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Publications (14)

  • James R. Prudent · Chad A. Hall · David J. Marshall · [...] · Scott Harried
    Article · Jul 2016 · Cancer Research
  • James R. Prudent · Chad Hall · David J. Marshall · [...] · John Murphy
    Article · Jul 2016 · Cancer Research
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    David J Marshall · Scott S Harried · John L Murphy · [...] · James R Prudent
    [Show abstract] [Hide abstract] ABSTRACT: The human Na(+)/K(+)-ATPase (NKA) is a plasma membrane ion pump that uses ATP to help maintain the resting potential of all human cells. Inhibition of the NKA leads to cell swelling and death. The results of this investigation show that on cancer cells, the NKA either comes in close proximity to, associate with or complexes to important cancer-related proteins, and thus can be targeted with a new type of precision therapy called the extracellular drug conjugate or EDC. The EDCs reported here exhibit EC50 values in the low to mid-picomolar range, and signal to noise ratios > 1,000:1, both of which are dependent on the cell surface expression of the NKA and corresponding cancer-related target. We demonstrate that a potent small molecule inhibitor of the NKA can be covalently attached to antibodies targeting CD20, CD38, CD56, CD147, or dysadherin, to create a series of selective and powerful EDCs that kill cancer cells extracellularly by a mechanism resembling necrosis. This is therefore a framework for the development of a new type of precision therapy wherein exquisite selectivity is achieved for targeting extracellular disease-related proteins.Molecular Therapy (2016); doi:10.1038/mt.2016.119.
    Full-text available · Article · Jun 2016 · Molecular Therapy
  • James R. Prudent · David J. Marshall · John Murphy · Fabio Malavasi
    Article · Aug 2015 · Cancer Research
  • [Show abstract] [Hide abstract] ABSTRACT: Despite advances in treatment modalities, head and neck squamous cell carcinoma (HNSCC) remains a challenge to treat with poor survival and high morbidity, necessitating a therapy with greater efficacy. EDC22 is an extracellular drug conjugate of the monoclonal antibody targeting CD147 (glycoprotein highly expressed on HNSCC cells) linked with a small drug molecule inhibitor of Na, K-ATPase. In this study, EDC22's potential as a treatment modality for HNSCC was performed. HNSCC cell lines (FADU, OSC-19, Cal27, SCC-1) were cultured in vitro and proliferation and cell viability were assessed following treatment with a range of concentrations of EDC22 (0.25-5.00μg/mL). Mice bearing HNSCC xenografts (OSC-19, SCC-1) were treated with either EDC22 (3-10mg/kg), anti-CD147 monoclonal antibody, cisplatin (1mg/kg) or radiation therapy (2Gy/week) monotherapy or in combination. In vitro, treatment with minimal concentration of EDC22 (0.25μg/mL) significantly decreased cellular proliferation and cell viability (p<0.0001). In vivo, systemic treatment with EDC22 significantly decreased primary tumor growth rate in both an orthotopic mouse model (OSC-19) and a flank tumor mouse model (SCC-1) (p<0.05). In addition, EDC22 therapy resulted in a greater reduction in tumor growth in vivo compared to radiation monotherapy (p<0.05) and a similar reduction in tumor growth compared to cisplatin monotherapy. Combination therapy provided no significant further reduction in tumor growth relative to EDC22 monotherapy. EDC22 is a potent inhibitor of HNSCC cell proliferation in vitro and in vivo, warranting further investigations of its clinical potential in the treatment of HNSCC.
    Article · Aug 2013 · Oral Oncology
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    David J Marshall · Erik Reisdorf · Gerda Harms · [...] · James R Prudent
    [Show abstract] [Hide abstract] ABSTRACT: There are numerous viral and bacterial causes of respiratory disease. To enable rapid and sensitive detection of even the most prevalent causes, there is a need for more-simplified testing systems that enable researchers and clinicians to perform multiplexed molecular diagnostics quickly and easily. To this end, a new multiplexed molecular test called the MultiCode-PLx respiratory virus panel (PLx-RVP) was developed and then implemented in a public health laboratory setting. A total of 687 respiratory samples were analyzed for the presence of 17 viruses that commonly cause respiratory disease. As a comparator, the samples were also tested using a standard testing algorithm that included the use of a real-time influenza virus A and B reverse transcription-PCR test and routine viral culture identification. The standard testing algorithm identified 503 (73%) samples as positive and 184 as negative. Analyzing the same 687 samples, the PLx-RVP assay detected one or more targets in 528 (77%) samples and found 159 samples negative for all targets. There were 25 discordant results between the two systems; 14 samples were positive for viruses not routinely tested for by the Wisconsin State Laboratory of Hygiene, and 13 of these were confirmed by real-time PCR. When the results of the standard testing algorithm were considered "true positives," the PLx-RVP assay showed an overall sensitivity of 99% and an overall specificity of 87%. In total, the PLx-RVP assay detected an additional 40 viral infections, of which 11 were mixed infections.
