132 results on '"Stephen J. Salipante"'
Search Results
2. Bartonella spp. Infections Identified by Molecular Methods, United States
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David W. McCormick, Sara L. Rassoulian-Barrett, Daniel R. Hoogestraat, Stephen J. Salipante, Dhruba SenGupta, Elizabeth A. Dietrich, Brad T. Cookson, Grace E. Marx, and Joshua A. Lieberman
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Microbiology (medical) ,Infectious Diseases ,Epidemiology - Published
- 2023
3. Windows into leishmaniasis epidemiology in the United States: September 2021 through August 2022
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Thao T. Truong, Karissa Crawford, Ichih Wang-McGuire, Kendal Jensen, Aisha Mushtaq, Nicole A.P. Lieberman, Frederick S. Buckner, Wesley C. Van Voorhis, Brad T. Cookson, Stephen J. Salipante, and Joshua A. Lieberman
- Abstract
Leishmaniasis is a rare disease in the United States, with an estimated annual incidence of dozens of cases occurring primarily in travelers, migrants, and military personnel. True disease incidence is unknown, since leishmaniasis is not a nationally notifiable condition. Here, we describe the results of molecular leishmaniasis over a 1-year interval (September 2021 to August 2022) when our laboratory served as the primary national reference laboratory for molecular diagnosis of civilian leishmaniasis. We tested 218 specimens submitted from 36 states yielding 94/186 (50.5%) positive cases with species or species complex-level identification and 18 novel mini-exon alleles. Most species belonged to subgenusViannia(75.6%) and associated with cutaneous or mucocutaneous disease. Cases were associated with recent travel (18.1%), travel timing unspecified (7.4%), migration (7.4%), remote travel (2.1%), military (1.1%), or unknown history (63.8%). These data illustrate the clinical utility of molecular testing for leishmaniasis and provide unique insight into disease epidemiology.
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- 2023
4. Diagnosis of Ovarian Carcinoma Homologous Recombination DNA Repair Deficiency From Targeted Gene Capture Oncology Assays
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Niklas Krumm, Nithisha S. Khasnavis, Marc Radke, Kalyan Banda, Helen R. Davies, Christopher Pennil, Kathryn McLean, Vera A. Paulson, Eric Q. Konnick, Winslow C. Johnson, Grogan Huff, Serena Nik-Zainal, Elizabeth M. Swisher, Christina M. Lockwood, and Stephen J. Salipante
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Cancer Research ,Oncology - Abstract
PURPOSE Homologous recombination DNA repair deficiency (HRD) is a therapeutic biomarker for sensitivity to platinum and poly(ADP-ribose) polymerase inhibitor therapies in breast and ovarian cancers. Several molecular phenotypes and diagnostic strategies have been developed to assess HRD; however, their clinical implementation remains both technically challenging and methodologically unstandardized. METHODS We developed and validated an efficient and cost-effective strategy for HRD determination on the basis of calculation of a genome-wide loss of heterozygosity (LOH) score through targeted, hybridization capture and next-generation DNA sequencing augmented with 3,000 common, polymorphic single-nucleotide polymorphism (SNP) sites distributed genome-wide. This approach requires minimal sequence reads and can be readily integrated into targeted gene capture workflows already in use for molecular oncology. We interrogated 99 ovarian neoplasm-normal pairs using this method and compared results with patient mutational genotypes and orthologous predictors of HRD derived from whole-genome mutational signatures. RESULTS LOH scores of ≥11% had >86% sensitivity for identifying tumors with HRD-causing mutations in an independent validation set (90.6% sensitivity for all specimens). We found strong agreement of our analytic approach with genome-wide mutational signature assays for determining HRD, yielding an estimated 96.7% sensitivity and 50% specificity. We observed poor concordance with mutational signatures inferred using only mutations detected by the targeted gene capture panel, suggesting inadequacy of the latter approach. LOH score did not significantly correlate with treatment outcomes. CONCLUSION Targeted sequencing of genome-wide polymorphic SNP sites can be used to infer LOH events and subsequently diagnose HRD in ovarian tumors. The methods presented here are readily generalizable to other targeted gene oncology assays and could be adapted for HRD diagnosis in other tumor types.
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- 2023
5. MLH1 Loss in Primary Prostate Cancer
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Sanaz Nourmohammadi Abadchi, Laura A. Sena, Emmanuel S. Antonarakis, Colin C. Pritchard, James R. Eshleman, Eric Q. Konnick, Stephen J. Salipante, Eugene Shenderov, and Tamara L. Lotan
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Cancer Research ,Oncology - Abstract
PURPOSE Among mismatch repair-deficient (MMRd) prostate cancers, loss of MLH1 is relatively uncommon and few cases have been reported in detail. METHODS Here, we describe the molecular features of two cases of primary prostate cancer with MLH1 loss detected by immunohistochemistry, and in one case, confirmed via transcriptomic profiling. RESULTS Both cases were microsatellite stable on standard polymerase chain reaction (PCR)–based microsatellite instability (MSI) testing, but showed evidence of MSI on a newer PCR-based long mononucleotide repeat (LMR) assay and by next-generation sequencing. Germline testing was negative for Lynch syndrome–associated mutations in both cases. Targeted or whole-exome tumor sequencing using multiple commercial/academic platforms (Foundation, Tempus, JHU, and UW-OncoPlex) showed modestly elevated, though variable, tumor mutation burden estimates (2.3-10 mutations/Mb) consistent with MMRd, but without identifiable pathogenic single-nucleotide or indel mutations in MLH1. Copy-number analysis confirmed biallelic MLH1 loss in one case and monoallelic MLH1 loss in the second case, without evidence of MLH1 promoter hypermethylation in either. The second patient was treated with single-agent pembrolizumab and demonstrated a short-lived prostate-specific antigen response. CONCLUSION These cases highlight the challenges in identifying MLH1-deficient prostate cancers using standard MSI testing and commercial sequencing panels, and support the utility of immunohistochemical assays and LMR- or sequencing-based MSI testing for detection of MMRd prostate cancers.
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- 2023
6. Supp. Table S22 from Genetic Mechanisms of Immune Evasion in Colorectal Cancer
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Ulrike Peters, Shuji Ogino, Antoni Ribas, Charles S. Fuchs, Thomas J. Hudson, Levi A. Garraway, Eric S. Lander, Stacey B. Gabriel, Jesse M. Zaretsky, Syed H. Zaidi, Ming Yu, Catherine J. Wu, David A. Wheeler, Alexander Upfill-Brown, Jennifer Tsoi, Wei Sun, Janet L. Stanford, Sachet Shukla, Brian Shirts, Eve Shinbrot, Daniel Sanghoon Shin, Stephen J. Salipante, Ben J. Raphael, Elleanor H. Quist, Cristina Puig-Saus, Colin C. Pritchard, Matteo Pellegrini, Brian B. Nadel, Dennis J. Montoya, Mark D.M. Leiserson, Paige Krystofinski, Yeon Joo Kim, Jeroen R. Huyghe, Siwen Hu-Lieskovan, Li Hsu, William M. Grady, Milan S. Geybels, Helena Escuin-Ordinas, Charles Connolly, Gabriel Abril-Rodriguez, Katsuhiko Nosho, Teppei Morikawa, Kentaro Inamura, Zhi Rong Qian, Reiko Nishihara, Jonathan A. Nowak, Michael Quist, Xinmeng Jasmine Mu, Tsuyoshi Hamada, Daniel K. Wells, Marios Giannakis, and Catherine S. Grasso
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Supplementary Table S22: Segmented b-allele deficits
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- 2023
7. Supp. Tables S16-S19 from Genetic Mechanisms of Immune Evasion in Colorectal Cancer
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Ulrike Peters, Shuji Ogino, Antoni Ribas, Charles S. Fuchs, Thomas J. Hudson, Levi A. Garraway, Eric S. Lander, Stacey B. Gabriel, Jesse M. Zaretsky, Syed H. Zaidi, Ming Yu, Catherine J. Wu, David A. Wheeler, Alexander Upfill-Brown, Jennifer Tsoi, Wei Sun, Janet L. Stanford, Sachet Shukla, Brian Shirts, Eve Shinbrot, Daniel Sanghoon Shin, Stephen J. Salipante, Ben J. Raphael, Elleanor H. Quist, Cristina Puig-Saus, Colin C. Pritchard, Matteo Pellegrini, Brian B. Nadel, Dennis J. Montoya, Mark D.M. Leiserson, Paige Krystofinski, Yeon Joo Kim, Jeroen R. Huyghe, Siwen Hu-Lieskovan, Li Hsu, William M. Grady, Milan S. Geybels, Helena Escuin-Ordinas, Charles Connolly, Gabriel Abril-Rodriguez, Katsuhiko Nosho, Teppei Morikawa, Kentaro Inamura, Zhi Rong Qian, Reiko Nishihara, Jonathan A. Nowak, Michael Quist, Xinmeng Jasmine Mu, Tsuyoshi Hamada, Daniel K. Wells, Marios Giannakis, and Catherine S. Grasso
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Supplementary Tables S16-S19, describing IHC results on NHS/HPFS data
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- 2023
8. Supp. Table S21 from Genetic Mechanisms of Immune Evasion in Colorectal Cancer
