Negative Regulation of Human Pregnane X Receptor by MicroRNA-18a-5p: Evidence for Suppression of MicroRNA-18a-5p Expression by Rifampin and Rilpivirine.

Small noncoding microRNAs act as post-transcriptional regulators of gene expression involved in diverse biologic functions. Pregnane X receptor (PXR, NR1I2), a member of the superfamily of nuclear receptors, is a transcription factor governing the transport and biotransformation of various drugs and other chemicals. In the present study, we identified a specific microRNA (miR) involved in regulating the expression and functionality of human PXR (hPXR). According to bioinformatics analysis employing three commonly used algorithms (TargetScan, miRanda, and DIANA-microT-CDS), miR-18a-5p was predicted to be the top candidate microRNA regulator of hPXR. Consequently, this microRNA was selected for detailed experimental investigation. As shown in cell-based dual-luciferase reporter gene assays, functional interaction occurred between miR-18a-5p and the microRNA recognition element of miR-18a-5p in the 3'-untranslated region of hPXR mRNA. Transfection of LS180 human colorectal adenocarcinoma cells with an miR-18a-5p mimic decreased hPXR mRNA and protein expression, whereas transfection of LS180 cells with an miR-18a-5p inhibitor increased hPXR mRNA and protein expression. The decrease in hPXR expression by the miR-18a-5p mimic was associated with a reduction in the extent of hPXR target gene (CYP3A4) induction by rifampin and rilpivirine. Treatment of untransfected LS180 cells with either of these hPXR agonists decreased endogenous expression of miR-18a-5p, and this preceded the onset of CYP3A4 induction. In conclusion, miR-18a-5p is a negative regulator of hPXR expression and the hPXR agonists rifampin and rilpivirine are chemical suppressors of miR-18a-5p expression.

Recent gene duplication and subfunctionalization produced a mitochondrial GrpE, the nucleotide exchange factor of the Hsp70 complex, specialized in thermotolerance to chronic heat stress in Arabidopsis.

The duplication and divergence of heat stress (HS) response genes might help plants adapt to varied HS conditions, but little is known on the topic. Here, we examined the evolution and function of Arabidopsis (Arabidopsis thaliana) mitochondrial GrpE (Mge) proteins. GrpE acts as a nucleotide-exchange factor in the Hsp70/DnaK chaperone machinery. Genomic data show that AtMge1 and AtMge2 arose from a recent whole-genome duplication event. Phylogenetic analysis indicated that duplication and preservation of Mges occurred independently in many plant species, which suggests a common tendency in the evolution of the genes. Intron retention contributed to the divergence of the protein structure of Mge paralogs in higher plants. In both Arabidopsis and tomato (Solanum lycopersicum), Mge1 is induced by ultraviolet B light and Mge2 is induced by heat, which suggests regulatory divergence of the genes. Consistently, AtMge2 but not AtMge1 is under the control of HsfA1, the master regulator of the HS response. Heterologous expression of AtMge2 but not AtMge1 in the temperature-sensitive Escherichia coli grpE mutant restored its growth at 43°C. Arabidopsis T-DNA knockout lines under different HS regimes revealed that Mge2 is specifically required for tolerating prolonged exposure to moderately high temperature, as compared with the need of the heat shock protein 101 and the HS-associated 32-kD protein for short-term extreme heat. Therefore, with duplication and subfunctionalization, one copy of the Arabidopsis Mge genes became specialized in a distinct type of HS. We provide direct evidence supporting the connection between gene duplication and adaptation to environmental stress.

Myofibroblast YAP/TAZ activation is a key step in organ fibrogenesis.

Fibrotic diseases account for nearly half of all deaths in the developed world. Despite its importance, the pathogenesis of fibrosis remains poorly understood. Recently, the two mechanosensitive transcription cofactors YAP and TAZ have emerged as important profibrotic regulators in multiple murine tissues. Despite this growing recognition, a number of important questions remain unanswered, including which cell types require YAP/TAZ activation for fibrosis to occur and the time course of this activation. Here, we present a detailed analysis of the role that myofibroblast YAP and TAZ play in organ fibrosis and the kinetics of their activation. Using analyses of cells, as well as multiple murine and human tissues, we demonstrated that myofibroblast YAP and TAZ were activated early after organ injury and that this activation was sustained. We further demonstrated the critical importance of myofibroblast YAP/TAZ in driving progressive scarring in the kidney, lung, and liver, using multiple transgenic models in which YAP and TAZ were either deleted or hyperactivated. Taken together, these data establish the importance of early injury-induced myofibroblast YAP and TAZ activation as a key event driving fibrosis in multiple organs. This information should help guide the development of new antifibrotic YAP/TAZ inhibition strategies.