    Full-text available · Article · Jan 2008 · Journal of Clinical Microbiology
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    Frederick S Nolte · David J Marshall · Christopher Rasberry · [...] · James R Prudent
    [Show abstract] [Hide abstract] ABSTRACT: The MultiCode-PLx system (EraGen Biosciences, Inc., Madison, WI) for the detection of respiratory viruses uses an expanded genetic alphabet, multiplex PCR chemistry, and microsphere flow cytometry to rapidly detect and specifically identify 17 different respiratory viruses directly in clinical specimens. The MultiCode-PLx system was tested in parallel with direct fluorescent-antibody (DFA) staining and rapid shell vial culture (R-mix cells; Diagnostic Hybrids, Inc. Athens, OH) with 354 respiratory specimens from adult patients that were submitted to the clinical virology laboratory at the Emory University Hospital. Single-target PCRs were performed with retained samples to confirm the positive results obtained with the MultiCode-PLx system for viruses not covered by DFA and R-mix culture (metapneumovirus, coronaviruses [CoV], parainfluenza viruses 4a and 4b, and rhinoviruses) and to resolve any discrepancies between the DFA and R-mix culture and the MultiCode-PLx results for viruses common to both systems. Respiratory viruses were detected in 77 (21.8%) and 116 (32.7%) specimens by DFA and R-mix culture and with the MultiCode-PLx system, respectively. Among the viruses common to both systems, the MultiCode-PLx system detected significantly more influenza A viruses (P = 0.0026). An additional increased diagnostic yield with the MultiCode-PLx system resulted from the detection of human metapneumovirus (HMPV) in 9 specimens, human CoV (HCoV) in 3 specimens, and human rhinovirus (HRV) in 16 specimens. Also, two mixed viral infections were detected by the MultiCode-PLx system (HCoV OC43 and HRV infections and HMPV and HRV infections), but none were detected by DFA and R-mix culture. Single-target PCRs verified the results obtained with the MultiCode-PLx system for 73 of 81 (90.1%) specimens that had discordant results or that were not covered by DFA and R-mix culture. The MultiCode-PLx system provides clinical laboratories with a practical, rapid, and sensitive means for the massively multiplexed molecular detection of common respiratory viruses.
    Full-text available · Article · Oct 2007 · Journal of Clinical Microbiology
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    [Show abstract] [Hide abstract] ABSTRACT: Human respiratory viruses are a diverse group of pathogens composed of hundreds of virus strains, and this presents a major challenge for diagnostic laboratories. To efficiently detect numerous viruses in a large epidemiologic study, we developed a fast, multitarget, sensitive, and specific assay named the Respiratory MultiCode-PLx Assay (RMA). The RMA utilizes improved multiplex PCR chemistry (EraGen MultiCode-PLx technology) coupled with high-throughput microsphere flow cytometry (Luminex). Eighteen sets of virus-specific multiplex PCR primers were developed based on the conserved sequences of all available respiratory-virus sequences for eight distinct groups: human rhinovirus (HRV), respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus (InfV), metapneumovirus, adenovirus (Ad), coronavirus, and enterovirus. Each primer set detected 20 cDNA copies of the intended target per sample and had no reaction with 60,000 copies of human genomic DNA. The accuracy and sensitivity of the RMA for detecting respiratory viruses in human samples were tested with two sets of clinical specimens. First, 101 nasal-wash specimens that were positive for HRV, RSV, InfV, PIV, or Ad by traditional techniques were reanalyzed by RMA, and all target viruses were detected with an overall sensitivity of 94% and specificity of 99%. Second, 103 nasal-wash samples from 5-year-old children with asthma and respiratory symptoms were analyzed; RMA detected viruses in 74 specimens (71.8%) compared to only 24 (23.3%) by traditional culture and immunofluorescent-staining techniques. These results show that RMA is an accurate, sensitive, and practical test for respiratory-virus infections.
    Full-text available · Article · Sep 2007 · Journal of Clinical Microbiology
  • John B Biggins · James R Prudent · David J Marshall · Jon S Thorson
    [Show abstract] [Hide abstract] ABSTRACT: Exploring the properties of molecules that cleave DNA (i.e., enzymatic nucleases, chemical footprinting agents, and naturally occurring DNA cleaving antibiotics) has been an ongoing process with benefits extending toward both laboratory and clinical applications. Despite the progress that has been made toward understanding the mechanics of DNA cleavage, a simple and continuous assay for detecting DNA cleavage has been lacking. Herein, we describe the molecular break light assay, wherein a single oligo-nucleotide modified by a 5'-fluorophore-3'-quencher pair adopting a stem-loop structure with an appropriate DNA recognition site, provides for the rapid assaying of DNA cleavage with high sensitivity. Furthermore, the described methodology is highly convenient in that it is readily adaptable to common laboratory fluorometers and multi-well plate/ array systems, which may provide the basis for high-throughput screening of novel DNA cleaving agents. This assay may also be further extended to natural or "unnatural" transcription factor protection assay systems.
