Publications by authors named "Michael T Lam"

31 Publications

Increased peripheral blood neutrophil activation phenotypes and NETosis in critically ill COVID-19 patients: a case series and review of the literature.

Clin Infect Dis 2021 May 14. Epub 2021 May 14.

Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of California San Diego (UCSD), La Jolla, CA 92093, USA.

Background: Increased inflammation has been well defined in COVID-19, while definitive pathways driving severe forms of this disease remain uncertain. Neutrophils are known to contribute to immunopathology in infections, inflammatory diseases and acute respiratory distress syndrome (ARDS), a primary cause of morbidity and mortality in COVID-19. Changes in neutrophil function in COVID-19 may give insight into disease pathogenesis and identify therapeutic targets.

Methods: Blood was obtained serially from critically ill COVID-19 patients for eleven days. Neutrophil extracellular trap formation (NETosis), oxidative burst, phagocytosis and cytokine levels were assessed. Lung tissue was obtained immediately post-mortem for immunostaining. Pubmed searches for neutrophils, lung and COVID-19 yielded ten peer-reviewed research articles in English.

Results: Elevations in neutrophil-associated cytokines IL-8 and IL-6, and general inflammatory cytokines IP-10, GM-CSF, IL-1b, IL-10 and TNF, were identified both at first measurement and across hospitalization (p<0.0001). COVID neutrophils had exaggerated oxidative burst (p<0.0001), NETosis (p<0.0001) and phagocytosis (p<0.0001) relative to controls. Increased NETosis correlated with leukocytosis and neutrophilia, and neutrophils and NETs were identified within airways and alveoli in lung parenchyma of 40% of SARS-CoV-2 infected lungs available for examination (2 out of 5). While elevations in IL-8 and ANC correlated with disease severity, plasma IL-8 levels alone correlated with death.

Conclusions: Literature to date demonstrates compelling evidence of increased neutrophils in the circulation and lungs of COVID-19 patients. importantly, neutrophil quantity and activation correlates with severity of disease. Similarly, our data shows that circulating neutrophils in COVID-19 exhibit an activated phenotype with enhanced NETosis and oxidative burst.
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http://dx.doi.org/10.1093/cid/ciab437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8241438PMC
May 2021

ATS Core Curriculum 2020. Adult Sleep Medicine.

ATS Sch 2020 Dec 30;1(4):476-494. Epub 2020 Dec 30.

Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of California, Los Angeles, Los Angeles, California.

The American Thoracic Society Core Curriculum updates clinicians annually in adult and pediatric pulmonary disease, medical critical care, and sleep medicine, in a 3-4-year recurring cycle of topics. These topics will be presented at the 2020 Virtual Conference. Below is the adult sleep medicine core that includes topics pertinent to sleep-disordered breathing and insomnia.
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http://dx.doi.org/10.34197/ats-scholar.2020-0017REDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015760PMC
December 2020

Design and Implementation of NK Cell-Based Immunotherapy to Overcome the Solid Tumor Microenvironment.

Cancers (Basel) 2020 Dec 21;12(12). Epub 2020 Dec 21.

Department of Immunology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.

Natural killer (NK) cells are innate immune effectors capable of broad cytotoxicity via germline-encoded receptors and can have conferred cytotoxic potential via the addition of chimeric antigen receptors. Combined with their reduced risk of graft-versus-host disease (GvHD) and cytokine release syndrome (CRS), NK cells are an attractive therapeutic platform. While significant progress has been made in treating hematological malignancies, challenges remain in using NK cell-based therapy to combat solid tumors due to their immunosuppressive tumor microenvironments (TMEs). The development of novel strategies enabling NK cells to resist the deleterious effects of the TME is critical to their therapeutic success against solid tumors. In this review, we discuss strategies that apply various genetic and non-genetic engineering approaches to enhance receptor-mediated NK cell cytotoxicity, improve NK cell resistance to TME effects, and enhance persistence in the TME. The successful design and application of these strategies will ultimately lead to more efficacious NK cell therapies to treat patients with solid tumors. This review outlines the mechanisms by which TME components suppress the anti-tumor activity of endogenous and adoptively transferred NK cells while also describing various approaches whose implementation in NK cells may lead to a more robust therapeutic platform against solid tumors.
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http://dx.doi.org/10.3390/cancers12123871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767468PMC
December 2020

Display of Self-Peptide on Adeno-Associated Virus Capsid Decreases Phagocytic Uptake .