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Ulrike Peters, Shuji Ogino, Antoni Ribas, Charles S. Fuchs, Thomas J. Hudson, Levi A. Garraway, Eric S. Lander, Stacey B. Gabriel, Jesse M. Zaretsky, Syed H. Zaidi, Ming Yu, Catherine J. Wu, David A. Wheeler, Alexander Upfill-Brown, Jennifer Tsoi, Wei Sun, Janet L. Stanford, Sachet Shukla, Brian Shirts, Eve Shinbrot, Daniel Sanghoon Shin, Stephen J. Salipante, Ben J. Raphael, Elleanor H. Quist, Cristina Puig-Saus, Colin C. Pritchard, Matteo Pellegrini, Brian B. Nadel, Dennis J. Montoya, Mark D.M. Leiserson, Paige Krystofinski, Yeon Joo Kim, Jeroen R. Huyghe, Siwen Hu-Lieskovan, Li Hsu, William M. Grady, Milan S. Geybels, Helena Escuin-Ordinas, Charles Connolly, Gabriel Abril-Rodriguez, Katsuhiko Nosho, Teppei Morikawa, Kentaro Inamura, Zhi Rong Qian, Reiko Nishihara, Jonathan A. Nowak, Michael Quist, Xinmeng Jasmine Mu, Tsuyoshi Hamada, Daniel K. Wells, Marios Giannakis, and Catherine S. Grasso
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Supplementary Table S21: Segmented copy-number ratios
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- 2023
9. Supp. Table S2 from Genetic Mechanisms of Immune Evasion in Colorectal Cancer
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Ulrike Peters, Shuji Ogino, Antoni Ribas, Charles S. Fuchs, Thomas J. Hudson, Levi A. Garraway, Eric S. Lander, Stacey B. Gabriel, Jesse M. Zaretsky, Syed H. Zaidi, Ming Yu, Catherine J. Wu, David A. Wheeler, Alexander Upfill-Brown, Jennifer Tsoi, Wei Sun, Janet L. Stanford, Sachet Shukla, Brian Shirts, Eve Shinbrot, Daniel Sanghoon Shin, Stephen J. Salipante, Ben J. Raphael, Elleanor H. Quist, Cristina Puig-Saus, Colin C. Pritchard, Matteo Pellegrini, Brian B. Nadel, Dennis J. Montoya, Mark D.M. Leiserson, Paige Krystofinski, Yeon Joo Kim, Jeroen R. Huyghe, Siwen Hu-Lieskovan, Li Hsu, William M. Grady, Milan S. Geybels, Helena Escuin-Ordinas, Charles Connolly, Gabriel Abril-Rodriguez, Katsuhiko Nosho, Teppei Morikawa, Kentaro Inamura, Zhi Rong Qian, Reiko Nishihara, Jonathan A. Nowak, Michael Quist, Xinmeng Jasmine Mu, Tsuyoshi Hamada, Daniel K. Wells, Marios Giannakis, and Catherine S. Grasso
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Supplementary Table S2: Somatic mutations
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- 2023
10. Supp. Tables S1, S3-S15, S20, and S23 from Genetic Mechanisms of Immune Evasion in Colorectal Cancer
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Ulrike Peters, Shuji Ogino, Antoni Ribas, Charles S. Fuchs, Thomas J. Hudson, Levi A. Garraway, Eric S. Lander, Stacey B. Gabriel, Jesse M. Zaretsky, Syed H. Zaidi, Ming Yu, Catherine J. Wu, David A. Wheeler, Alexander Upfill-Brown, Jennifer Tsoi, Wei Sun, Janet L. Stanford, Sachet Shukla, Brian Shirts, Eve Shinbrot, Daniel Sanghoon Shin, Stephen J. Salipante, Ben J. Raphael, Elleanor H. Quist, Cristina Puig-Saus, Colin C. Pritchard, Matteo Pellegrini, Brian B. Nadel, Dennis J. Montoya, Mark D.M. Leiserson, Paige Krystofinski, Yeon Joo Kim, Jeroen R. Huyghe, Siwen Hu-Lieskovan, Li Hsu, William M. Grady, Milan S. Geybels, Helena Escuin-Ordinas, Charles Connolly, Gabriel Abril-Rodriguez, Katsuhiko Nosho, Teppei Morikawa, Kentaro Inamura, Zhi Rong Qian, Reiko Nishihara, Jonathan A. Nowak, Michael Quist, Xinmeng Jasmine Mu, Tsuyoshi Hamada, Daniel K. Wells, Marios Giannakis, and Catherine S. Grasso
- Abstract
Supplementary Tables S1, S3 through S15, S20, and S23
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- 2023
11. Data from Genetic Mechanisms of Immune Evasion in Colorectal Cancer
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Ulrike Peters, Shuji Ogino, Antoni Ribas, Charles S. Fuchs, Thomas J. Hudson, Levi A. Garraway, Eric S. Lander, Stacey B. Gabriel, Jesse M. Zaretsky, Syed H. Zaidi, Ming Yu, Catherine J. Wu, David A. Wheeler, Alexander Upfill-Brown, Jennifer Tsoi, Wei Sun, Janet L. Stanford, Sachet Shukla, Brian Shirts, Eve Shinbrot, Daniel Sanghoon Shin, Stephen J. Salipante, Ben J. Raphael, Elleanor H. Quist, Cristina Puig-Saus, Colin C. Pritchard, Matteo Pellegrini, Brian B. Nadel, Dennis J. Montoya, Mark D.M. Leiserson, Paige Krystofinski, Yeon Joo Kim, Jeroen R. Huyghe, Siwen Hu-Lieskovan, Li Hsu, William M. Grady, Milan S. Geybels, Helena Escuin-Ordinas, Charles Connolly, Gabriel Abril-Rodriguez, Katsuhiko Nosho, Teppei Morikawa, Kentaro Inamura, Zhi Rong Qian, Reiko Nishihara, Jonathan A. Nowak, Michael Quist, Xinmeng Jasmine Mu, Tsuyoshi Hamada, Daniel K. Wells, Marios Giannakis, and Catherine S. Grasso
- Abstract
To understand the genetic drivers of immune recognition and evasion in colorectal cancer, we analyzed 1,211 colorectal cancer primary tumor samples, including 179 classified as microsatellite instability–high (MSI-high). This set includes The Cancer Genome Atlas colorectal cancer cohort of 592 samples, completed and analyzed here. MSI-high, a hypermutated, immunogenic subtype of colorectal cancer, had a high rate of significantly mutated genes in important immune-modulating pathways and in the antigen presentation machinery, including biallelic losses of B2M and HLA genes due to copy-number alterations and copy-neutral loss of heterozygosity. WNT/β-catenin signaling genes were significantly mutated in all colorectal cancer subtypes, and activated WNT/β-catenin signaling was correlated with the absence of T-cell infiltration. This large-scale genomic analysis of colorectal cancer demonstrates that MSI-high cases frequently undergo an immunoediting process that provides them with genetic events allowing immune escape despite high mutational load and frequent lymphocytic infiltration and, furthermore, that colorectal cancer tumors have genetic and methylation events associated with activated WNT signaling and T-cell exclusion.Significance: This multi-omic analysis of 1,211 colorectal cancer primary tumors reveals that it should be possible to better monitor resistance in the 15% of cases that respond to immune blockade therapy and also to use WNT signaling inhibitors to reverse immune exclusion in the 85% of cases that currently do not. Cancer Discov; 8(6); 730–49. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663
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- 2023
12. The Vaginal Microbiome of Transgender and Gender Nonbinary Individuals
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Gabrielle Winston McPherson, Zil Goldstein, Stephen J. Salipante, Jessica Rongitsch, Noah G. Hoffman, Geolani W. Dy, Kelsi Penewit, and Dina N. Greene
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Gender Studies ,Medicine (miscellaneous) - Published
- 2023
13. In Vitro Selection Identifies Staphylococcus aureus Genes Influencing Biofilm Formation
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Dustin R. Long, Kelsi Penewit, Hsin-Yu Lo, Jared Almazan, Elizabeth A. Holmes, Andrew B. Bryan, Daniel J. Wolter, Janessa D. Lewis, Adam Waalkes, and Stephen J. Salipante
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Infectious Diseases ,Immunology ,Parasitology ,Microbiology - Abstract
Staphylococcus aureus generates biofilms during many chronic human infections, which contributes to its growth and persistence in the host. Multiple genes and pathways necessary for S. aureus biofilm production have been identified, but knowledge is incomplete, and little is known about spontaneous mutations that increase biofilm formation as infection progresses.
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- 2023
14. Discovery of a unique pathway for glutathione utilization in Francisella
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Yaxi Wang, Hannah E. Ledvina, Catherine A. Tower, Stanimir Kambarev, Elizabeth Liu, James C. Charity, Lieselotte S.M. Kreuk, Qiwen Chen, Larry A. Gallagher, Matthew C. Radey, Guilhem F. Rerolle, Yaqiao Li, Kelsi M. Penewit, Serdar Turkarslan, Shawn J. Skerrett, Stephen J. Salipante, Nitin S. Baliga, Joshua J. Woodward, Simon L. Dove, S. Brook Peterson, Jean Celli, and Joseph D. Mougous
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Glutathione (GSH) is an abundant metabolite that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella spp. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.
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- 2023
15. Identifying indels from WGS short reads of haploid genomes distinguishes variant-calling algorithms
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Yee Mey Seah, Mary K. Stewart, Daniel Hoogestraat, Molly Ryder, Brad T. Cookson, Stephen J. Salipante, and Noah G. Hoffman
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Identification of clinically relevant strains of bacteria increasingly relies on whole genome sequencing. The downstream bioinformatics steps necessary for calling variants from short read sequences are well-established but seldom validated against haploid genomes. We devised anin silicoworkflow to introduce single nucleotide polymorphisms (SNP) and indels into bacterial reference genomes, and computationally generate sequencing reads based on the mutated genomes. We then applied the method toMycobacterium tuberculosisH37Rv and used the synthetic reads as truth sets for evaluating several popular variant callers. Insertions proved especially challenging for most variant callers to correctly identify, relative to deletions and single nucleotide polymorphisms. With adequate read depth, however, variant callers that use high quality soft-clipped reads and base mismatches to perform local realignment consistently had the highest precision and recall in identifying medium-length insertions and deletions.
