Publications by authors named "Daniel R Salomon"

77 Publications

Extended survival versus accelerated rejection of nonhuman primate islet allografts: Effect of mesenchymal stem cell source and timing.

Am J Transplant 2021 11 2;21(11):3524-3537. Epub 2021 Jul 2.

Diabetes Research Institute, University of Miami, Miami, Florida, USA.

Mesenchymal stem cells (MSC) have been shown to be immunomodulatory, tissue regenerative, and graft promoting; however, several questions remain with regard to ideal MSC source and timing of administration. In this study, we utilized a rigorous preclinical model of allogeneic islet cell transplantation, incorporating reduced immune suppression and near to complete mismatch of major histocompatibility antigens between the diabetic cynomolgus monkey recipient and the islet donor, to evaluate both the graft promoting impact of MSC source, that is, derived from the islet recipient, the islet donor or an unrelated third party as well as the impact of timing. Co-transplant of MSC and islets on post-operative day 0, followed by additional IV MSC infusions in the first posttransplant month, resulted in prolongation of rejection free and overall islet survival and superior metabolic control for animals treated with recipient as compared to donor or third-party MSC. Immunological analyses demonstrated that infusion of MSC from either source did not prevent alloantibody formation to the islet or MSC donor; however, treatment with recipient MSC resulted in significant downregulation of memory T cells, decreased anti-donor T cell proliferation, and a trend toward increased Tregulatory:Tconventional ratios.
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http://dx.doi.org/10.1111/ajt.16693DOI Listing
November 2021

Discovery and cross-validation of peripheral blood and renal biopsy gene expression signatures from ethnically diverse kidney transplant populations.

Am J Transplant 2019 12 8;19(12):3356-3366. Epub 2019 Jul 8.

Scripps Center for Organ Transplantation, Scripps Green Hospital, La Jolla, California.

We determined peripheral blood (PB) and biopsy (Bx) RNA expression signatures in a Brazilian and US cohort of kidney transplant patients. Phenotypes assigned by precise histology were: acute rejection (AR), interstitial fibrosis/tubular atrophy/chronic rejection (CR), excellent functioning transplants (TX), and glomerulonephritis recurrence (GN). Samples were analyzed on microarrays and profiles from each cohort were cross-validated on the other cohort with similar phenotypes. We discovered signatures for each tissue: (1) AR vs TX, (2) CR vs TX, and (3) GN vs TX using the Random Forests algorithm. We validated biopsies signatures of AR vs TX (area under the curve [AUC] 0.97) and CR vs TX (AUC 0.87). We also validated both PB and Bx signatures of AR vs TX and CR vs TX with varying degrees of accuracy. Several biological pathways were shared between AR and CR, suggesting similar rejection mechanisms in these 2 clinical phenotypes. Thus, we identified gene expression signatures for AR and CR in transplant patients and validated them in independent cohorts of significantly different racial/ethnic backgrounds. These results reveal that there are strong unifying immune mechanisms driving transplant diseases and identified in the signatures discovered in each cohort, suggesting that molecular diagnostics across populations are feasible despite ethnic and environmental differences.
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http://dx.doi.org/10.1111/ajt.15482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883121PMC
December 2019

Expression of the miR-150 tumor suppressor is restored by and synergizes with rapamycin in a human leukemia T-cell line.

Leuk Res 2018 11 22;74:1-9. Epub 2018 Sep 22.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, United States.

miR-150 functions as a tumor suppressor in malignancies of the lymphocyte lineage and its expression is significantly reduced in these cells. However, the mechanism of miR-150 repression is unknown and so are pharmacological interventions that can reverse it. Here, we report that reduced expression of miR-150 in human Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells is mediated by constitutive mTOR signaling, a common characteristic of T-ALL cell lines and clinical isolates. Activating mTOR signaling in non-malignant T cells also resulted in a significant miR-150 down-regulation. Conversely, treatment with a pharmacological mTOR inhibitor, rapamycin, increased miR-150 expression in a dose-dependent manner in Jurkat cells, as well as in other leukemia cells. Interestingly, ectopic over-expression of miR-150 acted in a feed-forward loop and further sensitized Jurkat cells to a rapamycin-induced cell cycle arrest by targeting a large network of cell cycle genes. These findings suggest that miR-150 is normally expressed in quiescent T lymphocytes to reinforce an anti-proliferative state, and that mTOR signaling promotes cell proliferation in part by inhibiting miR-150 expression. Restoration of the miR-150-dependent anti-proliferative loop constitutes a novel mechanism underlying the efficacy of rapamycin in a T-ALL cell line. Further investigation of this mechanism in clinical isolates of T-ALL and other hematopoietic malignancies could help better guide development of targeted therapies.
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http://dx.doi.org/10.1016/j.leukres.2018.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290994PMC
November 2018

Development and clinical validity of a novel blood-based molecular biomarker for subclinical acute rejection following kidney transplant.

