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Hepatocyte nuclear factor 4 alpha antisense 1 ( HNF4A-AS1 ) is a long noncoding RNA (lncRNA) gene physically located next to the transcription factor HNF4A gene in the human genome. Its transcription products have been reported to inhibit the progression of hepatocellular carcinoma (HCC) and negatively regulate the expression of cytochrome P450s (CYPs), including CYP1A2, 2B6, 2C9, 2C19, 2E1, and 3A4. By altering CYP expression, lncRNA HNF4A-AS1 also contributes to the susceptibility of drug-induced liver injury. Thus, HNF4A-AS1 lncRNA is a promising target for controlling HCC and modulating drug metabolism. However, HNF4A-AS1 has four annotated alternative transcripts in the human genome browsers, and it is unclear which transcripts the small interfering RNAs or small hairpin RNAs used in the previous studies are silenced and which transcripts should be used as the target. In this study, four annotated and two newly identified transcripts were confirmed. These six transcripts showed different expression levels in different liver disease conditions, including metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, and obesity. The expression patterns of all HNF4A-AS1 transcripts were further investigated in liver cell growth from human embryonic stem cells to matured hepatocyte-like cells, HepaRG differentiation, and exposure to rifampicin treatment. Several HNF4A-AS1 transcripts highly displayed correlations with these situations. In addition, some of the HNF4A-AS1 transcripts also showed a strong correlation with CYP3A4 during HepaRG maturation and rifampicin exposure. Our findings provide valuable insights into the specific roles of HNF4A-AS1 transcripts, paving the way for more targeted therapeutic strategies for liver diseases and drug metabolism. SIGNIFICANCE STATEMENT: This study explores the alternative transcripts of HNF4A-AS1, showing how their expression changes in different biological conditions, from various liver diseases to the growth and differentiation of hepatocytes and drug metabolism. The generated knowledge is essential for understanding the independent roles of different transcripts from the same lncRNA in different liver diseases and drug metabolism situations., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Antisense oligonucleotides (ASOs) are single stranded nucleic acids that target RNA. The US Food and Drug Administration has approved ASOs for several diseases. ASOs utilize three principal modes of action (MOA). The first MOA is initiated by base-pairing between the ASO and its target mRNA, followed by RNase H-dependent mRNA degradation. The second MOA is triggered by ASOs that occlude splice acceptor sites in pre-mRNAs leading to skipping of a mutation-bearing exon. The third MOA involves ASOs that sterically hinder mRNA function, often inhibiting translation. ASOs contain a variety of modifications to the sugar-phosphate backbone and bases that stabilize the ASO or render them resistant to RNase activity. RNase H-dependent ASOs include inotersen and eplontersen (for hereditary transthyretin amyloidosis), fomiversen (for opportunistic cytomegalovirus infection), mipomersen (for familial hypercholesterolemia), and tofersen [for amyotrophic lateral sclerosis (ALS)]. Splice modulating ASOs include nursinersen (for spinal muscular atrophy) and eteplirsen, golodirsen, viltolarsen, and casimersen (all for the treatment of Duchenne muscular dystrophy). In addition, a designer ASO, milasen, was used to treat a single individual afflicted with Batten disease. Since ASO design relies principally upon knowledge of mRNA sequence, the bench to bedside pipeline for ASOs is expedient compared with protein-directed drugs. [Graphical abstract available.]., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The long non-coding RNA (lncRNA) hepatocyte nuclear factor-1 alpha (HNF1A) antisense RNA 1 (HNF1A-AS1) is an important lncRNA for liver growth, development, cell differentiation, and drug metabolism. Like many lncRNAs, HNF1A-AS1 has multiple annotated alternative transcripts in the human genome. Several fundamental biological questions are still not solved: (1) How many transcripts really exist in biological samples, such as liver samples and liver cell lines? (2) What are the expression patterns of different alternative HNF1A-AS1 transcripts at different conditions, including during cell growth and development, after exposure to xenobiotics (such as drugs), and in disease conditions, such as metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD) cirrhosis, and obesity? (3) Does the siRNA used in previous studies knock down one or multiple transcripts? (4) Do different transcripts have the same or different functions for gene regulation? The presented data confirm the existence of several annotated HNF1A-AS1 transcripts in liver samples and cell lines, but also identify some new transcripts, which are not annotated in the Ensembl genome database. Expression patterns of the identified HNF1A-AS1 transcripts are highly correlated with the cell differentiation of matured hepatocyte-like cells from human embryonic stem cells (hESC), growth and differentiation of HepaRG cells, in response to rifampicin induction, and in various liver disease conditions. The expression levels of the HNF1A-AS1 transcripts are also highly correlated to the expression of cytochrome P450 enzymes, such as CYP3A4, during HepaRG growth, differentiation, and in response to rifampicin induction.

Entangled photons are a key resource in quantum technologies. While intense laser light propagating in nonlinear crystals is conventionally used to generate entangled photons, such schemes have low efficiency due to the weak nonlinear response of known materials and losses associated with in/out photon coupling. Here, we show how to generate entangled polariton pairs directly within optical waveguides using free electrons. The measured energy loss of undeflected electrons heralds the production of counter-propagating polariton pairs entangled in energy and emission direction. For illustration, we study the excitation of plasmon polaritons in metal strip waveguides that strongly enhance light-matter interactions, rendering two-plasmon generation dominant over single-plasmon excitation. We demonstrate that electron energy losses detected within optimal frequency ranges can reliably signal the generation of plasmon pairs entangled in energy and momentum. Our proposed scheme is directly applicable to other types of optical waveguides for in situ generation of entangled photon pairs.