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- 2022
16. Genome Capture Sequencing Selectively Enriches Bacterial DNA and Enables Genome-Wide Measurement of Intrastrain Genetic Diversity in Human Infections
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Hillary S. Hayden, Snehal Joshi, Matthew C. Radey, Anh T. Vo, Cara Forsberg, Sarah J. Morgan, Adam Waalkes, Elizabeth A. Holmes, Sara M. Klee, Mary J. Emond, Pradeep K. Singh, and Stephen J. Salipante
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DNA, Bacterial ,Cystic Fibrosis ,Virology ,Pseudomonas aeruginosa ,Humans ,Genetic Variation ,Pseudomonas Infections ,Sequence Analysis, DNA ,Microbiology ,Genome, Bacterial ,Anti-Bacterial Agents - Abstract
Within-host evolution produces genetic diversity in bacterial strains that cause chronic human infections. However, the lack of facile methods to measure bacterial allelic variation in clinical samples has limited understanding of intrastrain diversity's effects on disease. Here, we report a new method termed genome capture sequencing (GenCap-Seq) in which users inexpensively make hybridization probes from genomic DNA or PCR amplicons to selectively enrich and sequence targeted bacterial DNA from clinical samples containing abundant human or nontarget bacterial DNA. GenCap-Seq enables accurate measurement of allele frequencies over targeted regions and is scalable from specific genes to entire genomes, including the strain-specific accessory genome. The method is effective with samples in which target DNA is rare and inhibitory and DNA-degrading substances are abundant, including human sputum and feces. In proof-of-principle experiments, we used GenCap-Seq to investigate the responses of diversified Pseudomonas aeruginosa populations chronically infecting the lungs of people with cystic fibrosis to
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- 2022
17. Isolated MLH1 Loss by Immunohistochemistry Because of Benign Germline
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Dustin E, Bosch, Matthew M, Yeh, Stephen J, Salipante, Angela, Jacobson, Stacey A, Cohen, Eric Q, Konnick, and Vera A, Paulson
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Germ Cells ,Humans ,Female ,Microsatellite Instability ,Colorectal Neoplasms ,MutL Protein Homolog 1 ,Immunohistochemistry ,Endometrial Neoplasms ,Retrospective Studies - Abstract
Mismatch repair (MMR) immunohistochemistry (IHC) is frequently used to inform prognosis, select (immuno-)therapy, and identify patients for heritable cancer syndrome testing. However, false-negative and false-positive MMR IHC interpretations have been described.Following identification of discordant MMR IHC and DNA-based microsatellite instability testing in a patient with colorectal carcinoma, we retrospectively reviewed institutional archives to identify patient samples with similar discrepancies.We report a patient with metastatic colorectal carcinoma who initially received immunotherapy on the basis of apparent isolated loss of MLH1 by IHC; notably,This study confirms that rare germline polymorphisms can result in incorrect IHC results, potentially affecting selection of optimal therapy and the decision to pursue germline testing. This case further highlights the need for expert molecular pathologic review and communication between clinical and molecular oncology teams.
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- 2022
18. Contaminated Incubators: Source of a Multispecies Enterobacter Outbreak of Neonatal Sepsis
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Enrique Hernandez-Alonso, Nadège Bourgeois-Nicolaos, Margaux Lepainteur, Véronique Derouin, Simon Barreault, Adam Waalkes, Luis A. Augusto, Stuti Gera, Orane Gleizes, Pierre Tissieres, Stephen J. Salipante, Daniele de Luca, and Florence Doucet-Populaire
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Microbiology (medical) ,Cross Infection ,General Immunology and Microbiology ,Ecology ,Physiology ,Enterobacter ,Infant, Newborn ,Infant ,Bacteremia ,Cell Biology ,Disease Outbreaks ,Incubators ,Infectious Diseases ,Genetics ,Humans ,Neonatal Sepsis - Abstract
The genus Enterobacter includes species responsible for nosocomial outbreaks in fragile patients, especially in neonatal intensive care units (NICUs). Determining the primary source of infection is critical to outbreak management and patient outcomes. In this investigation, we report the management and control measures implemented during an Enterobacter outbreak of bloodstream infections in premature babies. The study was conducted in a French NICU over a 3-year period (2016 to 2018) and included 20 premature infants with bacteremia. The clinical and microbiological characteristics were identified, and whole-genome sequencing (WGS) was performed on bacteremia isolates. Initially, several outbreak containment strategies were carried out with no success. Next, outbreak investigation pinpointed the neonatal incubators as the primary reservoir and source of contamination in this outbreak. A new sampling methodology during "on" or "in use" conditions enabled its identification, which led to their replacement, thus resulting in the containment of the outbreak. WGS analysis showed a multiclonal outbreak. Some clones were identified in different isolation sources, including patients and neonatal incubators. In addition, microbiological results showed a multispecies outbreak with a high prevalence of Enterobacter bugandensis and Enterobacter xiangfangensis. We conclude that the NICU health care environment represents an important reservoir for Enterobacter transmission and infection. Finally, extracting samples from the neonatal incubator during active use conditions improves the recovery of bacteria from contaminated equipment. This method should be used more frequently to achieve better monitoring of the NICU for HAIs prevention.
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- 2022
19. SCORe: SARS-CoV-2 Omicron Variant RBD-Binding DNA Aptamer for Multiplexed Rapid Detection and Pseudovirus Neutralization
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Lucy F. Yang, Nataly Kacherovsky, Joey Liang, Stephen J. Salipante, and Suzie H. Pun
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SARS-CoV-2 ,COVID-19 ,Humans ,RNA Viruses ,Angiotensin-Converting Enzyme 2 ,Aptamers, Nucleotide ,Antibodies, Viral ,Analytical Chemistry - Abstract
During the COVID-19 (coronavirus disease 2019) pandemic, several SARS-CoV-2 variants of concern emerged, including the Omicron variant, which has enhanced infectivity and immune invasion. Many antibodies and aptamers that bind the spike (S) of previous strains of SARS-CoV-2 either do not bind or bind with low affinity to Omicron S. In this study, we report a high-affinity
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- 2022
20. Isolated MLH1 Loss by Immunohistochemistry Because of Benign Germline MLH1 Polymorphisms
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Dustin E. Bosch, Matthew M. Yeh, Stephen J. Salipante, Angela Jacobson, Stacey A. Cohen, Eric Q. Konnick, and Vera A. Paulson
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Cancer Research ,Oncology - Abstract
PURPOSE Mismatch repair (MMR) immunohistochemistry (IHC) is frequently used to inform prognosis, select (immuno-)therapy, and identify patients for heritable cancer syndrome testing. However, false-negative and false-positive MMR IHC interpretations have been described. MATERIALS AND METHODS Following identification of discordant MMR IHC and DNA-based microsatellite instability testing in a patient with colorectal carcinoma, we retrospectively reviewed institutional archives to identify patient samples with similar discrepancies. RESULTS We report a patient with metastatic colorectal carcinoma who initially received immunotherapy on the basis of apparent isolated loss of MLH1 by IHC; notably, MLH1 promoter hypermethylation was negative. Subsequent evaluation of neoplastic tissue on a DNA-based targeted next-generation sequencing panel demonstrated microsatellite stability, low tumor mutational burden, and a benign MLH1 variant, MLH1 p.V384D, accompanied by loss of heterozygosity. The constellation of findings and repeat MLH1 IHC demonstrating retained expression using a different antibody-clone, supported reclassification of the neoplasm as MMR-proficient. Immunotherapy was discontinued, and cytotoxic chemotherapy was initiated. This index case of apparent discordance between MMR IHC and DNA-based microsatellite instability prompted a retrospective review of institutional archives to identify patient samples with similar discrepancies. Further evaluation of neoplasms harboring MLH1 p.V384D with loss of heterozygosity revealed systematic antibody-dependent interference. The review also identified a second IHC-interference candidate, MLH1 p.A441T. CONCLUSION This study confirms that rare germline polymorphisms can result in incorrect IHC results, potentially affecting selection of optimal therapy and the decision to pursue germline testing. This case further highlights the need for expert molecular pathologic review and communication between clinical and molecular oncology teams.
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- 2022
21. Polyclonality, Shared Strains, and Convergent Evolution in Chronic Cystic Fibrosis Staphylococcus aureus Airway Infection
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Stephen J. Salipante, Dustin R. Long, Kathryn McLean, Kelsi Penewit, Daniel J. Wolter, Adam Waalkes, Michael Lee, Elizabeth E. Holmes, Lucas R. Hoffman, and Mimi R. Precit
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Pulmonary and Respiratory Medicine ,Whole genome sequencing ,Molecular epidemiology ,business.industry ,Respiratory pathogen ,Critical Care and Intensive Care Medicine ,medicine.disease_cause ,medicine.disease ,Cystic fibrosis ,Microbiology ,Staphylococcus aureus ,Convergent evolution ,medicine ,Airway ,business ,Genetic adaptation - Abstract
Rationale: Staphylococcus aureus is the most common respiratory pathogen isolated from patients with cystic fibrosis (CF) in the United States. Although modes of acquisition and genetic adaptation ...
- Published
- 2021
22. Recombineering in Staphylococcus aureus
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Kelsi, Penewit and Stephen J, Salipante
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Gene Editing ,Recombinases ,Staphylococcus aureus ,Humans ,CRISPR-Cas Systems ,Staphylococcal Infections ,Genetic Engineering - Abstract
Recombineering has proven to be an extraordinarily powerful and versatile approach for the modification of bacterial genomes, but has historically not been possible in the important opportunistic pathogen Staphylococcus aureus. After evaluating the activity of various recombinases in S. aureus, we developed methods for recombineering in that organism using synthetic, single-stranded DNA oligonucleotides. This approach can be coupled to CRISPR/Cas9-mediated lethal counterselection in order to improve the efficiency with which recombinant S. aureus are recovered, which is especially useful in instances where mutants lack a selectable phenotype. These methods provide a rapid, scalable, precise, and inexpensive means to engineer point mutations, variable-length deletions, and short insertions into the S. aureus genome.
