Noncanonical Wnt signaling maintains hematopoietic stem cells in the niche.

Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin(+) osteoblasts (N-cad(+)OBs that enrich osteoprogenitors) in the niche. We further found that N-cad(+)OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca(2+)-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.

Targeting the Liver with Nucleic Acid Therapeutics for the Treatment of Systemic Diseases of Liver Origin.

Systemic diseases of liver origin (SDLO) are complex diseases in multiple organ systems, such as cardiovascular, musculoskeletal, endocrine, renal, respiratory, and sensory organ systems, caused by irregular liver metabolism and production of functional factors. Examples of such diseases discussed in this article include primary hyperoxaluria, familial hypercholesterolemia, acute hepatic porphyria, hereditary transthyretin amyloidosis, hemophilia, atherosclerotic cardiovascular diseases, α-1 antitrypsin deficiency-associated liver disease, and complement-mediated diseases. Nucleic acid therapeutics use nucleic acids and related compounds as therapeutic agents to alter gene expression for therapeutic purposes. The two most promising, fastest-growing classes of nucleic acid therapeutics are antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs). For each listed SDLO disease, this article discusses epidemiology, symptoms, genetic causes, current treatment options, and advantages and disadvantages of nucleic acid therapeutics by either ASO or siRNA drugs approved or under development. Furthermore, challenges and future perspectives on adverse drug reactions and toxicity of ASO and siRNA drugs for the treatment of SDLO diseases are also discussed. In summary, this review article will highlight the clinical advantages of nucleic acid therapeutics in targeting the liver for the treatment of SDLO diseases. SIGNIFICANCE STATEMENT: Systemic diseases of liver origin (SDLO) contain rare and common complex diseases caused by irregular functions of the liver. Nucleic acid therapeutics have shown promising clinical advantages to treat SDLO. This article aims to provide the most updated information on targeting the liver with antisense oligonucleotides and small interfering RNA drugs. The generated knowledge may stimulate further investigations in this growing field of new therapeutic entities for the treatment of SDLO, which currently have no or limited options for treatment.

CslA and GlxA from Streptomyces lividans form a functional cellulose synthase complex.

Filamentous growth of streptomycetes coincides with the synthesis and deposition of an uncharacterized protective glucan at hyphal tips. Synthesis of this glucan depends on the integral membrane protein CslA and the radical copper oxidase GlxA, which are part of a presumably large multiprotein complex operating at growing tips. Here, we show that CslA and GlxA interact by forming a protein complex that is sufficient to synthesize cellulose in vitro. Mass spectrometry analysis revealed that the purified complex produces cellulose chains with a degree of polymerization of at least 80 residues. Truncation analyses demonstrated that the removal of a significant extracellular segment of GlxA had no impact on complex formation, but significantly diminished activity of CslA. Altogether, our work demonstrates that CslA and GlxA form the active core of the cellulose synthase complex and provide molecular insights into a unique cellulose biosynthesis system that is conserved in streptomycetes. IMPORTANCE: Cellulose stands out as the most abundant polysaccharide on Earth. While the synthesis of this polysaccharide has been extensively studied in plants and Gram-negative bacteria, the mechanisms in Gram-positive bacteria have remained largely unknown. Our research unveils a novel cellulose synthase complex formed by the interaction between the cellulose synthase-like protein CslA and the radical copper oxidase GlxA from Streptomyces lividans, a soil-dwelling Gram-positive bacterium. This discovery provides molecular insights into the distinctive cellulose biosynthesis machinery. Beyond expanding our understanding of cellulose biosynthesis, this study also opens avenues for exploring biotechnological applications and ecological roles of cellulose in Gram-positive bacteria, thereby contributing to the broader field of microbial cellulose biosynthesis and biofilm research.

Epigenetic Mechanisms Contribute to Intraindividual Variations of Drug Metabolism Mediated by Cytochrome P450 Enzymes.

Significant interindividual and intraindividual variations on cytochrome P450 (CYP)-mediated drug metabolism exist in the general population globally. Genetic polymorphisms are one of the major contribution factors for interindividual variations, but epigenetic mechanisms mainly contribute to intraindividual variations, including DNA methylation, histone modifications, microRNAs, and long non-coding RNAs. The current review provides analysis of advanced knowledge in the last decade on contributions of epigenetic mechanisms to intraindividual variations on CYP-mediated drug metabolism in several situations, including (1) ontogeny, the developmental changes of CYP expression in individuals from neonates to adults; (2) increased activities of CYP enzymes induced by drug treatment; (3) increased activities of CYP enzymes in adult ages induced by drug treatment at neonate ages; and (4) decreased activities of CYP enzymes in individuals with drug-induced liver injury (DILI). Furthermore, current challenges, knowledge gaps, and future perspective of the epigenetic mechanisms in development of CYP pharmacoepigenetics are discussed. In conclusion, epigenetic mechanisms have been proven to contribute to intraindividual variations of drug metabolism mediated by CYP enzymes in age development, drug induction, and DILI conditions. The knowledge has helped understanding how intraindividual variation are generated. Future studies are needed to develop CYP-based pharmacoepigenetics to guide clinical applications for precision medicine with improved therapeutic efficacy and reduced risk of adverse drug reactions and toxicity. SIGNIFICANCE STATEMENT: Understanding epigenetic mechanisms in contribution to intraindividual variations of CYP-mediated drug metabolism may help to develop CYP-based pharmacoepigenetics for precision medicine to improve therapeutic efficacy and reduce adverse drug reactions and toxicity for drugs metabolized by CYP enzymes.

