Publications by authors named "Steven R Head"

81 Publications

Krüppel-like factor-4 and Krüppel-like factor-2 are important regulators of joint tissue cells and protect against tissue destruction and inflammation in osteoarthritis.

Ann Rheum Dis 2022 May 9. Epub 2022 May 9.

Department of Molecular Medicine, Scripps Research, La Jolla, California, USA

Objectives: Analysing expression patterns of Krüppel-like factor (KLF) transcription factors in normal and osteoarthritis (OA) human cartilage, and determining functions and mechanisms of KLF4 and KLF2 in joint homoeostasis and OA pathogenesis.

Methods: Experimental approaches included human joint tissues cells, transgenic mice and mouse OA model with viral KLF4 gene delivery to demonstrate therapeutic benefit in structure and pain improvement. Mechanistic studies applied global gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq).

Results: Several KLF genes were significantly decreased in OA cartilage. Among them, KLF4 and KLF2 were strong inducers of cartilage collagen genes and Proteoglycan-4. Cartilage-specific deletion of in mature mice aggravated severity of experimental OA. Transduction of human chondrocytes with Adenovirus (Ad) expressing KLF4 or KLF2 enhanced expression of major cartilage extracellular matrix (ECM) genes and SRY-box transcription factor-9, and suppressed mediators of inflammation and ECM-degrading enzymes. Ad-KLF4 and Ad-KLF2 enhanced similar protective functions in meniscus cells and synoviocytes, and promoted chondrocytic differentiation of human mesenchymal stem cells. Viral KLF4 delivery into mouse knees reduced severity of OA-associated changes in cartilage, meniscus and synovium, and improved pain behaviours. ChIP-seq analysis suggested that KLF4 directly bound cartilage signature genes. Ras-related protein-1 signalling was the most enriched pathway in KLF4-transduced cells, and its signalling axis was involved in upregulating cartilage ECM genes by KLF4 and KLF2.

Conclusions: KLF4 and KLF2 may be central transcription factors that increase protective and regenerative functions in joint tissue cells, suggesting that KLF gene transfer or molecules upregulating KLFs are therapeutic candidates for OA.
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http://dx.doi.org/10.1136/annrheumdis-2021-221867DOI Listing
May 2022

Real-time digital polymerase chain reaction (PCR) as a novel technology improves limit of detection for rare allele assays.

Transl Lung Cancer Res 2021 Dec;10(12):4336-4352

State Key Laboratory of Molecular Oncology, Department of Medical Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

Background: Tumor heterogeneity may lead to false negative test results for tissue biopsy-based companion diagnostic tests. Real-time polymerase chain reaction (PCR) and digital PCR assays are used to detect rare alleles in cell-free circulating DNA for liquid biopsies; however, those tests lack strong sensitivity at low allele frequencies. We show here a novel real-time digital PCR instrument that utilizes cycle-based amplification curves to further improve the sensitivity and quantification accuracy of digital PCR.

Methods: The novel real-time digital PCR instrument was compared to an endpoint digital PCR system to determine the sensitivity and quantification accuracy of both instruments. Samples were all thermal cycled on the real-time digital PCR instrument but were analyzed on both endpoint and real-time digital PCR instruments to compare the performance without introducing other variables. Contrived samples for epidermal growth factor receptor () exon 19 deletion, T790M, and L858R point mutations as well as human epidermal growth factor receptor 2 () amplification were tested. Different mutant allele frequencies and wildtype to mutant gene copy number ratios were tested for and , respectively.

Results: By removing false positive datapoints using real-time amplification curves, real-time digital PCR improved sensitivity by lowering the baseline for wildtype samples. For 19del assay, samples with 2 or more fluorescein amidite (FAM) labeled positive wells are determined positive by real-time digital PCR, while a minimum of 5 FAM positive datapoints is needed by endpoint digital PCR. Improved limit of detection for 19del mutation was also observed. Real-time digital PCR also had better quantification accuracy and sensitivity, resulting in the mutant allele frequencies being closer to the expected values for all mutations, especially at very low allele frequencies. However, at high allele frequencies or for gene amplification assays, real-time digital PCR is comparable with endpoint digital PCR.