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- 2022
23. Aptamer Sandwich Lateral Flow Assay (AptaFlow) for Antibody-Free SARS-CoV-2 Detection
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Lucy F. Yang, Nataly Kacherovsky, Nuttada Panpradist, Ruixuan Wan, Joey Liang, Bo Zhang, Stephen J. Salipante, Barry R. Lutz, and Suzie H. Pun
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COVID-19 Vaccines ,SARS-CoV-2 ,Spike Glycoprotein, Coronavirus ,COVID-19 ,Humans ,Aptamers, Nucleotide ,Antibodies, Viral ,Pandemics ,Analytical Chemistry - Abstract
The COVID-19 pandemic is among the greatest health and socioeconomic crises in recent history. Although COVID-19 vaccines are being distributed, there remains a need for rapid testing to limit viral spread from infected individuals. We previously identified the SARS-CoV-2 spike protein N-terminal domain (NTD) binding DNA aptamer 1 (SNAP1) for detection of SARS-CoV-2 virus by aptamer-antibody sandwich enzyme-linked immunoassay (ELISA) and lateral flow assay (LFA). In this work, we identify a new aptamer that also binds at the NTD, named SARS-CoV-2 spike protein NTD-binding DNA aptamer 4 (SNAP4). SNAP4 binds with high affinity (30 nM) for the SARS-CoV-2 spike protein, a 2-fold improvement over SNAP1. Furthermore, we utilized both SNAP1 and SNAP4 in an aptamer sandwich LFA (AptaFlow), which detected SARS-CoV-2 UV-inactivated virus at concentrations as low as 10
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- 2022
24. Emergence of Dalbavancin, Vancomycin, and Daptomycin Nonsusceptible Staphylococcus aureus in a Patient Treated With Dalbavancin: Case Report and Isolate Characterization
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Rutan Zhang, Hari Polenakovik, Ismael A Barreras Beltran, Adam Waalkes, Stephen J Salipante, Libin Xu, and Brian J Werth
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Microbiology (medical) ,Methicillin-Resistant Staphylococcus aureus ,Staphylococcus aureus ,Infectious Diseases ,Daptomycin ,Vancomycin ,Brief Report ,Humans ,Microbial Sensitivity Tests ,Teicoplanin ,Staphylococcal Infections ,Anti-Bacterial Agents - Abstract
A patient with end-stage renal disease received 2 doses of dalbavancin for methicillin-resistant Staphylococcus aureus (MRSA) arteriovenous fistula infection and presented 5 weeks later with infective endocarditis secondary to vancomycin, daptomycin, and dalbavancin nonsusceptible MRSA. Resistance was associated with walK and scrA mutations, reduced long-chain lipid content, and reduced membrane fluidity.
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- 2022
25. Identification of a novel tedizolid resistance mutation in rpoB of MRSA after in vitro serial passage
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Libin Xu, Tianwei Shen, Brian J. Werth, Adam Waalkes, Stephen J. Salipante, Kelsi Penewit, and Abhinav Nath
- Subjects
Methicillin-Resistant Staphylococcus aureus ,0301 basic medicine ,Microbiology (medical) ,Retapamulin ,medicine.medical_treatment ,030106 microbiology ,Tetrazoles ,Dalfopristin ,Microbial Sensitivity Tests ,Biology ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Serial passage ,medicine ,Pharmacology (medical) ,Serial Passage ,Oxazoles ,Oxazolidinones ,Original Research ,Pharmacology ,Quinupristin ,Resistance mutation ,rpoB ,Organophosphates ,Anti-Bacterial Agents ,030104 developmental biology ,Infectious Diseases ,chemistry ,Mutation ,Linezolid ,Tedizolid - Abstract
Objectives Tedizolid is an oxazolidinone antimicrobial with activity against Gram-positive bacteria, including MRSA. Tedizolid resistance is uncommon and tedizolid’s capacity to select for cross-resistance to other antimicrobials is incompletely understood. The objective of this study was to further explore the phenotypic and genetic basis of tedizolid resistance in MRSA. Methods We selected for tedizolid resistance in an MRSA laboratory strain, N315, by serial passage until an isolate with an MIC ≥1 log2 dilution above the breakpoint for resistance (≥2 mg/L) was recovered. This isolate was subjected to WGS and susceptibility to a panel of related and unrelated antimicrobials was tested in order to determine cross-resistance. Homology modelling was performed to evaluate the potential impact of the mutation on target protein function. Results After 10 days of serial passage we recovered a phenotypically stable mutant with a tedizolid MIC of 4 mg/L. WGS revealed only one single nucleotide variant (A1345G) in rpoB, corresponding to amino acid substitution D449N. MICs of linezolid, chloramphenicol, retapamulin and quinupristin/dalfopristin increased by ≥2 log2 dilutions, suggesting the emergence of the so-called ‘PhLOPSa’ resistance phenotype. Susceptibility to other drugs, including rifampicin, was largely unchanged. Homology models revealed that the mutated residue of RNA polymerase would be unlikely to directly affect oxazolidinone action. Conclusions To the best of our knowledge, this is the first time that an rpoB mutation has been implicated in resistance to PhLOPSa antimicrobials. The mechanism of resistance remains unclear, but is likely indirect, involving σ-factor binding or other alterations in transcriptional regulation.
- Published
- 2020
26. Identifying Optimal Loci for the Molecular Diagnosis of Microsatellite Instability
- Author
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Varun P Panicker, Dustin R. Long, Stephen J. Salipante, Ronald J. Hause, and Adam Waalkes
- Subjects
0301 basic medicine ,Clinical Biochemistry ,Locus (genetics) ,Diagnostic accuracy ,Computational biology ,Biology ,03 medical and health sciences ,0302 clinical medicine ,Breast cancer ,Neoplasms ,Biomarkers, Tumor ,medicine ,Humans ,Exome ,Whole genome sequencing ,Biochemistry (medical) ,Microsatellite instability ,DNA ,Articles ,medicine.disease ,030104 developmental biology ,Genetic Loci ,030220 oncology & carcinogenesis ,Mutation ,Microsatellite ,Microsatellite Instability ,DNA mismatch repair - Abstract
BackgroundMicrosatellite instability (MSI) predicts oncological response to checkpoint blockade immunotherapies. Although microsatellite mutation is pathognomonic for the condition, loci have unequal diagnostic value for predicting MSI within and across cancer types.MethodsTo better inform molecular diagnosis of MSI, we examined 9438 tumor-normal exome pairs and 901 whole genome sequence pairs from 32 different cancer types and cataloged genome-wide microsatellite instability events. Using a statistical framework, we identified microsatellite mutations that were predictive of MSI within and across cancer types. The diagnostic accuracy of different subsets of maximally informative markers was estimated computationally using a dedicated validation set.ResultsTwenty-five cancer types exhibited hypermutated states consistent with MSI. Recurrently mutated microsatellites associated with MSI were identifiable in 15 cancer types, but were largely specific to individual cancer types. Cancer-specific microsatellite panels of 1 to 7 loci were needed to attain ≥95% diagnostic sensitivity and specificity for 11 cancer types, and in 8 of the cancer types, 100% sensitivity and specificity were achieved. Breast cancer required 800 loci to achieve comparable performance. We were unable to identify recurrent microsatellite mutations supporting reliable MSI diagnosis in ovarian tumors. Features associated with informative microsatellites were cataloged.ConclusionsMost microsatellites informative for MSI are specific to particular cancer types, requiring the use of tissue-specific loci for optimal diagnosis. Limited numbers of markers are needed to provide accurate MSI diagnosis in most tumor types, but it is challenging to diagnose breast and ovarian cancers using predefined microsatellite locus panels.
- Published
- 2020
27. Identification of a DNA Aptamer That Binds to Human Monocytes and Macrophages
- Author
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Nataly Kacherovsky, Stephen J. Salipante, Meilyn Sylvestre, Suzie H. Pun, Christopher P. Saxby, and Heather H. Gustafson
- Subjects
Aptamer ,medicine.medical_treatment ,CD14 ,Cell ,Population ,Biomedical Engineering ,Pharmaceutical Science ,Bioengineering ,02 engineering and technology ,CD16 ,01 natural sciences ,Monocytes ,Mice ,Drug Delivery Systems ,Neoplasms ,medicine ,Animals ,Humans ,Macrophage ,education ,Pharmacology ,education.field_of_study ,010405 organic chemistry ,Chemistry ,Macrophages ,SELEX Aptamer Technique ,Organic Chemistry ,Immunotherapy ,Aptamers, Nucleotide ,021001 nanoscience & nanotechnology ,Ligand (biochemistry) ,0104 chemical sciences ,Cell biology ,medicine.anatomical_structure ,0210 nano-technology ,Biotechnology - Abstract
As cancer strategies shift toward immunotherapy, the need for new binding ligands to target and isolate specific immune cell populations has soared. Based on prior work identifying a peptide specific for murine M2-like macrophages, we sought to identify an aptamer that could bind human M2-like macrophages. Tumor-associated macrophages (TAMs) adopt an M2-like phenotype and support tumor progression and dissemination. Here, we employed cell-SELEX to identify an aptamer ligand that targets this cell population over tissue resident (M0-like) or tumoricidal (M1-like) macrophages. Instead, we identified an aptamer that binds both human M0- and M2-like macrophages and monocytes, with highest binding affinity to M2-like macrophage (Kd ∼ 20 nM) and monocytes (Kd ∼ 45 nM) and minimal binding to other leukocytes. The aptamer binds to CD14+ but not CD16+ monocytes, and is rapidly internalized by these cells. We also demonstrate that this aptamer is able to bind human monocytes when both are administered in vivo to mice. Thus, binding to these cell populations (monocytes, M0-like and M2-like macrophages), this aptamer lends itself toward monocyte-specific applications, such as monocyte-targeted drug delivery or column selection.
- Published
- 2020
28. Occurrence of cross-resistance and β-lactam seesaw effect in glycopeptide-, lipopeptide- and lipoglycopeptide-resistant MRSA correlates with membrane phosphatidylglycerol levels
- Author
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Tianwei Shen, Brian J. Werth, Libin Xu, Elizabeth A. Holmes, Kelly M. Hines, Kathryn McLean, Nathaniel K Ashford, Adam Waalkes, Stephen J. Salipante, and Kelsi Penewit
- Subjects
Methicillin-Resistant Staphylococcus aureus ,Microbiology (medical) ,Lipoglycopeptide ,Membrane lipids ,Microbial Sensitivity Tests ,beta-Lactams ,medicine.disease_cause ,Microbiology ,Lipopeptides ,chemistry.chemical_compound ,medicine ,Pharmacology (medical) ,Original Research ,Pharmacology ,Glycopeptides ,Lipoglycopeptides ,Dalbavancin ,Lipopeptide ,Phosphatidylglycerols ,biochemical phenomena, metabolism, and nutrition ,Methicillin-resistant Staphylococcus aureus ,Glycopeptide ,Anti-Bacterial Agents ,Infectious Diseases ,chemistry ,Vancomycin ,Daptomycin ,medicine.drug - Abstract
Background Glycopeptides (GPs), lipopeptides (LPs) and lipoglycopeptides (LGPs) are related antimicrobials important for the management of invasive MRSA infections. Cross-resistance among these antibiotics in MRSA is well documented, as is the observation that susceptibility of MRSA to β-lactams increases as susceptibility to GPs and LPs decreases (i.e. the seesaw effect). Efforts to understand the relationship between GP/LP/LGP cross-resistance and the seesaw effect have focused on the PBPs, but the role of lipid metabolism has not been investigated. Objectives Since the cell membrane is structurally and metabolically integrated with the cell wall and anchors associated proteins, including PBPs, we examined the relationship between membrane lipid composition and the phenomena of cross-resistance among GPs/LPs/LGPs and the β-lactam seesaw effect. Methods We selected for daptomycin, vancomycin and dalbavancin resistance using the USA300 strain JE2 and evaluated the resulting mutants by WGS, MS-based lipidomics and antimicrobial susceptibility testing to assess the relationship between membrane composition, cross-resistance, and the seesaw effect. Results We observed cross-resistance to GPs/LPs/LGPs among the selected strains and the seesaw effect against various β-lactams, depending on the PBP targets of the particular β-lactam. We found that modification of membrane composition occurs not only in daptomycin-selected strains, but also vancomycin- and dalbavancin-selected strains. Significantly, we observed that the abundance of most phosphatidylglycerols positively correlates with MICs of GPs/LPs/LGPs and negatively correlates with the MICs of β-lactams. Conclusions These studies demonstrate a major association between membrane remodelling, cross-resistance and the seesaw effect.
