Publications by authors named "Jennifer M Johnson"

50 Publications

Commentary: SWS Brain-Wave Music May Improve the Quality of Sleep: An EEG Study.

Front Neurosci 2021 1;15:609169. Epub 2021 Feb 1.

Lincoln Sleep Research Centre, University of Lincoln, Lincoln, United Kingdom.

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http://dx.doi.org/10.3389/fnins.2021.609169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7882482PMC
February 2021

Discordant Responses Between Primary Head and Neck Tumors and Nodal Metastases Treated With Neoadjuvant Nivolumab: Correlation of Radiographic and Pathologic Treatment Effect.

Front Oncol 2020 2;10:566315. Epub 2020 Dec 2.

Department of Otolaryngology - Head and Neck Surgery, Thomas Jefferson University, Philadelphia, PA, United States.

PD-1 blockade represents a promising treatment in patients with head and neck squamous cell carcinoma (HNSCC). We analyzed results of a neoadjuvant randomized window-of-opportunity trial of nivolumab plus/minus tadalafil to investigate whether immunotherapy-mediated treatment effects vary by site of involvement (primary tumor, lymph nodes) and determine how radiographic tumor shrinkage correlates with pathologic treatment effect.

Patients And Methods: Forty-four patients enrolled in trial NCT03238365 were treated with nivolumab 240 mg intravenously on days 1 and 15 with or without oral tadalafil, as determined by random assignment, followed by surgery on day 31. Radiographic volumetric response (RVR) was defined as percent change in tumor volume from pretreatment to posttreatment CT scan. Responders were defined as those with a 10% reduction in the volume of the primary tumor or lymph nodes (LN). Pathologic treatment effect (PTE) was defined as the area showing fibrosis or lymphohistiocytic inflammation divided by total tumor area.

Results: Sixteen of 32 patients (50%) with pathologic evidence of LN involvement exhibited discordant PTE between primary sites and LN. In four patients with widely discordant adjacent LN, increased PTE was associated with increased infiltration of tumor CD8 T cells and CD163 macrophages, whereas stromal regulatory T cells were associated with low nodal PTE. RVR correlated with PTE at both primary tumor (slope = 0.55, < 0.001) and in LN (slope = 0.62, < 0.05). 89% (16/18) of radiographic non-responders with T1-T3 primary sites had no (n = 7) or minimal PTE (n = 9), whereas 15/17 (88%) of radiographic responders had moderate (n = 12) or complete (n = 3) PTE.

Conclusion: Nivolumab often induces discordant treatment effects between primary tumor sites and metastatic lymph nodes within subjects. This treatment discordance was also demonstrated in adjacent lymph nodes, which may correlate with local immune cell makeup. Finally, although these data were generated by a relatively small population size, our data support the use of early radiographic response to assess immunotherapy treatment effect in HNSCC.
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http://dx.doi.org/10.3389/fonc.2020.566315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738605PMC
December 2020

Mutation signature analysis identifies increased mutation caused by tobacco smoke associated DNA adducts in larynx squamous cell carcinoma compared with oral cavity and oropharynx.

Sci Rep 2019 12 17;9(1):19256. Epub 2019 Dec 17.

Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, 19107, USA.

Squamous cell carcinomas of the head and neck (HNSCC) arise from mucosal keratinocytes of the upper aero-digestive tract. Despite a common cell of origin and similar driver-gene mutations which divert cell fate from differentiation to proliferation, HNSCC are considered a heterogeneous group of tumors categorized by site of origin within the aero-digestive mucosa, and the presence or absence of HPV infection. Tobacco use is a major driver of carcinogenesis in HNSCC and is a poor prognosticator that has previously been associated with poor immune cell infiltration and higher mutation numbers. Here, we study patterns of mutations in HNSCC that are derived from the specific nucleotide changes and their surrounding nucleotide context (also known as mutation signatures). We identify that mutations linked to DNA adducts associated with tobacco smoke exposure are predominantly found in the larynx. Presence of this class of mutation, termed COSMIC signature 4, is responsible for the increased burden of mutation in this anatomical sub-site. In addition, we show that another mutation pattern, COSMIC signature 5, is positively associated with age in HNSCC from non-smokers and that larynx SCC from non-smokers have a greater number of signature 5 mutations compared with other HNSCC sub-sites. Immunohistochemistry demonstrates a significantly lower Ki-67 proliferation index in size matched larynx SCC compared with oral cavity SCC and oropharynx SCC. Collectively, these observations support a model where larynx SCC are characterized by slower growth and increased susceptibility to mutations from tobacco carcinogen DNA adducts.
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http://dx.doi.org/10.1038/s41598-019-55352-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917707PMC
December 2019