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., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Decreased peak sodium current (I Na ) and increased late sodium current (I Na,L ), through the cardiac sodium channel Na V 1.5 encoded by SCN5A, cause arrhythmias. Many Na V 1.5 posttranslational modifications have been reported. A recent report concluded that acute hypoxia increases I Na,L by increasing a small ubiquitin-like modifier (SUMOylation) at K442-Na V 1.5., Objective: The purpose of this study was to determine whether and by what mechanisms SUMOylation alters I Na , I Na,L , and cardiac electrophysiology., Methods: SUMOylation of Na V 1.5 was detected by immunoprecipitation and immunoblotting. I Na was measured by patch clamp with/without SUMO1 overexpression in HEK293 cells expressing wild-type (WT) or K442R-Na V 1.5 and in neonatal rat cardiac myocytes (NRCMs). SUMOylation effects were studied in vivo by electrocardiograms and ambulatory telemetry using Scn5a heterozygous knockout (SCN5A +/- ) mice and the de-SUMOylating protein SENP2 (AAV9-SENP2), AAV9-SUMO1, or the SUMOylation inhibitor anacardic acid. Na V 1.5 trafficking was detected by immunofluorescence., Results: Na V 1.5 was SUMOylated in HEK293 cells, NRCMs, and human heart tissue. HyperSUMOylation at Na V 1.5-K442 increased I Na in NRCMs and in HEK cells overexpressing WT but not K442R-Na v 1.5. SUMOylation did not alter other channel properties including I Na,L . AAV9-SENP2 or anacardic acid decreased I Na , prolonged QRS duration, and produced heart block and arrhythmias in SCN5A +/- mice, whereas AAV9-SUMO1 increased I Na and shortened QRS duration. SUMO1 overexpression enhanced membrane localization of Na V 1.5., Conclusion: SUMOylation of K442-Na v 1.5 increases peak I Na without changing I Na,L , at least in part by altering membrane abundance. Our findings do not support SUMOylation as a mechanism for changes in I Na,L. Na v 1.5 SUMOylation may modify arrhythmic risk in disease states and represents a potential target for pharmacologic manipulation., (Copyright © 2023 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

The ability of plasmons to focus light on nanometer length scales opens a wide range of enticing applications in optics and photonics, among which the enhancement of nonlinear light-matter interactions for all-optical modulation and spectral diversification emerges as a prominent theme. However, the subwavelength plasmonic near-field enhancement in good plasmonic materials such as noble metals is hindered by large ohmic losses, while conventional phase-matching of fields in bulk nonlinear crystals is not suitable for realizing nonlinear optical phenomena on the nanoscale. In contrast, anharmonic electron motion of free charge carriers in highly-doped graphene, which supports long-lived, highly-confined, and actively-tunable plasmons, renders the carbon monolayer an excellent platform for both plasmonics and nonlinear optics. Here we theoretically explore the enhancement in nonlinear response that can be achieved by interfacing multiple graphene nanostructures in close proximity to trigger nonlocal effects associated with large gradients in the electromagnetic near field. Focusing on second- and third-harmonic generation, we introduce a semianalytical formalism to describe interacting graphene nanoribbons with independent width, location, and electrical doping, so as to realize configurations in which plasmonic resonances may simultaneously enhance both the fundamental optical excitation frequency and harmonic intermediary and/or output frequencies. Our findings reveal the importance of both passive and active tuning in the design of atomically-thin nanostructures for nonlinear optical applications, and in particular emphasize the role played by nonlocal effects in generating an even-ordered nonlinear response that may contribute to other nonlinear optical processes through a cascaded interaction. We anticipate that our findings can aid in the design of actively-tunable nonlinear plasmonic resonators and metasurfaces.

The field of antisense oligonucleotide (ASO)-based therapies have been making strides in precision medicine due to their potent therapeutic application. Early successes in treating some genetic diseases are now attributed to an emerging class of antisense drugs. After two decades, the US Food and Drug Administration (FDA) has approved a considerable number of ASO drugs, primarily to treat rare diseases with optimal therapeutic outcomes. However, safety is one of the biggest challenges to the therapeutic utility of ASO drugs. Due to patients' and health care practitioners' urgent demands for medicines for untreatable conditions, many ASO drugs have been approved. However, a complete understanding of the mechanisms of adverse drug reactions (ADRs) and toxicities of ASOs still need to be resolved. The range of ADRs is unique to a specific drug, while few ADRs are common to a section of drugs as a whole. Nephrotoxicity is an important concern that needs to be addressed considering the clinical translation of any drug candidates ranging from small molecules to ASO-based drugs. This article encompasses what is known about the nephrotoxicity of ASO drugs, the potential mechanisms of action(s), and recommendations for future investigations on the safety of ASO drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Competing Interests: Declaration of interests No interests are declared.

Due to the lack of treatment options for the genetic disease primary hyperoxaluria (PH), including three subtypes PH1, PH2, and PH3, caused by accumulation of oxalate forming kidney stones, there is an urgent need for the development of a drug therapy aside from siRNA drug lumasiran for patients with PH1. After the recent success of drug therapies based on small interfering RNA (siRNA), nedosiran is currently being developed for the treatment of three types of PH as a siRNA-based modality. Through specific inhibition of lactate dehydrogenase enzyme, the key enzyme in biosynthesis of oxalate in liver, phase 1, 2, and 3 clinical trials of nedosiran have achieved the desired primary end point of reduction of urinary oxalate levels in patients with PH1. More PH2 and PH3 patients need to be tested for efficacy. It has also produced a favorable secondary end point on safety and toxicity in PH patients. In addition to common injection site reactions that resolved spontaneously, no severe nedosiran treatment-associated adverse events were reported. Based on the positive results in the clinical studies, nedosiran is a candidate siRNA drug to treat PH patients., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

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., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