- Published
- 2020
29. Recombineering in Staphylococcus aureus
- Author
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Kelsi Penewit and Stephen J. Salipante
- Published
- 2022
30. Open-Sourced CIViC Annotation Pipeline to Identify and Annotate Clinically Relevant Variants Using Single-Molecule Molecular Inversion Probes
- Author
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Obi L. Griffith, Nicholas C. Spies, Kelsy C. Cotto, Damian T. Rieke, Malachi Griffith, Kelsi Penewit, Adam Waalkes, Katie M. Campbell, Lana Sheta, Colin Pritchard, Ravindra Uppaluri, Zachary L. Skidmore, Matt C. Mosior, Kilannin Krysiak, Arpad Danos, Stephen J. Salipante, Todd A. Fehniger, Erica K. Barnell, and Ramaswamy Govindan
- Subjects
0301 basic medicine ,Computer science ,DNA Mutational Analysis ,Computational biology ,03 medical and health sciences ,Annotation ,0302 clinical medicine ,Software Design ,Neoplasms ,Original Reports ,Databases, Genetic ,Biomarkers, Tumor ,Humans ,Molecular Targeted Therapy ,Computational Biology ,Genetic Variation ,High-Throughput Nucleotide Sequencing ,Molecular Sequence Annotation ,General Medicine ,Pipeline (software) ,030104 developmental biology ,ROC Curve ,Neoplasms diagnosis ,030220 oncology & carcinogenesis ,Special Series: Informatics Tools for Cancer Research and Care ,DNA Probes - Abstract
PURPOSE Clinical targeted sequencing panels are important for identifying actionable variants for patients with cancer; however, existing approaches do not provide transparent and rationally designed clinical panels to accommodate the rapidly growing knowledge within oncology. MATERIALS AND METHODS We used the Clinical Interpretations of Variants in Cancer (CIViC) database to develop an Open-Sourced CIViC Annotation Pipeline (OpenCAP). OpenCAP provides methods to identify variants within the CIViC database, build probes for variant capture, use probes on prospective samples, and link somatic variants to CIViC clinical relevance statements. OpenCAP was tested using a single-molecule molecular inversion probe (smMIP) capture design on 27 cancer samples from 5 tumor types. In total, 2,027 smMIPs were designed to target 111 eligible CIViC variants (61.5 kb of genomic space). RESULTS When compared with orthogonal sequencing, CIViC smMIP sequencing demonstrated a 95% sensitivity for variant detection (n = 61 of 64 variants). Variant allele frequencies for variants identified on both sequencing platforms were highly concordant (Pearson’s r = 0.885; n = 61 variants). Moreover, for individuals with paired tumor and normal samples (n = 12), 182 clinically relevant variants missed by orthogonal sequencing were discovered by CIViC smMIP sequencing. CONCLUSION The OpenCAP design paradigm demonstrates the utility of an open-source and open-access database built on attendant community contributions with peer-reviewed interpretations. Use of a public repository for variant identification, probe development, and variant interpretation provides a transparent approach to build dynamic next-generation sequencing–based oncology panels.
- Published
- 2019
31. A population-level strain genotyping method to study pathogen strain dynamics in human infections
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Sarah J. Morgan, Samantha L. Durfey, Sumedha Ravishankar, Peter Jorth, Wendy Ni, Duncan T. Skerrett, Moira L. Aitken, Edward F. McKone, Stephen J. Salipante, Matthew C. Radey, and Pradeep K. Singh
- Subjects
Infectious disease ,Genotyping Techniques ,Bacterial infections ,Resource and Technical Advance ,Disease Transmission, Infectious ,Humans ,General Medicine ,Molecular genetics ,Infections ,Microbiology - Abstract
A hallmark of chronic bacterial infections is the long-term persistence of 1 or more pathogen species at the compromised site. Repeated detection of the same bacterial species can suggest that a single strain or lineage is continually present. However, infection with multiple strains of a given species, strain acquisition and loss, and changes in strain relative abundance can occur. Detecting strain-level changes and their effects on disease is challenging because most methods require labor-intensive isolate-by-isolate analyses, and thus, only a few cells from large infecting populations can be examined. Here, we present a population-level method for enumerating and measuring the relative abundance of strains called population multi-locus sequence typing (PopMLST). The method exploits PCR amplification of strain-identifying polymorphic loci, next-generation sequencing to measure allelic variants, and informatic methods to determine whether variants arise from sequencing errors or low-abundance strains. These features enable PopMLST to simultaneously interrogate hundreds of bacterial cells that are cultured en masse from patient samples or are present in DNA directly extracted from clinical specimens without ex vivo culture. This method could be used to detect epidemic or super-infecting strains, facilitate understanding of strain dynamics during chronic infections, and enable studies that link strain changes to clinical outcomes.
- Published
- 2021
32. High Clinical Impact of Broad-Range Fungal PCR in Suspected Fungal Sinusitis
- Author
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Lori Bourassa, Kyoko Kurosawa, Karen Stephens, Joshua A. Lieberman, Dhruba J. Sengupta, Patrick C. Mathias, Brad T. Cookson, Andrew Bryan, Stephen J. Salipante, and James A. Mays
- Subjects
Microbiology (medical) ,medicine.medical_specialty ,business.industry ,Mycology ,medicine.disease ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Fungal sinusitis ,Fungal disease ,Invasive fungal disease ,Mycoses ,Molecular microbiology ,Chart review ,Internal medicine ,Humans ,Medicine ,Sinusitis ,DNA, Fungal ,business ,Anatomic Location ,Retrospective Studies - Abstract
Broad-range fungal PCR is a powerful tool for identifying pathogens directly from patient specimens; however, reported estimates of clinical utility vary and costs discourage universal testing. We investigated the diagnostic and clinical utility of broad-range fungal PCR by examining 9 years of results from sinonasal specimens, hypothesizing that this anatomic location would identify immunocompromised patients at high risk for invasive fungal disease. We retrospectively identified 644 PCRs and 1,446 fungal cultures from sinus sites. To determine the relative performance of each testing modality, we performed chart review on 52 patients having specimens submitted for culture and PCR on the same day. Positivity rates were significantly higher for PCR (37.1%) than culture (13.7%) but similar for formalin-fixed and fresh tissues (42.3% versus 34.6%). Relative to culture, PCR had significantly faster turnaround time to both preliminary (94.5 versus 108.8 h) and final positive (137.9 versus 278.5 h) results. Among chart-reviewed patients, 88% were immunocompromised, 65% had proven or probable fungal disease, and testing sensitivities for culture and PCR (67.5% and 85.0%) were not statistically different. Nevertheless, PCR identified pathogens not recovered by culture in 14.9% of cases and informed clinical decision-making in 16.7% of all reviewed cases, and sensitivity of PCR combined with culture (90.0%) was higher than that of culture alone. We conclude that broad-range fungal PCR is frequently informative for patients at risk of serious fungal disease and is complementary to and has faster turnaround time than culture. Formalin-fixed tissue does not adversely affect diagnostic yield, but anatomic site may impact assay positivity rates.
- Published
- 2021
33. Discovery of coordinately regulated pathways that provide innate protection against interbacterial antagonism
- Author
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See-Yeun Ting, Kaitlyn D LaCourse, Hannah E Ledvina, Rutan Zhang, Matthew C Radey, Hemantha D Kulasekara, Rahul Somavanshi, Savannah K Bertolli, Larry A Gallagher, Jennifer Kim, Kelsi M Penewit, Stephen J Salipante, Libin Xu, S Brook Peterson, and Joseph D Mougous
- Subjects
General Immunology and Microbiology ,Bacteria ,Bacterial Proteins ,General Neuroscience ,Pseudomonas aeruginosa ,Eukaryota ,General Medicine ,General Biochemistry, Genetics and Molecular Biology ,Immunity, Innate - Abstract
Bacterial survival is fraught with antagonism, including that deriving from viruses and competing bacterial cells. It is now appreciated that bacteria mount complex antiviral responses; however, whether a coordinated defense against bacterial threats is undertaken is not well understood. Previously, we showed that Pseudomonas aeruginosa possess a danger-sensing pathway that is a critical fitness determinant during competition against other bacteria. Here, we conducted genome-wide screens in P. aeruginosa that reveal three conserved and widespread interbacterial antagonism resistance clusters (arc1-3). We find that although arc1-3 are coordinately activated by the Gac/Rsm danger-sensing system, they function independently and provide idiosyncratic defense capabilities, distinguishing them from general stress response pathways. Our findings demonstrate that Arc3 family proteins provide specific protection against phospholipase toxins by preventing the accumulation of lysophospholipids in a manner distinct from previously characterized membrane repair systems. These findings liken the response of P. aeruginosa to bacterial threats to that of eukaryotic innate immunity, wherein threat detection leads to the activation of specialized defense systems.