Conclusions: This novel technology with improved sensitivity is important and needed because it addresses current issues with liquid biopsy tests. Due to limited amounts of circulating tumor DNA (ctDNA) obtained for liquid biopsy tests, few copies of mutant alleles are expected. With the lower baseline of real-time digital PCR, false negative test results from tissue biopsy would be more effectively reduced, leading to more patients receiving the targeted therapy they need for better survival.
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http://dx.doi.org/10.21037/tlcr-21-728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8743530PMC
December 2021

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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9034438PMC
November 2021

Increased sensitivity using real-time dPCR for detection of SARS-CoV-2.

Biotechniques 2021 01 23;70(1):7-20. Epub 2020 Nov 23.

Gnomegen, 6440 Lusk Blvd, D207, San Diego, CA 92121, USA.

A real-time dPCR system was developed to improve the sensitivity, specificity and quantification accuracy of end point dPCR. We compared three technologies - real-time qPCR, end point dPCR and real-time dPCR - in the context of SARS-CoV-2. Some improvement in limit of detection was obtained with end point dPCR compared with real-time qPCR, and the limit of detection was further improved with the newly developed real-time dPCR technology through removal of false-positive signals. Real-time dPCR showed increased linear dynamic range compared with end point dPCR based on quantitation from amplification curves. Real-time dPCR can improve the performance of TaqMan assays beyond real-time qPCR and end point dPCR with better sensitivity and specificity, absolute quantification and a wider linear range of detection.
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http://dx.doi.org/10.2144/btn-2020-0133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888512PMC
January 2021

Feasibility and Comparison Study of Fecal Sample Collection Methods in Healthy Volunteers and Solid Organ Transplant Recipients Using 16S rRNA and Metagenomics Approaches.

Biopreserv Biobank 2020 Oct 21;18(5):425-440. Epub 2020 Aug 21.

Scripps Clinic Bio-Repository and Bio-Informatics Core, La Jolla, California, USA.

The human microbiome encompasses a variety of microorganisms that change dynamically and are in close contact with the body. The microbiome influences health and homeostasis, as well as the immune system, and any significant change in this equilibrium (dysbiosis) triggers both acute and chronic health conditions. Microbiome research has surged, in part, due to advanced sequencing technologies enabling rapid, accurate, and cost-effective identification of the microbiome. A major prerequisite for stool sample collection to study the gut microbiome in longitudinal prospective studies requires standardized protocols that can be easily replicated. However, there are still significant bottlenecks to stool specimen collection that contribute to low patient retention rates in microbiome studies. These barriers are further exacerbated in solid organ transplant recipients where diarrhea is estimated to occur in up to half the patient population. We sought to test two relatively easy sample collection methods (fecal swab and wipes) and compare them to the more cumbersome "gold" standard collection method (scoop) using two different sequencing technologies (16S ribosomal RNA sequencing and shotgun metagenomics). Our comparison of the collection methods shows that both the swabs and the wipes are comparable to the scoop method in terms of bacterial abundance and diversity. The swabs, however, were closer in representation to the scoop and were easier to collect and process compared to the wipes. Potential contamination of the swab and the wipe samples by abundant skin commensals was low in our analysis. Comparison of the two sequencing technologies showed that they were complementary, and that 16S sequencing provided enough coverage to detect and differentiate between bacterial species identified in the collected samples. Our pilot study demonstrates that alternative collection methods for stool sampling are a viable option in clinical applications, such as organ transplant studies. The use of these methods may result in better patient retention recruitment rates in serial microbiome studies.
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http://dx.doi.org/10.1089/bio.2020.0032DOI Listing
October 2020

Identification of Key Determinants of Staphylococcus aureus Vaginal Colonization.

mBio 2019 12 24;10(6). Epub 2019 Dec 24.

Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA

is an important pathogen responsible for nosocomial and community-acquired infections in humans, and methicillin-resistant (MRSA) infections have continued to increase despite widespread preventative measures. can colonize the female vaginal tract, and reports have suggested an increase in MRSA infections in pregnant and postpartum women as well as outbreaks in newborn nurseries. Currently, little is known about specific factors that promote MRSA vaginal colonization and subsequent infection. To study colonization of the female reproductive tract in a mammalian system, we developed a mouse model of vaginal carriage and demonstrated that both hospital-associated and community-associated MRSA isolates can colonize the murine vaginal tract. Immunohistochemical analysis revealed an increase in neutrophils in the vaginal lumen during MRSA colonization. Additionally, we observed that a mutant lacking fibrinogen binding adhesins exhibited decreased persistence within the mouse vagina. To further identify novel factors that promote vaginal colonization, we performed RNA sequencing to determine the transcriptome of MRSA growing during vaginal carriage at 5 h, 1 day, and 3 days postinoculation. Over 25% of the bacterial genes were differentially regulated at all time points during colonization compared to laboratory cultures. The most highly induced genes were those involved in iron acquisition, including the Isd system and siderophore transport systems. Mutants deficient in these pathways did not persist as well during colonization. These results reveal that fibrinogen binding and the capacity to overcome host nutritional limitation are important determinants of MRSA vaginal colonization. is an opportunistic pathogen able to cause a wide variety of infections in humans. Recent reports have suggested an increasing prevalence of MRSA in pregnant and postpartum women, coinciding with the increased incidence of MRSA infections in neonatal intensive care units (NICUs) and newborn nurseries. Vertical transmission from mothers to infants at delivery is a likely route of MRSA acquisition by the newborn; however, essentially nothing is known about host and bacterial factors that influence MRSA carriage in the vagina. Here, we established a mouse model of vaginal colonization and observed that multiple MRSA strains can persist in the vaginal tract. Additionally, we determined that MRSA interactions with fibrinogen and iron uptake can promote vaginal persistence. This study is the first to identify molecular mechanisms which govern vaginal colonization by MRSA, the critical initial step preceding infection and neonatal transmission.
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http://dx.doi.org/10.1128/mBio.02321-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935855PMC
December 2019

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage.

Nat Microbiol 2019 10 8;4(10):1727-1736. Epub 2019 Jul 8.

National Food Institute, Research Group for Genomic Epidemiology, Technical University of Denmark, Kongens Lyngby, Denmark.

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.
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http://dx.doi.org/10.1038/s41564-019-0494-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7440971PMC
October 2019

Seasonal dynamics of DNA and RNA viral bioaerosol communities in a daycare center.

Microbiome 2019 04 1;7(1):53. Epub 2019 Apr 1.

Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA, 24061, USA.

Background: Viruses play an important role in ecosystems, including the built environment (BE). While numerous studies have characterized bacterial and fungal microbiomes in the BE, few have focused on the viral microbiome (virome). Longitudinal microbiome studies provide insight into the stability and dynamics of microbial communities; however, few such studies exist for the microbiome of the BE, and most have focused on bacteria. Here, we present a longitudinal, metagenomic-based analysis of the airborne DNA and RNA virome of a children's daycare center. Specifically, we investigate how the airborne virome varies as a function of season and human occupancy, and we identify possible sources of the viruses and their hosts, mainly humans, animals, plants, and insects.

Results: Season strongly influenced the airborne viral community composition, and a single sample collected when the daycare center was unoccupied suggested that occupancy also influenced the community. The pattern of influence differed between DNA and RNA viromes. Human-associated viruses were much more diverse and dominant in the winter, while the summertime virome contained a high relative proportion and diversity of plant-associated viruses.

Conclusions: This airborne microbiome in this building exhibited seasonality in its viral community but not its bacterial community. Human occupancy influenced both types of communities. By adding new data about the viral microbiome to complement burgeoning information about the bacterial and fungal microbiomes, this study contributes to a more complete understanding of the airborne microbiome.
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http://dx.doi.org/10.1186/s40168-019-0672-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444849PMC
April 2019

Loss of GCNT2/I-branched glycans enhances melanoma growth and survival.

Nat Commun 2018 08 22;9(1):3368. Epub 2018 Aug 22.

Department of Dermatology, Brigham and Women's Hospital, Boston, MA, 02115, USA.

Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.
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http://dx.doi.org/10.1038/s41467-018-05795-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105653PMC
August 2018

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

Primer Extension, Capture, and On-Bead cDNA Ligation: An Efficient RNAseq Library Prep Method for Determining Reverse Transcription Termination Sites.

Methods Mol Biol 2018 ;1712:253-261

Next Generation Sequencing and Microarray Core Facility, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA, 92037, USA.

In this chapter, we describe a method for making Illumina-compatible sequencing libraries from RNA. This protocol can be used for standard RNAseq analysis for detecting differentially expressed genes. In addition, this protocol is ideally suited for adapting to RIPseq, 5'-RACE, RNA structural probing, nascent RNA sequencing, and other protocols where polymerase termination sites need to be profiled. The utilization of solid-phase bead chemistries facilitates simple workflow and efficient library yields.
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http://dx.doi.org/10.1007/978-1-4939-7514-3_16DOI Listing
July 2018

Allele-specific wild-type TP53 expression in the unaffected carrier parent of children with Li-Fraumeni syndrome.