A Pilot Study of Radiation Therapy in Combination With Pembrolizumab in Patients With Metastatic Renal Cell Cancer.

Am J Clin Oncol 2020 02;43(2):82-86

Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA.

Objectives: There is no study published regarding the benefit of radiation therapy (RT) in combination with immune checkpoint inhibitors (ICIs) for the treatment of metastatic renal cell cancer (mRCC). This report is part of an exploratory study aiming to determine the immunomodulatory activity of RT alone or in combination with pembrolizumab in solid tumors.

Materials And Methods: mRCC patients were treated with a combination of RT (8 Gy×1 or 4 Gy×5) followed by pembrolizumab with or without lead-in dose of pembrolizumab. Treatment response was measured based on the modified Response Evaluation Criteria in Solid Tumors criteria. Adverse events were monitored and graded. Pre-RT and post-RT tumor biopsies were obtained to evaluate programmed death-ligand 1 expression. Immune markers from peripheral blood before, during, and after treatment were analyzed using flow cytometry.

Results: Twelve mRCC patients who progressed on prior antiangiogenic therapy were enrolled. Half had 2 lines of prior therapy. Two patients (16.7%) had partial responses and were on study for 12.4 and 14.5 months. Three patients had stable disease for a period ranging from 4.2 to 10.4 months, whereas 7 patients had progressive disease. Median progression-free survival was 8.6 months and median overall survival was 32.3 months. Three patients had grade ≥3 events (hyperglycemia, thrombocytopenia, transaminitis). Biopsied tissue programmed death-ligand 1 expression and tumor-infiltrating lymphocytes were numerically higher in responders comparing to nonresponders (Modified Proportion Score 45% vs. 30.45%; tumor-infiltrating lymphocytes odds ratio 4.92).

Conclusion: Combining RT with pembrolizumab in pretreated mRCC is well-tolerated and appears to have comparable efficacy with single-agent nivolumab.
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http://dx.doi.org/10.1097/COC.0000000000000636DOI Listing
February 2020

Single institution implementation of permanent Cs interstitial brachytherapy for previously irradiated patients with resectable recurrent head and neck carcinoma.

J Contemp Brachytherapy 2019 Jun 28;11(3):227-234. Epub 2019 Jun 28.

Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, PA, United States.

Purpose: Permanent interstitial brachytherapy is an appealing treatment modality for patients with locoregional recurrent, resectable head and neck carcinoma (HNC), having previously received radiation. Cesium-131 (Cs) is a permanent implant brachytherapy isotope, with a low average photon energy of 30 keV and a short half-life of 9.7 days. Exposure to medical staff and family members is low; patient isolation and patient room shielding are not required. This work presents a single institution's implementation process of utilizing an intraoperative, permanent Cs implant for patients with completely resected recurrent HNC.

Material And Methods: Fifteen patients receiving Cs permanent seed brachytherapy were included in this analysis. The process of pre-planning, selecting the dose prescription, seed ordering, intraoperative procedures, post-implant planning, and radiation safety protocols are described.