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., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Importance: Although survival for in-hospital cardiac arrest (IHCA) has improved substantially over the last 2 decades, survival rates have plateaued in recent years. A better understanding of hospital differences in IHCA incidence may provide important insights regarding best practices for prevention of IHCA., Objective: To determine the incidence of IHCA among Medicare beneficiaries, and evaluate hospital variation in incidence of IHCA., Design, Setting, and Participants: This observational cohort study analyzes 2014 to 2017 data from 170 hospitals participating in the Get With The Guidelines-Resuscitation registry, linked to Medicare files. Participants were adults aged 65 years and older. Statistical analysis was performed from January to December 2021., Exposures: Case-mix index, teaching status, and nurse-staffing., Main Outcomes and Measures: Hospital incidence of IHCA among Medicare beneficiaries was estimated as the number of IHCA patients divided by the total number of hospital admissions. Multivariable hierarchical regression models were used to calculate hospital incidence rates adjusted for differences in patient case-mix and evaluate the association of hospital variables with IHCA incidence., Results: Among a total of 4.5 million admissions at 170 hospitals, 38 630 patients experienced an IHCA during 2014 to 2017. Among the 38 630 patients with IHCAs, 7571 (19.6%) were non-Hispanic Black, 26 715 (69.2%) were non-Hispanic White, and 16 732 (43.3%) were female; the mean (SD) age at admission was 76.3 (7.8) years. The median risk-adjusted IHCA incidence was 8.5 per 1000 admissions (95% CI, 8.2-9.0 per 1000 admissions). After adjusting for differences in case-mix index, IHCA incidence varied markedly across hospitals ranging from 2.4 per 1000 admissions to 25.5 per 1000 admissions (IQR, 6.6-11.4; median odds ratio, 1.51 [95% CI, 1.44-1.58]). Among hospital variables, a higher case-mix index, higher nurse staffing, and teaching status were associated with a lower hospital incidence of IHCA., Conclusions and Relevance: This cohort study found that the incidence of IHCA varies markedly across hospitals, and hospitals with higher nurse staffing and teaching status had lower IHCA incidence rates. Future studies are needed to better understand processes of care at hospitals with exceptionally low IHCA incidence to identify best practices for cardiac arrest prevention.

Epithelial to mesenchymal transition (EMT) is a cellular process in which cells composing epithelial tissue lose requirements for physical contact with neighboring cells and acquire mesenchymal characteristics consisting of increased migratory and invasive behaviors. EMT is a fundamental process that is required for initial and later events during embryogenesis. Cancer stem cells (CSCs) possess multipotency sufficient for their differentiation into bulk tumor cells and also have the capacity to undergo EMT. When CSCs initiate EMT programs the resulting cancerous mesenchymal cells become invasive and this migratory behavior also poises them for metastatic activity. Long noncoding RNAs (lncRNAs) are functional RNA molecules that do not encode proteins, yet regulate the expression of protein-coding genes through recruitment or sequestration of gene-regulatory proteins and microRNAs. lncRNA exhibit tissue-specific patterns of gene expression during development and specific sets of lncRNAs are also involved in various cancer types. This review considers the interplay between lncRNAs and the biogenesis of CSCs. We also review function of lncRNAs in EMT in CSCs. In addition, we discuss the utility of lncRNAs as biomarkers of cancer progression, and their potential use as therapeutic targets for treatment of cancer., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

More than two decades after the natural gene-silencing mechanism of RNA interference was elucidated, small interfering RNA (siRNA)-based therapeutics have finally broken into the pharmaceutical market. With three agents already approved and many others in advanced stages of the drug development pipeline, siRNA drugs are on their way to becoming a standard modality of pharmacotherapy. The majority of late-stage candidates are indicated for rare or orphan diseases, whose patients have an urgent need for novel and effective therapies. Additionally, there are agents that have the potential to meet the need of a broader population. Inclisiran, for instance, is being developed for hypercholesterolemia and has shown benefit in patients who are uncontrolled even after maximal statin therapy. This review provides a brief overview of mechanisms of siRNA action, physiological barriers to its delivery and activity, and the most common chemical modifications and delivery platforms used to overcome these barriers. Furthermore, this review presents comprehensive profiles of the three approved siRNA drugs (patisiran, givosiran, and lumasiran) and the seven other siRNA candidates in Phase 3 clinical trials (vutrisiran, nedosiran, inclisiran, fitusiran, teprasiran, cosdosiran, and tivanisiran), summarizing their modifications and delivery strategies, disease-specific mechanisms of action, updated clinical trial status, and future outlooks., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: The final stage of liver development is the production of hepatocytes and cholangiocytes (biliary epithelial cells) from bipotent hepatic progenitor cells. We used HepaRG cells, which are bipotent and able to differentiate into both hepatocytes and cholangiocytes, as a model to study the action of a novel lncRNA (lnc-RHL) and its role in the regulation of bipotency leading to hepatocytes and cholangiocytes., Materials and Methods: Differentiation of HepaRG cells was assessed by marker expression and morphology which revealed their ability to differentiate into hepatocytes and cholangiocytes (modelling the behaviour of hepatoblasts in vivo). Using a qRT-PCR and RACE, we cloned a novel lncRNA (lnc-RHL; regulator of hepatic lineages) that is upregulated upon HepaRG differentiation. Using inducible knockdown of lnc-RHL concurrently with differentiation, we show that lnc-RHL is required for proper HepaRG cell differentiation resulting in diminution of the hepatocyte lineage., Results: Here, we report the discovery of lnc-RHL, a spliced and polyadenylated 670 base lncRNA expressed from the 11q23.3 apolipoprotein gene cluster. lnc-RHL expression is confined to hepatic lineages and is upregulated when bipotent HepaRG cells are caused to differentiate. HepaRG cells made deficient for lnc-RHL have reduced ability to differentiate into hepatocytes, but retain their ability to differentiate into cholangiocytes., Conclusions: Deficiency for lnc-RHL in HepaRG cells converts them from bipotent progenitor cells to unipotent progenitor cells with impaired ability to yield hepatocytes. We conclude that lnc-RHL is a key regulator of bipotency in HepaRG cells., (© 2021 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

