Publications by authors named "S Timothy Motley"

54 Publications

Multiplexed and Extraction-Free Amplification for Simplified SARS-CoV-2 RT-PCR Tests.

Anal Chem 2021 03 25;93(9):4160-4165. Epub 2021 Feb 25.

Global Health Labs, Bellevue, Washington 98007, United States.

The rapid onset of the global COVID-19 pandemic has led to challenges for accurately diagnosing the disease, including supply shortages for sample collection, preservation, and purification. Currently, most diagnostic tests require RNA extraction and detection by RT-PCR; however, extraction is expensive and time-consuming and requires technical expertise. With these challenges in mind, we report extraction-free, multiplexed amplification of SARS-CoV-2 RNA from 246 clinical samples, resulting in 86% sensitivity and 100% specificity. The multiplex RT-PCR uses the CDC singleplex targets and has an LoD of 2 c/μL. We also report on amplification using a range of master mixes in different transport media. This work can help guide which combinations of reagents will enable accurate results when availability of supplies changes throughout the pandemic. Implementing these methods can reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.
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http://dx.doi.org/10.1021/acs.analchem.0c03918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927279PMC
March 2021

Transcriptomic Profiling of Human Effector and Regulatory T Cell Subsets Identifies Predictive Population Signatures.

Immunohorizons 2020 10 9;4(10):585-596. Epub 2020 Oct 9.

Immunology Program, Benaroya Research Institute, Seattle, WA 98101;

After activation, CD4 Th cells differentiate into functionally specialized populations that coordinate distinct immune responses and protect against different types of pathogens. In humans, these effector and memory Th cell subsets can be readily identified in peripheral blood based on their differential expression of chemokine receptors that govern their homeostatic and inflammatory trafficking. Foxp3 regulatory T (Treg) cells can also be divided into subsets that phenotypically mirror each of these effector populations and share expression of key transcription factors and effector cytokines. In this study, we performed comprehensive transcriptional profiling of 11 phenotypically distinct Th and Treg cell subsets sorted from peripheral blood of healthy individuals. Despite their shared phenotypes, we found that mirror Th and Treg subsets were transcriptionally dissimilar and that Treg cell populations showed limited transcriptional diversity compared with Th cells. We identified core transcriptional signatures shared across all Th and Treg cell populations and unique signatures that define each of the Th or Treg populations. Finally, we applied these signatures to bulk Th and Treg RNA-sequencing data and found enrichment of specific Th and Treg cell populations in different human tissues. These results further define the molecular basis for the functional specialization and differentiation of Th and Treg cell populations and provide a new resource for examining Th and Treg specialization in RNA-sequencing data.
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http://dx.doi.org/10.4049/immunohorizons.2000037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085975PMC
October 2020

An Antisense Oligonucleotide Leads to Suppressed Transcription of Hdac2 and Long-Term Memory Enhancement.

Mol Ther Nucleic Acids 2020 Mar 27;19:1399-1412. Epub 2020 Feb 27.

Department of Pharmacology, Vanderbilt University, Nashville, TN, USA. Electronic address:

Knockout of the memory suppressor gene histone deacetylase 2 (Hdac2) in mice elicits cognitive enhancement, and drugs that block HDAC2 have potential as therapeutics for disorders affecting memory. Currently available HDAC2 catalytic activity inhibitors are not fully isoform specific and have short half-lives. Antisense oligonucleotides (ASOs) are drugs that elicit extremely long-lasting, specific inhibition through base pairing with RNA targets. We utilized an ASO to reduce Hdac2 messenger RNA (mRNA) in mice and determined its longevity, specificity, and mechanism of repression. A single injection of the Hdac2-targeted ASO in the central nervous system produced persistent reduction in HDAC2 protein and Hdac2 mRNA levels for 16 weeks. It enhanced object location memory for 8 weeks. RNA sequencing (RNA-seq) analysis of brain tissues revealed that the repression was specific to Hdac2 relative to related Hdac isoforms, and Hdac2 reduction caused alterations in the expression of genes involved in extracellular signal-regulated kinase (ERK) and memory-associated immune signaling pathways. Hdac2-targeted ASOs also suppress a nonpolyadenylated Hdac2 regulatory RNA and elicit direct transcriptional suppression of the Hdac2 gene through stalling RNA polymerase II. These findings identify transcriptional suppression of the target gene as a novel mechanism of action of ASOs.
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http://dx.doi.org/10.1016/j.omtn.2020.01.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047133PMC
March 2020

Rapid Light-Dependent Degradation of Fluorescent Dyes in Formulated Serum-Free Media.

Immunohorizons 2019 12 16;3(12):585-592. Epub 2019 Dec 16.

Center for Basic Discovery, Benaroya Research Institute, Seattle, WA 98101; and.

Chemically defined serum-free media are increasingly used as a tool to help standardize experiments by eliminating the potential variability contributed by pooled serum. These media are formulated for the culture and expansion of specific cell types, maintaining cell viability without the need for exogenous animal proteins. Formulated serum-free media could thus help improve viability and reduce variability during sample preparation for flow cytometry, yet a thorough analysis of how such media impact fluorochrome-Ab conjugates has not been performed. In this study, we expose fluorescent Ab-labeled cells or Ab capture beads to white light in the presence of various hematopoietic cell culture media and provide evidence that formulated serum-free media permit rapid light-initiated fluorescent dye degradation in a cell-independent manner. We observed fluorescence signal loss of several dyes, which included fluorescence spillover into adjacent detectors. Finally, photostability of Ab-fluorochrome conjugates in formulated serum-free media is partially restored in the presence of either serum or vitamin C, implicating reactive oxygen species in the observed signal loss. Thus, our data indicate that formulated serum-free media designed to standardize cell culture are not currently optimized for use with fluorochrome-Ab conjugates, and thus, extreme caution should be exercised when using these media in cytometric experiments.
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http://dx.doi.org/10.4049/immunohorizons.1900080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244342PMC
December 2019

Cardiac Manifestations of Idiopathic Inflammatory Myopathy Treated With Rituximab: A Single-Center Case Series and Review of the Literature.

J Clin Rheumatol 2019 Jul 26. Epub 2019 Jul 26.

Department of Rheumatology San Antonio Uniformed Services Health Education Consortium Ft Sam Houston, TX Department of Clinical and Applied Science Education University of the Incarnate Word School of Osteopathic Medicine San Antonio, TX. Rheumatology Service Division of Medicine Landstuhl Regional Medical Center Landstuhl, Germany.

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http://dx.doi.org/10.1097/RHU.0000000000001141DOI Listing
July 2019