- Published
- 2021
34. Evolution of cefiderocol resistance in
- Author
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Brian J, Werth, Nathaniel K, Ashford, Kelsi, Penewit, Adam, Waalkes, Elizabeth A, Holmes, Andrew, Bryan, and Stephen J, Salipante
- Abstract
Cefiderocol is a siderophore cephalosporin active against MDR Gram-negatives includingWe selected cefiderocol resistance in three clinical strains ofEach parent strain was initially susceptible to cefiderocol (MICs of 0.03125, 0.03125 and 0.125 mg/L), and one initially tested susceptible to ceftazidime/avibactam (MIC 4 mg/L). We recovered evolved isolates achieving cefiderocol resistance at MICs of 8-32 mg/L from each parental strain. Some cefiderocol resistant isolates reverted following one to four drug-free passages. Ceftazidime/avibactam MICs of passaged isolates repeatedly increased to ≥256 mg/L, and while other MICs were largely unchanged, trimethoprim/sulfamethoxazole MICs declined 4-fold in two strains. WGS revealed one evolved isolate carrying six coding mutations, while four were isogenic mutants of
- Published
- 2021
35. Neutrophilic inflammation in gallbladder carcinoma correlates with patient survival: A case-control study
- Author
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Andrew Bryan, Matthew M. Yeh, Stephen J. Salipante, Rodney A. Schmidt, Dustin E. Bosch, Dhruba J. Sengupta, Camtu D. Truong, and Paul E. Swanson
- Subjects
Male ,Pathology ,medicine.medical_specialty ,medicine.medical_treatment ,Perforation (oil well) ,Malignancy ,Pathology and Forensic Medicine ,Carcinoma ,Cholecystitis ,Medicine ,Humans ,Cholecystectomy ,High-power field ,Aged ,business.industry ,Gallbladder ,Case-control study ,General Medicine ,Middle Aged ,medicine.disease ,Survival Rate ,medicine.anatomical_structure ,Neutrophil Infiltration ,Case-Control Studies ,Gallbladder Neoplasms ,business - Abstract
Gallbladder carcinoma is an uncommon malignancy with an overall 5-year survival of less than 5%. Gallbladder carcinoma has been strongly linked with cholelithiasis and chronic inflammation. Case reports and series have described cholecystitis with acute (neutrophilic) inflammation in association with gallbladder carcinoma, although a clear relationship to patient outcome has not been established. Our series included 8 cases of gallbladder carcinoma with high tumor-associated neutrophils (>25 per high power field) that were associated with shorter patient survival (Cox regression coefficient 6.2, p = 0.004) than age- and stage-matched controls. High tumor-associated neutrophils were not associated with gallbladder rupture/perforation or increased bacterial load measured by 16S PCR. Neutrophilic inflammation with gallbladder carcinoma correlates to shorter survival, independent of patient age and stage of carcinoma. The findings suggest that the degree of neutrophilic inflammation may have prognostic significance in specimens from patients with gallbladder carcinoma after cholecystectomy. Further studies with larger case numbers are needed to confirm and generalize these findings.
- Published
- 2021
36. Coordinately regulated interbacterial antagonism defense pathways constitute a bacterial innate immune system
- Author
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Matthew C. Radey, Rutan Zhang, Savannah K. Bertolli, Stephen J. Salipante, Kaitlyn D. LaCourse, Joseph D. Mougous, Libin Xu, Hannah E. Ledvina, Jennifer Kim, See-Yeun Ting, Larry A. Gallagher, Kelsi Penewit, Rahul Somavanshi, S. Brook Peterson, and Hemantha D. Kulasekara
- Subjects
Fight-or-flight response ,Innate immune system ,biology ,Pseudomonas aeruginosa ,medicine ,Phospholipase ,biology.organism_classification ,medicine.disease_cause ,Antagonism ,Sensing system ,Bacteria ,Function (biology) ,Cell biology - Abstract
Bacterial survival is fraught with antagonism, including that deriving from viruses and competing bacterial cells1–3 4. It is now appreciated that bacteria mount complex antiviral responses; however, whether a coordinated defense against bacterial threats is undertaken is not well understood. Previously we showed thatPseudomonas aeruginosapossess a danger sensing pathway that is a critical fitness determinant during competition against other bacteria5, 6. Here, we conducted genome-wide screens inP. aeruginosathat reveal three conserved and widespread interbacterial antagonism resistance clusters (arc1-3). We find that althougharc1-3are coordinately activated by the Gac/Rsm danger sensing system, they function independently and provide idiosyncratic defense capabilities, distinguishing them from general stress response pathways. Our findings demonstrate that Arc3 family proteins provide specific protection against phospholipase toxins by preventing the accumulation of lysophospholipids in a manner distinct from previously characterized membrane repair systems. These findings liken the response ofP. aeruginosato bacterial threats to that of eukaryotic innate immunity, wherein threat detection leads to the activation of specialized defense systems.
- Published
- 2021
37. Ultrasensitive Quantitation of Genomic Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms
- Author
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Adam Waalkes, Francesca Urselli, J. Lee Nelson, Stephen J. Salipante, David Wu, Kelsi Penewit, Sami B. Kanaan, and Jerald P. Radich
- Subjects
DNA Copy Number Variations ,Genotype ,Chemistry ,Residual cancer ,Hematopoietic Stem Cell Transplantation ,High-Throughput Nucleotide Sequencing ,Regular Article ,Computational biology ,Genomics ,Molecular Inversion Probe ,Inversion (discrete mathematics) ,Chimerism ,DNA sequencing ,Pathology and Forensic Medicine ,Molecular Medicine ,Molecule ,Humans - Abstract
Genomic chimerism represents co-existing cells with different genotypes and has diagnostic significance in transplant engraftment monitoring, residual cancer detection, and other contexts. We previously described an approach to chimerism detection by interrogating variably present or absent genomic loci using single-molecule molecular inversion probes (smMIPs) and next-generation sequencing, which provided ultrasensitive limits of detection (
- Published
- 2021
38. Genome-wide protein-DNA interaction site mapping using a double strand DNA-specific cytosine deaminase
- Author
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Michael J. Gebhardt, Larry A. Gallagher, Joseph D. Mougous, Pia A. Andrade, FoSheng Hsu, S. Brook Peterson, Paul A. Wiggins, Kelsi Penewit, Stephen J. Salipante, Víctor de Lorenzo, Thomas LaFramboise, Jennifer Kim, James C. Charity, Matthew C. Radey, Elena Velazquez, Marcos H. de Moraes, and Simon L. Dove
- Subjects
Protein family ,Transcription (biology) ,Chemistry ,Binding protein ,Uracil-DNA glycosylase ,Cytosine deaminase ,Protein–DNA interaction ,Computational biology ,Transcription factor ,DNA sequencing - Abstract
DNA–protein interactions (DPIs) are central to such fundamental cellular processes as transcription and chromosome maintenance and organization. The spatiotemporal dynamics of these interactions dictate their functional consequences; therefore, there is great interest in facile methods for defining the sites of DPI within cells. Here, we present a general method for mapping DPI sites in vivo using the double stranded DNA-specific cytosine deaminase toxin DddA. Our approach, which we term DddA-sequencing (3D-seq), entails generating a translational fusion of DddA to a DNA binding protein of interest, inactivating uracil DNA glycosylase, modulating DddA activity via its natural inhibitor protein, and DNA sequencing for genome-wide DPI detection. We successfully applied this method to three Pseudomonas aeruginosa transcription factors that represent divergent protein families and bind variable numbers of chromosomal locations. 3D-seq offers several advantages over existing technologies including ease of implementation and the possibility to measure DPIs at single-cell resolution.
- Published
- 2021
39. Genome-wide protein-DNA interaction site mapping in bacteria using a double-stranded DNA-specific cytosine deaminase
- Author
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Larry A. Gallagher, Elena Velazquez, S. Brook Peterson, James C. Charity, Matthew C. Radey, Michael J. Gebhardt, FoSheng Hsu, Lauren M. Shull, Kevin J. Cutler, Keven Macareno, Marcos H. de Moraes, Kelsi M. Penewit, Jennifer Kim, Pia A. Andrade, Thomas LaFramboise, Stephen J. Salipante, Michelle L. Reniere, Victor de Lorenzo, Paul A. Wiggins, Simon L. Dove, and Joseph D. Mougous
- Subjects
Microbiology (medical) ,Genome ,Bacteria ,Immunology ,Protein Interaction Mapping ,Genetics ,Cell Biology ,DNA ,Applied Microbiology and Biotechnology ,Microbiology ,Cytosine Deaminase - Abstract
DNA–protein interactions are central to fundamental cellular processes, yet widely implemented technologies for measuring these interactions on a genome scale in bacteria are laborious and capture only a snapshot of binding events. We devised a facile method for mapping DNA–protein interaction sites in vivo using the double-stranded DNA-specific cytosine deaminase toxin DddA. In 3D-seq (DddA-sequencing), strains containing DddA fused to a DNA-binding protein of interest accumulate characteristic mutations in DNA sequence adjacent to sites occupied by the DNA-bound fusion protein. High-depth sequencing enables detection of sites of increased mutation frequency in these strains, yielding genome-wide maps of DNA–protein interaction sites. We validated 3D-seq for four transcription regulators in two bacterial species, Pseudomonas aeruginosa and Escherichia coli. We show that 3D-seq offers ease of implementation, the ability to record binding event signatures over time and the capacity for single-cell resolution.
- Published
- 2021
40. Genomic attributes of homology-directed DNA repair deficiency in metastatic prostate cancer
- Author
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Navonil De Sarkar, Sayan Dasgupta, Payel Chatterjee, Ilsa Coleman, Gavin Ha, Lisa S. Ang, Emily A. Kohlbrenner, Sander B. Frank, Talina A. Nunez, Stephen J. Salipante, Eva Corey, Colm Morrissey, Eliezer Van Allen, Michael T. Schweizer, Michael C. Haffner, Radhika Patel, Brian Hanratty, Jared M. Lucas, Ruth F. Dumpit, Colin C. Pritchard, Robert B. Montgomery, and Peter S. Nelson
- Subjects
Male ,Disease Models, Animal ,Mice ,Oncology ,Animals ,Humans ,Prostatic Neoplasms ,DNA repair ,General Medicine ,Genomics ,Neoplasm Metastasis ,DNA Repair-Deficiency Disorders ,Research Article - Abstract
Cancers with homology-directed DNA repair (HRR) deficiency exhibit high response rates to poly(ADP-ribose) polymerase inhibitors (PARPi) and platinum chemotherapy. Though mutations disrupting BRCA1 and BRCA2 associate with HRR deficiency (HRRd), patterns of genomic aberrations and mutation signatures may be more sensitive and specific indicators of compromised repair. Here, we evaluated whole-exome sequences from 418 metastatic prostate cancers (mPCs) and determined that one-fifth exhibited genomic characteristics of HRRd that included Catalogue Of Somatic Mutations In Cancer mutation signature 3. Notably, a substantial fraction of tumors with genomic features of HRRd lacked biallelic loss of a core HRR-associated gene, such as BRCA2. In this subset, HRRd associated with loss of chromodomain helicase DNA binding protein 1 but not with mutations in serine-protein kinase ATM, cyclin dependent kinase 12, or checkpoint kinase 2. HRRd genomic status was strongly correlated with responses to PARPi and platinum chemotherapy, a finding that supports evaluating biomarkers reflecting functional HRRd for treatment allocation.