SETDB1 is a histone methyltransferase that converts H3K9me2 to H3K9me3. SETDB1 activity and H3K9me3 are crucial for the formation of obligately silenced heterochromatin such as that of centromeres. Here we show that a microRNA, miR-152-3p, is involved in the regulation of SETDB1 protein levels, but surprisingly, miR-152-3p plays a positive regulatory role for SETDB1 expression. Inhibition of miR-152-3p by anti-miR treatment resulted in a robust reduction in SETDB1 protein levels, though SETDB1 mRNA levels were unaffected. This was also accompanied by a blockade of the biochemical pathway proceeding from H3K9me2 to H3K9me3 as evidenced by quantitative nucleosome ELISA assays that showed that H3K9me2 accumulates in cells treated with an anti-miR that targets miR-152-3p. In addition, the action of a miR-152-3p mimic increased flux of the reaction leading to H3K9me3. We also performed site-directed mutagenesis of three predicted miR-152-3p target recognition sequences to yield three precise deletions. Deletion of one of the three sites recapitulated the positive regulatory aspect of the action of miR-152-3p upon SETDB1 expression in a luciferase reporter assay. Previous studies have shown that miR-152-3p negatively regulates DNMT1, the sole maintenance DNA methyltransferase which is required for levels of 5-methylcytosine levels within DNA. Our results shown that miR-152-3p positively regulates the production of H3K9me3 by regulating the production of SETDB1. Therefore, our findings provide strong evidence that miR-152-3p can serve as a toggle switch that regulates the balance between DNA methylation and H3K9 histone methylation in constitutive heterochromatin., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The novel SARS-CoV-2 virus has quickly spread worldwide, bringing the whole world as well as the economy to a standstill. As the world is struggling to minimize the transmission of this devastating disease, several strategies are being actively deployed to develop therapeutic interventions. Pharmaceutical companies and academic researchers are relentlessly working to investigate experimental, repurposed or FDA-approved drugs on a compassionate basis and novel biologics for SARS-CoV-2 prophylaxis and treatment. Presently, a tremendous surge of COVID-19 clinical trials are advancing through different stages. Among currently registered clinical efforts, ~86% are centered on testing small molecules or antibodies either alone or in combination with immunomodulators. The rest ~14% of clinical efforts are aimed at evaluating vaccines and convalescent plasma-based therapies to mitigate the disease's symptoms. This review provides a comprehensive overview of current therapeutic modalities being evaluated against SARS-CoV-2 virus in clinical trials.

Purpose of Review: Cardiac arrest is a common condition associated with high mortality and a substantial risk of neurological injury among survivors. Targeted temperature management (TTM) is the only strategy shown to reduce the risk of neurologic disability cardiac arrest patients. In this article, we provide a comprehensive review of TTM with an emphasis on recent trials., Recent Findings: After early studies demonstrating the benefit of TTM in out-of-hospital cardiac arrest due to a shockable rhythm, newer studies have extended the benefit of TTM to patients with a nonshockable rhythm and in-hospital cardiac arrest. A target temperature of 33 °C was not superior to 36 °C, suggesting that a lenient targeted temperature may be appropriate especially for patients unable to tolerate lower temperatures. Although early initiation of TTM appears to be beneficial, the benefit of prehospital cooling has not been shown and use of intravenous cold saline in the prehospital setting may be harmful., Summary: There is substantial risk of neurological injury in cardiac arrest survivors who remain comatose. TTM is an effective treatment that can lower the risk of neurological disability in such patients and ideally delivered as part of a comprehensive, goal-directed post-resuscitation management by a multidisciplinary team in a tertiary medical center., Competing Interests: Conflict of InterestT. P. Rasmussen declares that he has no conflict of interest. T.C. Bullis declares that he has no conflict of interest . S. Girotra declares that he has no conflict of interest., (© Springer Science+Business Media, LLC, part of Springer Nature 2020.)

The cumulative incidence of systolic heart failure is similar in men and women. However, major prognostic differences exist between genders. We sought to measure gender differences in furosemide prescribing patterns for patients with preexisting heart failure with reduced ejection fraction (HFrEF) admitted with Stage C acute decompensation, regardless of the underlying cause. We conducted a single-center retrospective analysis of patients admitted between 2015 and 2018 for acute on chronic decompensated HFrEF. Primary outcomes were differences in initial furosemide dose, total dose over the first 24 hours of hospitalization, and total dose during the entire hospitalization between women and men. Secondary outcomes included acute kidney injury (AKI), intubation, noninvasive ventilation (NIV), and in-hospital 30-day and 1-year mortality. We studied 434 patients (31% female) with similar baseline characteristics. Females received significantly less furosemide compared to men for the initial dose, over the first 24 hours, and throughout their hospitalization. However, AKI was more prevalent in women versus men ( p =0.008). Females admitted for acute on chronic decompensated HFrEF receive significantly less furosemide when compared to men, but developed more AKI prior to discharge., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Tyler P. Rasmussen et al.)

Cellular identity is established and maintained by the interplay of cell type-specific transcription factors and epigenetic regulation of the genome. During development in vivo and differentiation in vitro, transitions from one cell type to the next are triggered by cell signaling events culminating in modifications of chromatin that render genes accessible or inaccessible to the transcriptional apparatus. In recent years it has become apparent that cellular identity is plastic, and technological reprogramming methods such as somatic cell nuclear transfer and induced pluripotency can yield reprogrammed cells that have been restored to a state of developmental potency. Long noncoding RNAs (lncRNAs) are untranslated functional RNA molecules that are intimately involved in the regulation of the chromatin of protein-coding genes. In fact, recent evidence shows that there are more lncRNA species in the cell than mRNA species and that most protein-coding genes are likely to be under epigenetic regulation mediated by lncRNAs. This review examines lncRNA function in reprogrammed pluripotent cells and cancer stem cells. Because cancer stem cells arise from normal cells, their biogenesis can be viewed as a reprogramming process that occurs in vivo, and parallels between artificial reprogramming and cancer stem cell biogenesis are discussed., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Precision medicine is a rapidly-developing modality of medicine in human healthcare. Based on each patient׳s unique characteristics, more accurate dosages and drug selection can be made to achieve better therapeutic efficacy and less adverse reactions in precision medicine. A patient׳s individual parameters that affect drug transporter action can be used to develop a precision medicine guidance, due to the fact that therapeutic efficacy and adverse reactions of drugs can both be affected by expression and function of drug transporters on the cell membrane surface. The purpose of this review is to summarize unique characteristics of human breast cancer resistant protein (BCRP) and the genetic variability in the BCRP encoded gene ABCG2 in the development of precision medicine. Inter-individual variability of BCRP/ ABCG2 can impact choices and outcomes of drug treatment for several diseases, including cancer chemotherapy. Several factors have been implicated in expression and function of BCRP, including genetic, epigenetic, physiologic, pathologic, and environmental factors. Understanding the roles of these factors in controlling expression and function of BCRP is critical for the development of precision medicine based on BCRP-mediated drug transport.