Cancer Genet 2017 02 9;211:9-17. Epub 2017 Jan 9.

Hematology Research and Advanced Diagnostics Laboratories, CHOC Children's Hospital of Orange County, Orange, CA, USA; Division of Hematology, CHOC Children's Hospital of Orange County, Orange, CA, USA; Division of Pediatric Hematology, School of Medicine, University of California at Irvine, Orange, CA, USA.

Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder where an oncogenic TP53 germline mutation is passed from parent to child. Tumor protein p53 is a key tumor suppressor regulating cell cycle arrest in response to DNA damage. Paradoxically, some mutant TP53 carriers remain unaffected, while their children develop cancer within the first few years of life. To address this paradox, response to UV stress was compared in dermal fibroblasts (dFb) from an affected LFS patient vs. their unaffected carrier parent. UV induction of CDKN1A/p21, a regulatory target of p53, in LFS patient dFb was significantly reduced compared to the unaffected parent. UV exposure also induced significantly greater p53[Ser15]-phosphorylation in LFS patient dFb, a reported property of some mutant p53 variants. Taken together, these results suggested that unaffected parental dFb may express an increased proportion of wild-type vs. mutant p53. Indeed, a significantly increased ratio of wild-type to mutant TP53 allele-specific expression in the unaffected parent dFb was confirmed by RT-PCR-RFLP and RNA-seq analysis. Hence, allele-specific expression of wild-type TP53 may allow an unaffected parent to mount a response to genotoxic stress more characteristic of homozygous wild-type TP53 individuals than their affected offspring, providing protection from the oncogenesis associated with LFS.
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http://dx.doi.org/10.1016/j.cancergen.2017.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347445PMC
February 2017

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92.

PLoS Genet 2017 02 27;13(2):e1006623. Epub 2017 Feb 27.

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America.

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.
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http://dx.doi.org/10.1371/journal.pgen.1006623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5348049PMC
February 2017

p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish.

Proc Natl Acad Sci U S A 2016 07 8;113(30):8460-5. Epub 2016 Jul 8.

The Scripps Laboratories for tRNA Synthetase Research, The Scripps Research Institute, La Jolla, CA 92037; Department of Cell and Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037; Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037; Department of Metabolism and Aging, The Scripps Research Institute, Jupiter, FL 33458;

Brain and heart pathologies are caused by editing defects of transfer RNA (tRNA) synthetases, which preserve genetic code fidelity by removing incorrect amino acids misattached to tRNAs. To extend understanding of the broader impact of synthetase editing reactions on organismal homeostasis, and based on effects in bacteria ostensibly from small amounts of mistranslation of components of the replication apparatus, we investigated the sensitivity to editing of the vertebrate genome. We show here that in zebrafish embryos, transient overexpression of editing-defective valyl-tRNA synthetase (ValRS(ED)) activated DNA break-responsive H2AX and p53-responsive downstream proteins, such as cyclin-dependent kinase (CDK) inhibitor p21, which promotes cell-cycle arrest at DNA damage checkpoints, and Gadd45 and p53R2, with pivotal roles in DNA repair. In contrast, the response of these proteins to expression of ValRS(ED) was abolished in p53-deficient fish. The p53-activated downstream signaling events correlated with suppression of abnormal morphological changes caused by the editing defect and, in adults, reversed a shortened life span (followed for 2 y). Conversely, with normal editing activities, p53-deficient fish have a normal life span and few morphological changes. Whole-fish deep sequencing showed genomic mutations associated with the editing defect. We suggest that the sensitivity of p53 to expression of an editing-defective tRNA synthetase has a critical role in promoting genome integrity and organismal homeostasis.
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http://dx.doi.org/10.1073/pnas.1608139113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968768PMC
July 2016

Regulatory T-cell-intrinsic amphiregulin is dispensable for suppressive function.

J Allergy Clin Immunol 2016 06 31;137(6):1907-1909. Epub 2016 Mar 31.

Department of Clinical Science and Services, Immune Regulation Laboratory, Royal Veterinary College, London, United Kingdom. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2016.01.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889774PMC
June 2016

Transfection of microRNA Mimics Should Be Used with Caution.