Am J Transplant 2019 01 31;19(1):98-109. Epub 2018 Aug 31.

Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Noninvasive biomarkers are needed to monitor stable patients after kidney transplant (KT), because subclinical acute rejection (subAR), currently detectable only with surveillance biopsies, can lead to chronic rejection and graft loss. We conducted a multicenter study to develop a blood-based molecular biomarker for subAR using peripheral blood paired with surveillance biopsies and strict clinical phenotyping algorithms for discovery and validation. At a predefined threshold, 72% to 75% of KT recipients achieved a negative biomarker test correlating with the absence of subAR (negative predictive value: 78%-88%), while a positive test was obtained in 25% to 28% correlating with the presence of subAR (positive predictive value: 47%-61%). The clinical phenotype and biomarker independently and statistically correlated with a composite clinical endpoint (renal function, biopsy-proved acute rejection, ≥grade 2 interstitial fibrosis, and tubular atrophy), as well as with de novo donor-specific antibodies. We also found that <50% showed histologic improvement of subAR on follow-up biopsies despite treatment and that the biomarker could predict this outcome. Our data suggest that a blood-based biomarker that reduces the need for the indiscriminate use of invasive surveillance biopsies and that correlates with transplant outcomes could be used to monitor KT recipients with stable renal function, including after treatment for subAR, potentially improving KT outcomes.
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http://dx.doi.org/10.1111/ajt.15011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6387870PMC
January 2019

A genome-wide survey of mutations in the Jurkat cell line.

BMC Genomics 2018 May 8;19(1):334. Epub 2018 May 8.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, 92037, USA.

Background: The Jurkat cell line has an extensive history as a model of T cell signaling. But at the turn of the 21st century, some expression irregularities were observed, raising doubts about how closely the cell line paralleled normal human T cells. While numerous expression deficiencies have been described in Jurkat, genetic explanations have only been provided for a handful of defects.

Results: Here, we report a comprehensive catolog of genomic variation in the Jurkat cell line based on whole-genome sequencing. With this list of all detectable, non-reference sequences, we prioritize potentially damaging mutations by mining public databases for functional effects. We confirm documented mutations in Jurkat and propose links from detrimental gene variants to observed expression abnormalities in the cell line.

Conclusions: The Jurkat cell line harbors many mutations that are associated with cancer and contribute to Jurkat's unique characteristics. Genes with damaging mutations in the Jurkat cell line are involved in T-cell receptor signaling (PTEN, INPP5D, CTLA4, and SYK), maintenance of genome stability (TP53, BAX, and MSH2), and O-linked glycosylation (C1GALT1C1). This work ties together decades of molecular experiments and serves as a resource that will streamline both the interpretation of past research and the design of future Jurkat studies.
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http://dx.doi.org/10.1186/s12864-018-4718-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5941560PMC
May 2018

Microdroplet PCR for Highly Multiplexed Targeted Bisulfite Sequencing.

Methods Mol Biol 2018 ;1708:333-348

Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA, 92037, USA.

Many methods exist for examining CpG DNA methylation. However, many of these are qualitative, laborious to apply to a large number of genes simultaneously, or are not easy to target to specific regions of interest. Microdroplet PCR-based bisulfite sequencing allows for quantitative single base resolution analysis of investigator selected regions of interest. Following bisulfite conversion of genomic DNA, targeted microdroplet PCR is conducted with custom primer libraries. Samples are then fragmented, concatenated, and sequenced by high-throughput sequencing. The most recent technology allows for this method to be conducted with as little as 250 ng of bisulfite-converted DNA. The primary advantage of this method is the ability to hand-select the targeted regions covered by up to 10,000 amplicons of 500-600 bp. Moreover, the nature of microdroplet PCR virtually eliminates PCR bias and allows for the amplification of all targets simultaneously in a single tube.
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http://dx.doi.org/10.1007/978-1-4939-7481-8_17DOI Listing
July 2018

Gene expression signature predicts human islet integrity and transplant functionality in diabetic mice.

PLoS One 2017 2;12(10):e0185331. Epub 2017 Oct 2.

Department of Translational Research and Cellular Therapeutics, Diabetes, and Metabolism Research Institute, City of Hope National Medical Center, Duarte, California, United States of America.