The role of the mitochondrial Ca 2+ uniporter (MCU) in physiologic cell proliferation remains to be defined. Here, we demonstrated that the MCU was required to match mitochondrial function to metabolic demands during the cell cycle. During the G 1 -S transition (the cycle phase with the highest mitochondrial ATP output), mitochondrial fusion, oxygen consumption, and Ca 2+ uptake increased in wild-type cells but not in cells lacking MCU. In proliferating wild-type control cells, the addition of the growth factors promoted the activation of the Ca 2+ /calmodulin-dependent kinase II (CaMKII) and the phosphorylation of the mitochondrial fission factor Drp1 at Ser 616 The lack of the MCU was associated with baseline activation of CaMKII, mitochondrial fragmentation due to increased Drp1 phosphorylation, and impaired mitochondrial respiration and glycolysis. The mitochondrial fission/fusion ratio and proliferation in MCU-deficient cells recovered after MCU restoration or inhibition of mitochondrial fragmentation or of CaMKII in the cytosol. Our data highlight a key function for the MCU in mitochondrial adaptation to the metabolic demands during cell cycle progression. Cytosolic CaMKII and the MCU participate in a regulatory circuit, whereby mitochondrial Ca 2+ uptake affects cell proliferation through Drp1., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Among individuals diagnosed with epilepsy, as many as one in three develop resistance to antiepileptic drugs (AEDs) thus rendering their seizures refractory to treatment. Despite current antiepileptic drugs (AEDs) having a variety of modes of action, seizures in drug-resistant individuals often persist even after treatment with two or more drugs. The underlying cause of this broad resistance is currently under debate, but two dominant theories have emerged and have been widely studied. Here we discuss current literature investigating the "transporter theory", the idea that individuals present with drug resistance due to genetic variability in the ABCB1 gene encoding the efflux transporter multidrug resistance protein 1 (MDR1). Results of in vitro and in vivo studies suggest that variability in the expression of the MDR1 transporter may be closely tied to drug resistance. While there is much support for this hypothesis from molecular and mechanistic studies, population-based studies of ABCB1 polymorphisms are divergent in their conclusions, and there is need for additional investigations., Competing Interests: The authors declare no competing financial interests.

Purpose of This Review: In order to combat the development of drug resistance, the clinical treatment of tuberculosis requires the combined use of several anti-tuberculosis (anti-TB) drugs, including isoniazid and rifampicin. Combinational treatment approaches are suggested by the World Health Organization (WHO) and are widely accepted throughout the world. Unfortunately, a major side effect of the treatment is the development of anti-tuberculosis drug-induced liver injury (AT-DILI). Many factors contribute to isoniazid- and rifampicin-mediated AT-DILI and genetic variations are among the most common factors. The purpose of this review is to provide information on genetic variations associated with isoniazid- and rifampicin-mediated AT-DILI., Recent Findings: The genetic variations associated with AT-DILI have been identified in the genomic regions within or near genes encoding proteins in the following pathways: drug metabolizing enzymes ( NAT2, CYP2E1, and GSTs ), accumulation of bile acids, lipids, and heme metabolites ( CYP7A1, BSEP, UGTs, and PXR ), immune adaptation ( HLAs and TNF-α ), and oxidant challenge ( TXNRD1, SOD1, BACH1, and MAFK )., Summary: The information summarized in this review considers the genetic bases of risk factors contributing to AT-DILI and provides information that may help for future studies. Some of the implicated genetic variations can be used in the design of genetic tests and serve as biomarkers for the prediction of isoniazid- and rifampicin-mediated AT-DILI risk in personalized medicine., Competing Interests: Conflict of Interest: None of the authors has a conflict of interest to declare in relation to this work.

Human birth defects are relatively common and can be caused by exposure to environmental teratogens or to pharmaceuticals with teratogenic activities. Human embryonic stem cells (hESCs), by virtue of their pluripotent nature, provide an excellent cellular platform for teratogen detection and risk assessment. This unit describes detailed protocols for the preparation and validation of highly pluripotent hESCs, the production of large quantities of aggregated multicellular spheroids composed of hESCs, and these spheroids' differentiation into embryoid bodies (EBs). EBs contain a variety of cells of endodermal, ectodermal, and mesodermal origin and can be subjected to compound exposure in vitro. Hence, they are useful for the detection of chemicals with teratogenic activities. Beyond describing protocols to assemble and culture EBs, this unit details methods to exploit the EB system for teratological assessment. In addition, strategies to distinguish compounds with bona fide teratogenic activity versus simple toxicity are discussed. © 2018 by John Wiley & Sons, Inc., (Copyright © 2018 John Wiley & Sons, Inc.)

Long non-coding RNAs (lncRNAs) constitute the largest class of non-coding transcripts in the human genome. Results from next-generation sequencing and bioinformatics advances indicate that the human genome contains more non-coding RNA genes than protein-coding genes. Validated functions of lncRNAs suggest that they are master regulators of gene expression and often exert their influences via epigenetic mechanisms by modulating chromatin structure. Specific lncRNAs can regulate transcription in gene clusters. Since the functions of protein-coding genes in clusters are often tied to specific pathways, lncRNAs constitute attractive pharmacological targets. Here we review the current knowledge of lncRNA functions in human cells and their roles in disease processes. We also present forward-looking perspectives on how they might be manipulated pharmacologically for the treatment of a variety of human diseases, in which regulation of gene expression by epigenetic mechanisms plays a major role.

