Publications by authors named "Daniel R Caffrey"

32 Publications

PFRED: A computational platform for siRNA and antisense oligonucleotides design.

PLoS One 2021 22;16(1):e0238753. Epub 2021 Jan 22.

Simulation and Modeling Sciences, Pfizer, Cambridge, MA, United States of America.

PFRED a software application for the design, analysis, and visualization of antisense oligonucleotides and siRNA is described. The software provides an intuitive user-interface for scientists to design a library of siRNA or antisense oligonucleotides that target a specific gene of interest. Moreover, the tool facilitates the incorporation of various design criteria that have been shown to be important for stability and potency. PFRED has been made available as an open-source project so the code can be easily modified to address the future needs of the oligonucleotide research community. A compiled version is available for downloading at https://github.com/pfred/pfred-gui/releases/tag/v1.0 as a java Jar file. The source code and the links for downloading the precompiled version can be found at https://github.com/pfred.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0238753PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822268PMC
April 2021

Inflammasome-derived cytokine IL18 suppresses amyloid-induced seizures in Alzheimer-prone mice.

Proc Natl Acad Sci U S A 2018 09 20;115(36):9002-9007. Epub 2018 Aug 20.

Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA 01605;

Alzheimer's disease (AD) is characterized by the progressive destruction and dysfunction of central neurons. AD patients commonly have unprovoked seizures compared with age-matched controls. Amyloid peptide-related inflammation is thought to be an important aspect of AD pathogenesis. We previously reported that NLRP3 inflammasome KO mice, when bred into APPswe/PS1ΔE9 (APP/PS1) mice, are completely protected from amyloid-induced AD-like disease, presumably because they cannot produce mature IL1β or IL18. To test the role of IL18, we bred IL18KO mice with APP/PS1 mice. Surprisingly, IL18KO/APP/PS1 mice developed a lethal seizure disorder that was completely reversed by the anticonvulsant levetiracetam. IL18-deficient AD mice showed a lower threshold in chemically induced seizures and a selective increase in gene expression related to increased neuronal activity. IL18-deficient AD mice exhibited increased excitatory synaptic proteins, spine density, and basal excitatory synaptic transmission that contributed to seizure activity. This study identifies a role for IL18 in suppressing aberrant neuronal transmission in AD.
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http://dx.doi.org/10.1073/pnas.1801802115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130368PMC
September 2018

Synonymous Mutations at the Beginning of the Influenza A Virus Hemagglutinin Gene Impact Experimental Fitness.

J Mol Biol 2018 04 18;430(8):1098-1115. Epub 2018 Feb 18.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA. Electronic address:

The fitness effects of synonymous mutations can provide insights into biological and evolutionary mechanisms. We analyzed the experimental fitness effects of all single-nucleotide mutations, including synonymous substitutions, at the beginning of the influenza A virus hemagglutinin (HA) gene. Many synonymous substitutions were deleterious both in bulk competition and for individually isolated clones. Investigating protein and RNA levels of a subset of individually expressed HA variants revealed that multiple biochemical properties contribute to the observed experimental fitness effects. Our results indicate that a structural element in the HA segment viral RNA may influence fitness. Examination of naturally evolved sequences in human hosts indicates a preference for the unfolded state of this structural element compared to that found in swine hosts. Our overall results reveal that synonymous mutations may have greater fitness consequences than indicated by simple models of sequence conservation, and we discuss the implications of this finding for commonly used evolutionary tests and analyses.
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http://dx.doi.org/10.1016/j.jmb.2018.02.009DOI Listing
April 2018

SLC46 Family Transporters Facilitate Cytosolic Innate Immune Recognition of Monomeric Peptidoglycans.

J Immunol 2017 07 24;199(1):263-270. Epub 2017 May 24.

Program in Innate Immunity, Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605; and

Tracheal cytotoxin (TCT), a monomer of DAP-type peptidoglycan from , causes cytopathology in the respiratory epithelia of mammals and robustly triggers the Imd pathway. PGRP-LE, a cytosolic innate immune sensor in , directly recognizes TCT and triggers the Imd pathway, yet the mechanisms by which TCT accesses the cytosol are poorly understood. In this study, we report that CG8046, a SLC46 family transporter, is a novel transporter facilitating cytosolic recognition of TCT, and plays a crucial role in protecting flies against systemic infection. In addition, mammalian SLC46A2s promote TCT-triggered NOD1 activation in human epithelial cell lines, indicating that SLC46As is a conserved group of peptidoglycan transporter contributing to cytosolic immune recognition.
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http://dx.doi.org/10.4049/jimmunol.1600409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513181PMC
July 2017

miR-718 represses proinflammatory cytokine production through targeting phosphatase and tensin homolog (PTEN).