There is growing evidence that transplantation of cadaveric human islets is an effective therapy for type 1 diabetes. However, gauging the suitability of islet samples for clinical use remains a challenge. We hypothesized that islet quality is reflected in the expression of specific genes. Therefore, gene expression in 59 human islet preparations was analyzed and correlated with diabetes reversal after transplantation in diabetic mice. Analysis yielded 262 differentially expressed probesets, which together predict islet quality with 83% accuracy. Pathway analysis revealed that failing islet preparations activated inflammatory pathways, while functional islets showed increased regeneration pathway gene expression. Gene expression associated with apoptosis and oxygen consumption showed little overlap with each other or with the 262 probeset classifier, indicating that the three tests are measuring different aspects of islet cell biology. A subset of 36 probesets surpassed the predictive accuracy of the entire set for reversal of diabetes, and was further reduced by logistic regression to sets of 14 and 5 without losing accuracy. These genes were further validated with an independent cohort of 16 samples. We believe this limited number of gene classifiers in combination with other tests may provide complementary verification of islet quality prior to their clinical use.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185331PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624587PMC
October 2017

H3K27 Methylation Dynamics during CD4 T Cell Activation: Regulation of JAK/STAT and IL12RB2 Expression by JMJD3.

J Immunol 2017 11 25;199(9):3158-3175. Epub 2017 Sep 25.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037.

The changes to the epigenetic landscape in response to Ag during CD4 T cell activation have not been well characterized. Although CD4 T cell subsets have been mapped globally for numerous epigenetic marks, little has been done to study their dynamics early after activation. We have studied changes to promoter H3K27me3 during activation of human naive and memory CD4 T cells. Our results show that these changes occur relatively early (1 d) after activation of naive and memory cells and that demethylation is the predominant change to H3K27me3 at this time point, reinforcing high expression of target genes. Additionally, inhibition of the H3K27 demethylase JMJD3 in naive CD4 T cells demonstrates how critically important molecules required for T cell differentiation, such as JAK2 and IL12RB2, are regulated by H3K27me3. Our results show that H3K27me3 is a dynamic and important epigenetic modification during CD4 T cell activation and that JMJD3-driven H3K27 demethylation is critical for CD4 T cell function.
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http://dx.doi.org/10.4049/jimmunol.1700475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679303PMC
November 2017

Biomarker Guidelines for High-Dimensional Genomic Studies in Transplantation: Adding Method to the Madness.

Transplantation 2017 03;101(3):457-463

1 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA. 2 Department of Surgery, University of Virginia, Charlottesville, VA. 3 Department of Transplant Nephrology, Mayo Clinic, Phoenix, AZ. 4 Comprehensive Transplant Center, Feinberg School of Medicine, Northwestern University, Chicago, IL. 5 Department of Surgery, Mayo Clinic, Phoenix, AZ.

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http://dx.doi.org/10.1097/TP.0000000000001622DOI Listing
March 2017

Peripheral Blood Cell Gene Expression Diagnostic for Identifying Symptomatic Transthyretin Amyloidosis Patients: Male and Female Specific Signatures.

Theranostics 2016 18;6(11):1792-809. Epub 2016 Jul 18.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037.

Background: Early diagnosis of familial transthyretin (TTR) amyloid diseases remains challenging because of variable disease penetrance. Currently, patients must have an amyloid positive tissue biopsy to be eligible for disease-modifying therapies. Endomyocardial biopsies are typically amyloid positive when cardiomyopathy is suspected, but this disease manifestation is generally diagnosed late. Early diagnosis is often difficult because patients exhibit apparent symptoms of polyneuropathy, but have a negative amyloid biopsy. Thus, there is a pressing need for an additional early diagnostic strategy for TTR-aggregation-associated polyneuropathy and cardiomyopathy.

Methods And Findings: Global peripheral blood cell mRNA expression profiles from 263 tafamidis-treated and untreated V30M Familiar Amyloid Neuropathy patients, asymptomatic V30M carriers, and healthy, age- and sex-matched controls without TTR mutations were used to differentiate symptomatic from asymptomatic patients. We demonstrate that blood cell gene expression patterns reveal sex-independent, as well as male- and female-specific inflammatory signatures in symptomatic FAP patients, but not in asymptomatic carriers. These signatures differentiated symptomatic patients from asymptomatic V30M carriers with >80% accuracy. There was a global downregulation of the eIF2 pathway and its associated genes in all symptomatic FAP patients. We also demonstrated that the molecular scores based on these signatures significantly trended toward normalized values in an independent cohort of 46 FAP patients after only 3 months of tafamidis treatment.