Results: Tumor volumes were contoured on the available preoperative PET/CT scans and a pre-implant treatment plan was created using uniform source strength and uniform 1 cm seed spacing. Implants were performed intraoperatively, following tumor resection. In five of the fifteen cases, intraoperative findings necessitated a change from the planned number of seeds and recalculation of the pre-implant plan. The average prescription dose was 56.1 ±6.6 Gy (range, 40-60 Gy). The average seed strength used was 2.2 ±0.2 U (3.5 ±0.3 mCi). Patients returned to a recovery room on a standard surgical floor and remained inpatients, without radiation safety restrictions, based on standard surgical recovery protocols. A post-implant treatment plan was generated based on immediate post-operative CT imaging to verify the seed distribution and confirm delivery of the prescription dose. Patients were provided educational information regarding radiation safety recommendations.

Conclusions: Cesium-131 interstitial brachytherapy is feasible and does not pose major radiation safety concerns; it should be considered as a treatment option for previously irradiated patients with recurrent, resectable HNC.
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http://dx.doi.org/10.5114/jcb.2019.85778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701381PMC
June 2019

High-Throughput Protein Production of Membrane Proteins in Saccharomyces cerevisiae.

Methods Mol Biol 2019 ;2025:227-259

Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.

This chapter outlines a protocol to assess viability for large-scale protein production and purification for selected targets from an initial medium-throughput cloning strategy. Thus, one can assess a broad number of potential candidate proteins, mutants, or expression variants using an empirically minimalistic approach. In addition, a key output from this protocol is utilization of Saccharomyces cerevisiae as a means for the efficient screening and production of purified proteins. The primary focus in this protocol is overexpression of polytopic integral membrane proteins though methods can be equally applied to soluble proteins. The protocol starts with outlining high-throughput (sans robotics) cloning of expression proteins into a dual-tag yeast expression plasmid. These membrane proteins are then screened for expression level, detergent solubilization, initial purity, and chromatography characteristics. Both small- and large-scale expression methods are discussed along with fermentation.
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http://dx.doi.org/10.1007/978-1-4939-9624-7_11DOI Listing
March 2020

Combining Radiation and Immune Checkpoint Blockade in the Treatment of Head and Neck Squamous Cell Carcinoma.

Front Oncol 2019 6;9:122. Epub 2019 Mar 6.

Department of Medical Oncology, Thomas Jefferson University, Philadelphia, PA, United States.

Head and neck squamous cell carcinoma (HNSCC) is a significant cause of morbidity and mortality worldwide. Current treatment options, even though potentially curative, have many limitations including a high rate of complications. Over the past few years immune checkpoint inhibitors (ICI) targeting cytotoxic lymphocyte antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed cell death ligand 1 (PD-L1) have changed treatment paradigms in many malignancies and are currently under investigation in HNSCC as well. Despite improvements in treatment outcomes and the implementation of combined modality approaches long-term survival rates in patients with locally advanced HNSCC remain suboptimal. Accumulating evidence suggests that under certain conditions, radiation may be delivered in conjunction with ICI to augment efficacy. In this review, we will discuss the immune modulating mechanisms of ICI and radiation, how changing the dose, fractionation, and field of radiation may alter the tumor microenvironment (TME), and how these two treatment modalities may work in concert to generate durable treatment responses against HNSCC.
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http://dx.doi.org/10.3389/fonc.2019.00122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414812PMC
March 2019

Metformin as a Therapeutic Target in Endometrial Cancers.

Front Oncol 2018 28;8:341. Epub 2018 Aug 28.

Department of Medical Oncology, Thomas Jefferson University, Philadelphia, PA, United States.