J Biol Chem 2017 04 16;292(14):5634-5644. Epub 2017 Feb 16.

From the Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 and.

Bacterial sepsis involves a complex interaction between the host immune response and bacterial LPS. LPS binds Toll-like receptor (TLR) 4, which leads to the release of proinflammatory cytokines that are essential for a potent innate immune response against pathogens. The innate immune system is tightly regulated, as excessive inflammation can lead to organ failure and death. MicroRNAs have recently emerged as important regulators of the innate immune system. Here we determined the function of miR-718, which is conserved across mammals and overlaps with the 5' UTR of the interleukin 1 receptor-associated kinase () gene. As IRAK1 is a key component of innate immune signaling pathways that are downstream of most TLRs, we hypothesized that miR-718 helps regulate the innate immune response. Activation of TLR4, but not TLR3, induced the expression of miR-718 in macrophages. miR-718 expression was also induced in the spleens of mice upon LPS injection. miR-718 modulates PI3K/Akt signaling by directly down-regulating phosphatase and tensin homolog (PTEN), thereby promoting phosphorylation of Akt, which leads to a decrease in proinflammatory cytokine production. Phosphorylated Akt induces let-7e expression, which, in turn, down-regulates TLR4 and further diminishes TLR4-mediated proinflammatory signals. Decreased miR-718 expression is associated with bacterial burden during infection and alters the infection dynamics of Furthermore, miR-718 regulates the induction of LPS tolerance in macrophages. We propose a role for miR-718 in controlling TLR4 signaling and inflammatory cytokine signaling through a negative feedback regulation loop involving down-regulation of TLR4, IRAK1, and NF-κB.
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http://dx.doi.org/10.1074/jbc.M116.749325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392559PMC
April 2017

Immunobiology of Long Noncoding RNAs.

Annu Rev Immunol 2017 04 11;35:177-198. Epub 2017 Jan 11.

Program in Innate Immunity, Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605; email:

The discovery of long noncoding RNAs (lncRNA) has provided a new perspective on gene regulation in diverse biological contexts. lncRNAs are remarkably versatile molecules that interact with RNA, DNA, or proteins to promote or restrain the expression of protein-coding genes. Activation of immune cells is associated with dynamic changes in expression of genes, the products of which combat infectious microorganisms, initiate repair, and resolve inflammatory responses in cells and tissues. Recent evidence indicates that lncRNAs play important roles in directing the development of diverse immune cells and controlling the dynamic transcriptional programs that are a hallmark of immune cell activation. The importance of these molecules is underscored by their newly recognized roles in inflammatory diseases. In this review, we discuss the contribution of lncRNAs in the development and activation of immune cells and their roles in immune-related diseases. We also discuss challenges faced in identifying biological functions for this large and complex class of genes.
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http://dx.doi.org/10.1146/annurev-immunol-041015-055459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449690PMC
April 2017

A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation.

Cell 2016 Jun;165(7):1672-1685

Program in Innate Immunity, University of Massachusetts Medical School, Worcester, MA 01605, USA; Centre for Molecular Inflammation Research, Department of Cancer Research and Molecular Medicine, NTNU, 7491 Trondheim, Norway. Electronic address:

Long intergenic noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here, we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS localizes at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3' end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses, highlighting the importance of lincRNAs in the immune system.
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http://dx.doi.org/10.1016/j.cell.2016.05.075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289747PMC
June 2016

Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Nat Commun 2016 Feb 9;7:10507. Epub 2016 Feb 9.

Department of Medicine, Division of Infectious Diseases, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.
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http://dx.doi.org/10.1038/ncomms10507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4748124PMC
February 2016

A Balance between Inhibitor Binding and Substrate Processing Confers Influenza Drug Resistance.

J Mol Biol 2016 Feb 4;428(3):538-553. Epub 2015 Dec 4.