Conclusions: This study identifies novel molecular signatures that differentiate symptomatic FAP patients from asymptomatic V30M carriers as well as affected males and females. We envision using this approach, initially in parallel with amyloid biopsies, to identify individuals who are asymptomatic gene carriers that may convert to FAP patients. Upon further validation, peripheral blood cell mRNA expression profiling could become an independent early diagnostic. This quantitative gene expression signature for symptomatic FAP could also become a biomarker to demonstrate significant disease-modifying effects of drugs and drug candidates. For example, when new disease modifiers are being evaluated in a FAP clinical trial, such surrogate biomarkers have the potential to provide an objective, quantitative and mechanistic molecular diagnostic of disease response to therapy.
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http://dx.doi.org/10.7150/thno.14584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4997237PMC
September 2017

Genome-wide expression profiling in the peripheral blood of patients with fibromyalgia.

Clin Exp Rheumatol 2016 Mar-Apr;34(2 Suppl 96):S89-98. Epub 2016 Feb 12.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA.

Objectives: Fibromyalgia (FM) is a common pain disorder characterized by nociceptive dysregulation. The basic biology of FM is poorly understood. Herein we have used agnostic gene expression as a potential probe for informing its underlying biology and the development of a proof-of-concept diagnostic gene expression signature.

Methods: We analyzed RNA expression in 70 FM patients and 70 healthy controls. The isolated RNA was amplified and hybridized to Affymetrix® Human Gene 1.1 ST Peg arrays. The data was analyzed using Partek Genomics Suite version 6.6.

Results: Fibromyalgia patients exhibited a differential expression of 421 genes (p<0.001), several relevant to pathways for pain processing, such as glutamine/glutamate signaling and axonal development. There was also an upregulation of several inflammatory pathways and downregulation of pathways related to hypersensitivity and allergy. Using rigorous diagnostic modeling strategies, we show "locked" gene signatures discovered on Training and Test cohorts, that have a mean Area Under the Curve (AUC) of 0.81 on randomized, independent external data cohorts. Lastly, we identified a subset of 10 probesets that provided a diagnostic sensitivity for FM of 95% and a specificity of 96%. We also show that the signatures for FM were very specific to FM rather than common FM comorbidities.

Conclusions: These findings provide new insights relevant to the pathogenesis of FM, and provide several testable hypotheses that warrant further exploration and also establish the foundation for a first blood-based molecular signature in FM that needs to be validated in larger cohorts of patients.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888802PMC
July 2016

Comparative top down proteomics of peripheral blood mononuclear cells from kidney transplant recipients with normal kidney biopsies or acute rejection.

Proteomics 2016 07;16(14):2048-58

Proteomics Center of Excellence, Northwestern University, Evanston, IL, USA.

Recent studies utilizing transcriptomics, metabolomics, and bottom up proteomics have identified molecular signatures of kidney allograft pathology. Although these results make significant progress toward non-invasive differential diagnostics of dysfunction of a transplanted kidney, they provide little information on the intact, often modified, protein molecules present during progression of this pathology. Because intact proteins underpin diverse biological processes, measuring the relative abundance of their modified forms promises to advance mechanistic understanding, and might provide a new class of biomarker candidates. Here, we used top down proteomics to inventory the modified forms of whole proteins in peripheral blood mononuclear cells (PBMCs) taken at the time of kidney biopsy for 40 kidney allograft recipients either with healthy transplants or those suffering acute rejection. Supported by gas-phase fragmentation of whole protein ions during tandem mass spectrometry, we identified 344 proteins mapping to 2905 distinct molecular forms (proteoforms). Using an initial implementation of a label-free approach to quantitative top down proteomics, we obtained evidence suggesting relative abundance changes in 111 proteoforms between the two patient groups. Collectively, our work is the first to catalog intact protein molecules in PBMCs and suggests differentially abundant proteoforms for further analysis.
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http://dx.doi.org/10.1002/pmic.201600008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956503PMC
July 2016

UHM-ULM interactions in the RBM39-U2AF65 splicing-factor complex.

Acta Crystallogr D Struct Biol 2016 Apr 24;72(Pt 4):497-511. Epub 2016 Mar 24.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein-protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39-U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemical shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35-U2AF65 and RBM39-SF3b155, the RBM39-UHM-U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM-ULM binding interface, providing a rationale for the known specificity of UHM-ULM interactions. This study therefore establishes a structural basis for specific UHM-ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.
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http://dx.doi.org/10.1107/S2059798316001248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822562PMC
April 2016

A CRISPR Way to Block PERVs--Engineering Organs for Transplantation.

Authors:
Daniel R Salomon

N Engl J Med 2016 Mar;374(11):1089-91

From the Department of Molecular and Experimental Medicine, Scripps Research Institute, San Diego, CA.

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http://dx.doi.org/10.1056/NEJMcibr1515623DOI Listing
March 2016

Transplant-induced reactivation of murine cytomegalovirus immediate early gene expression is associated with recruitment of NF-κB and AP-1 to the major immediate early promoter.