Formation of wall-less cells in Kitasatospora viridifaciens requires cytoskeletal protein FilP in oxygen-limiting conditions.

The cell wall is considered an essential component for bacterial survival, providing structural support, and protection from environmental insults. Under normal growth conditions, filamentous actinobacteria insert new cell wall material at the hyphal tips regulated by the coordinated activity of cytoskeletal proteins and cell wall biosynthetic enzymes. Despite the importance of the cell wall, some filamentous actinobacteria can produce wall-deficient S-cells upon prolonged exposure to hyperosmotic stress. Here, we performed cryo-electron tomography and live cell imaging to further characterize S-cell extrusion in Kitasatospora viridifaciens. We show that exposure to hyperosmotic stress leads to DNA compaction, membrane and S-cell extrusion, and thinning of the cell wall at hyphal tips. Additionally, we find that the extrusion of S-cells is abolished in a cytoskeletal mutant strain that lacks the intermediate filament-like protein FilP. Furthermore, micro-aerobic culturing promotes the formation of S-cells in the wild type, but the limited oxygen still impedes S-cell formation in the ΔfilP mutant. These results demonstrate that S-cell formation is stimulated by oxygen-limiting conditions and dependent on functional cytoskeleton remodeling.

Alterations of Cytochrome P450-Mediated Drug Metabolism during Liver Repair and Regeneration after Acetaminophen-Induced Liver Injury in Mice.

Acetaminophen (APAP)-induced liver injury (AILI) is the leading cause of acute liver failure in the United States, but its impact on metabolism, therapeutic efficacy, and adverse drug reactions (ADRs) of co- and/or subsequent administered drugs are not fully investigated. The current work explored this field with a focus on the AILI-mediated alterations of cytochrome P450-mediated drug metabolism. Various levels of liver injury were induced in mice by treatment with APAP at 0, 200, 400, and 600 mg/kg. Severity of liver damage was determined at 24, 48, 72, and 96 hours by plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), microRNA miR122, and tissue staining. The expression and activities of CYP3A11, 1A2, 2B10, 2C29, and 2E1 were measured. Sedation efficacy and ADRs of midazolam, a CYP3A substrate, were monitored after APAP treatment. ALT, AST, and miR122 increased at 24 hours after APAP treatment with all APAP doses, whereas only groups treated with 200 and 400 mg/kg recovered back to normal levels at 72 and 96 hours. The expression and activity of the cytochromes P450 significantly decreased at 24 hours with all APAP doses but only recovered back to normal at 72 and 96 hours with 200 and 400, but not 600, mg/kg of APAP. The alterations of cytochrome P450 activities resulted in altered sedation efficacy and ADRs of midazolam, which were corrected by dose justification of midazolam. Overall, this work illustrated a low cytochrome P450 expression window after AILI, which can decrease drug metabolism and negatively impact drug efficacy and ADRs. SIGNIFICANCE STATEMENT: The data generated in the mouse model demonstrated that expression and activities of cytochrome P450 enzymes and correlated drug efficacy and ADRs are altered during the time course of liver repair and regeneration after liver is injured by treatment with APAP. Dose justifications based on predicted changes of cytochrome P450 activities can achieve desired therapeutic efficacy and avoid ADRs. The generated data provide fundamental knowledge for translational research to drug treatment for patients during liver recovery and regeneration who have experienced AILI.

Long Noncoding RNAs Hepatocyte Nuclear Factor 4A Antisense RNA 1 and Hepatocyte Nuclear Factor 1A Antisense RNA 1 Are Involved in Ritonavir-Induced Cytotoxicity in Hepatoma Cells.