NUAK1 promotes organ fibrosis via YAP and TGF-ß/SMAD signaling.

Fibrosis is a central pathway that drives progression of multiple chronic diseases, yet few safe and effective clinical antifibrotic therapies exist. In most fibrotic disorders, transforming growth factor-ß (TGF-ß)-driven scarring is an important pathologic feature and a key contributor to disease progression. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are two closely related transcription cofactors that are important for coordinating fibrogenesis after organ injury, but how they are activated in response to tissue injury has, so far, remained unclear. Here, we describe NUAK family kinase 1 (NUAK1) as a TGF-ß-inducible profibrotic kinase that is up-regulated in multiple fibrotic organs in mice and humans. Mechanistically, we show that TGF-ß induces a rapid increase in NUAK1 in fibroblasts. NUAK1, in turn, can promote profibrotic YAP and TGF-ß/SMAD signaling, ultimately leading to organ scarring. Moreover, activated YAP and TAZ can induce further NUAK1 expression, creating a profibrotic positive feedback loop that enables persistent fibrosis. Using mouse models of kidney, lung, and liver fibrosis, we demonstrate that this fibrogenic signaling loop can be interrupted via fibroblast-specific loss of NUAK1 expression, leading to marked attenuation of fibrosis. Pharmacologic NUAK1 inhibition also reduced scarring, either when initiated immediately after injury or when initiated after fibrosis was already established. Together, our data suggest that NUAK1 plays a critical, previously unrecognized role in fibrogenesis and represents an attractive target for strategies that aim to slow fibrotic disease progression.

Performing nutrition assessment remotely via telehealth.

Performing nutrition assessment remotely via telehealth is a topic of significant interest given the global pandemic in 2020 that has necessitated physical distancing and virtual communications. This review presents an evidence-based approach to conducting nutrition assessments remotely. The authors present suggestions for adaptations that can be used to perform a remote nutrition-focused physical exam. Direct-to-consumer technologies that can be used in remote nutrition assessment are discussed and compared. Practice tips for conducting a telehealth visit are also presented. The aim of this publication is to provide interdisciplinary clinicians a set of guidelines and best practices for performing nutrition assessments in the era of telehealth.

Completeness of reporting of quality improvement studies in neonatology is inadequate: a systematic literature survey.

INTRODUCTION: Quality improvement (QI) is a growing field of inquiry in healthcare, but the reporting quality of QI studies in neonatology remains unclear. We conducted a systematic survey of the literature to assess the reporting quality of QI studies and factors associated with reporting quality. METHODS: We searched Medline for publications of QI studies from 2016 to 16 April 2020. Pairs of reviewers independently screened citations and assessed reporting quality using a 31-item modified Standards for Quality Improvement Reporting Excellence, 2nd edition (SQUIRE 2.0) checklist. We reported the number (percentage) of studies that reported each item and their corresponding 95% CIs. We used Poisson regression to explore factors associated with reporting quality, namely, journal endorsement of SQUIRE 2.0, declaration of funding sources, year of publication and number of authors. The results were reported as incidence rate ratio (IRR) and 95% CI. RESULTS: Of 1921 citations, 336 were eligible; among them, we randomly selected 100 articles to assess reporting quality. The mean (standard deviation) number of SQUIRE 2.0 items adhered to was 22.0 (4.5). Percentage of articles reporting each item varied from 26% to 100%. Journal endorsement of SQUIRE 2.0 (IRR=1.11, 95% CI 1.02 to 1.21, p=0.015), declaration of funding sources and increasing number of authors were significantly associated with better reporting. CONCLUSIONS: Reporting quality of QI studies in neonatology is inadequate. Endorsing the SQUIRE 2.0 guideline is a step that journals can implement to enhance the completeness of reporting.

Differences in effects on myocardium and mitochondria by angiogenic inhibitors suggest separate mechanisms of cardiotoxicity.