ACS Synth Biol 2020 09 31;9(9):2246-2251. Epub 2020 Aug 31.

Department of Bioengineering, Rice University, Houston, Texas 77005, United States.

Adeno-associated virus (AAV) vectors are currently investigated as gene transfer agents for the treatment of a variety of diseases. However, activation of the host immune response upon vector administration limits the use of AAV in the clinical setting. To decrease host detection of AAVs, we tested the CD47-based "don't-eat-me" signal in the context of the AAV capsid. We genetically incorporated the bioactive region of CD47, named "self-peptide" (SP), onto the surface of the AAV2 capsid. AAV mutants were structurally and functionally characterized for vector production, SP and linker incorporation into the capsid, transduction efficiency, and phagocytic susceptibility. We demonstrate that utilizing linkers improves the AAV2 capsid's tolerance to SP insertion. Notably, the SP significantly decreases the phagocytic susceptibility of AAV2 Collectively, these results suggest that display of the SP motif on the AAV capsid surface can inhibit phagocytosis of the vector the "don't-eat-me" signaling.
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http://dx.doi.org/10.1021/acssynbio.0c00203DOI Listing
September 2020

OSA as a probable risk factor for severe COVID-19.

J Clin Sleep Med 2020 09;16(9):1649

San Diego School of Medicine, University of California, San Diego, California.

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http://dx.doi.org/10.5664/jcsm.8708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7970598PMC
September 2020

A research-driven approach to the identification of novel natural killer cell deficiencies affecting cytotoxic function.

Blood 2020 02;135(9):629-637

Department of Pediatrics, Columbia University Medical Center, New York, NY; and.

Natural killer cell deficiencies (NKDs) are an emerging phenotypic subtype of primary immune deficiency. NK cells provide a defense against virally infected cells using a variety of cytotoxic mechanisms, and patients who have defective NK cell development or function can present with atypical, recurrent, or severe herpesviral infections. The current pipeline for investigating NKDs involves the acquisition and clinical assessment of patients with a suspected NKD followed by subsequent in silico, in vitro, and in vivo laboratory research. Evaluation involves initially quantifying NK cells and measuring NK cell cytotoxicity and expression of certain NK cell receptors involved in NK cell development and function. Subsequent studies using genomic methods to identify the potential causative variant are conducted along with variant impact testing to make genotype-phenotype connections. Identification of novel genes contributing to the NKD phenotype can also be facilitated by applying the expanding knowledge of NK cell biology. In this review, we discuss how NKDs that affect NK cell cytotoxicity can be approached in the clinic and laboratory for the discovery of novel gene variants.
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http://dx.doi.org/10.1182/blood.2019000925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046607PMC
February 2020

A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.

J Exp Med 2019 12 10;216(12):2778-2799. Epub 2019 Oct 10.

Baylor-Hopkins Center for Mendelian Genomics, Houston, TX.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.
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http://dx.doi.org/10.1084/jem.20190147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888978PMC
December 2019

Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells.

Elife 2017 06 6;6. Epub 2017 Jun 6.

Department of Cellular and Molecular Medicine, University of Arizona, Tucson, United States.

Endothelial cells (ECs) are critical determinants of vascular homeostasis and inflammation, but transcriptional mechanisms specifying their identities and functional states remain poorly understood. Here, we report a genome-wide assessment of regulatory landscapes of primary human aortic endothelial cells (HAECs) under basal and activated conditions, enabling inference of transcription factor networks that direct homeostatic and pro-inflammatory programs. We demonstrate that 43% of detected enhancers are EC-specific and contain SNPs associated to cardiovascular disease and hypertension. We provide evidence that AP1, ETS, and GATA transcription factors play key roles in HAEC transcription by co-binding enhancers associated with EC-specific genes. We further demonstrate that exposure of HAECs to oxidized phospholipids or pro-inflammatory cytokines results in signal-specific alterations in enhancer landscapes and associate with coordinated binding of CEBPD, IRF1, and NFκB. Collectively, these findings identify cis-regulatory elements and corresponding trans-acting factors that contribute to EC identity and their specific responses to pro-inflammatory stimuli.
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http://dx.doi.org/10.7554/eLife.22536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461113PMC
June 2017

Application of magic in healthcare: A scoping review.