Endometrial cancer is the most common gynecologic malignancy in developed countries. Its increasing incidence is thought to be related in part to the rise of metabolic syndrome, which has been shown to be a risk factor for the development of hyperestrogenic and hyperinsulinemic states. This has consequently lead to an increase in other hormone-responsive cancers as well e.g., breast and ovarian cancer. The correlation between obesity, hyperglycemia, and endometrial cancer has highlighted the important role of metabolism in cancer establishment and persistence. Tumor-mediated reprogramming of the microenvironment and macroenvironment can range from induction of cytokines and growth factors to stimulation of surrounding stromal cells to produce energy-rich catabolites, fueling the growth, and survival of cancer cells. Such mechanisms raise the prospect of the metabolic microenvironment itself as a viable target for treatment of malignancies. Metformin is a biguanide drug that is a first-line treatment for type 2 diabetes that has beneficial effects on various markers of the metabolic syndrome. Many studies suggest that metformin shows potential as an adjuvant treatment for uterine and other cancers. Here, we review the evidence for metformin as a treatment for cancers of the endometrium. We discuss the available clinical data and the molecular mechanisms by which it may exert its effects, with a focus on how it may alter the tumor microenvironment. The pleiotropic effects of metformin on cellular energy production and usage as well as intercellular and hormone-based interactions make it a promising candidate for reprogramming of the cancer ecosystem. This, along with other treatments aimed at targeting tumor metabolic pathways, may lead to novel treatment strategies for endometrial cancer.
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http://dx.doi.org/10.3389/fonc.2018.00341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121131PMC
August 2018

type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation.

Proc Natl Acad Sci U S A 2018 09 4;115(38):E8977-E8986. Epub 2018 Sep 4.

Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210;

, an obligatory intracellular bacterium, infects monocytes/macrophages by sequestering a regulator of endosomal traffic, the small GTPase RAB5, on its membrane-bound inclusions to avoid routing to host-cell phagolysosomes. How RAB5 is sequestered on ehrlichial inclusions is poorly understood, however. We found that native translocated factor-2 (Etf-2), a previously predicted effector of the type IV secretion system, and recombinant Etf-2 (cloned into the genome) are secreted into the host-cell cytoplasm and localize to ehrlichial inclusions. Ectopically expressed Etf-2-GFP also localized to inclusions and membranes of early endosomes marked with RAB5 and interacted with GTP-bound RAB5 but not with a GDP-bound RAB5. Etf-2, although lacking a RAB GTPase-activating protein (GAP) Tre2-Bub2-Cdc16 (TBC) domain, contains two conserved TBC domain motifs, namely an Arg finger and a Gln finger, and site-directed mutagenesis revealed that both Arg and Gln are required for Etf-2 localization to early endosomes. The yeast two-hybrid assay and microscale thermophoresis revealed that Etf-2 binds tightly to GTP-bound RAB5 but not to GDP-bound RAB5. However, Etf-2 lacks RAB5-specific GAP activity. Etf-2 localized to bead-containing phagosomes as well as endosomes containing beads coated with the C-terminal fragment of EtpE (entry-triggering protein of ), an outer-membrane invasin, and significantly delayed RAB5 dissociation from and RAB7 localization to phagosomes/endosomes and RABGAP5 localization to endosomes. Thus, binding of Etf-2 to RAB5-GTP appears to delay RAB5 inactivation by impeding RABGAP5 localization to endosomes. This suggests a unique mechanism by which RAB5 is sequestered on ehrlichial inclusions to benefit bacterial survival and replication.
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http://dx.doi.org/10.1073/pnas.1806904115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156607PMC
September 2018

The effect of cathodal transcranial direct current stimulation during rapid eye-movement sleep on neutral and emotional memory.

R Soc Open Sci 2018 Jul 18;5(7):172353. Epub 2018 Jul 18.

School of Psychology, University of Lincoln, Brayford Pool, Lincoln LN6 7TS, UK.