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655, USA. Electronic address:

The therapeutic benefits of the neuraminidase (NA) inhibitor oseltamivir are dampened by the emergence of drug resistance mutations in influenza A virus (IAV). To investigate the mechanistic features that underlie resistance, we developed an approach to quantify the effects of all possible single-nucleotide substitutions introduced into important regions of NA. We determined the experimental fitness effects of 450 nucleotide mutations encoding positions both surrounding the active site and at more distant sites in an N1 strain of IAV in the presence and absence of oseltamivir. NA mutations previously known to confer oseltamivir resistance in N1 strains, including H275Y and N295S, were adaptive in the presence of drug, indicating that our experimental system captured salient features of real-world selection pressures acting on NA. We identified mutations, including several at position 223, that reduce the apparent affinity for oseltamivir in vitro. Position 223 of NA is located adjacent to a hydrophobic portion of oseltamivir that is chemically distinct from the substrate, making it a hotspot for substitutions that preferentially impact drug binding relative to substrate processing. Furthermore, two NA mutations, K221N and Y276F, each reduce susceptibility to oseltamivir by increasing NA activity without altering drug binding. These results indicate that competitive expansion of IAV in the face of drug pressure is mediated by a balance between inhibitor binding and substrate processing.
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http://dx.doi.org/10.1016/j.jmb.2015.11.027DOI Listing
February 2016

The domain structure and distribution of Alu elements in long noncoding RNAs and mRNAs.

RNA 2016 Feb 10;22(2):254-64. Epub 2015 Dec 10.

Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

Approximately 75% of the human genome is transcribed and many of these spliced transcripts contain primate-specific Alu elements, the most abundant mobile element in the human genome. The majority of exonized Alu elements are located in long noncoding RNAs (lncRNAs) and the untranslated regions of mRNA, with some performing molecular functions. To further assess the potential for Alu elements to be repurposed as functional RNA domains, we investigated the distribution and evolution of Alu elements in spliced transcripts. Our analysis revealed that Alu elements are underrepresented in mRNAs and lncRNAs, suggesting that most exonized Alu elements arising in the population are rare or deleterious to RNA function. When mRNAs and lncRNAs retain exonized Alu elements, they have a clear preference for Alu dimers, left monomers, and right monomers. mRNAs often acquire Alu elements when their genes are duplicated within Alu-rich regions. In lncRNAs, reverse-oriented Alu elements are significantly enriched and are not restricted to the 3' and 5' ends. Both lncRNAs and mRNAs primarily contain the Alu J and S subfamilies that were amplified relatively early in primate evolution. Alu J subfamilies are typically overrepresented in lncRNAs, whereas the Alu S dimer is overrepresented in mRNAs. The sequences of Alu dimers tend to be constrained in both lncRNAs and mRNAs, whereas the left and right monomers are constrained within particular Alu subfamilies and classes of RNA. Collectively, these findings suggest that Alu-containing RNAs are capable of forming stable structures and that some of these Alu domains might have novel biological functions.
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http://dx.doi.org/10.1261/rna.048280.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712675PMC
February 2016

Cutting Edge: A Natural Antisense Transcript, AS-IL1α, Controls Inducible Transcription of the Proinflammatory Cytokine IL-1α.

J Immunol 2015 Aug 15;195(4):1359-63. Epub 2015 Jul 15.

Program in Innate Immunity, Division of Infectious Diseases, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605;

Natural antisense transcripts (NATs) are a class of long noncoding RNAs (lncRNAs) that are complementary to other protein-coding genes. Although thousands of NATs are encoded by mammalian genomes, their functions in innate immunity are unknown. In this study, we identified and characterized a novel NAT, AS-IL1α, which is partially complementary to IL-1α. Similar to IL-1α, AS-IL1α is expressed at low levels in resting macrophages and is induced following infection with Listeria monocytogenes or stimulation with TLR ligands (Pam3CSK4, LPS, polyinosinic-polycytidylic acid). Inducible expression of IL-1α mRNA and protein were significantly reduced in macrophages expressing shRNA that target AS-IL1α. AS-IL1α is located in the nucleus and did not alter the stability of IL-1α mRNA. Instead, AS-IL1α was required for the recruitment of RNA polymerase II to the IL-1α promoter. In summary, our studies identify AS-IL1α as an important regulator of IL-1α transcription during the innate immune response.
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http://dx.doi.org/10.4049/jimmunol.1500264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4530055PMC
August 2015

Positive Selection Drives Preferred Segment Combinations during Influenza Virus Reassortment.

Mol Biol Evol 2015 Jun 23;32(6):1519-32. Epub 2015 Feb 23.

Department of Medicine, University of Massachusetts Medical School, Worcester.