Front Genet 2015 2;6:340. Epub 2015 Dec 2.

Department of Immunology and Microbial Science, The Scripps Research Institute La Jolla, CA, USA.

Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5'- and 3'-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics.
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http://dx.doi.org/10.3389/fgene.2015.00340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4667072PMC
December 2015

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

Phenotypic and Functional Properties of Human Steady State CD14+ and CD1a+ Antigen Presenting Cells and Epidermal Langerhans Cells.

PLoS One 2015 25;10(11):e0143519. Epub 2015 Nov 25.

Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands.

Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143519PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659545PMC
June 2016

Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes.

Methods Mol Biol 2016 ;1352:67-83

DNA Array Core, Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA.

With the increasing need for understanding antibody specificity in antibody and vaccine research, pepscan assays provide a rapid method for mapping and profiling antibody responses to continuous epitopes. We have developed a relatively low-cost method to generate peptide microarray slides for studying antibody binding. Using a setup of an IntavisAG MultiPep RS peptide synthesizer, a Digilab MicroGrid II 600 microarray printer robot, and an InnoScan 1100 AL scanner, the method allows the interrogation of up to 1536 overlapping, alanine-scanning, and mutant peptides derived from the target antigens. Each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface.
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http://dx.doi.org/10.1007/978-1-4939-3037-1_6DOI Listing
August 2016

Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection.

J Virol 2015 Nov 2;89(22):11473-86. Epub 2015 Sep 2.

Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research Demokritos, Aghia Paraskevi, Athens, Greece.

Unlabelled: The lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera.

Importance: This work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented double-stranded RNA (dsRNA) virus (CPV; Reoviridae) and highlights the importance of viral small-RNA (vsRNA) analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20-nucleotide vsRNAs or based on the activity of an uncharacterized endo-RNase that cleaves the viral RNA substrate at a degenerate motif. The analysis also indicates the existence of a degradation pathway that targets the positive strand of segment 10.
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http://dx.doi.org/10.1128/JVI.01695-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4645660PMC
November 2015

ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3'-Azido cDNAs.

J Mol Biol 2015 Aug 24;427(16):2610-6. Epub 2015 Jun 24.

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

We present a simple method called "ClickSeq" for NGS (next-generation sequencing) library synthesis that uses click chemistry rather than enzymatic reactions for the ligation of Illumina sequencing adaptors. In ClickSeq, randomly primed reverse transcription reactions are supplemented with azido-2',3'-dideoxynucleotides that randomly terminate DNA synthesis and release 3'-azido-blocked cDNA fragments in a process akin to dideoxy-Sanger sequencing. Purified fragments are "click ligated" via copper-catalyzed alkyne-azide cycloaddition to DNA oligos modified with a 5'-alkyne group. This generates ssDNA molecules containing an unnatural triazole-linked DNA backbone that is sufficiently biocompatible for PCR amplification to generate a cDNA library for RNAseq. Here, we analyze viral RNAs and mRNA to demonstrate that ClickSeq produces unbiased NGS libraries with low error rates comparable to standard methods. Importantly, ClickSeq is robust against common artifacts of NGS such as chimera formation and artifactual recombination with fewer than 3 aberrant events detected per million reads.
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http://dx.doi.org/10.1016/j.jmb.2015.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523409PMC
August 2015

A novel histone deacetylase complex in the control of transcription and genome stability.

Mol Cell Biol 2014 Sep 7;34(18):3500-14. Epub 2014 Jul 7.

Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA

The acetylation state of histones, controlled by histone acetyltransferases (HATs) and deacetylases (HDACs), profoundly affects DNA transcription and repair by modulating chromatin accessibility to the cellular machinery. The Schizosaccharomyces pombe HDAC Clr6 (human HDAC1) binds to different sets of proteins that define functionally distinct complexes: I, I', and II. Here, we determine the composition, architecture, and functions of a new Clr6 HDAC complex, I'', delineated by the novel proteins Nts1, Mug165, and Png3. Deletion of nts1 causes increased sensitivity to genotoxins and deregulated expression of Tf2 elements, long noncoding RNA, and subtelomeric and stress-related genes. Similar, but more pervasive, phenotypes are observed upon Clr6 inactivation, supporting the designation of complex I'' as a mediator of a key subset of Clr6 functions. We also reveal that with the exception of Tf2 elements, the genome-wide loading sites and loci regulated by Clr6 I″ do not correlate. Instead, Nts1 loads at genes that are expressed in midmeiosis, following oxidative stress, or are periodically expressed. Collective data suggest that Clr6 I'' has (i) indirect effects on gene expression, conceivably by mediating higher-order chromatin organization of subtelomeres and Tf2 elements, and (ii) direct effects on the transcription of specific genes in response to certain cellular or environmental stimuli.
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http://dx.doi.org/10.1128/MCB.00519-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135615PMC
September 2014