    Article · Feb 2006 · Methods in Molecular Biology
  • Christopher B. Sherrill · David J. Marshall · Michael J. Moser · [...] · James R. Prudent
    Article · Oct 2005 · Journal of the American Chemical Society
  • Scott C Johnson · David J Marshall · Gerda Harms · [...] · James R Prudent
    [Show abstract] [Hide abstract] ABSTRACT: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expansion of newborn screening. We describe the development and technical evaluation of a multiplex platform that may foster increased newborn genetic screening. MultiCode PLx involves three major steps: PCR, target-specific extension, and liquid chip decoding. Each step is performed in the same reaction vessel, and the test is completed in approximately 3 h. For site-specific labeling and room-temperature decoding, we use an additional base pair constructed from isoguanosine and isocytidine. We used the method to test for mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The developed test was performed manually and by automated liquid handling. Initially, 225 samples with a range of genotypes were tested retrospectively with the method. A prospective study used samples from >400 newborns. In the retrospective study, 99.1% of samples were correctly genotyped with no incorrect calls made. In the perspective study, 95% of the samples were correctly genotyped for all targets, and there were no incorrect calls. The unique genetic multiplexing platform was successfully able to test for 31 targets within the CFTR gene and provides accurate genotype assignments in a clinical setting.
    Article · Nov 2004 · Clinical Chemistry
  • Christopher B Sherrill · David J Marshall · Michael J Moser · [...] · James R Prudent
    [Show abstract] [Hide abstract] ABSTRACT: Organic chemistry has made possible the synthesis of molecules that expand on Nature's genetic alphabet. Using the previously described nonstandard DNA base pair constructed from isoguanine and 5-methylisocytosine, we report a highly specific and sensitive method that allows for the fast and specific quantitation of genetic sequences in a closed tube format. During PCR amplification, enzymatic site-specific incorporation of a quencher covalently linked to isoguanine allows for the simultaneous detection and identification of multiple targets. The specificity of method is then established by analysis of thermal denaturation or melting of the amplicons. The appropriate functions of all reactions are further verified by incorporation of an independent target into the reaction mixture. We report that the method is sensitive down to the single copy level, and specificity is demonstrated by multiplexed end-point genotypic analysis of four targets simultaneously using four separate fluorescent reporters. The method is general enough for quantitative and qualitative analysis of both RNA and DNA using previously developed primer sets. Though the method described employs the commonly used PCR, the enzymatic incorporation of reporter groups into DNA site-specifically should find broad utility throughout molecular biology.
    Article · May 2004 · Journal of the American Chemical Society
  • Scott C Johnson · Christopher B Sherrill · David J Marshall · [...] · James R Prudent
    [Show abstract] [Hide abstract] ABSTRACT: Two additional bases (isoguanosine and isocytosine), generating a third base pair, have been implemented in PCR. Enzyme fidelity for the third base pair is demonstrated using molecular thermodynamic melting, chemical cleavage and molecular beacons. When amplifying as few as 15 targets containing multiple non‐natural base pairs with 40 cycles of amplification, our results confirm sequence conservation. The additional sequence space provided by three base pairs allows for the construction of molecular tools that achieve higher complexity and better discrimination than those possible with natural DNA alone.
    Article · Feb 2004 · Nucleic Acids Research
  • Michael J Moser · David J Marshall · Jennifer K Grenier · [...] · James R Prudent
    [Show abstract] [Hide abstract] ABSTRACT: With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensitivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target. We developed a novel real-time PCR technology that uses universal energy transfer probes constructed from An Expanded Genetic Information System (AEGIS) for both quantification and genotyping analyses. RNA quantification by reverse transcription-PCR was linear over four orders of magnitude for the simultaneous analysis of beta-actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was performed by endpoint analysis for factor V Leiden and prothrombin 20210A mutation detection. There was concordance for 173 samples between the genotyping results from Invader tests and the AEGIS universal energy transfer probe system for both factor V Leiden and prothrombin G20210A. Two prothrombin and one factor V sample gave indeterminate results (no calls). The AEGIS universal probe system allows for rapid development of PCR assays for nucleic acid quantification and genotyping.
    Article · Apr 2003 · Clinical Chemistry