Influenza A virus (IAV) has a segmented genome that allows for the exchange of genome segments between different strains. This reassortment accelerates evolution by breaking linkage, helping IAV cross species barriers to potentially create highly virulent strains. Challenges associated with monitoring the process of reassortment in molecular detail have limited our understanding of its evolutionary implications. We applied a novel deep sequencing approach with quantitative analysis to assess the in vitro temporal evolution of genomic reassortment in IAV. The combination of H1N1 and H3N2 strains reproducibly generated a new H1N2 strain with the hemagglutinin and nucleoprotein segments originating from H1N1 and the remaining six segments from H3N2. By deep sequencing the entire viral genome, we monitored the evolution of reassortment, quantifying the relative abundance of all IAV genome segments from the two parent strains over time and measuring the selection coefficients of the reassorting segments. Additionally, we observed several mutations coemerging with reassortment that were not found during passaging of pure parental IAV strains. Our results demonstrate how reassortment of the segmented genome can accelerate viral evolution in IAV, potentially enabled by the emergence of a small number of individual mutations.
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http://dx.doi.org/10.1093/molbev/msv044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462674PMC
June 2015

Influenza virus drug resistance: a time-sampled population genetics perspective.

PLoS Genet 2014 Feb 27;10(2):e1004185. Epub 2014 Feb 27.

School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland ; Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland.

The challenge of distinguishing genetic drift from selection remains a central focus of population genetics. Time-sampled data may provide a powerful tool for distinguishing these processes, and we here propose approximate Bayesian, maximum likelihood, and analytical methods for the inference of demography and selection from time course data. Utilizing these novel statistical and computational tools, we evaluate whole-genome datasets of an influenza A H1N1 strain in the presence and absence of oseltamivir (an inhibitor of neuraminidase) collected at thirteen time points. Results reveal a striking consistency amongst the three estimation procedures developed, showing strongly increased selection pressure in the presence of drug treatment. Importantly, these approaches re-identify the known oseltamivir resistance site, successfully validating the approaches used. Enticingly, a number of previously unknown variants have also been identified as being positively selected. Results are interpreted in the light of Fisher's Geometric Model, allowing for a quantification of the increased distance to optimum exerted by the presence of drug, and theoretical predictions regarding the distribution of beneficial fitness effects of contending mutations are empirically tested. Further, given the fit to expectations of the Geometric Model, results suggest the ability to predict certain aspects of viral evolution in response to changing host environments and novel selective pressures.
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http://dx.doi.org/10.1371/journal.pgen.1004185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937227PMC
February 2014

Long noncoding RNAs in innate and adaptive immunity.

Curr Opin Immunol 2014 Feb 22;26:140-6. Epub 2013 Dec 22.

Program in Innate Immunity, Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA. Electronic address:

The differentiation and activation of both innate and adaptive immune cells is highly dependent on a coordinated set of transcriptional and post-transcriptional events. Chromatin-modifiers and transcription factors regulate the accessibility and transcription of immune genes, respectively. Immune cells also express miRNA and RNA-binding proteins that provide an additional layer of regulation at the mRNA level. However, long noncoding RNAs (lncRNAs), which have been primarily studied in the context of genomic imprinting, cancer, and cell differentiation, are now emerging as important regulators of immune cell differentiation and activation. In this review, we provide a brief overview of lncRNAs, their known functions in immunity, and discuss their potential to be more broadly involved in other aspects of the immune response.
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http://dx.doi.org/10.1016/j.coi.2013.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3932021PMC
February 2014

The transcriptional repressor BLIMP1 curbs host defenses by suppressing expression of the chemokine CCL8.

J Immunol 2014 Mar 29;192(5):2291-304. Epub 2014 Jan 29.

Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605;

The transcriptional repressor B lymphocyte-induced maturation protein 1 (BLIMP1) is a master regulator of B and T cell differentiation. To examine the role of BLIMP1 in innate immunity, we used a conditional knockout (CKO) of Blimp1 in myeloid cells and found that Blimp1 CKO mice were protected from lethal infection induced by Listeria monocytogenes. Transcriptome analysis of Blimp1 CKO macrophages identified the murine chemokine (C-C motif) ligand 8, CCL8, as a direct target of Blimp1-mediated transcriptional repression in these cells. BLIMP1-deficient macrophages expressed elevated levels of Ccl8, and consequently Blimp1 CKO mice had higher levels of circulating CCL8, resulting in increased neutrophils in the peripheral blood, promoting a more aggressive antibacterial response. Mice lacking the Ccl8 gene were more susceptible to L. monocytogenes infection than were wild-type mice. Although CCL8 failed to recruit neutrophils directly, it was chemotactic for γ/δ T cells, and CCL8-responsive γ/δ T cells were enriched for IL-17F. Finally, CCL8-mediated enhanced clearance of L. monocytogenes was dependent on γ/δ T cells. Collectively, these data reveal an important role for BLIMP1 in modulating host defenses by suppressing expression of the chemokine CCL8.
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http://dx.doi.org/10.4049/jimmunol.1301799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943885PMC
March 2014

Distinct cellular origins for serotonin-expressing and enterochromaffin-like cells in the gastric corpus.