Several multikinase angiogenesis inhibitors demonstrate mitochondrial and/or cardiovascular toxicity, suggesting an on-target pharmacologic effect. To evaluate whether cardiotoxicity is directly related to vascular endothelial growth factor receptor inhibition, we investigated the effects of sunitinib, sorafenib, and pazopanib on myocardial function and structure. We used a rat model to assess myocardial effects of the inhibitors concurrently exposed to the cardiac stressor dobutamine. Echocardiographic abnormalities including premature ventricular contractions, decreases in heart rate, circumferential strain, and radial and circumferential strain rates were noted with sorafenib, but not with sunitinib or pazopanib. Ultrastructural analysis of ventricular cardiomyocytes by transmission electron microscopy revealed mitochondrial swelling, dense deposits, and matrix cavitation in rats given sunitinib and disrupted mitochondrial cristae in rats given sorafenib, but there were no effects with pazopanib. Effects on neonatal rat cardiomyocyte cultures were assessed, which identified decreases in mitochondrial membrane potential with sunitinib treatment, but not with sorafenib or pazopanib. Intracellular adenosine triphosphate depletion was observed with sunitinib and sorafenib, but not pazopanib. Our results show that cardiotoxicity is not necessarily related to a pharmacologic classwide effect of vascular endothelial growth factor receptor inhibition, and the rat myocardial structural and functional changes identified in this study may be instead a result of inhibition of other kinase pathways, the mechanism of which may be associated with mitochondrial toxicity.

"When you first walk out the gates where do [you] go?": Barriers and opportunities to achieving continuity of health care at the time of release from a provincial jail in Ontario.

We aimed to explore continuity of health care and health barriers, facilitators, and opportunities for people at the time of release from a provincial correctional facility in Ontario, Canada. We conducted focus groups in community-based organizations in a city in Ontario, Canada: a men's homeless shelter, a mental health service organization, and a social service agency with programs for people with substance use disorders. We included adults who spoke English well enough to participate in the discussion and who had been released from the provincial correctional facility in the previous year. We conducted three focus groups with 18 total participants. Participants had complex health needs on release, including ongoing physical and psychological impacts of time in custody. They identified lack of access to high quality health care; lack of housing, employment, social services, and social supports; and discrimination on the basis of incarceration history as barriers to health on release. Access to health care, housing, social services, and social supports all facilitated health on release. To address health needs on release, participants suggested providing health information in jail, improving discharge planning, and developing accessible clinics in the community. This pilot study identified opportunities to support health at the time of release from jail, including delivery of programs in jail, linkage with and development of programs in the community, and efforts to support structural changes to prevent and address discrimination. These data will inform ongoing work to support health and continuity of care on release from a provincial correctional facility.

Comparative analysis of ginsenosides in human glucocorticoid receptor binding, transactivation, and transrepression.

Conflicting data exist on the effect of ginsenosides on transactivation of human glucocorticoid receptor α (herein referred to as glucocorticoid receptor), and relatively little is known regarding the effect of these chemicals on transrepression of this receptor. We investigated the effect of 20(S)-protopanaxadiol (PPD), PPD-type ginsenosides (Rb1, Rb2, Rc, Rd, Rh2, and Compound K), 20(S)-protopanaxatriol (PPT), and PPT-type ginsenosides (Re, Rf, Rg1, and Rh1) on glucocorticoid receptor binding, transactivation, and transrepression. Each ginsenoside was less efficacious than dexamethasone (positive control) in binding to the ligand-binding domain of glucocorticoid receptor. Among the ginsenosides investigated, Rh2 had the smallest IC50 value (15 ± 1µM), whereas it was 0.02 ± 0.01µM for dexamethasone. In contrast to dexamethasone, none of the ginsenosides influenced glucocorticoid receptor transactivation or transrepression in LS180 human colorectal adenocarcinoma cells, as assessed in a dual-luciferase reporter gene assay. Rh2 did not affect the endogenous mRNA level of tyrosine aminotransferase (marker for glucocorticoid receptor transactivation) or corticosteroid-binding globulin (marker for glucocorticoid receptor transrepression) in HepG2 human hepatocellular carcinoma cells. This chemical also did not alter the response by a glucocorticoid receptor agonist (dexamethasone or Compound A) in the dual-luciferase reporter gene assay or target gene expression assay. In conclusion, ginsenosides were less efficacious and less potent than dexamethasone in binding to the ligand-binding domain of glucocorticoid receptor. The number of glycosylated groups was associated with a decrease in receptor binding potency. PPD-type and PPT-type ginsenosides are not modulators of glucocorticoid receptor transactivation or transrepression in LS180 and HepG2 cells.