J Gen Virol 2016 Apr 20;97(4):941-954. Epub 2016 Jan 20.

Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Reactivation of latent human cytomegalovirus is a significant infectious complication of organ transplantation and current therapies target viral replication once reactivation of latent virus has already occurred. The specific molecular pathways that activate viral gene expression in response to transplantation are not well understood. Our studies aim to identify these factors, with the goal of developing novel therapies that prevent transcriptional reactivation in transplant recipients. Murine cytomegalovirus (MCMV) is a valuable model for studying latency and reactivation of CMV in vivo. We previously demonstrated that transplantation of MCMV-latently infected kidneys into allogeneic recipients induces reactivation of immediate early (IE) gene expression and epigenetic reprogramming of the major immediate early promoter (MIEP) within 48 h. We hypothesize that these events are mediated by activation of signalling pathways that lead to binding of transcription factors to the MIEP, including AP-1 and NF-κB. Here we show that transplantation induces rapid activation of several members of the AP-1 and NF-κB transcription factor family and we demonstrate that canonical NF-κB (p65/p50), the junD component of AP-1, and nucleosome remodelling complexes are recruited to the MIEP following transplantation. Proteomic analysis of recipient plasma and transcriptome analysis of kidney RNA identified five extracellular ligands, including TNF, IL-1β, IL-18, CD40L and IL-6, and three intracellular signalling pathways associated with reactivation of IE gene expression. Identification of the factors that mediate activation of these signalling pathways may eventually lead to new therapies to prevent reactivation of CMV and its sequelae.
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http://dx.doi.org/10.1099/jgv.0.000407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854367PMC
April 2016

The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.

PLoS One 2015 7;10(12):e0144409. Epub 2015 Dec 7.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, United States of America.

Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as "central" interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, "peripheral" interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144409PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4671683PMC
June 2016

Suppression of transcriptional drift extends C. elegans lifespan by postponing the onset of mortality.

Elife 2015 Dec 1;4:e08833. Epub 2015 Dec 1.

Department of Chemical Physiology, The Scripps Research Institute, La Jolla, United States.

Longevity mechanisms increase lifespan by counteracting the effects of aging. However, whether longevity mechanisms counteract the effects of aging continually throughout life, or whether they act during specific periods of life, preventing changes that precede mortality is unclear. Here, we uncover transcriptional drift, a phenomenon that describes how aging causes genes within functional groups to change expression in opposing directions. These changes cause a transcriptome-wide loss in mRNA stoichiometry and loss of co-expression patterns in aging animals, as compared to young adults. Using Caenorhabditis elegans as a model, we show that extending lifespan by inhibiting serotonergic signals by the antidepressant mianserin attenuates transcriptional drift, allowing the preservation of a younger transcriptome into an older age. Our data are consistent with a model in which inhibition of serotonergic signals slows age-dependent physiological decline and the associated rise in mortality levels exclusively in young adults, thereby postponing the onset of major mortality.
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http://dx.doi.org/10.7554/eLife.08833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4720515PMC
December 2015

Defining CD4 T cell memory by the epigenetic landscape of CpG DNA methylation.

J Immunol 2015 Feb 9;194(4):1565-79. Epub 2015 Jan 9.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037

Memory T cells are primed for rapid responses to Ag; however, the molecular mechanisms responsible for priming remain incompletely defined. CpG methylation in promoters is an epigenetic modification, which regulates gene transcription. Using targeted bisulfite sequencing, we examined methylation of 2100 genes (56,000 CpGs) mapped by deep sequencing of T cell activation in human naive and memory CD4 T cells. Four hundred sixty-six CpGs (132 genes) displayed differential methylation between naive and memory cells. Twenty-one genes exhibited both differential methylation and gene expression before activation, linking promoter DNA methylation states to gene regulation; 6 of 21 genes encode proteins closely studied in T cells, whereas 15 genes represent novel targets for further study. Eighty-four genes demonstrated differential methylation between memory and naive cells that correlated to differential gene expression following activation, of which 39 exhibited reduced methylation in memory cells coupled with increased gene expression upon activation compared with naive cells. These reveal a class of primed genes more rapidly expressed in memory compared with naive cells and putatively regulated by DNA methylation. These findings define a DNA methylation signature unique to memory CD4 T cells that correlates with activation-induced gene expression.
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http://dx.doi.org/10.4049/jimmunol.1401162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364524PMC
February 2015

Plasma protein biomarkers enhance the clinical prediction of kidney injury recovery in patients undergoing liver transplantation.

Hepatology 2014 Dec 29;60(6):2017-26. Epub 2014 Oct 29.

Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, IL; Division of Gastroenterology and Hepatology, Northwestern University Feinberg School of Medicine, Chicago, IL.

Unlabelled: Biomarkers predictive of recovery from acute kidney injury (AKI) after liver transplantation (LT) could enhance decision algorithms regarding the need for liver-kidney transplantation or renal sparing regimens. Multianalyte plasma/urine kidney injury protein panels were performed immediately before and 1 month post-LT in an initial test group divided by reversible pre-LT AKI (rAKI = post-LT renal recovery) versus no AKI (nAKI). This was followed by a larger validation set that included an additional group: irreversible pre-LT AKI (iAKI = no post-LT renal recovery). In the test group (n = 16), six pre-LT plasma (not urine) kidney injury proteins (osteopontin [OPN], neutrophil gelatinase-associated lipocalin, cystatin C, trefoil factor 3, tissue inhibitor of metalloproteinase [TIMP]-1, and β-2-microglobulin) were higher in rAKI versus nAKI (P < 0.05) and returned to normal values with renal recovery post-LT. In the validation set (n = 46), a number of proteins were significantly higher in both rAKI and iAKI versus nAKI. However, only pre-LT plasma OPN (P = 0.009) and TIMP-1 (P = 0.019) levels were significantly higher in rAKI versus iAKI. Logistic regression modeling was used to correlate the probability of post-LT rAKI, factoring in both pre-LT protein markers and clinical variables. A combined model including elevated OPN and TIMP-1 levels, age <57, and absence of diabetes had the highest area under the curve of 0.82, compared to protein-only and clinical variable-only models.

Conclusion: These data suggest that plasma protein profiles might improve the prediction of pre-LT kidney injury recovery after LT. However, multicenter, prospective studies are needed to validate these findings and ultimately test the value of such protein panels in perioperative management and decision making.
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http://dx.doi.org/10.1002/hep.27346DOI Listing
December 2014

A structurally distinct human mycoplasma protein that generically blocks antigen-antibody union.

Science 2014 Feb;343(6171):656-661

Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

We report the discovery of a broadly reactive antibody-binding protein (Protein M) from human mycoplasma. The crystal structure of the ectodomain of transmembrane Protein M differs from other known protein structures, as does its mechanism of antibody binding. Protein M binds with high affinity to all types of human and nonhuman immunoglobulin G, predominantly through attachment to the conserved portions of the variable region of the κ and λ light chains. Protein M blocks antibody-antigen union, likely because of its large C-terminal domain extending over the antibody-combining site, blocking entry to large antigens. Similar to the other immunoglobulin-binding proteins such as Protein A, Protein M as well as its orthologs in other Mycoplasma species could become invaluable reagents in the antibody field.
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http://dx.doi.org/10.1126/science.1246135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3987992PMC
February 2014

Library construction for next-generation sequencing: overviews and challenges.

Biotechniques 2014 1;56(2):61-4, 66, 68, passim. Epub 2014 Feb 1.

NGS and Microarray Core Facility, The Scripps Research Institute, La Jolla, CA.

High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed.
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http://dx.doi.org/10.2144/000114133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351865PMC
September 2014

Finding the active genes in deep RNA-seq gene expression studies.

BMC Genomics 2013 Nov 11;14:778. Epub 2013 Nov 11.

Donnelly Centre, Banting & Best Department of Medical Research, University of Toronto, Toronto, Canada.

Background: Early application of second-generation sequencing technologies to transcript quantitation (RNA-seq) has hinted at a vast mammalian transcriptome, including transcripts from nearly all known genes, which might be fully measured only by ultradeep sequencing. Subsequent studies suggested that low-abundance transcripts might be the result of technical or biological noise rather than active transcripts; moreover, most RNA-seq experiments did not provide enough read depth to generate high-confidence estimates of gene expression for low-abundance transcripts. As a result, the community adopted several heuristics for RNA-seq analysis, most notably an arbitrary expression threshold of 0.3 - 1 FPKM for downstream analysis. However, advances in RNA-seq library preparation, sequencing technology, and informatic analysis have addressed many of the systemic sources of uncertainty and undermined the assumptions that drove the adoption of these heuristics. We provide an updated view of the accuracy and efficiency of RNA-seq experiments, using genomic data from large-scale studies like the ENCODE project to provide orthogonal information against which to validate our conclusions.

Results: We show that a human cell's transcriptome can be divided into active genes carrying out the work of the cell and other genes that are likely the by-products of biological or experimental noise. We use ENCODE data on chromatin state to show that ultralow-expression genes are predominantly associated with repressed chromatin; we provide a novel normalization metric, zFPKM, that identifies the threshold between active and background gene expression; and we show that this threshold is robust to experimental and analytical variations.