Gastroenterology 2014 Mar 4;146(3):754-764.e3. Epub 2013 Dec 4.

Division of Gastroenterology, University of Massachusetts Medical School, Worcester, Massachusetts; Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts. Electronic address:

Background & Aims: The alimentary tract contains a diffuse endocrine system comprising enteroendocrine cells that secrete peptides or biogenic amines to regulate digestion, insulin secretion, food intake, and energy homeostasis. Lineage analysis in the stomach revealed that a significant fraction of endocrine cells in the gastric corpus did not arise from Neurogenin3 (Neurog3)-expressing cells, unlike enteroendocrine cells elsewhere in the digestive tract. We aimed to isolate enriched serotonin-secreting and enterochromaffin-like (ECL) cells from the stomach and to clarify their cellular origin.

Methods: We used Neurogenic differentiation 1 (NeuroD1) and Neurog3 lineage analysis and examined the differentiation of serotonin-producing and ECL cells in stomach tissues of NeuroD1-cre;ROSA(tdTom), tryptophan hydroxylase 1 (Tph1)-cyan fluorescent protein (CFP), c-Kit(wsh/wsh), and Neurog3Cre;ROSA(tdTom) mice by immunohistochemistry. We used fluorescence-activated cell sorting to isolate each cell type for gene expression analysis. We also performed RNA sequencing analysis of ECL cells.

Results: Neither serotonin-secreting nor ECL cells of the corpus arose from cells expressing NeuroD1. Serotonin-secreting cells expressed a number of mast cell genes but not genes associated with endocrine differentiation; they did not develop in c-Kit(wsh/wsh) mice and were labeled with transplanted bone marrow cells. RNA sequencing analysis of ECL cells revealed high expression levels of many genes common to endocrine cells, including transcription factors, hormones, ion channels, and solute transporters but not markers of bone marrow cells.

Conclusions: Serotonin-expressing cells of the gastric corpus of mice appear to be bone marrow-derived mucosal mast cells. Gene expression analysis of ECL cells indicated that they are endocrine cells of epithelial origin that do not express the same transcription factors as their intestinal enteroendocrine cell counterparts.
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http://dx.doi.org/10.1053/j.gastro.2013.11.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943955PMC
March 2014

Evolution of the influenza A virus genome during development of oseltamivir resistance in vitro.

J Virol 2014 Jan 23;88(1):272-81. Epub 2013 Oct 23.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y resistance mutation fixed reproducibly within the population. The presence of the H274Y mutation in the viral population, at either a low or a high frequency, led to measurable changes in the neuraminidase inhibition assay. Surprisingly, fixation of the resistance mutation was not accompanied by alterations of viral population diversity or differentiation, and oseltamivir did not alter the selective environment. While the neighboring K248E mutation was also a target of positive selection prior to H274Y fixation, H274Y was the primary beneficial mutation in the population. In addition, once evolved, the H274Y mutation persisted after the withdrawal of the drug, even when not fixed in viral populations. We conclude that only selection of H274Y is required for oseltamivir resistance and that H274Y is not deleterious in the absence of the drug. These collective results could offer an explanation for the recent reproducible rise in oseltamivir resistance in seasonal H1N1 IAV strains in humans.
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http://dx.doi.org/10.1128/JVI.01067-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911755PMC
January 2014

Overexpression of membrane-bound fas ligand (CD95L) exacerbates autoimmune disease and renal pathology in pristane-induced lupus.

J Immunol 2013 Sep 5;191(5):2104-14. Epub 2013 Aug 5.

Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Loss-of-function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-tetramethylpentadecane (TMPD), a hydrocarbon oil commonly known as pristane, which induces systemic lupus erythematosus-like disease. Although Fas/Fas ligand (FasL) interactions have been strongly implicated in the activation-induced cell death of both lymphocytes and other APCs, FasL can also trigger the production of proinflammatory cytokines. FasL is a transmembrane protein with a matrix metalloproteinase cleavage site in the ectodomain. Matrix metalloproteinase cleavage inactivates membrane-bound FasL and releases a soluble form reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the proapoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation to produce the deleted cleavage site (ΔCS) mouse line. ΔCS mice express higher levels of membrane-bound FasL than do wild-type mice and fail to release soluble FasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and ΔCS BALB/c mice were compared with control TMPD-injected BALB/c mice. We found that FasL deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. In contrast, ΔCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and macrophages, a profound change in anti-nuclear Ab specificity, and markedly increased proteinuria and kidney pathology compared with controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL proinflammatory activity.
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http://dx.doi.org/10.4049/jimmunol.1300341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4219920PMC
September 2013

A long noncoding RNA mediates both activation and repression of immune response genes.

Science 2013 Aug 1;341(6147):789-92. Epub 2013 Aug 1.

Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.

An inducible program of inflammatory gene expression is central to antimicrobial defenses. This response is controlled by a collaboration involving signal-dependent activation of transcription factors, transcriptional co-regulators, and chromatin-modifying factors. We have identified a long noncoding RNA (lncRNA) that acts as a key regulator of this inflammatory response. Pattern recognition receptors such as the Toll-like receptors induce the expression of numerous lncRNAs. One of these, lincRNA-Cox2, mediates both the activation and repression of distinct classes of immune genes. Transcriptional repression of target genes is dependent on interactions of lincRNA-Cox2 with heterogeneous nuclear ribonucleoprotein A/B and A2/B1. Collectively, these studies unveil a central role of lincRNA-Cox2 as a broad-acting regulatory component of the circuit that controls the inflammatory response.
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http://dx.doi.org/10.1126/science.1240925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4376668PMC
August 2013

RNA helicase signaling is critical for type i interferon production and protection against Rift Valley fever virus during mucosal challenge.

J Virol 2013 May 13;87(9):4846-60. Epub 2013 Feb 13.

Center for Global Health and Diseases, Case Western Reserve University, Cleveland, Ohio, USA.

Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both in vitro assays and in vivo mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV.
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http://dx.doi.org/10.1128/JVI.01997-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624317PMC
May 2013

Evolution of specific protein-protein interaction sites following gene duplication.

J Mol Biol 2012 Oct 9;423(2):257-72. Epub 2012 Jul 9.

Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Gene duplication is a common evolutionary process that leads to the expansion and functional diversification of protein subfamilies. The evolutionary events that cause paralogous proteins to bind different protein ligands (functionally diverged interfaces) are investigated and compared to paralogous proteins that bind the same protein ligand (functionally preserved interfaces). We find that functionally diverged interfaces possess more subfamily-specific residues than functionally preserved interfaces. These subfamily-specific residues are usually partially buried at the interface rim and achieve specific binding through optimized hydrogen bond geometries. In addition to optimized hydrogen bond geometries, side-chain modeling experiments suggest that steric effects are also important for binding specificity. Residues that are completely buried at the interface hub are also less conserved in functionally diverged interfaces than in functionally preserved interfaces. Consistent with this finding, hub residues contribute less to free energy of binding in functionally diverged interfaces than in functionally preserved interfaces. Therefore, we propose that protein binding is a delicate balance between binding affinity that primarily occurs at the interface hub and binding specificity that primarily occurs at the interface rim.
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http://dx.doi.org/10.1016/j.jmb.2012.06.039DOI Listing
October 2012

Immunology. Select inflammasome assembly.

Science 2012 Apr;336(6080):420-1

Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.

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http://dx.doi.org/10.1126/science.1222362DOI Listing
April 2012

siRNA off-target effects can be reduced at concentrations that match their individual potency.

PLoS One 2011 5;6(7):e21503. Epub 2011 Jul 5.

Pfizer Inc, Cambridge, Massachusetts, United States of America.

Small interfering RNAs (siRNAs) are routinely used to reduce mRNA levels for a specific gene with the goal of studying its function. Several studies have demonstrated that siRNAs are not always specific and can have many off-target effects. The 3' UTRs of off-target mRNAs are often enriched in sequences that are complementary to the seed-region of the siRNA. We demonstrate that siRNA off-targets can be significantly reduced when cells are treated with a dose of siRNA that is relatively low (e.g. 1 nM), but sufficient to effectively silence the intended target. The reduction in off-targets was demonstrated for both modified and unmodified siRNAs that targeted either STAT3 or hexokinase II. Low concentrations reduced silencing of transcripts with complementarity to the seed region of the siRNA. Similarly, off-targets that were not complementary to the siRNA were reduced at lower doses, including up-regulated genes that are involved in immune response. Importantly, the unintended induction of caspase activity following treatment with a siRNA that targeted hexokinase II was also shown to be a concentration-dependent off-target effect. We conclude that off-targets and their related phenotypic effects can be reduced for certain siRNA that potently silence their intended target at low concentrations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021503PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3130022PMC
December 2011

AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC.