Complement Ther Clin Pract 2017 Feb 2;26:5-11. Epub 2016 Nov 2.

Department of Family Medicine, Schulich School of Medicine & Denistry, Western University, Canada. Electronic address:

Study Design: Scoping review.

Introduction: The art and science of magic traces back to ancient days. Physicians are often compared to magicians metaphorically. Nonetheless, there exist various genuine applications of magic in the healthcare setting.

Purpose: To explore and summarize the literature reporting the applications of magic tricks or any derived techniques in healthcare or clinical environments.

Methods: A literature search was performed on ten databases: Medline, Embase, CINAHL, Cochrane Central, Cochrane Database of Systematic Reviews, PEDro, Scopus, the International Index to Performing Arts, General OneFile and Newstand, to identify references related to the application of magic in healthcare. Relevant studies were charted, categorized, and summarized.

Results: 29 relevant references were found, consisting of 20 peer-reviewed publications and nine popular literature articles. Five distinct applications of magic in the clinical setting were identified. The literature showed an overall lack of academic evidence.
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http://dx.doi.org/10.1016/j.ctcp.2016.11.002DOI Listing
February 2017

SREBP1 Contributes to Resolution of Pro-inflammatory TLR4 Signaling by Reprogramming Fatty Acid Metabolism.

Cell Metab 2017 02 29;25(2):412-427. Epub 2016 Dec 29.

Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Macrophages play pivotal roles in both the induction and resolution phases of inflammatory processes. Macrophages have been shown to synthesize anti-inflammatory fatty acids in an LXR-dependent manner, but whether the production of these species contributes to the resolution phase of inflammatory responses has not been established. Here, we identify a biphasic program of gene expression that drives production of anti-inflammatory fatty acids 12-24 hr following TLR4 activation and contributes to downregulation of mRNAs encoding pro-inflammatory mediators. Unexpectedly, rather than requiring LXRs, this late program of anti-inflammatory fatty acid biosynthesis is dependent on SREBP1 and results in the uncoupling of NFκB binding from gene activation. In contrast to previously identified roles of SREBP1 in promoting production of IL1β during the induction phase of inflammation, these studies provide evidence that SREBP1 also contributes to the resolution phase of TLR4-induced gene activation by reprogramming macrophage lipid metabolism.
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http://dx.doi.org/10.1016/j.cmet.2016.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568699PMC
February 2017

Increasing number of databases searched in systematic reviews and meta-analyses between 1994 and 2014.

J Med Libr Assoc 2016 Oct;104(4):284-289

Objectives: The purpose of this study was to determine whether the number of bibliographic databases used to search the health sciences literature in individual systematic reviews (SRs) and meta-analyses (MAs) changed over a twenty-year period related to the official 1995 launch of the Cochrane Database of Systematic Reviews (CDSR).

Methods: Ovid MEDLINE was searched using a modified version of a strategy developed by the Scottish Intercollegiate Guidelines Network to identify SRs and MAs. Records from 3 milestone years were searched: the year immediately preceding (1994) and 1 (2004) and 2 (2014) decades following the CDSR launch. Records were sorted with randomization software. Abstracts or full texts of the records were examined to identify database usage until 100 relevant records were identified from each of the 3 years.

Results: The mean and median number of bibliographic databases searched in 1994, 2004, and 2014 were 1.62 and 1, 3.34 and 3, and 3.73 and 4, respectively. Studies that searched only 1 database decreased over the 3 milestone years (60% in 1994, 28% in 2004, and 10% in 2014).

Conclusions: The number of bibliographic databases searched in individual SRs and MAs increased from 1994 to 2014.
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http://dx.doi.org/10.3163/1536-5050.104.4.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079489PMC
October 2016

Lungs can tell time-a highlight from 2016 ATS session on clock genes, inflammation, immunology, and sleep.

J Thorac Dis 2016 Jul;8(Suppl 7):S579-81

Division of Pulmonary, Critical Care & Sleep Medicine, La Jolla, CA 92037, USA.

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http://dx.doi.org/10.21037/jtd.2016.07.36DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990684PMC
July 2016

It's Just Old Age.

Authors:
Michael T Lam

Med Humanit 2016 Sep;42(3):210

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http://dx.doi.org/10.1136/medhum-2016-010906DOI Listing
September 2016

Osteoarthritis.