Teratogens are compounds that can induce birth defects upon exposure of the developing fetus. To date, most teratogen studies utilize pregnant rodents to determine compound teratogenicity in vivo. However, this is a low throughput approach that cannot easily meet the need for comprehensive high-volume teratogen assessment, a goal of the US Environmental Protection Agency. In addition, rodent and human development differ substantially, and therefore the use of assays using relevant human cells has utility. For these reasons, interest has recently focused on the use of human embryonic stem cells for teratogen assessment. Here we present a highly standardized and quantitative system for the detection and analysis of teratogens that utilizes well-characterized and purified highly pluripotent stem cells. We have devised strategies to mass-produce thousands of uniformly sized spheroids of human ESCs (hESCs) that can be caused to undergo synchronous differentiation to yield embryoid bodies (EBs) in the presence and absence of suspected teratogens. The system uses all human cells and rigorously controlled and standardized EB culture conditions. Furthermore, the approach has been made quantitative by using high-content imaging approaches. Our system offers distinct advantages over earlier EB systems that rely heavily on the use on mouse ESCs and EB aggregates of stochastic sizes. Together, our results show that thousands of suspected teratogens could be assessed using human EB-based approaches., Competing Interests: The authors have declared that no competing interests exist.

The primary purpose of the study was to evaluate whether a pericardiectomy (PERI) alters training- or myocardial infarction (MI)-induced left ventricular hypertrophy (LVH), chamber geometry, gene expression and/or running performance. Mice were randomized into 6 groups: naïve control (CONT)-sedentary (Sed), CONT-trained (Tr), PERI-Sed, PERI-Tr, MI-Sed and MI-Tr. MI mice also received a pericardiectomy as part of the MI surgical procedure. 10 weeks of treadmill running resulted in enhanced running performance-to-exhaustion in all 3 trained groups (CONT-Tr, PERI-Tr, MI-Tr) compared to sedentary cohorts ( P <0.001). Training also resulted in similar increases in normalized LVH (LV/BW) in CONT-Tr and PERI-Tr mice. 2D-echocardiographic evaluation of LV internal chamber dimensions revealed that stroke diameter (SD) was larger in PERI compared to MI ( P <0.01) but not CONT mice. Ventricular B-type natriuretic peptide mRNA (BNP) was elevated only in the 2 MI groups. Left ventricle β 1 -adrenergic receptor (β 1 -AR) and melusin transcripts both demonstrated an overall increase in trained compared to sedentary mice (both P <0.05). Additionally long-term pericardiectomy did not further enhance running performance or increase LV/BW in either sedentary or trained mice., (© Georg Thieme Verlag KG Stuttgart · New York.)

In this article we outline how ultra-compact, yet high performance spectrometers can be designed and built with highly dispersive transmission gratings. By using fused silica as the grating material, and by careful design of the detailed grating structure, we demonstrate an ultraviolet spectrometer with a high and nearly flat efficiency from 178 to 409 nm, a resolution of 0.2 nm, and dimensions of only 61 mm × 64 mm × 19 mm. We tested this spectrometer in a laser-induced breakdown spectroscopy experiment and showed that the spectral information gathered with the spectrometer can be used to obtain quantitative results for sulfur., (© The Author(s) 2016.)

Myocardial mitochondrial Ca(2+) entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca(2+) are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca(2+) uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca(2+) entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU-permeabilized myocardial fibers or isolated mitochondria in low Ca(2+) were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca(2+) homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca(2+)] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca(2+) homeostasis. Mitochondrial Ca(2+) overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca(2+) homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.

Heart rate increases are a fundamental adaptation to physiological stress, while inappropriate heart rate increases are resistant to current therapies. However, the metabolic mechanisms driving heart rate acceleration in cardiac pacemaker cells remain incompletely understood. The mitochondrial calcium uniporter (MCU) facilitates calcium entry into the mitochondrial matrix to stimulate metabolism. We developed mice with myocardial MCU inhibition by transgenic expression of a dominant-negative (DN) MCU. Here, we show that DN-MCU mice had normal resting heart rates but were incapable of physiological fight or flight heart rate acceleration. We found that MCU function was essential for rapidly increasing mitochondrial calcium in pacemaker cells and that MCU-enhanced oxidative phoshorylation was required to accelerate reloading of an intracellular calcium compartment before each heartbeat. Our findings show that MCU is necessary for complete physiological heart rate acceleration and suggest that MCU inhibition could reduce inappropriate heart rate increases without affecting resting heart rate.

Three-dimensional chromosomal conformations regulate transcription by moving enhancers and regulatory elements into spatial proximity with target genes. Here we describe activity-regulated long-range loopings bypassing up to 0.5 Mb of linear genome to modulate NMDA glutamate receptor GRIN2B expression in human and mouse prefrontal cortex. Distal intronic and 3' intergenic loop formations competed with repressor elements to access promoter-proximal sequences, and facilitated expression via a "cargo" of AP-1 and NRF-1 transcription factors and TALE-based transcriptional activators. Neuronal deletion or overexpression of Kmt2a/Mll1 H3K4- and Kmt1e/Setdb1 H3K9-methyltransferase was associated with higher-order chromatin changes at distal regulatory Grin2b sequences and impairments in working memory. Genetic polymorphisms and isogenic deletions of loop-bound sequences conferred liability for cognitive performance and decreased GRIN2B expression. Dynamic regulation of chromosomal conformations emerges as a novel layer for transcriptional mechanisms impacting neuronal signaling and cognition., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The host-dependent nature of idiosyncratic drug-induced liver injury (iDILI) suggests that rare genetic polymorphisms may contribute to the disease. Indeed, a few mutations in key genes have already been identified using conventional human genetics approaches. Over 50 commonly used drugs can precipitate iDILI, making this a substantial medical problem. Only recently have human induced pluripotent stem cells been used as a research tool to discover novel iDILI genes and to study the mechanisms of iDILI in vitro. Here we review the current state of stem cell use in the investigation of iDILI, with a special focus on genetics. In addition, the concerns and difficulties associated with genetics and animal model research are discussed. We then present the features of patient-specific pluripotent stem cells (which may be derived from iDILI patients themselves), and explain why these cells may be of great utility. A variety of recent approaches to produce hepatocyte-like cells from pluripotent cells and the associated advantages and limitations of such cells are discussed. Future directions for the use of stem cell science to investigate iDILI include novel ways to identify new iDILI genes, a consideration of epigenetic impacts on iDILI, and the development of new and improved strategies for the production of hepatocytes from human pluripotent cells.