Ritonavir (RTV), a pharmacoenhancer used in anti-HIV regimens, can induce liver damage. RTV is primarily metabolized by cytochrome P450 3A4 (CYP3A4) in the liver. HNF4A antisense RNA 1 (HNF4A-AS1) and HNF1A antisense RNA 1 (HNF1A-AS1) are long noncoding RNAs that regulate the expression of pregnane X receptor (PXR) and CYP3A4. This study investigated the role and underlying mechanisms of HNF4A-AS1 and HNF1A-AS1 in RTV-induced hepatotoxicity. HNF4A-AS1 and HNF1A-AS1 were knocked down by small hairpin RNAs in Huh7 and HepG2 cells. Lactate dehydrogenase and reactive oxygen species assays were performed to assess RTV-induced hepatotoxicity. Chromatin immunoprecipitation quantitative real-time polymerase chain reaction was used to detect PXR enrichment and histone modifications in the CYP3A4 promoter. HNF4A-AS1 knockdown increased PXR and CYP3A4 expression and exacerbated RTV-induced cytotoxicity, whereas HNF1A-AS1 knockdown generated the opposite phenotype. Mechanistically, enrichment of PXR and trimethylation of histone 3 lysine 4 (H3K4me3) in the CYP3A4 promoter was increased, and trimethylation of histone 3 lysine 27 (H3K27me3) was decreased after HNF4A-AS1 knockdown. However, PXR and H3K4me3 enrichment decreased after HNF1A-AS1 knockdown. Alterations in RTV-induced hepatotoxicity caused by decreasing HNF4A-AS1 or HNF1A-AS1 were reversed by knockdown or overexpression of PXR. Increased susceptibility to RTV-induced liver injury caused by the PXR activator rifampicin was attenuated by HNF4A-AS1 overexpression or HNF1A-AS1 knockdown. Taken together, these results revealed that HNF4A-AS1 and HNF1A-AS1 modulated RTV-induced hepatotoxicity by regulating CYP3A4 expression, primarily by affecting the binding of PXR and histone modification status in the CYP3A4 promoter. SIGNIFICANCE STATEMENT: HNF4A-AS1 and HNF1A-AS1, transcribed separately from neighboring antisense genes of the human transcription factor genes HNF4A and HNF1A, were identified as long noncoding RNAs that can affect RTV-induced hepatotoxicity and susceptibility to RTV-induced hepatotoxicity caused by rifampicin exposure, mainly by affecting the expression of CY3A4 via alterations in PXR enrichment and histone modification status in the CYP3A4 promoter. This discovery provides directions for further research on the mechanisms of RTV-induced liver injury.

Absorption, Distribution, Metabolism, and Excretion of US Food and Drug Administration-Approved Antisense Oligonucleotide Drugs.

Absorption, distribution, metabolism, and excretion (ADME) are the key biologic processes for determination of a drug's pharmacokinetic parameters, which have direct impacts on efficacy and adverse drug reactions (ADRs). The chemical structures, dosage forms, and sites and routes of administration are the principal determinants of ADME profiles and consequent impacts on their efficacy and ADRs. Newly developed large molecule biologic antisense oligonucleotide (ASO) drugs have completely unique ADME that is not fully defined. ASO-based drugs are single-stranded synthetic antisense nucleic acids with diverse modes of drug actions from induction of mRNA degradation, exon skipping and restoration, and interactions with proteins. ASO drugs have a great potential to treat certain human diseases that have remained untreatable with small molecule-based drugs. The ADME of ASO drugs contributes to their unique set of ADRs and toxicity. In this review, to better understand their ADME, the 10 US Food and Drug Administration (FDA)-approved ASO drugs were selected: fomivirsen, pegaptanib, mipomersen, nusinersen, inotersen, defibrotide, eteplirsen, golodirsen, viltolarsen, and casimersen. A meta-analysis was conducted on their formulation, dosage, sites of administration, local and systematic distribution, metabolism, degradation, and excretion. Membrane permeabilization through endocytosis and nucleolytic degradation by endonucleases and exonucleases are major ADME features of the ASO drugs that differ from small-molecule drugs. The information summarized here provides comprehensive ADME characteristics of FDA-approved ASO drugs, leading to a better understanding of their therapeutic efficacy and their potential ADRs and toxicity. Numerous knowledge gaps, particularly on cellular uptake and subcellular trafficking and distribution, are identified, and future perspectives and directions are discussed. SIGNIFICANCE STATEMENT: Through a systematic analysis of the existing information of absorption, distribution, metabolism, and excretion (ADME) parameters for 10 US Food and Drug Administration (FDA)-approved antisense oligonucleotide (ASO) drugs, this review provides an overall view of the unique ADME characteristics of ASO drugs, which are distinct from small chemical drug ADME. This knowledge is useful for discovery and development of new ASO drugs as well as clinical use of current FDA-approved ASO drugs.

Adverse Drug Reactions and Toxicity of the Food and Drug Administration-Approved Antisense Oligonucleotide Drugs.