Authors:
Michael T Lam

Med Humanit 2016 Sep;42(3):165

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http://dx.doi.org/10.1136/medhum-2016-010906.1DOI Listing
September 2016

Tissue damage drives co-localization of NF-κB, Smad3, and Nrf2 to direct Rev-erb sensitive wound repair in mouse macrophages.

Elife 2016 07 27;5. Epub 2016 Jul 27.

Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, United States.

Although macrophages can be polarized to distinct phenotypes in vitro with individual ligands, in vivo they encounter multiple signals that control their varied functions in homeostasis, immunity, and disease. Here, we identify roles of Rev-erb nuclear receptors in regulating responses of mouse macrophages to complex tissue damage signals and wound repair. Rather than reinforcing a specific program of macrophage polarization, Rev-erbs repress subsets of genes that are activated by TLR ligands, IL4, TGFβ, and damage-associated molecular patterns (DAMPS). Unexpectedly, a complex damage signal promotes co-localization of NF-κB, Smad3, and Nrf2 at Rev-erb-sensitive enhancers and drives expression of genes characteristic of multiple polarization states in the same cells. Rev-erb-sensitive enhancers thereby integrate multiple damage-activated signaling pathways to promote a wound repair phenotype.
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http://dx.doi.org/10.7554/eLife.13024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963201PMC
July 2016

Timing Matters: Circadian Rhythm in Sepsis, Obstructive Lung Disease, Obstructive Sleep Apnea, and Cancer.

Ann Am Thorac Soc 2016 07;13(7):1144-54

5 Division of Pulmonary, Critical Care, and Sleep Medicine, University of California, San Diego Health Sciences, San Diego, California.

Physiological and cellular functions operate in a 24-hour cyclical pattern orchestrated by an endogenous process known as the circadian rhythm. Circadian rhythms represent intrinsic oscillations of biological functions that allow for adaptation to cyclic environmental changes. Key clock genes that affect the persistence and periodicity of circadian rhythms include BMAL1/CLOCK, Period 1, Period 2, and Cryptochrome. Remarkable progress has been made in our understanding of circadian rhythms and their role in common medical conditions. A critical review of the literature supports the association between circadian misalignment and adverse health consequences in sepsis, obstructive lung disease, obstructive sleep apnea, and malignancy. Circadian misalignment plays an important role in these disease processes and can affect disease severity, treatment response, and survivorship. Normal inflammatory response to acute infections, airway resistance, upper airway collapsibility, and mitosis regulation follows a robust circadian pattern. Disruption of normal circadian rhythm at the molecular level affects severity of inflammation in sepsis, contributes to inflammatory responses in obstructive lung diseases, affects apnea length in obstructive sleep apnea, and increases risk for cancer. Chronotherapy is an underused practice of delivering therapy at optimal times to maximize efficacy and minimize toxicity. This approach has been shown to be advantageous in asthma and cancer management. In asthma, appropriate timing of medication administration improves treatment effectiveness. Properly timed chemotherapy may reduce treatment toxicities and maximize efficacy. Future research should focus on circadian rhythm disorders, role of circadian rhythm in other diseases, and modalities to restore and prevent circadian disruption.
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http://dx.doi.org/10.1513/AnnalsATS.201602-125FRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015754PMC
July 2016

Effective Gene Delivery to Valvular Interstitial Cells Using Adeno-Associated Virus Serotypes 2 and 3.

Tissue Eng Part C Methods 2015 Aug 3;21(8):808-15. Epub 2015 Apr 3.

1 Department of Bioengineering, Rice University , Houston, Texas.

Currently, curative therapies for heart valve diseases do not exist, thus motivating the need for new therapeutics, regenerative and tissue-engineered valves, and further basic research into pathological mechanisms. For studying valve diseases and developing valve therapies, effective methods to manipulate gene expression in primary valvular interstitial cells (VICs), which promote calcification in disease, would be valuable. Unfortunately, there is little information reported about effective gene delivery methods for VICs. Adeno-associated virus (AAV) is a clinically proven gene delivery vector capable of transducing many cell types and tissues, but has not yet been reported to infect valvular cells. In this study, AAV serotypes 1-9 were tested for their ability to deliver a green fluorescent protein (GFP) reporter into VICs in vitro. Flow cytometry results indicate AAV2 and AAV3 are capable of transducing VICs more efficiently than other serotypes. Furthermore, transduction efficiencies can be optimized by increasing the multiplicity of infection (MOI) and using self-complementary, double-stranded genomes, yielding up to 98% successfully transduced cells. Transduction of VICs by AAV2 or AAV3 in the presence of competing soluble heparin significantly reduces delivery efficiencies, suggesting heparan sulfate proteoglycans act as the primary VIC receptors of these two serotypes. Overall, this study establishes AAV2 and AAV3 as efficient gene delivery vehicles for primary VICs. Such effective delivery vectors for valve cells may be broadly useful for numerous applications, including the study of valvular cell biology, development of valve disease therapies, and regulation of genes for tissue engineering heart valves.
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http://dx.doi.org/10.1089/ten.TEC.2014.0493DOI Listing
August 2015