Effects of 6-hydroxydopamine on mitochondrial function and glutathione status in SH-SY5Y human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells were incubated with 6-hydroxydopamine (6-OHDA) for 4 and 24 h to examine the mechanism of cell death and to determine the time-dependent effects of 6-OHDA on cellular glutathione status. After 4 h, 6-OHDA significantly depleted cellular ATP and GSH concentrations with only slight increases in cell death. GSH:GSSG ratios and mitochondrial membrane potential (Deltapsim) were significantly decreased during 4 h incubations with 6-OHDA. High concentrations of 6-OHDA (100 microM) induced oxidative stress and mitochondrial dysfunction in SH-SY5Y cells within 4 h leading to cell death. In 24 h incubations, 25 and 50 microM 6-OHDA significantly decreased ATP concentrations; however, significant increases in cell death were only observed with 50 microM 6-OHDA. 6-OHDA induced a concentration-dependent increase in GSH and total glutathione concentrations after 24 h. After exposure to 50 microM 6-OHDA, GSH concentrations were increased up to 12-fold after 24 h with no change in the GSH:GSSG ratio. Gene analysis suggests that the increase in GSH concentration was due to increased expression of the GSH synthesis genes glutamate cysteine ligase modifier and catalytic subunits. Our results suggest that 6-OHDA induces oxidative stress in SH-SY5Y cells resulting in an adaptive increase in cellular GSH concentrations.

Effects of troglitazone on HepG2 viability and mitochondrial function.

Troglitazone (TRO), a member of the thiazolidinedione class of drugs, has been associated with hepatotoxicity in patients. The following in vitro study was conducted to investigate the effects of TRO on mitochondrial function and viability in a human hepatoma cell line, HepG2. TRO induced a concentration- and time-dependent increase in cell death, as measured by lactate dehydrogenase release. Exposure to 50 or 100 micro M TRO produced total loss of cell viability within 5 h. Preincubation of HepG2 cells with P450 inhibitors did not significantly protect against TRO-induced cell death suggesting that P450 metabolism was not required to induce cell death. Preincubation with the mitochondrial permeability transition inhibitor, cyclosporin A, provided complete protection against TRO-induced cell death. Our results also indicated that TRO produced concentration-dependent decreases in cellular ATP levels and mitochondrial membrane potential (MMP). Ultrastructural analysis demonstrated that TRO induced mitochondrial changes at concentrations of > or =10 micro M after 2 h. Decreased MMP and altered mitochondrial morphology occurred at time points that preceded cell death and at sublethal concentrations of TRO. These observations in HepG2 cells suggest that TRO disrupts mitochondrial function, leading to mitochondrial permeability transition and cell death.

Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically.

Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf1)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIalpha (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h postdosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir.

Comparison of health status and health care services utilization between migrants and natives of the same ethnic origin--the case of Hong Kong.

Based on the 2009 Thematic Household Survey in Hong Kong, this study compared health status and utilization of health care services in Hong Kong between migrants from Mainland China and natives. Overall, Mainland migrants reported lower socioeconomic conditions, worse health status, and less health care services utilization than the natives. After controlling for socio-demographic factors, we found that the migrants were 1.2 times more likely to report fair or poor health and 0.78 times less likely to report having a usual source of care, compared with the natives. Mainland migrants also had fewer physician visits and relied more on the public sector. Within the migrant group, those who had language advantage had more visits, and the recent arrivals who stayed in Hong Kong for three years or less had fewer visits and were far less likely to have a usual source of care. The findings underscore migration as an important social determinant of health in Hong Kong. A combination of targeted social and health policies is needed to help Mainland migrants better integrate into society and to improve their access to care. Programs should be tailored to address varying needs from different subgroups among migrants.

Some like it hot, some like it warm: phenotyping to explore thermotolerance diversity.

Plants have evolved overlapping but distinct cellular responses to different aspects of high temperature stress. These responses include basal thermotolerance, short- and long-term acquired thermotolerance, and thermotolerance to moderately high temperatures. This 'thermotolerance diversity' means that multiple phenotypic assays are essential for fully describing the functions of genes involved in heat stress responses. A large number of genes with potential roles in heat stress responses have been identified using genetic screens and genome wide expression studies. We examine the range of phenotypic assays that have been used to characterize thermotolerance phenotypes in both Arabidopsis and crop plants. Three major variables differentiate thermotolerance assays: (1) the heat stress regime used, (2) the developmental stage of the plants being studied, and (3) the actual phenotype which is scored. Consideration of these variables will be essential for deepening our understanding of the molecular genetics of plant thermotolerance.