Nature 2009 Mar 21;458(7237):514-8. Epub 2009 Jan 21.

Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

The innate immune system senses nucleic acids by germline-encoded pattern recognition receptors. RNA is sensed by Toll-like receptor members TLR3, TLR7 and TLR8, or by the RNA helicases RIG-I (also known as DDX58) and MDA-5 (IFIH1). Little is known about sensors for cytoplasmic DNA that trigger antiviral and/or inflammatory responses. The best characterized of these responses involves activation of the TANK-binding kinase (TBK1)-interferon regulatory factor 3 (IRF3) signalling axis to trigger transcriptional induction of type I interferon genes. A second, less well-defined pathway leads to the activation of an 'inflammasome' that, via caspase-1, controls the catalytic cleavage of the pro-forms of the cytokines IL1beta and IL18 (refs 6, 7). Using mouse and human cells, here we identify the PYHIN (pyrin and HIN domain-containing protein) family member absent in melanoma 2 (AIM2) as a receptor for cytosolic DNA, which regulates caspase-1. The HIN200 domain of AIM2 binds to DNA, whereas the pyrin domain (but not that of the other PYHIN family members) associates with the adaptor molecule ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain) to activate both NF-kappaB and caspase-1. Knockdown of Aim2 abrogates caspase-1 activation in response to cytoplasmic double-stranded DNA and the double-stranded DNA vaccinia virus. Collectively, these observations identify AIM2 as a new receptor for cytoplasmic DNA, which forms an inflammasome with the ligand and ASC to activate caspase-1.
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http://dx.doi.org/10.1038/nature07725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726264PMC
March 2009

Prediction of specificity-determining residues for small-molecule kinase inhibitors.

BMC Bioinformatics 2008 Nov 25;9:491. Epub 2008 Nov 25.

Pfizer Research Technology Center, 620 Memorial Drive, Cambridge, MA 02139, USA.

Background: Designing small-molecule kinase inhibitors with desirable selectivity profiles is a major challenge in drug discovery. A high-throughput screen for inhibitors of a given kinase will typically yield many compounds that inhibit more than one kinase. A series of chemical modifications are usually required before a compound exhibits an acceptable selectivity profile. Rationalizing the selectivity profile for a small-molecule inhibitor in terms of the specificity-determining kinase residues for that molecule can be an important step toward the goal of developing selective kinase inhibitors.

Results: Here we describe S-Filter, a method that combines sequence and structural information to predict specificity-determining residues for a small molecule and its kinase selectivity profile. Analysis was performed on seven selective kinase inhibitors where a structural basis for selectivity is known. S-Filter correctly predicts specificity determinants that were described by independent groups. S-Filter also predicts a number of novel specificity determinants that can often be justified by further structural comparison.

Conclusion: S-Filter is a valuable tool for analyzing kinase selectivity profiles. The method identifies potential specificity determinants that are not readily apparent, and provokes further investigation at the structural level.
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http://dx.doi.org/10.1186/1471-2105-9-491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655090PMC
November 2008

PFAAT version 2.0: a tool for editing, annotating, and analyzing multiple sequence alignments.

BMC Bioinformatics 2007 Oct 11;8:381. Epub 2007 Oct 11.

Pfizer Global Research and Development, 620 Memorial Drive, Cambridge, MA 02139, USA.

Background: By virtue of their shared ancestry, homologous sequences are similar in their structure and function. Consequently, multiple sequence alignments are routinely used to identify trends that relate to function. This type of analysis is particularly productive when it is combined with structural and phylogenetic analysis.

Results: Here we describe the release of PFAAT version 2.0, a tool for editing, analyzing, and annotating multiple sequence alignments. Support for multiple annotations is a key component of this release as it provides a framework for most of the new functionalities. The sequence annotations are accessible from the alignment and tree, where they are typically used to label sequences or hyperlink them to related databases. Sequence annotations can be created manually or extracted automatically from UniProt entries. Once a multiple sequence alignment is populated with sequence annotations, sequences can be easily selected and sorted through a sophisticated search dialog. The selected sequences can be further analyzed using statistical methods that explicitly model relationships between the sequence annotations and residue properties. Residue annotations are accessible from the alignment viewer and are typically used to designate binding sites or properties for a particular residue. Residue annotations are also searchable, and allow one to quickly select alignment columns for further sequence analysis, e.g. computing percent identities. Other features include: novel algorithms to compute sequence conservation, mapping conservation scores to a 3D structure in Jmol, displaying secondary structure elements, and sorting sequences by residue composition.