Conclusions: The zFPKM normalization method accurately separates the biologically relevant genes in a cell, which are associated with active promoters, from the ultralow-expression noisy genes that have repressed promoters. A read depth of twenty to thirty million mapped reads allows high-confidence quantitation of genes expressed at this threshold, providing important guidance for the design of RNA-seq studies of gene expression. Moreover, we offer an example for using extensive ENCODE chromatin state information to validate RNA-seq analysis pipelines.
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http://dx.doi.org/10.1186/1471-2164-14-778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3870982PMC
November 2013

MicroRNA regulation of T-lymphocyte immunity: modulation of molecular networks responsible for T-cell activation, differentiation, and development.

Crit Rev Immunol 2013 ;33(5):435-76

Laboratory for Functional Genomics, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA.

MicroRNAs (miRNA) are a class of small non-coding RNAs that constitute an essential and evolutionarily conserved mechanism for post-transcriptional gene regulation. Multiple miRNAs have been described to play key roles in T-lymphocyte development, differentiation, and function. In this review, we highlight the current literature regarding the differential expression of miRNAs in various models of murine and human T-cell biology. We emphasize mechanistic understandings of miRNA regulation of thymocyte development, T-cell activation, and differentiation into effector and memory subsets. We describe the participation of miRNAs in complex regulatory circuits shaping T-cell proteomes in a context-dependent manner. It is striking that some miRNAs regulate multiple processes, while others only appear in limited functional contexts. It is also evident that the expression and function of specific miRNAs can differ between murine and human systems. Ultimately, it is not always correct to simplify the complex events of T-cell biology into a model driven by only one or two master regulator miRNAs. In reality, T-cell activation and differentiation involve the expression of multiple miRNAs with many mRNA targets; thus, the true extent of miRNA regulation of T-cell biology is likely far more vast than currently appreciated.
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http://dx.doi.org/10.1615/critrevimmunol.2013006858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4185288PMC
January 2014

Genomic biomarkers correlate with HLA-identical renal transplant tolerance.

J Am Soc Nephrol 2013 Sep 20;24(9):1376-85. Epub 2013 Jun 20.

Comprehensive Transplant Center, Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.

The ability to achieve immunologic tolerance after transplantation is a therapeutic goal. Here, we report interim results from an ongoing trial of tolerance in HLA-identical sibling renal transplantation. The immunosuppressive regimen included alemtuzumab induction, donor hematopoietic stem cells, tacrolimus/mycophenolate immunosuppression converted to sirolimus, and complete drug withdrawal by 24 months post-transplantation. Recipients were considered tolerant if they had normal biopsies and renal function after an additional 12 months without immunosuppression. Of the 20 recipients enrolled, 10 had at least 36 months of follow-up after transplantation. Five of these 10 recipients had immunosuppression successfully withdrawn for 16-36 months (tolerant), 2 had disease recurrence, and 3 had subclinical rejection in protocol biopsies (nontolerant). Microchimerism disappeared after 1 year, and CD4(+)CD25(high)CD127(-)FOXP3(+) regulatory T cells and CD19(+)IgD/M(+)CD27(-) B cells were increased through 5 years post-transplantation in both tolerant and nontolerant recipients. Immune/inflammatory gene expression pathways in the peripheral blood and urine, however, were differentially downregulated between tolerant and nontolerant recipients. In summary, interim results from this trial of tolerance in HLA-identical renal transplantation suggest that predictive genomic biomarkers, but not immunoregulatory phenotyping, may be able to discriminate tolerant from nontolerant patients.
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http://dx.doi.org/10.1681/ASN.2013010068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3752953PMC
September 2013

RNA purification and expression analysis using microarrays and RNA deep sequencing.

Methods Mol Biol 2013 ;1034:385-403

Microarray and Next Generation Sequencing Core Facility, The Scripps Research Institute, La Jolla, CA, USA.

Transcriptome analysis or global gene expression profiling is a powerful tool for discovery as well as -understanding biological mechanisms in health and disease. We present in this chapter a description of methods used to isolate mRNA from cells and tissues that has been optimized for preservation of RNA quality using clinical materials and implemented successfully in several large, multicenter studies by the authors. In addition, two methods, gene expression microarrays and RNAseq, are described for mRNA profiling of cells and tissues from clinical or laboratory sources.
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http://dx.doi.org/10.1007/978-1-62703-493-7_25DOI Listing
February 2014

Genomic meta-analysis of growth factor and integrin pathways in chronic kidney transplant injury.

BMC Genomics 2013 Apr 23;14:275. Epub 2013 Apr 23.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.