The market for large molecule biologic drugs has grown rapidly, including antisense oligonucleotide (ASO) drugs. ASO drugs work as single-stranded synthetic oligonucleotides that reduce production or alter functions of disease-causing proteins through various mechanisms, such as mRNA degradation, exon skipping, and ASO-protein interactions. Since the first ASO drug, fomivirsen, was approved in 1998, the U.S. Food and Drug Administration (FDA) has approved 10 ASO drugs to date. Although ASO drugs are efficacious in treating some diseases that are untargetable by small-molecule chemical drugs, concerns on adverse drug reactions (ADRs) and toxicity cannot be ignored. Illustrative of this, mipomersen was recently taken off the market due to its hepatotoxicity risk. This paper reviews ADRs and toxicity from FDA drug labeling, preclinical studies, clinical trials, and postmarketing real-world studies on the 10 FDA-approved ASO drugs, including fomivirsen and pegaptanib, mipomersen, nusinersen, inotersen, defibrotide, eteplirsen, golodirsen, viltolarsen, and casimersen. Unique and common ADRs and toxicity for each ASO drug are summarized here. The risk of developing hepatotoxicity, kidney toxicity, and hypersensitivity reactions co-exists for multiple ASO drugs. Special precautions need to be in place when certain ASO drugs are administrated. Further discussion is extended on studying the mechanisms of ADRs and toxicity of these drugs, evaluating the existing physiologic and pathologic states of patients, optimizing the dose and route of administration, and formulating personalized treatment plans to improve the clinical utility of FDA-approved ASO drugs and discovery and development of new ASO drugs with reduced ADRs. SIGNIFICANCE STATEMENT: The current review provides a comprehensive analysis of unique and common ADRs and the toxicity of FDA-approved ASO drugs. The information can help better manage the risk of severe hepatotoxicity, kidney toxicity, and hypersensitivity reactions in the usage of currently approved ASO drugs and the discovery and development of new and safer ASO drugs.

Effects of Overexpression of Fibroblast Growth Factor 15/19 on Hepatic Drug Metabolizing Enzymes.

Fibroblast growth factors 15 (FGF15) and 19 (FGF19) are endocrine growth factors that play an important role in maintaining bile acid homeostasis. FGF15/19-based therapies are currently being tested in clinical trials for the treatment of nonalcoholic steatohepatitis and cholestatic liver diseases. To determine the physiologic impact of long-term elevations of FGF15/19, a transgenic mouse model with overexpression of Fgf15 (Fgf15 Tg) was used in the current study. The RNA sequencing (RNA-seq) analysis revealed elevations of the expression of several genes encoding phase I drug metabolizing enzymes (DMEs), including Cyp2b10 and Cyp3a11, in Fgf15 Tg mice. We found that the induction of several Cyp2b isoforms resulted in increased function of CYP2B in microsomal metabolism and pharmacokinetics studies. Because the CYP2B family is known to be induced by constitutive androstane receptor (CAR), to determine the role of CAR in the observed inductions, we crossed Fgf15 Tg mice with CAR knockout mice and found that CAR played a minor role in the observed alterations in DME expression. Interestingly, we found that the overexpression of Fgf15 in male mice resulted in a phenotypical switch from the male hepatic expression pattern of DMEs to that of female mice. Differences in secretion of growth hormone (GH) between male and female mice are known to drive sexually dimorphic, STAT5b-dependent expression patterns of hepatic genes. We found that male Fgf15 Tg mice presented with many features similar to GH deficiency, including lowered body length and weight, Igf-1 and Igfals expression, and STAT5 signaling. SIGNIFICANCE STATEMENT: The overexpression of Fgf15 in mice causes an alteration in DMEs at the mRNA, protein, and functional levels, which is not entirely due to CAR activation but associated with lower GH signaling.

Simultaneous Targeting of Multiple oncomiRs with Phosphorothioate or PNA-Based Anti-miRs in Lymphoma Cell Lines.

PURPOSE: MicroRNAs (miRNAs) are short (~ 22 nts) RNAs that regulate gene expression via binding to mRNA. MiRNAs promoting cancer are known as oncomiRs. Targeting oncomiRs is an emerging area of cancer therapy. OncomiR-21 and oncomiR-155 are highly upregulated in lymphoma cells, which are dependent on these oncomiRs for survival. Targeting specific miRNAs and determining their effect on cancer cell progression and metastasis have been the focus of various studies. Inhibiting a single miRNA can have a limited effect, as there may be other overexpressed miRNAs present that may promote tumor proliferation. Herein, we target miR-21 and miR-155 simultaneously using nanoparticles delivered two different classes of antimiRs: phosphorothioates (PS) and peptide nucleic acids (PNAs) and compared their efficacy in lymphoma cell lines. METHODS: Poly-Lactic-co-Glycolic acid (PLGA) nanoparticles (NPs) containing PS and PNA-based antimiR-21 and -155 were formulated, and comprehensive NP characterizations: morphology (scanning electron microscopy), size (differential light scattering), and surface charge (zeta potential) were performed. Cellular uptake analysis was performed using a confocal microscope and flow cytometry analysis. The oncomiR knockdown and the effect on downstream targets were confirmed by gene expression (real time-polymerase chain reaction) assay. RESULTS: We demonstrated that simultaneous targeting with NP delivered PS and PNA-based antimiRs resulted in significant knockdown of miR-21 and miR-155, as well as their downstream target genes followed by reduced cell viability ex vivo. CONCLUSIONS: This project demonstrated that targeting miRNA-155 and miR-21 simultaneously using nanotechnology and a diverse class of antisense oligomers can be used as an effective approach for lymphoma therapy.