Enhancer RNAs.

Cell Cycle 2014 ;13(20):3151-2

a Department of Medicine ; University of California San Diego; La Jolla , CA USA.

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http://dx.doi.org/10.4161/15384101.2014.962860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613994PMC
September 2015

Synthetic virology: engineering viruses for gene delivery.

Wiley Interdiscip Rev Nanomed Nanobiotechnol 2014 Nov-Dec;6(6):548-58. Epub 2014 Sep 4.

Department of Bioengineering, Rice University, Houston, TX, USA.

The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or 'bionic' viruses, feature engineered components, or 'parts', that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies--rational, combinatorial, and pseudo-rational--have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavors will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine.
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http://dx.doi.org/10.1002/wnan.1287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227300PMC
June 2015

Enhancer RNAs and regulated transcriptional programs.

Trends Biochem Sci 2014 Apr 24;39(4):170-82. Epub 2014 Mar 24.

Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, USA; Howard Hughes Medical Institute, Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, USA. Electronic address:

A large portion of the human genome is transcribed into RNAs without known protein-coding functions, far outnumbering coding transcription units. Extensive studies of long noncoding RNAs (lncRNAs) have clearly demonstrated that they can play critical roles in regulating gene expression, development, and diseases, acting both as transcriptional activators and repressors. More recently, enhancers have been found to be broadly transcribed, resulting in the production of enhancer-derived RNAs, or eRNAs. Here, we review emerging evidence suggesting that at least some eRNAs contribute to enhancer function. We discuss these findings with respect to potential mechanisms of action of eRNAs and other ncRNAs in regulated gene expression.
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http://dx.doi.org/10.1016/j.tibs.2014.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4266492PMC
April 2014

25-Hydroxycholesterol activates the integrated stress response to reprogram transcription and translation in macrophages.

J Biol Chem 2013 Dec 4;288(50):35812-23. Epub 2013 Nov 4.

From the Departments of Cellular and Molecular Medicine.

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.
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http://dx.doi.org/10.1074/jbc.M113.519637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861632PMC
December 2013

Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription.

Nature 2013 Jun 2;498(7455):511-5. Epub 2013 Jun 2.

Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.

Rev-Erb-α and Rev-Erb-β are nuclear receptors that regulate the expression of genes involved in the control of circadian rhythm, metabolism and inflammatory responses. Rev-Erbs function as transcriptional repressors by recruiting nuclear receptor co-repressor (NCoR)-HDAC3 complexes to Rev-Erb response elements in enhancers and promoters of target genes, but the molecular basis for cell-specific programs of repression is not known. Here we present evidence that in mouse macrophages Rev-Erbs regulate target gene expression by inhibiting the functions of distal enhancers that are selected by macrophage-lineage-determining factors, thereby establishing a macrophage-specific program of repression. Remarkably, the repressive functions of Rev-Erbs are associated with their ability to inhibit the transcription of enhancer-derived RNAs (eRNAs). Furthermore, targeted degradation of eRNAs at two enhancers subject to negative regulation by Rev-Erbs resulted in reduced expression of nearby messenger RNAs, suggesting a direct role of these eRNAs in enhancer function. By precisely defining eRNA start sites using a modified form of global run-on sequencing that quantifies nascent 5' ends, we show that transfer of full enhancer activity to a target promoter requires both the sequences mediating transcription-factor binding and the specific sequences encoding the eRNA transcript. These studies provide evidence for a direct role of eRNAs in contributing to enhancer functions and suggest that Rev-Erbs act to suppress gene expression at a distance by repressing eRNA transcription.
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http://dx.doi.org/10.1038/nature12209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839578PMC
June 2013

Regulation of circadian behaviour and metabolism by REV-ERB-α and REV-ERB-β.