Approximately 200 cell types and multiple tissues are established throughout the development of the zygote to an adult mammal. During this process, the cellular genome remains fixed, yet the transcriptome of each of the cell types become widely divergent. This review discusses the epigenetics of preimplantation embryos and the use of embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) as cell-culture models for the inner cell mass (ICM) and epiblast, respectively. Differential patterns of transcription are set up during development by the action of key transcription factors and epigenetics, which are involved in the establishment and maintenance of stable transcriptional states during development. In early embryos, for example, changes in the epigenome consist of alterations to the methylation of CpG dinucleotides and post-translational modification of histones within chromatin. In addition, histone replacement occurs broadly in zygotes. The ICM of the blastocyst, on the other hand, has the amazing ability to contribute to every tissue and cell type present in the adult body. Therefore, ESCs are arguably the most important cell-culture model available to developmental biologists. The advantages and risks of using ESCs to model ICM pluripotency are therefore discussed., (© 2013 Wiley Periodicals, Inc.)

Hepatocytes play a central and crucial role in cholesterol and lipid homeostasis, and their proper function is of key importance for cardiovascular health. In particular, hepatocytes (especially periportal hepatocytes) endogenously synthesize large amounts of cholesterol and secrete it into circulating blood via apolipoprotein particles. Cholesterol-secreting hepatocytes are also the clinically-relevant cells targeted by statin treatment in vivo. The study of cholesterol homeostasis is largely restricted to the use of animal models and immortalized cell lines that do not recapitulate those key aspects of normal human hepatocyte function that result from genetic variation of individuals within a population. Hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells can provide a cell culture model for the study of cholesterol homeostasis, dyslipidemias, the action of statins and other pharmaceuticals important for cardiovascular health. We have analyzed expression of core components for cholesterol homeostasis in untreated human iPS cells and in response to pravastatin. Here we show the production of differentiated cells resembling periportal hepatocytes from human pluripotent stem cells. These cells express a broad range of apolipoproteins required for secretion and elimination of serum cholesterol, actively secrete cholesterol into the medium, and respond functionally to statin treatment by reduced cholesterol secretion. Our research shows that HLCs derived from human pluripotent cells provide a robust cell culture system for the investigation of the hepatic contribution to human cholesterol homeostasis at both cellular and molecular levels. Importantly, it permits for the first time to also functionally assess the impact of genetic polymorphisms on cholesterol homeostasis. Finally, the system will also be useful for mechanistic studies of heritable dyslipidemias, drug discovery, and investigation of modes of action of cholesterol-modulatory drugs.

Analyses of mature adipocytes have shown that they possess a reprogramming ability in vitro, which is associated with dedifferentiation. The subsequent dedifferentiated fat cells (DFAT cells) are multipotent and can differentiate into adipocytes and other cell types as well. Mature adipocytes can be easily obtained by biopsy, and the cloned progeny cells are homogeneous in vitro. Therefore, DFAT cells (a new type of stem cell) may provide an excellent source of cells for tissue regeneration, engineering and disease treatment. The dedifferentiation of mature adipocytes, the multipotent capacity of DFAT cells and comparisons and contrasts with mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPS) are discussed in this review.

Multiple methods exist that can reprogram differentiated cells to a pluripotent state similar to that of embryonic stem cells (ESCs). These include somatic cell nuclear transfer (SCNT), fusion-mediated reprogramming (FMR) of somatic cells with ESCs, and the production of induced pluripotent stem cells (iPSCs). All of these methods yield cells in which the endogenous Oct4 gene is reactivated. We were interested in comparing the activity of the Oct4 promoter in three different classes of pluripotent cells, including normal ESCs, FMR cells (FMRCs), and iPSCs. We prepared cells of all three types that harbor a transgene composed of the mouse Oct4 promoter driving green fluorescent protein (Oct4-GFP). All cell derivations started with a characterized transgenic Oct4-GFP mouse, and from this we derived ESCs, FMRCs, and iPSCs with the Oct4-GFP transgene present in an identical genomic integration site in all three cell types. Using flow cytometry we assessed Oct4 promoter expression, cell cycle behavior, and differentiation kinetics. We found similar levels of GFP expression in all three cell types and no significant alterations in pluripotency or differentiation. Our results suggest that the pluripotent condition is a potent "local attractor" state, because it can be achieved through three vastly different avenues.

Rationale: The sodium-calcium exchanger 1 (NCX1) is predominantly expressed in the heart and is implicated in controlling automaticity in isolated sinoatrial node (SAN) pacemaker cells, but the potential role of NCX1 in determining heart rate in vivo is unknown., Objective: To determine the role of Ncx1 in heart rate., Methods and Results: We used global myocardial and SAN-targeted conditional Ncx1 knockout (Ncx1(-/-)) mice to measure the effect of the NCX current on pacemaking activity in vivo, ex vivo, and in isolated SAN cells. We induced conditional Ncx1(-/-) using a Cre/loxP system. Unexpectedly, in vivo and ex vivo hearts and isolated SAN cells showed that basal rates in Ncx1(-/-) (retaining ≈20% of control level NCX current) and control mice were similar, suggesting that physiological NCX1 expression is not required for determining resting heart rate. However, increases in heart rate and SAN cell automaticity in response to isoproterenol or the dihydropyridine Ca(2+) channel agonist BayK8644 were significantly blunted or eliminated in Ncx1(-/-) mice, indicating that NCX1 is important for fight or flight heart rate responses. In contrast, the pacemaker current and L-type Ca(2+) currents were equivalent in control and Ncx1(-/-) SAN cells under resting and isoproterenol-stimulated conditions. Ivabradine, a pacemaker current antagonist with clinical efficacy, reduced basal SAN cell automaticity similarly in control and Ncx1(-/-) mice. However, ivabradine decreased automaticity in SAN cells isolated from Ncx1(-/-) mice more effectively than in control SAN cells after isoproterenol, suggesting that the importance of NCX current in fight or flight rate increases is enhanced after pacemaker current inhibition., Conclusions: Physiological Ncx1 expression is required for increasing sinus rates in vivo, ex vivo, and in isolated SAN cells, but not for maintaining resting heart rate.