An evolutionarily conserved long noncoding RNA TUNA controls pluripotency and neural lineage commitment.

Mol Cell 2014 Mar 13;53(6):1005-19. Epub 2014 Feb 13.

Program for RNA Biology, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA; Department of Pediatrics, Rady Children's Hospital San Diego and University of California San Diego, La Jolla, CA 92093, USA; Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, CA 92093, USA. Electronic address:

Here, we generated a genome-scale shRNA library targeting long intergenic noncoding RNAs (lincRNAs) in the mouse. We performed an unbiased loss-of-function study in mouse embryonic stem cells (mESCs) and identified 20 lincRNAs involved in the maintenance of pluripotency. Among these, TUNA (Tcl1 Upstream Neuron-Associated lincRNA, or megamind) was required for pluripotency and formed a complex with three RNA-binding proteins (RBPs). The TUNA-RBP complex was detected at the promoters of Nanog, Sox2, and Fgf4, and knockdown of TUNA or the individual RBPs inhibited neural differentiation of mESCs. TUNA showed striking evolutionary conservation of both sequence- and CNS-restricted expression in vertebrates. Accordingly, knockdown of tuna in zebrafish caused impaired locomotor function, and TUNA expression in the brains of Huntington's disease patients was significantly associated with disease grade. Our results suggest that the lincRNA TUNA plays a vital role in pluripotency and neural differentiation of ESCs and is associated with neurological function of adult vertebrates.
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http://dx.doi.org/10.1016/j.molcel.2014.01.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010157PMC
March 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

Technical variations in low-input RNA-seq methodologies.

Sci Rep 2014 Jan 14;4:3678. Epub 2014 Jan 14.

1] Bioinformatics and Systems Biology Graduate Program, University of California at San Diego, La Jolla, California, USA [2] Department of Bioengineering, University of California at San Diego, La Jolla, California, USA [3] Departments of Cellular and Molecular Medicine and Chemistry and Biochemistry, University of California at San Diego, La Jolla, California, USA.

Recent advances in RNA-seq methodologies from limiting amounts of mRNA have facilitated the characterization of rare cell-types in various biological systems. So far, however, technical variations in these methods have not been adequately characterized, vis-à-vis sensitivity, starting with reduced levels of mRNA. Here, we generated sequencing libraries from limiting amounts of mRNA using three amplification-based methods, viz. Smart-seq, DP-seq and CEL-seq, and demonstrated significant technical variations in these libraries. Reduction in mRNA levels led to inefficient amplification of the majority of low to moderately expressed transcripts. Furthermore, noise in primer hybridization and/or enzyme incorporation was magnified during the amplification step resulting in significant distortions in fold changes of the transcripts. Consequently, the majority of the differentially expressed transcripts identified were either high-expressed and/or exhibited high fold changes. High technical variations ultimately masked subtle biological differences mandating the development of improved amplification-based strategies for quantitative transcriptomics from limiting amounts of mRNA.
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http://dx.doi.org/10.1038/srep03678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890974PMC
January 2014

The long noncoding RNA THRIL regulates TNFα expression through its interaction with hnRNPL.

Proc Natl Acad Sci U S A 2014 Jan 26;111(3):1002-7. Epub 2013 Dec 26.

Program for RNA Biology, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037.