Conclusion: PFAAT provides a framework whereby end-users can specify knowledge for a protein family in the form of annotation. The annotations can be combined with sophisticated analysis to test hypothesis that relate to sequence, structure and function.
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http://dx.doi.org/10.1186/1471-2105-8-381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2092438PMC
October 2007

Structure-based maximal affinity model predicts small-molecule druggability.

Nat Biotechnol 2007 Jan;25(1):71-5

Department of Molecular Informatics, Research Technology Center, Pfizer Global Research & Development, Cambridge, Massachusetts 02139, USA.

Lead generation is a major hurdle in small-molecule drug discovery, with an estimated 60% of projects failing from lack of lead matter or difficulty in optimizing leads for drug-like properties. It would be valuable to identify these less-druggable targets before incurring substantial expenditure and effort. Here we show that a model-based approach using basic biophysical principles yields good prediction of druggability based solely on the crystal structure of the target binding site. We quantitatively estimate the maximal affinity achievable by a drug-like molecule, and we show that these calculated values correlate with drug discovery outcomes. We experimentally test two predictions using high-throughput screening of a diverse compound collection. The collective results highlight the utility of our approach as well as strategies for tackling difficult targets.
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http://dx.doi.org/10.1038/nbt1273DOI Listing
January 2007

Of mice and man: TLR11 (finally) finds profilin.

Trends Immunol 2005 Oct;26(10):509-11

Division of Infectious Disease & Immunology, Department of Medicine, The University of Massachusetts Medical School, Worcester, MA 01605, USA.

Toll-like receptors (TLRs) are primordial pathogen-recognition proteins that function as sentinels for the innate immune system. One of the TLR mysteries relates to TLR11, a receptor present in mice, but not humans, and known to recognize uropathogenic Escherichia coli. The first defined ligand for TLR11 has now been described as a profilin-like protein from Toxoplasma gondii. This discovery potentially gives us important clues as to how a gene expressed in mice, but not humans, actually relates to human infectious diseases.
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http://dx.doi.org/10.1016/j.it.2005.08.006DOI Listing
October 2005

Are protein-protein interfaces more conserved in sequence than the rest of the protein surface?

Protein Sci 2004 Jan;13(1):190-202

Pfizer Discovery Technology Center, Cambridge, Massachusetts 02139, USA.

Protein interfaces are thought to be distinguishable from the rest of the protein surface by their greater degree of residue conservation. We test the validity of this approach on an expanded set of 64 protein-protein interfaces using conservation scores derived from two multiple sequence alignment types, one of close homologs/orthologs and one of diverse homologs/paralogs. Overall, we find that the interface is slightly more conserved than the rest of the protein surface when using either alignment type, with alignments of diverse homologs showing marginally better discrimination. However, using a novel surface-patch definition, we find that the interface is rarely significantly more conserved than other surface patches when using either alignment type. When an interface is among the most conserved surface patches, it tends to be part of an enzyme active site. The most conserved surface patch overlaps with 39% (+/- 28%) and 36% (+/- 28%) of the actual interface for diverse and close homologs, respectively. Contrary to results obtained from smaller data sets, this work indicates that residue conservation is rarely sufficient for complete and accurate prediction of protein interfaces. Finally, we find that obligate interfaces differ from transient interfaces in that the former have significantly fewer alignment gaps at the interface than the rest of the protein surface, as well as having buried interface residues that are more conserved than partially buried interface residues.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2286531PMC
http://dx.doi.org/10.1110/ps.03323604DOI Listing
January 2004

LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF.

J Exp Med 2003 Oct 29;198(7):1043-55. Epub 2003 Sep 29.

Division of Infectious Disease and Immunology, Department of Medicine, The University of Massachusetts Medical School, Worcester, MA 01605, USA.

Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF)-related adaptor molecule (TRAM) is the fourth TIR domain-containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-kappaB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-alpha/beta, regulated on activation, normal T cell expressed and secreted (RANTES), and gamma interferon-inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor-like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.
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http://dx.doi.org/10.1084/jem.20031023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2194210PMC
October 2003