Background: Chronic Allograft Nephropathy (CAN) is a clinical entity of progressive kidney transplant injury. The defining histology is tubular atrophy with interstitial fibrosis (IFTA). Using a meta-analysis of microarrays from 84 kidney transplant biopsies, we revealed growth factor and integrin adhesion molecule pathways differentially expressed and correlated with histological progression. A bioinformatics approach mining independent datasets leverages new and existing data to identify correlative changes in integrin and growth factor signaling pathways.

Results: Analysis of CAN/IFTA Banff grades showed that hepatocyte growth factor (HGF), and epidermal growth factor (EGF) pathways are significantly differentially expressed in all classes of CAN/IFTA. MAPK-dependent pathways were also significant. However, the TGFβ pathways, albeit present, failed to differentiate CAN/IFTA progression. The integrin subunits β8, αv, αμ and β5 are differentially expressed, but β1, β6 and α6 specifically correlate with progression of chronic injury. Results were validated using our published proteomic profiling of CAN/IFTA.

Conclusions: CAN/IFTA with chronic kidney injury is characterized by expression of distinct growth factors and specific integrin adhesion molecules as well as their canonical signaling pathways. Drug target mapping suggests several novel candidates for the next generation of therapeutics to prevent or treat progressive transplant dysfunction with interstitial fibrosis.
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http://dx.doi.org/10.1186/1471-2164-14-275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644490PMC
April 2013

HLA identical non-chimeric and HLA disparate chimeric renal transplant tolerance.

Clin Transpl 2013 :145-56

In this chapter, we describe studies on non-chimeric human leukocyte antigen (HLA) identical tolerance and chimeric HLA disparate tolerance brought about by infusions of hematopoietic stem cells from the renal donor (DHSC). In our HLA identical series, 4 DHSC infusions were administered during the first 9 months posttransplant in a highly immunoregulatory environment using alemtuzumab induction and rapid conversion from early tacrolimus to mycophenolate and sirolimus. This resulted in the generation of recipient T regulatory cells accompanied by genomic indicators, but only transient chimerism. Seven of the first 12 recipients have been immunosuppression-free between 1 1/2 - 4 years with transplant biopsies free of rejection one year after immunosuppression withdrawal. The HLAdisparate group was treated by non-myeloablative conditioning consisting of: 200cGy whole body irradiation; fludarabine; cyclophosphamide; and, perioperative infusion of a product termed FCRx that contained DHSC, T cells, and a unique fraction of bone marrow derived CD8+TCR-alphabeta-negative cells. Five of the first 8 subjects became 100% chimeric in the peripheral blood and have been immunosuppression-free for 2 to 4 years without graft-versus-host-disease and with normal function and transplant biopsies. An additional 12 recipients with shorter follow-up have had similar courses. Those with non-durable chimerism have not been able to have immunosuppression withdrawn but maintain normal renal transplant function. We conclude that non-HLA disparities in renal transplants between HLA identical pairs may not need durable chimerism to induce tolerance provided by DHSC and temporary immunosuppression supporting the development of regulatory T cells. However, more intense conditioning and infusion of FCRx leading to durable chimerism in the absence of graft versus host disease is necessary to induce tolerance in HLA disparate pairs.
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September 2014

Hematopoietic stem cell gene therapy for the multisystemic lysosomal storage disorder cystinosis.

Mol Ther 2013 Feb 23;21(2):433-44. Epub 2012 Oct 23.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders (LSDs). The defective gene is CTNS encoding the lysosomal cystine transporter, cystinosin. Cystine accumulates in all tissues and leads to organ damage including end-stage renal disease. Using the Ctns(-/-) murine model for cystinosis, we tested the use of hematopoietic stem and progenitor cells (HSPC) genetically modified to express a functional CTNS transgene using a self-inactivating-lentiviral vector (SIN-LV). We showed that transduced cells were capable of decreasing cystine content in all tissues and improved kidney function. Transduced HSPC retained their differentiative capabilities, populating all tissue compartments examined and allowing long-term expression of the transgene. Direct correlation between the levels of lentiviral DNA present in the peripheral blood and the levels present in tissues were demonstrated, which could be useful to follow future patients. Using a new model of cystinosis, the DsRed Ctns(-/-) mice, and a LV driving the expression of the fusion protein cystinosin-enhanced green fluorescent protein (eGFP), we showed that cystinosin was transferred from CTNS-expressing cells to Ctns-deficient adjacent cells in vitro and in vivo. This transfer led to cystine decreases in Ctns-deficient cells in vitro. These data suggest that the mechanism of cross-correction is possible in cystinosis.
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http://dx.doi.org/10.1038/mt.2012.214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594011PMC
February 2013
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