Thousands of large intergenic noncoding RNAs (lincRNAs) have been identified in the mammalian genome, many of which have important roles in regulating a variety of biological processes. Here, we used a custom microarray to identify lincRNAs associated with activation of the innate immune response. A panel of 159 lincRNAs was found to be differentially expressed following innate activation of THP1 macrophages. Among them, linc1992 was shown to be expressed in many human tissues and was required for induction of TNFα expression. Linc1992 bound specifically to heterogenous nuclear ribonucleoprotein L (hnRNPL) and formed a functional linc1992-hnRNPL complex that regulated transcription of the TNFα gene by binding to its promoter. Transcriptome analysis revealed that linc1992 was required for expression of many immune-response genes, including other cytokines and transcriptional and posttranscriptional regulators of TNFα expression, and that knockdown of linc1992 caused dysregulation of these genes during innate activation of THP1 macrophages. Therefore, we named linc1992 THRIL (TNFα and hnRNPL related immunoregulatory LincRNA). Finally, THRIL expression was correlated with the severity of symptoms in patients with Kawasaki disease, an acute inflammatory disease of childhood. Collectively, our data provide evidence that lincRNAs and their binding proteins can regulate TNFα expression and may play important roles in the innate immune response and inflammatory diseases in humans.
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http://dx.doi.org/10.1073/pnas.1313768111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903238PMC
January 2014

Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua.

BMC Plant Biol 2013 Dec 20;13:220. Epub 2013 Dec 20.

Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.

Background: The medicinal plant Artemisia annua is covered with filamentous trichomes and glandular, artemisinin producing trichomes. A high artemisinin supply is needed at a reduced cost for treating malaria. Artemisinin production in bioreactors can be facilitated if a better insight is obtained in the biosynthesis of artemisinin and other metabolites. Therefore, metabolic activities of glandular and filamentous trichomes were investigated at the transcriptome level.

Results: By laser pressure catapulting, glandular and filamentous trichomes as well as apical and sub-apical cells from glandular trichomes were collected and their transcriptome was sequenced using Illumina RNA-Seq. A de novo transcriptome was assembled (Trinity) and studied with a differential expression analysis (edgeR).A comparison of the transcriptome from glandular and filamentous trichomes shows that MEP, MVA, most terpene and lipid biosynthesis pathways are significantly upregulated in glandular trichomes. Conversely, some transcripts coding for specific sesquiterpenoid and triterpenoid enzymes such as 8-epi-cedrol synthase and an uncharacterized oxidosqualene cyclase were significantly upregulated in filamentous trichomes. All known artemisinin biosynthesis genes are upregulated in glandular trichomes and were detected in both the apical and sub-apical cells of the glandular trichomes. No significant differential expression could be observed between the apical and sub-apical cells.

Conclusions: Our results underscore the vast metabolic capacities of A. annua glandular trichomes but nonetheless point to the existence of specific terpene metabolic pathways in the filamentous trichomes. Candidate genes that might be involved in artemisinin biosynthesis are proposed based on their putative function and their differential expression level.
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http://dx.doi.org/10.1186/1471-2229-13-220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3878173PMC
December 2013

Optimization of peptide arrays for studying antibodies to hepatitis C virus continuous epitopes.

J Immunol Methods 2014 Jan 19;402(1-2):35-42. Epub 2013 Nov 19.

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, United States. Electronic address:

Accurate and in-depth mapping of antibody responses is of great value in vaccine and antibody research. Using hepatitis C virus (HCV) as a model, we developed an affordable and high-throughput microarray-based assay for mapping antibody specificities to continuous antibody epitopes of HCV at high resolution. Important parameters in the chemistry for conjugating peptides/antigens to the array surface, the array layout, fluorophore choice and the methods for data analysis were investigated. Microscopic glass slide pre-coated with N-Hydroxysuccinimide (NHS)-ester (Slide H) was the preferred surface for conjugation of aminooxy-tagged peptides. This combination provides a simple chemical means to orient the peptides to the conjugation surface via an orthogonal covalent linkage at the N- or C-terminus of each peptide. The addition of polyvinyl alcohol to printing buffer gave uniform spot morphology and improved sensitivity and specificity of binding signals. Libraries of overlapping peptides covering the HCV E1 and E2 glycoprotein polypeptides (15-mer, 10 amino acids overlap) of 6 major HCV genotypes and the entire polypeptide sequence of the prototypic strain H77 were synthesized and printed in quadruplets in the assays. The utility of the peptide arrays was confirmed using HCV monoclonal antibodies (mAbs) specific to known continuous epitopes and immune sera of rabbits immunized with HCV antigens. The methods developed here can be easily adapted to studying antibody responses to antigens relevant in vaccine and autoimmune research.
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http://dx.doi.org/10.1016/j.jim.2013.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4009380PMC
January 2014
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