The impact of the globalization of cancer clinical trials on the enrollment of Black patients.

BACKGROUND: The enrollment of Black patients in cancer clinical trials continues to trend far below the true prevalence of disease in Black patients in the United States, limiting the generalizability of trial results. A potentially overlooked contributor to the underenrollment of Black patients may be the increasing enrollment of cancer trials in countries outside the United States. However, the impact of the globalization of cancer clinical trials on recruitment of racial minority patients has been understudied. METHODS: In this study, race and accrual location data for all cancer drugs approved by the US Food and Drug Administration (FDA) between 2015 and 2018 were analyzed. A disparity score was calculated for each approval, a metric comparing Black enrollment in clinical trials with the estimated burden of disease in Black patients. RESULTS: Of 49 global clinical trials supporting 35 FDA drug approvals with race data available, Black patients accounted for 2.5% of enrollment (range, 0%-10%), with a median disparity score of 0.19 (range, 0.01-0.98). In 21 clinical trials supporting 18 FDA drug approvals with both race and accrual location data available, 64% patients were enrolled outside the United States (range, 0%-100%). Black patients accounted for 3.2% of enrollment (range, 0.2%-10%), and the median disparity score was 0.23 (range, 0.01-0.98). There was a significant inverse correlation between the proportion of trial patients enrolled outside the United States and the disparity score (Pearson correlation, -0.61; P = .007). CONCLUSIONS: The globalization of cancer clinical trials is associated with a widening racial enrollment disparity gap in the United States. The impact of global trials on domestic clinical trial generalizability warrants further consideration from a regulatory and policy standpoint. LAY SUMMARY: Black patients continue to be underrepresented in cancer clinical trials; this disparity has worsened in recent years perhaps because of the globalization of cancer clinical trials. In an analysis of demographic information from 21 cancer clinical trials leading to US Food and Drug Administration approvals between 2015 and 2018, clinical trials conducted primarily outside the United States were 2-fold less likely to enroll Black participants than US clinical trials. Thus, the globalization of cancer clinical trials may have the unintended consequence of further exacerbating existing racial disparities in cancer clinical trial representation and ultimately the generalizability of trial results.

The Long Noncoding RNA Hepatocyte Nuclear Factor 4α Antisense RNA 1 Negatively Regulates Cytochrome P450 Enzymes in Huh7 Cells via Histone Modifications.

The maintenance of homeostasis of cytochromes P450 enzymes (P450s) under both physiologic and xenobiotic exposure conditions is ensured by the action of positive and negative regulators. In the current study, the hepatocyte nuclear factor 4α (HNF4A) antisense RNA 1 (HNF4A-AS1), an antisense long noncoding RNA of HNF4A, was found to be a negative regulator of the basal and rifampicin (RIF)-induced expression of nuclear receptors and downstream P450s. In Huh7 cells, knockdown of HNF4A-AS1 resulted in elevated expression of HNF4A, pregnane X receptor (PXR), and P450s (including CYP3A4) under both basal and RIF-induced conditions. Conversely, overexpression of HNF4A-AS1 led to decreased basal expression of constitutive androstane receptor, aryl hydrocarbon receptor, PXR, and all studied P450s. Of note, significantly diminished induction levels of PXR and CYP1A2, 2C8, 2C19, and 3A4 by RIF were also observed in HNF4A-AS1 plasmid-transfected Huh7 cells. Moreover, the negative feedback of HNF4A on HNF4A-AS1-mediated gene expression was validated using a loss-of-function experiment in this study. Strikingly, our data showed that increased enrichment levels of histone 3 lysine 4 trimethylation and HNF4A in the CYP3A4 promoter contribute to the elevated CYP3A4 expression after HNF4A-AS1 knockdown. Overall, the current study reveals that histone modifications contribute to the negative regulation of nuclear receptors and P450s by HNF4A-AS1 in basal and drug-induced levels. SIGNIFICANCE STATEMENT: Utilizing loss-of-function and gain-of-function experiments, the current study systematically investigated the negative regulation of HNF4A-AS1 on the expression of nuclear receptors (including HNF4A, constitutive androstane receptor, aryl hydrocarbon receptor, and pregnane X receptor) and P450s (including CYP1A2, 2E1, 2B6, 2D6, 2C8, 2C9, 2C19, and 3A4) in both basal and rifampicin-induced levels in Huh7 cells. Notably, this study is the first to reveal the contribution of histone modification to the HNF4A-AS1-mediated expression of CYP3A4 in Huh7 cells.