Nature 2012 Mar 29;485(7396):123-7. Epub 2012 Mar 29.

Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA.

The circadian clock acts at the genomic level to coordinate internal behavioural and physiological rhythms via the CLOCK-BMAL1 transcriptional heterodimer. Although the nuclear receptors REV-ERB-α and REV-ERB-β have been proposed to form an accessory feedback loop that contributes to clock function, their precise roles and importance remain unresolved. To establish their regulatory potential, we determined the genome-wide cis-acting targets (cistromes) of both REV-ERB isoforms in murine liver, which revealed shared recognition at over 50% of their total DNA binding sites and extensive overlap with the master circadian regulator BMAL1. Although REV-ERB-α has been shown to regulate Bmal1 expression directly, our cistromic analysis reveals a more profound connection between BMAL1 and the REV-ERB-α and REV-ERB-β genomic regulatory circuits than was previously suspected. Genes within the intersection of the BMAL1, REV-ERB-α and REV-ERB-β cistromes are highly enriched for both clock and metabolic functions. As predicted by the cistromic analysis, dual depletion of Rev-erb-α and Rev-erb-β function by creating double-knockout mice profoundly disrupted circadian expression of core circadian clock and lipid homeostatic gene networks. As a result, double-knockout mice show markedly altered circadian wheel-running behaviour and deregulated lipid metabolism. These data now unite REV-ERB-α and REV-ERB-β with PER, CRY and other components of the principal feedback loop that drives circadian expression and indicate a more integral mechanism for the coordination of circadian rhythm and metabolism.
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http://dx.doi.org/10.1038/nature11048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367514PMC
March 2012

PPARs and lipid ligands in inflammation and metabolism.

Chem Rev 2011 Oct;111(10):6321-40

Department of Medicine, Division of Digestive Diseases, University of California-Los Angeles, Los Angeles, California 90095, USA.

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http://dx.doi.org/10.1021/cr2001355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437919PMC
October 2011

Non-coding RNAs as regulators of gene expression and epigenetics.

Cardiovasc Res 2011 Jun 9;90(3):430-40. Epub 2011 May 9.

Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0651, USA.

Genome-wide studies have revealed that mammalian genomes are pervasively transcribed. This has led to the identification and isolation of novel classes of non-coding RNAs (ncRNAs) that influence gene expression by a variety of mechanisms. Here we review the characteristics and functions of regulatory ncRNAs in chromatin remodelling and at multiple levels of transcriptional and post-transcriptional regulation. We also describe the potential roles of ncRNAs in vascular biology and in mediating epigenetic modifications that might play roles in cardiovascular disease susceptibility. The emerging recognition of the diverse functions of ncRNAs in regulation of gene expression suggests that they may represent new targets for therapeutic intervention.
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http://dx.doi.org/10.1093/cvr/cvr097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096308PMC
June 2011

Artery and vein size is balanced by Notch and ephrin B2/EphB4 during angiogenesis.

Development 2008 Nov;135(22):3755-64

Department of Surgery, University of California, San Francisco, CA 94143, USA.

A mutual coordination of size between developing arteries and veins is essential for establishing proper connections between these vessels and, ultimately, a functional vasculature; however, the cellular and molecular regulation of this parity is not understood. Here, we demonstrate that the size of the developing dorsal aorta and cardinal vein is reciprocally balanced. Mouse embryos carrying gain-of-function Notch alleles show enlarged aortae and underdeveloped cardinal veins, whereas those with loss-of-function mutations show small aortae and large cardinal veins. Notch does not affect the overall number of endothelial cells but balances the proportion of arterial to venous endothelial cells, thereby modulating the relative sizes of both vessel types. Loss of ephrin B2 or its receptor EphB4 also leads to enlarged aortae and underdeveloped cardinal veins; however, endothelial cells with venous identity are mislocalized in the aorta, suggesting that ephrin B2/EphB4 signaling functions distinctly from Notch by sorting arterial and venous endothelial cells into their respective vessels. Our findings provide mechanistic insight into the processes underlying artery and vein size equilibration during angiogenesis.
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http://dx.doi.org/10.1242/dev.022475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2596923PMC
November 2008

Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice.

Proc Natl Acad Sci U S A 2008 Aug 30;105(31):10901-6. Epub 2008 Jul 30.

