixed model (voom). NB-fit and VST appear to be more robust than CP-fit for estimating and testing the heritability scores, while NB-fit is the most computationally expensive. CP-fit performed best in terms of the coverage of the confidence intervals. In addition, we applied the methods to both microRNA (miRNA) and messenger RNA (mRNA) sequencing datasets from a recombinant inbred mouse panel. We show that miRNA and mRNA expression can be a highly heritable molecular trait in mouse, and that some top heritable features coincide with expression quantitative trait loci.

Conclusions: The models and methods we investigated in this manuscript is applicable and extendable to sequencing experiments where some biological replicates are available and the environmental variation is properly controlled. The CP-fit approach for assessing heritability was implemented for the first time to our knowledge. All the methods presented, as well as the generation of simulated sequencing data under either negative binomial or compound Poisson mixed models, are provided in the R package HeritSeq.
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http://dx.doi.org/10.1186/s12859-017-1539-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333443PMC
March 2017

Uncovering the liver's role in immunity through RNA co-expression networks.

Mamm Genome 2016 10 11;27(9-10):469-84. Epub 2016 Jul 11.

Department of Pharmaceutical Sciences, University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, CO, 80045, USA.

Gene co-expression analysis has proven to be a powerful tool for ascertaining the organization of gene products into networks that are important for organ function. An organ, such as the liver, engages in a multitude of functions important for the survival of humans, rats, and other animals; these liver functions include energy metabolism, metabolism of xenobiotics, immune system function, and hormonal homeostasis. With the availability of organ-specific transcriptomes, we can now examine the role of RNA transcripts (both protein-coding and non-coding) in these functions. A systems genetic approach for identifying and characterizing liver gene networks within a recombinant inbred panel of rats was used to identify genetically regulated transcriptional networks (modules). For these modules, biological consensus was found between functional enrichment analysis and publicly available phenotypic quantitative trait loci (QTL). In particular, the biological function of two liver modules could be linked to immune response. The eigengene QTLs for these co-expression modules were located at genomic regions coincident with highly significant phenotypic QTLs; these phenotypes were related to rheumatoid arthritis, food preference, and basal corticosterone levels in rats. Our analysis illustrates that genetically and biologically driven RNA-based networks, such as the ones identified as part of this research, provide insight into the genetic influences on organ functions. These networks can pinpoint phenotypes that manifest through the interaction of many organs/tissues and can identify unannotated or under-annotated RNA transcripts that play a role in these phenotypes.
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http://dx.doi.org/10.1007/s00335-016-9656-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5002042PMC
October 2016
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