Developing an Institute for Health Care Delivery Science: successes, challenges, and solutions in the first five years.

Medical knowledge is increasing at an exponential rate. At the same time, unexplained variations in practice and patient outcomes and unacceptable rates of medical errors and inefficiencies in health care delivery have emerged. Our Institute for Health Care Delivery Science (I-HDS) began in 2014 as a novel platform to conduct multidisciplinary healthcare delivery research. We followed ten strategies to develop a successful institute with excellence in methodology and strong understanding of the value of team science. Our work was organized around five hubs: 1) Quality/Process Improvement and Systematic Review, 2) Comparative Effectiveness Research, Pragmatic Clinical Trials, and Predictive Analytics, 3) Health Economics and Decision Modeling, 4) Qualitative, Survey, and Mixed Methods, and 5) Training and Mentoring. In the first 5 years of the I-HDS, we have identified opportunities for change in clinical practice through research using our health system's electronic health record (EHR) data, and designed programs to educate clinicians in the value of research to improve patient care and recognize efficiencies in processes. Testing the value of several model interventions has guided prioritization of evidence-based quality improvements. Some of the changes in practice have already been embedded in the EHR workflow successfully. Development and sustainability of the I-HDS has been fostered by a mix of internal and external funding, including philanthropic foundations. Challenges remain due to the highly competitive funding environment and changes needed to adapt the EHR to healthcare delivery research. Further stakeholder engagement and culture change working with hospital leadership and I-HDS core and affiliate members continues.

Knockdown of Long Noncoding RNAs Hepatocyte Nuclear Factor 1α Antisense RNA 1 and Hepatocyte Nuclear Factor 4α Antisense RNA 1 Alters Susceptibility of Acetaminophen-Induced Cytotoxicity in HepaRG Cells.

Acetaminophen (APAP) is a commonly used over-the-counter drug for its analgesic and antipyretic effects. However, APAP overdose leads to severe APAP-induced liver injury (AILI) and even death as a result of the accumulation of N-acetyl-p-benzoquinone imine, the toxic metabolite of APAP generated by cytochrome P450s (P450s). Long noncoding RNAs HNF1α antisense RNA 1 (HNF1α-AS1) and HNF4α antisense RNA 1 (HNF4α-AS1) are regulatory RNAs involved in the regulation of P450 expression in both mRNA and protein levels. This study aims to determine the impact of HNF1α-AS1 and HNF4α-AS1 on AILI. Small hairpin RNAs were used to knock down HNF1α-AS1 and HNF4α-AS1 in HepaRG cells. Knockdown of these lncRNAs altered APAP-induced cytotoxicity, indicated by MTT and LDH assays. Specifically, HNF1α-AS1 knockdown decreased APAP toxicity with increased cell viability and decreased LDH release, whereas HNF4α-AS1 knockdown exacerbated APAP toxicity, with opposite effects in the MTT and LDH assays. Alterations on gene expression by knockdown of HNF1α-AS1 and HNF4α-AS1 were examined in several APAP metabolic pathways, including CYP1A2, CYP2E1, CYP3A4, UGT1A1, UGT1A9, SULT1A1, GSTP1, and GSTT1. Knockdown of HNF1α-AS1 decreased mRNA expression of CYP1A2, 2E1, and 3A4 by 0.71-fold, 0.35-fold, and 0.31-fold, respectively, whereas knockdown of HNF4α-AS1 induced mRNAs of CYP1A2, 2E1, and 3A4 by 1.3-fold, 1.95-fold, and 1.9-fold, respectively. These changes were also observed in protein levels. Knockdown of HNF1α-AS1 and HNF4α-AS1 had limited effects on the mRNA expression of UGT1A1, UGT1A9, SULT1A1, GSTP1, and GSTT1. Altogether, our study suggests that HNF1α-AS1 and HNF4α-AS1 affected AILI mainly through alterations of P450-mediated APAP biotransformation in HepaRG cells, indicating an important role of the lncRNAs in AILI. SIGNIFICANCE STATEMENT: The current research identified two lncRNAs, hepatocyte nuclear factor 1α antisense RNA 1 and hepatocyte nuclear factor 4α antisense RNA 1, which were able to affect susceptibility of acetaminophen (APAP)-induced liver injury in HepaRG cells, possibly through regulating the expression of APAP-metabolizing cytochrome P450 enzymes. This discovery added new factors, lncRNAs, which can be used to predict cytochrome P450-mediated drug metabolism and drug-induced toxicity.