- Published
- 2021
41. Unbiased sequencing of Mycobacterium tuberculosis urinary cell-free DNA reveals extremely short fragment lengths
- Author
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Barry R. Lutz, Adam Waalkes, Christopher Rosenthal, Amy Oreskovic, Adrienne E Shapiro, Stephen J. Salipante, Elizabeth A. Holmes, Paul K. Drain, and Douglas Wilson
- Subjects
Mycobacterium tuberculosis ,chemistry.chemical_compound ,biology ,Cell-free fetal DNA ,chemistry ,Urinary system ,Nucleic acid ,Urine ,biology.organism_classification ,Molecular biology ,DNA extraction ,DNA ,DNA sequencing - Abstract
Urine cell-free DNA (cfDNA) presents an attractive target for diagnosing pulmonary Mycobacterium tuberculosis (TB) infection but has not been thoroughly characterized. Here, we aimed to investigate the size and composition of TB-derived urine cfDNA with minimal bias using next-generation DNA sequencing (NGS). To enable analysis of highly fragmented urine cfDNA, we used a combination of DNA extraction (Q sepharose) and single-stranded sequence library preparation methods demonstrated to recover short, highly degraded cfDNA fragments. We examined urine cfDNA from ten HIV-positive patients with confirmed pulmonary TB (nine of which had TB cfDNA detectable by qPCR) and two TB-negative controls. TB-derived cfDNA was identifiable by NGS from all TB-positive patients. TB urine cfDNA was significantly shorter than human urine cfDNA, with median fragment lengths of ≤19–52 bp and 42–92 bp, respectively. TB cfDNA abundance increased exponentially with decreased fragment length, with a peak fragment length of ≤19 bp in most samples. Our methodology also revealed a larger fraction of short human genomic cfDNA than previously reported, with peak fragment lengths of 29–53 bp. Urine cfDNA fragments spanned the TB genome with relative uniformity, but nucleic acids derived from multicopy elements were proportionately overrepresented, providing regions of inherent signal amplification beneficial for molecular diagnosis. This study demonstrates the potential of urine cfDNA as a diagnostic biomarker for TB and will inform improved design of TB urine cfDNA assays. Methods capable of targeting the shortest cfDNA fragments possible will be critical to maximize TB urine cfDNA detection sensitivity.
- Published
- 2021
42. International Spread of Multidrug-Resistant Campylobacter coli in Men Who Have Sex With Men in Washington State and Québec, 2015–2018
- Author
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Brad T. Cookson, Christiane Gaudreau, Amin Addetia, Alexander L. Greninger, Ferric C. Fang, Mary K. Stewart, Robert J Cybulski, Stephen J. Salipante, Kimberly Starr, Andrew Bryan, and Sadjia Bekal
- Subjects
Male ,Washington ,0301 basic medicine ,Microbiology (medical) ,Washington State ,Sexual transmission ,030106 microbiology ,Campylobacter coli ,Microbial Sensitivity Tests ,medicine.disease_cause ,Men who have sex with men ,Sexual and Gender Minorities ,03 medical and health sciences ,Antibiotic resistance ,Campylobacter Infections ,Drug Resistance, Bacterial ,Humans ,Medicine ,Shigella ,MSM ,antimicrobial resistance ,Homosexuality, Male ,Articles and Commentaries ,Retrospective Studies ,Genetics ,biology ,business.industry ,Campylobacter ,Quebec ,Ribosomal RNA ,biology.organism_classification ,Anti-Bacterial Agents ,Québec ,Multiple drug resistance ,AcademicSubjects/MED00290 ,030104 developmental biology ,Infectious Diseases ,Macrolides ,business - Abstract
Background Campylobacter species are among the most common causes of enteric bacterial infections worldwide. Men who have sex with men (MSM) are at increased risk for sexually transmitted enteric infections, including globally distributed strains of multidrug-resistant Shigella species. Methods This was a retrospective study of MSM-associated Campylobacter in Seattle, Washington and Montréal, Québec with phenotypic antimicrobial resistance profiles and whole genome sequencing (WGS). Results We report the isolation of 2 clonal lineages of multidrug-resistant Campylobacter coli from MSM in Seattle and Montréal. WGS revealed nearly identical strains obtained from the 2 regions over a 4-year period. Comparison with the National Center for Biotechnology Information’s Pathogen Detection database revealed extensive Campylobacter species clusters carrying multiple drug resistance genes that segregated with these isolates. Examination of the genetic basis of antimicrobial resistance revealed multiple macrolide resistance determinants including a novel ribosomal RNA methyltransferase situated in a CRISPR (clustered regularly interspaced short palindromic repeats) array locus in a C. coli isolate. Conclusions As previously reported for Shigella, specific multidrug-resistant strains of Campylobacter are circulating by sexual transmission in MSM populations across diverse geographic locations, suggesting a need to incorporate sexual behavior in the investigation of clusters of foodborne pathogens revealed by WGS data., We show defined clonal lineages of likely sexually transmitted Campylobacter species associated with spread in men who have sex with men between Seattle and Montréal. We also report the first CRISPR-acquired antibiotic resistance gene in a clinical isolate.
- Published
- 2019
43. Traceless aptamer-mediated isolation of CD8+ T cells for chimeric antigen receptor T-cell therapy
- Author
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Ian I. Cardle, Jonathan L. Yu, Nataly Kacherovsky, Suzie H. Pun, Michael L. Baldwin, Stephen J. Salipante, Michael C. Jensen, and Emmeline L Cheng
- Subjects
0301 basic medicine ,Chemistry ,Oligonucleotide ,Aptamer ,medicine.medical_treatment ,Biomedical Engineering ,Medicine (miscellaneous) ,Bioengineering ,Immunotherapy ,Chimeric antigen receptor ,Computer Science Applications ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Cancer immunotherapy ,medicine ,Cytotoxic T cell ,Chimeric Antigen Receptor T-Cell Therapy ,030217 neurology & neurosurgery ,CD8 ,Biotechnology - Abstract
Chimeric antigen receptor T-cell therapies using defined product compositions require high-purity T-cell isolation systems that, unlike immunomagnetic positive enrichment, are inexpensive and leave no trace on the final cell product. Here, we show that DNA aptamers (generated with a modified cell−SELEX procedure to display low-nanomolar affinity for the T-cell marker CD8) enable the traceless isolation of pure CD8+ T cells at low cost and high yield. Captured CD8+ T cells are released label-free by complementary oligonucleotides that undergo toehold-mediated strand displacement with the aptamer. We also show that chimeric antigen receptor T cells manufactured from these cells are comparable to antibody-isolated chimeric antigen receptor T cells in proliferation, phenotype, effector function and antitumour activity in a mouse model of B-cell lymphoma. By employing multiple aptamers and the corresponding complementary oligonucleotides, aptamer-mediated cell selection could enable the fully synthetic, sequential and traceless isolation of desired lymphocyte subsets from a single system. DNA aptamers that specifically bind to the T-cell marker CD8 and can be displaced by a complementary oligonucleotide enable the isolation, at high purity and yield, of CD8+ T cells for chimeric antigen receptor T-cell therapies.
- Published
- 2019
44. The Vaginal Microbiome of Transgender Men
- Author
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Gabrielle Winston McPherson, Dina N. Greene, Karen Stephens, Jessica Rongitsch, Thomas J. Long, Noah G. Hoffman, Kelsi Penewit, and Stephen J. Salipante
- Subjects
Adult ,Male ,0301 basic medicine ,Adolescent ,medicine.drug_class ,Clinical Biochemistry ,Physiology ,030204 cardiovascular system & hematology ,Transgender Persons ,Cohort Studies ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,Surveys and Questionnaires ,Lactobacillus ,Transgender ,medicine ,Humans ,Testosterone ,Young adult ,biology ,Vaginal flora ,business.industry ,Microbiota ,Biochemistry (medical) ,Estrogens ,Testosterone (patch) ,Middle Aged ,biology.organism_classification ,030104 developmental biology ,Estrogen ,Vagina ,Cohort ,Female ,business ,Cohort study - Abstract
BACKGROUND Hormonal changes influence the composition of vaginal flora, which is directly related to the health of an individual. Transgender men prescribed testosterone experience a vaginal hormone composition that differs from cisgender women. To the author's knowledge, there are no clinical studies evaluating the influence that testosterone administration has on the vaginal microbiome. METHODS Vaginal swabs were self-collected by a cohort of self-identified healthy transgender men prescribed testosterone for at least 1 year (n = 28) and from cisgender women who were used as the comparator (n = 8). Participants completed a questionnaire to indicate the mode and dose of testosterone administration, sexual history, and vaginal health. Serum was collected for hormone analysis. Bacterial community profiles were assessed with broad-range PCR primers targeting the V3–V4 hypervariable region of the 16S bacterial rRNA, next-generation sequencing, and analysis by phylogenetic placement. RESULTS Compared to cisgender women, the vaginal floras of transgender men were less likely to have Lactobacillus as their primary genus. Intravaginal estrogen administration was positively associated with the presence of Lactobacillus in transgender men (P = 0.045). Transgender men had a significantly increased relative abundance of >30 species and a significantly higher α diversity (P = 0.0003). The presence of Lactobacillus was significantly associated with a lower α diversity index (P = 0.017). CONCLUSIONS The vaginal microbiome of transgender men who were assigned a female sex at birth and use testosterone may differ from that of cisgender women. Intravaginal estrogen administration may reduce these differences by promoting colonization with Lactobacillus species and decreasing α diversity.