Pacific Vascular Research Laboratory, Division of Vascular Surgery, Department of Surgery, University of California, San Francisco, CA 94143, USA.

Brain arteriovenous malformations (BAVMs) can cause devastating stroke in young people and contribute to half of all hemorrhagic stroke in children. Unfortunately, the pathogenesis of BAVMs is unknown. In this article we show that activation of Notch signaling in the endothelium during brain development causes BAVM in mice. We turned on constitutively active Notch4 (int3) expression in endothelial cells from birth by using the tetracycline-regulatable system. All mutants developed hallmarks of BAVMs, including cerebral arteriovenous shunting and vessel enlargement, by 3 weeks of age and died by 5 weeks of age. Twenty-five percent of the mutants showed signs of neurological dysfunction, including ataxia and seizure. Affected mice exhibited hemorrhage and neuronal cell death within the cerebral cortex and cerebellum. Strikingly, int3 repression resolved ataxia and reversed the disease progression, demonstrating that int3 is not only sufficient to induce, but also required to sustain the disease. We show that int3 expression results in widespread enlargement of the microvasculature, which coincided with a reduction in capillary density, linking vessel enlargement to Notch's known function of inhibiting vessel sprouting. Our data suggest that the Notch pathway is a molecular regulator of BAVM pathogenesis in mice, and offer hope that their regression might be possible by targeting the causal molecular lesion.
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http://dx.doi.org/10.1073/pnas.0802743105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2504798PMC
August 2008

Endothelial expression of constitutively active Notch4 elicits reversible arteriovenous malformations in adult mice.

Proc Natl Acad Sci U S A 2005 Jul 30;102(28):9884-9. Epub 2005 Jun 30.

Pacific Vascular Research Laboratory, Division of Vascular Surgery, Department of Surgery and Department of Anatomy, University of California-San Francisco, San Francisco, CA 94143, USA.

Direct communication between arteries and veins without intervening capillary beds is the primary pathology of arteriovenous malformations (AVMs). Although Notch signaling is implicated in embryonic arteriovenous (AV) differentiation, its function in the adult mammalian vasculature has not been established due to the embryonic lethality that often occurs in both gain- and loss-of-function mutants. We expressed a constitutively active Notch4, int3, in the adult mouse endothelium by using the tetracycline-repressible system to suppress int3 during embryogenesis. int3 caused profound blood vessel enlargement and AV shunting, which are hallmarks of AVM, and led to lethality within weeks of its expression. Vessel enlargement, a manifestation of AVM, occurred in an apparently tissue-specific fashion; the liver, uterus, and skin were affected. int3-mediated vascular defects were accompanied by arterialization, including ectopic venous expression of ephrinB2, increased smooth muscle cells, and up-regulation of endogenous Notch signaling. Remarkably, the defective vessels and illness were reversed upon repression of int3 expression. Finally, endothelial expression of a constitutively active Notch1 induced similar hepatic vascular lesions. Our results provide gain-of-function evidence that Notch signaling in the adult endothelium is sufficient to render arterial characteristics and lead to AVMs.
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http://dx.doi.org/10.1073/pnas.0504391102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1175015PMC
July 2005

Competitive immunoassay for vancomycin using capillary electrophoresis with laser-induced fluorescence detection.

Analyst 2002 Dec;127(12):1633-7

Environmental Health Sciences Program, Department of Public Health Sciences, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2G3.

A competitive immunoassay using capillary electrophoresis with laser-induced fluorescence was developed for vancomycin. Capillary electrophoresis using a Tris-glycine running buffer provided adequate separation of the antibody-bound from the unbound fluorescent probe (tracer) in less than 4 min. Laser-induced fluorescence polarization (LIFP) provided high sensitivity detection and simultaneous monitoring of fluorescence intensity and polarization. A fluorescence polarization value of 0.30 confirmed the formation of the antibody-tracer complex. Calibration curves showed a working linear range of 2-3 orders of magnitude with a minimum detectable concentration of 0.98 ng mL(-1) (or 1.1 fg vancomycin). Clinical samples obtained from patients undergoing vancomycin treatment were analyzed for vancomycin and the results correlated well with a standard immunoassay based on latex particle detection that was routinely used by a hospital laboratory. Only 1/10 of the reagents were needed as compared with the standard immunoassay.
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http://dx.doi.org/10.1039/b206531bDOI Listing
December 2002