Histone Methyltransferase G9a Regulates Expression of Nuclear Receptors and Cytochrome P450 Enzymes in HepaRG Cells at Basal Level and in Fatty Acid Induced Steatosis.

Obesity and nonalcoholic fatty liver disease (NAFLD) affect expression and function of cytochrome P450 genes (P450s). The increased expression of inflammatory cytokines is a major driver of the downregulation of P450 expression in NAFLD. Decrease in P450 expression could potentially lead to drug-drug interaction, inefficient pharmacological effect of a drug, or hepatotoxicity. An epigenetic modifier, histone 3 lysine 9 methyl transferase enzyme (G9a), known to increase histone 3 lysine 9 methylation, is downregulated in diet-induced obesity animal models. In a liver-specific G9a knockout animal model, expression of P450s was downregulated. Currently, the role of G9a in regulation of P450s in steatosis is unknown. Our hypothesis is that in steatosis G9a plays a role in downregulation of P450 expression. In this study, we used HepaRG cells to induce steatosis using a combination of free fatty acids oleic acid and palmitic acid. The G9a was knocked down and overexpressed using small interfering RNA and adenovirus mediated approaches, respectively. Knockdown and overexpression of G9a in the absence of steatosis decreased and increased expression of nuclear receptors constitutive androstane receptor (CAR), pregnane X receptor, small heterodimer partner, and CYP2B6, 2E1, 2C8, 2C9, and 3A4, respectively. In steatotic conditions, overexpression of G9a prevented fatty acid mediated decreased expression of CAR, CYP2C19, 2C8, 7A1, and 3A4. Our current study suggests that G9a might serve as a key regulator of P450 expression at both the basal level and in early steatotic conditions. Single nucleotide polymorphism of G9a leading to loss/gain of function could lead to the poor metabolizer or ultrarapid metabolizer phenotypes. SIGNIFICANCE STATEMENT: The current study demonstrates that histone modification enzyme G9a is involved in the regulation of expression of nuclear receptors constitutive androstane receptor, pregnane X receptor, and small heterodimer partner as well as drug-metabolizing cytochrome P450s (P450s) at basal conditions and in fatty acid induced cellular model of steatosis. Histone 3 lysine 9 methylation should be considered together with histone 3 lysine 4 and histone 3 lysine 27 methylation as the epigenetic mechanisms controlling gene expression of P450s.

Acetaminophen-Induced Liver Injury Alters Expression and Activities of Cytochrome P450 Enzymes in an Age-Dependent Manner in Mouse Liver.

Drug-induced liver injury (DILI) is a global medical problem. The risk of DILI is often related to expression and activities of drug-metabolizing enzymes, especially cytochrome P450s (P450s). However, changes on expression and activities of P450s after DILI have not been determined. The aim of this study is to fill this knowledge gap. Acetaminophen (APAP) was used as a model drug to induce DILI in C57BL/6J mice at different ages of days 10 (infant), 22 (child), and 60 (adult). DILI was assessed by levels of alanine aminotransferase and aspartate aminotransferase in plasma with a confirmation by H&E staining on liver tissue sections. The expression of selected P450s at mRNA and protein levels was measured by real-time polymerase chain reaction and liquid chromatography-tandem mass spectrometry, respectively. The activities of these P450s were determined by the formation of metabolites from probe drugs for each P450 using ultraperformance liquid chromatography-quadrupole time of flight mass spectrometry. DILI was induced at mild to severe levels in a dose-dependent manner in 200, 300, and 400 mg/kg APAP-treated groups at child and adult ages, but not at the infant age. Significantly decreased expression at mRNA and protein levels as well as enzymatic activities of CYP2E1, 3A11, 1A2, and 2C29 were found at child and adult ages. Adult male mice were more susceptible to APAP-induced liver injury than female mice with more decreased expression of P450s. These results suggest that altered levels of P450s in livers severely injured by drugs may affect the therapeutic efficacy of drugs, which are metabolized by P450s, more particularly for males. SIGNIFICANCE STATEMENT: The current study in an animal model demonstrates that acetaminophen-induced liver injury results in decreased expression and enzyme activities of several examined drug-metabolizing cytochrome P450s (P450s). The extent of such decreases is correlated to the degree of liver injury severity. The generated data may be translated to human health for patients who have drug-induced liver injury with decreased capability to metabolize drugs by certain P450s.

Cell Phones, Sexual Behaviors and HIV Prevalence in Rakai, Uganda: A Cross Sectional Analysis of Longitudinal Data.