- Published
- 2019
45. 476: Adaptive responses of Staphylococcus aureus to trimethoprim/sulfamethoxazole
- Author
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L. Gonsalves, Stephen J. Salipante, A. Dutta, Adam Waalkes, Lucas R. Hoffman, Daniel J. Wolter, and D. Long
- Subjects
Pulmonary and Respiratory Medicine ,Staphylococcus aureus ,business.industry ,Sulfamethoxazole ,Pediatrics, Perinatology and Child Health ,medicine ,medicine.disease_cause ,medicine.disease ,business ,Trimethoprim ,Cystic fibrosis ,medicine.drug ,Microbiology - Published
- 2021
46. Pattern associated leukemia immunophenotypes and measurable disease detection in acute myeloid leukemia or myelodysplastic syndrome with mutated NPM1
- Author
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Stephen J. Salipante, Brent L. Wood, David Wu, Jonathan R. Fromm, Xueyan Chen, Andres Moon, Lori Soma, Eric Hoyle, and Yi Zhou
- Subjects
0301 basic medicine ,NPM1 ,Histology ,Myeloid ,business.industry ,Myeloid leukemia ,Karyotype ,Cell Biology ,medicine.disease ,Leukemogenic ,Pathology and Forensic Medicine ,03 medical and health sciences ,Leukemia ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,Immunophenotyping ,hemic and lymphatic diseases ,030220 oncology & carcinogenesis ,medicine ,Cancer research ,business ,Cytometry - Abstract
Background The presence of measurable residual disease after therapy is a significant risk factor of relapse in patients with acute myeloid leukemia (AML). By detecting cells with leukemia-associated immunophenotype (LAIP), multiparameter flow cytometry (MFC) can detect residual leukemia at a level significantly lower than that detected by morphology. However, changes in LAIPs during or after therapy may pose a challenge to MRD testing. AML with mutated NPM1 represents the largest subtype of AML sharing a common leukemogenic mechanism and similar LAIPs. Here, we identified a common pattern of LAIPs in myeloid blasts with mutated NPM1, and studied its stability and limit of detection after therapy. Methods We summarized aberrancies of leukemic blasts with mutated NPM1 at diagnosis in 61 patients and paired relapse in 25 patients. In addition, we examined the detection of leukemic blasts in 590 specimens collected from 152 patients in complete remission after induction for AML/MDS-EB with mutated NPM1. Results Our findings demonstrate myeloid blasts with mutated NPM1 have a characteristic pattern of LAIPs that is present in nearly all cases of AML/MDS-EB with mutated NPM1 at initial diagnosis and relapse, regardless of morphologic variations, FLT3 ITD status, or karyotype abnormality. The myeloid blasts with mutated NPM1 can be detected at an approximate level of 0.1% of total leukocytes in morphologic remission with high specificity validated by clinical outcome. Conclusion The characteristic pattern of LAIPs of myeloid blasts with mutated NPM1 is common and stable, and allows sensitive and specific detection of AML or MDS with mutated NPM1 after therapy. © 2018 International Clinical Cytometry Society.
- Published
- 2018
47. Polyclonal BRCA2 Reversion Mutations Detected in Circulating Tumor DNA After Platinum Chemotherapy in a Patient With Metastatic Prostate Cancer
- Author
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Bruce Montgomery, Stephen J. Salipante, Heather H. Cheng, Colin Pritchard, and Peter S. Nelson
- Subjects
0301 basic medicine ,Cancer Research ,biology ,business.industry ,Reversion ,medicine.disease ,03 medical and health sciences ,Prostate cancer ,030104 developmental biology ,0302 clinical medicine ,Text mining ,Oncology ,Polyclonal antibodies ,Circulating tumor DNA ,030220 oncology & carcinogenesis ,Platinum chemotherapy ,biology.protein ,Cancer research ,Medicine ,business - Published
- 2018
48. Synergy Between Beta-Lactams and Lipo-, Glyco-, and Lipoglycopeptides is Independent of the Seesaw Effect in Methicillin-Resistant Staphylococcus aureus
- Author
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Libin Xu, Elizabeth A. Holmes, Kelly M. Hines, Ismael A Barreras Beltran, Rutan Zhang, Nathaniel K Ashford, Adam Waalkes, Brian J. Werth, Stephen J. Salipante, and Kelsi Penewit
- Subjects
Penicillin binding proteins ,QH301-705.5 ,daptomycin ,vancomycin ,synergy ,MRSA ,methicillin-resistant staphylococcus aureus ,medicine.disease_cause ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Biochemistry ,Meropenem ,Microbiology ,lipidomic analysis ,polycyclic compounds ,medicine ,Molecular Biosciences ,Cefoxitin ,Biology (General) ,Nafcillin ,Molecular Biology ,Original Research ,seesaw effect ,Chemistry ,General Commentary ,membrane fluidity ,Dalbavancin ,biochemical phenomena, metabolism, and nutrition ,seesaw ,Methicillin-resistant Staphylococcus aureus ,beta-lactam antibacterials ,Vancomycin ,lipidomics ,combination thera py ,Daptomycin ,dalbavancin ,medicine.drug - Abstract
Methicillin-resistant S. aureus (MRSA) are resistant to beta-lactams, but synergistic activity between beta-lactams and glycopeptides/lipopeptides is common. Many have attributed this synergy to the beta-lactam-glycopeptide seesaw effect; however, this association has not been rigorously tested. The objective of this study was to determine whether the seesaw effect is necessary for synergy and to measure the impact of beta-lactam exposure on lipid metabolism. We selected for three isogenic strains with reduced susceptibility to vancomycin, daptomycin, and dalbavancin by serial passaging the MRSA strain N315. We used whole genome sequencing to identify genetic variants that emerged and tested for synergy between vancomycin, daptomycin, or dalbavancin in combination with 6 beta-lactams with variable affinity for staphylococcal penicillin binding proteins (PBPs), including nafcillin, meropenem, ceftriaxone, ceftaroline, cephalexin, and cefoxitin, using time-kills. We observed that the seesaw effect with each beta-lactam was variable and the emergence of the seesaw effect for a particular beta-lactam was not necessary for synergy between that beta-lactam and vancomycin, daptomycin, or dalbavancin. Synergy was more commonly observed with vancomycin and daptomycin based combinations than dalbavancin in time-kills. Among the beta-lactams, cefoxitin and nafcillin were the most likely to exhibit synergy using the concentrations tested, while cephalexin was the least likely to exhibit synergy. Synergy was more common among the resistant mutants than the parent strain. Interestingly N315-D1 and N315-DAL0.5 both had mutations in vraTSR and walKR despite their differences in the seesaw effect. Lipidomic analysis of all strains exposed to individual beta-lactams at subinhibitory concentrations suggested that in general, the abundance of cardiolipins (CLs) and most free fatty acids (FFAs) positively correlated with the presence of synergistic effects while abundance of phosphatidylglycerols (PGs) and lysylPGs mostly negatively correlated with synergistic effects. In conclusion, the beta-lactam-glycopeptide seesaw effect and beta-lactam-glycopeptide synergy are distinct phenomena. This suggests that the emergence of the seesaw effect may not have clinical importance in terms of predicting synergy. Further work is warranted to characterize strains that don’t exhibit beta-lactam synergy to identify which strains should be targeted with combination therapy and which ones cannot and to further investigate the potential role of CLs in mediating synergy.
- Published
- 2021
49. Polyclonality, Shared Strains, and Convergent Evolution in Chronic Cystic Fibrosis
- Author
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Dustin R, Long, Daniel J, Wolter, Michael, Lee, Mimi, Precit, Kathryn, McLean, Elizabeth, Holmes, Kelsi, Penewit, Adam, Waalkes, Lucas R, Hoffman, and Stephen J, Salipante
- Subjects
Cohort Studies ,Male ,Molecular Epidemiology ,Staphylococcus aureus ,Adolescent ,Cystic Fibrosis ,Editorials ,Humans ,Female ,Staphylococcal Infections ,Child ,Respiratory Tract Infections ,Anti-Bacterial Agents - Published
- 2020
50. Sensitive Identification of Bacterial DNA in Clinical Specimens by Broad-Range 16S rRNA Gene Enrichment
- Author
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Joshua A. Lieberman, Stephen J. Salipante, Christopher Rosenthal, Vikas Peddu, Desiree A. Marshall, Noah G. Hoffman, Brad T. Cookson, Sara L. Rassoulian Barrett, Alexander L. Greninger, and Andrew Bryan
- Subjects
DNA, Bacterial ,0301 basic medicine ,Microbiology (medical) ,biology ,030106 microbiology ,Genes, rRNA ,Bacteriology ,Sequence Analysis, DNA ,Computational biology ,biology.organism_classification ,Genome ,DNA sequencing ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,Molecular microbiology ,Metagenomics ,RNA, Ribosomal, 16S ,Nucleic acid ,Humans ,Gene ,Bacteria ,DNA - Abstract
The broad-range detection and identification of bacterial DNA from clinical specimens are a foundational approach in the practice of molecular microbiology. However, there are circumstances under which conventional testing may yield false-negative or otherwise uninterpretable results, including the presence of multiple bacterial templates or degraded nucleic acids. Here, we describe an alternative, next-generation sequencing approach for the broad range detection of bacterial DNA using broad-range 16S rRNA gene hybrid capture (“16S Capture”). The method is able to deconvolute multiple bacterial species present in a specimen, is compatible with highly fragmented templates, and can be readily implemented when the overwhelming majority of nucleic acids in a specimen derive from the human host. We find that this approach is sensitive to detecting as few as 17 Staphylococcus aureus genomes from a background of 100 ng of human DNA, providing 19- to 189-fold greater sensitivity for identifying bacterial sequences than standard shotgun metagenomic sequencing, and is able to successfully recover organisms from across the eubacterial tree of life. Application of 16S Capture to a proof-of-principle case series demonstrated its ability to identify bacterial species that were consistent with histological evidence of infection, even when diagnosis could not be established using conventional broad range bacterial detection assays. 16S Capture provides a novel means for the efficient and sensitive detection of bacteria embedded in human tissues and for specimens containing highly fragmented template DNA.
- Published
- 2020
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