Histone posttranslational modifications (PTMs) are highly important molecular determinants of epigenetic regulatory mechanisms. Histone PTMs associated with nucleosomes are intimately tied to the transcriptional activity or silence of genes. In addition, nucleosomal PTMs participate in the organization of chromatin into higher-order structures and the progression through mitosis. Changes in histone PTMs are also regulated during the course of mammalian development and are altered in pathological states including cancer. Histone acetyl modifications (and also methylation and phosphorylation) are frequently assayed by western blotting (WB), mass spectrometry (MS), and chromatin immunoprecipitation (ChIP). Here we show that an enzyme-linked immunosorbent assay performed on nucleosomes (NU-ELISA) can quickly and effectively yield quantitative detection of global levels of histone acetylation on small samples such as single human embryonic stem cell colonies. The microscale NU-ELISA method presented here can be performed in most laboratories equipped with basic instrumentation for molecular and cellular biology.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H4. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Macrohistones (mH2As) are unusual histone variants found exclusively in vertebrate chromatin. In mice, the H2afy gene encodes two splice variants, mH2A1.1 and mH2A1.2 and a second gene, H2afy2, encodes an additional mH2A2 protein. Both mH2A isoforms have been found enriched on the inactive X chromosome (Xi) in differentiated mammalian female cells, and are incorporated into the chromatin of developmentally-regulated genes. To investigate the functional significance of mH2A isoforms for X chromosome inactivation (XCI), we produced male and female embryonic stem cell (ESC) lines with stably-integrated shRNA constructs that simultaneously target both mH2A1 and mH2A2. Surprisingly, we find that female ESCs deficient for both mH2A1 and mH2A2 readily execute and maintain XCI upon differentiation. Furthermore, male and female mH2A-deficient ESCs proliferate normally under pluripotency culture conditions, and respond to several standard differentiation procedures efficiently. Our results show that XCI can readily proceed with substantially reduced total mH2A content.

Meat animals are unique as experimental models for both lipid metabolism and adipocyte studies because of their direct economic value for animal production. This paper discusses the principles that regulate adipogenesis in major meat animals (beef cattle, dairy cattle, and pigs), the definition of adipose depot-specific regulation of lipid metabolism or adipogenesis, and introduces the potential value of these animals as models for metabolic research including mammary biology and the ontogeny of fatty livers.

Mammals, like all multicellular organisms, develop from a single cell--the totipotent zygote. During preimplantation development and subsequent development in utero, over 200 distinct cell types are established and integrated into the organ systems and tissues of the developing organism. Much of the field of mammalian developmental biology is devoted to investigation of mechanisms that govern the formation of complete organs and tissues. In contrast to later development, which consumes the vast majority of time associated with development in utero, preimplantation development and germ layer specification occur rapidly. Yet knowledge is limited regarding the regulatory mechanisms that specify the transient, but pluripotent, cellular lineages that form during the initial stages of mammalian development. Gametogenesis and preimplantation development are marked by dramatic and pervasive epigenetic changes rooted in chromatin dynamics. The fundamental mechanisms that specify subsequent cellular lineages of the conceptus are only now becoming understood, and tend to rely relatively heavily upon broad epigenetic mechanisms in addition to master transcription factors. This review considers epigenetic regulation in the very earliest stages of preimplantation development. In addition, recent advances which indicate that some epigenetic coding is imposed during gametogenesis and maintained during preimplantation development are considered., ((c) 2010 Wiley-Liss, Inc.)

Skeletal muscle stem cells from food-producing animals are of interest to agricultural life scientists seeking to develop a better understanding of the molecular regulation of lean tissue (skeletal muscle protein hypertrophy) and intramuscular fat (marbling) development. Enhanced understanding of muscle stem cell biology and function is essential for developing technologies and strategies to augment the metabolic efficiency and muscle hypertrophy of growing animals potentially leading to greater efficiency and reduced environmental impacts of animal production, while concomitantly improving product uniformity and consumer acceptance and enjoyment of muscle foods.

Importance of the Field: The establishment and maintenance of specialized chromatin is crucial for correct gene expression and chromosome stability in mammalian cells. Therefore, epigenetic insults are frequently observed in cancer. Several chromatin modifying enzymes have been implicated in leukemia, and are attractive candidates for the development of therapeutic agents., Areas Covered in This Review: The histone methyltransferase DOT1L is responsible for methylation of histone H3 at lysine 79 and is involved in the pathobiology of several leukemias, the majority of which are characterized by chromosomal translocations involving the mixed lineage leukemia (MLL) gene. Leukemic translocations yield fusion proteins involving MLL and other proteins that physically interact with DOT1L. These oncogenic fusion proteins recruit DOT1L to ectopic loci (including HOX gene clusters), whose mis-expression contributes to the transformed phenotype. Studies from stem cells and certain leukemias suggest a second mechanism of leukemogenesis, in which reduced or mistargeted DOT1L activity yields altered centromeric chromatin and consequent chromosomal instability. Targeting DOT1L enzymatic activity as well as interactions with leukemogenic fusion proteins is discussed as possible leads in therapeutic interventions., What the Reader Will Gain: In this review, we discuss the normal functions of DOT1L, its mechanistic roles in leukemogenesis, and possible strategies for targeting DOT1L in leukemia. DOT1L is an atypical histone lysine methyltransferase in that it does not contain an enzymatic domain common to all other lysine methyltranferases. This attribute makes DOT1L a unique and specifically targetable enzyme. An emerging role for DOT1L under normal cellular conditions as well as transformed conditions is emerging and shedding light on the biology and mechanisms of some translocation-induced leukemias., Take Home Message: DOT1L is critical in development, as shown in studies in mouse embryos and embryonic stem cells. DOT1L enzymatic activity is also required for the leukemic transformation capabilities of a number of oncogenic fusion proteins. In addition, interactions between DOT1L and oncogenic fusion proteins are necessary for the transformation process. Therefore, it may be possible to specifically target DOT1L enzymatic activity or DOT1L interactions with leukemogenic fusion proteins.