Cell phones have increased communication and connection across the globe and particularly in sub-Saharan Africa-with potential consequences for the HIV epidemic. We examined the association among ownership of cell phones, sexual behaviors (number of sexual partners, alcohol use before sex, inconsistent condom use), and HIV prevalence. Data were from four rounds (2010-2016) of the Rakai Community Cohort Study (N = 58,275). Sexual behaviors and HIV prevalence were compared between people who owned a cell phone to people who did not own a cell phone. We stratified analysis by younger (15-24 years) and older (25+ years) age groups and by gender. Using logistic regression and after adjusting for sociodemographic characteristics, we found cell phone ownership was independently associated with increased odds of having two or more sexual partners in the past 12 months across age and gender groups (young men AOR 1.67, 95% CI 1.47-1.90; young women AOR 1.28 95% CI 1.08-1.53; older men AOR 1.54 95% CI 1.41-1.69; older women AOR 1.44 95% CI 1.26-1.65). Interestingly, young men who owned cell phones had decreased odds of using condoms inconsistently (AOR 0.66, 95% CI 0.57-0.75). For young women, cell phone ownership was associated with increased odds of using alcohol before sex (AOR 1.38 95% CI 1.17-1.63) and increased odds of inconsistent condom use (AOR 1.40, 95% 1.17-1.67). After adjusting for sociodemographic characteristics, only young women who owned cell phones had increased odds of being HIV positive (AOR 1.27 95% CI 1.07-1.50). This association was not mediated by sexual behaviors (Adjusted for sociodemographic characteristics and sexual behaviors AOR 1.24, 95% CI 1.05-1.46). While cell phone ownership appears to be associated with increased HIV risk for young women, we also see a potential opportunity for future cell phone-based health interventions.

The HNF1α-Regulated LncRNA HNF1α-AS1 Is Involved in the Regulation of Cytochrome P450 Expression in Human Liver Tissues and Huh7 Cells.

Expression of cytochrome P450s (P450s) is regulated by epigenetic factors, such as DNA methylation, histone modifications, and noncoding RNAs through different mechanisms. Among these factors, long noncoding RNAs (lncRNAs) have been shown to play important roles in the regulation of gene expression; however, little is known about the effects of lncRNAs on the regulation of P450 expression. The aim of this study was to explore the role of lncRNAs in the regulation of P450 expression by using human liver tissues and hepatoma Huh7 cells. Through lncRNA microarray analysis and quantitative polymerase chain reaction in human liver tissues, we found that the lncRNA hepatocyte nuclear factor 1 alpha antisense 1 (HNF1α-AS1), an antisense RNA of HNF1α, is positively correlated with the mRNA expression of CYP2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 as well as pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Gain- and loss-of-function studies in Huh7 cells transfected with small interfering RNAs or overexpression plasmids showed that HNF1α not only regulated the expression of HNF1α-AS1 and P450s, but also regulated the expression of CAR, PXR, and aryl hydrocarbon receptor (AhR). In turn, HNF1α-AS1 regulated the expression of PXR and most P450s without affecting the expression of HNF1α, AhR, and CAR. Moreover, the rifampicin-induced expression of P450s was also affected by HNF1α and HNF1α-AS1. In summary, the results of this study suggested that HNF1α-AS1 is involved in the HNF1α-mediated regulation of P450s in the liver at both basal and drug-induced levels.

Fibroblast Growth Factor 15-Dependent and Bile Acid-Independent Promotion of Liver Regeneration in Mice.

The role of intestine-derived factors in promoting liver regeneration after partial hepatectomy (PHx) are not entirely known, but bile acids (BAs) and fibroblast growth factor 15 (Fgf15) that is highly expressed in the mouse ileum could promote hepatocyte proliferation. Fgf15 strongly suppresses the synthesis of BAs, and emerging evidence indicates that Fgf15 is important for liver regeneration. The mechanisms by which Fgf15 promotes liver regeneration are unclear, but Fgf15 may do so indirectly by reducing BA levels and/or directly by promoting cell proliferation. However, it remains undetermined whether these two mechanisms are independent or integrated. In this study, we aimed to clarify these relationships by generating Fgf15 Tet-Off, transgenic mice (Fgf15 Tg) that had very low BA levels as a result from overexpressed Fgf15-mediated suppression of BA synthesis. Compared with wild-type mice, the Fgf15 Tg mice showed increased hepatocyte proliferation even without surgery, and a further induction of the genes in cell-cycle progression after PHx. Moreover, overexpression of Fgf15 by adeno-associated virus (AAV)-Fgf15 transduction or treatment with the recombinant Fgf15 protein led to increased cell proliferation in vivo. Furthermore, Fgf15 Tg mice exhibited an earlier and greater activation of mitogen-activated protein kinase, signal transducer and activator of transcription 3, and NF-κB signaling pathways in the priming stage, and a disruption of the hippo signaling pathway in the termination stage of liver regeneration. Conclusion: Direct in vivo evidence demonstrates that Fgf15 is critical in stimulating the phases of priming and termination of liver regeneration that are critical for cell survival and liver-size determination, independent of BA levels. (Hepatology 2018; 00:000-000).