Sleep-dependent memory consolidation has been extensively studied. Neutral declarative memories and serial reaction time task (SRTT) performance can benefit from slow-wave activity, characterized by less than 1 Hz frequency cortical slow oscillations (SO). Emotional memories can benefit from theta activity, characterized by 4-8 Hz frequency cortical oscillations. Applying transcranial direct current stimulation (tDCS) during sleep entrains specific frequencies to alter sleep architecture. When applying cathodal tDCS (CtDCS), neural inhibition or excitation may depend on the waveform at the applied frequency. A double dissociation was predicted, with CtDCS at SO frequency improving neutral declarative memory and SRTT performance, and theta frequency CtDCS inhibiting negative emotional memory. Participants completed three CtDCS conditions (Theta: 5 Hz, SO: 0.75 Hz and control: sham) and completed an SRTT and word recognition task pre- and post-sleep, comprising emotional and neutral words to assess memory. In line with predictions, CtDCS improved neutral declarative memory when applied at SO frequency. When applied at theta frequency, no negative emotional word memory impairment was found but a positive association was found between post-stimulation theta power and emotional word recognition. SRTT performance was also not altered by either CtDCS frequency. Future studies should investigate overnight theta CtDCS and examine the effects of CtDCS during and after stimulation.
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http://dx.doi.org/10.1098/rsos.172353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6083708PMC
July 2018

Commentary: Effects of Sleep on Word Pair Memory in Children-Separating Item and Source Memory Aspects.

Front Psychol 2018 19;9:1022. Epub 2018 Jun 19.

School of Psychology, University of Lincoln, Lincoln, United Kingdom.

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http://dx.doi.org/10.3389/fpsyg.2018.01022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020786PMC
June 2018

"No Pumps Allowed": The "Problem" With Gender Expression and the Morehouse College "Appropriate Attire Policy".

J Homosex 2019 31;66(7):867-895. Epub 2018 Jul 31.

b Teaching/Instructional Policy, Organizational, & Leadership Studies Department , Temple University , Philadelphia , Pennsylvania , USA.

Within higher education literature, historically Black colleges and universities (HBCUs) have been lauded for their exceptional ability to provide African American students with culturally engaging academic and social environments. While the aforementioned may be true, much of this literature has presented HBCU students and these institutions as monolithic entities, with little regard to the ways social identities (e.g., gender, gender identity, sexual identities) shape students' undergraduate experiences. This investigation uses critical discourse analysis to explore the media's coverage and reactions to the Morehouse College appropriate attire policy in order to examine how their campus stakeholders problematized gender expression within this HBCU context. Implications for this research provides insights into how HBCU communities can both recognize and respond to the needs of their diverse queer student populations. This study concludes with highlighting new advancements being made on HBCU campuses that illustrate how they are making their campuses more inclusive of queer students.
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http://dx.doi.org/10.1080/00918369.2018.1486063DOI Listing
April 2019

Functional Characterization of the Saccharomyces cerevisiae Equilibrative Nucleoside Transporter 1 (ScENT1).

Molecules 2018 Mar 22;23(4). Epub 2018 Mar 22.

Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.

Equilibrative nucleoside transporters (ENTs) are polytopic membrane transporters responsible for the translocation of nucleosides, nucleobases-to a lesser extent-and nucleoside analog therapeutics across cellular membranes. ENTs function in a diffusion controlled bidirectional manner and are thought to utilize an alternating access transport mechanism. However, a detailed understanding of ENT function at the molecular level has remained elusive. ScENT1 (formerly known as Function Unknown Now 26 or FUN26) is the only known ENT ortholog endogenously expressed in , and a proteoliposome assay system was used to study homogenously overexpressed and purified ScENT1 (wildtype relative to L390A and F249I mutants). L390 and F249 are highly conserved residues and were found to alter transporter function. L390A produced a reduction of mean transport activity while F249I increased mean substrate translocation relative to wildtype protein. However, both mutations resulted in transport of UTP-a novel gain of function for any ENT. These residues were then mapped onto an ab initio model of FUN26 which suggests they function in substrate translocation (L390) or cytoplasmic gating (F249). Furthermore, wildtype, L390A, and F249I were found to be sensitive to the presence of alcohols. Ethanol attenuated ScENT1-mediated transport of uridine by ~50%. These findings further demonstrate functional similarities between ScENT1 and human ENT isoforms and support identification of FUN26 as ScENT1, the first ENT isoform in .
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http://dx.doi.org/10.3390/molecules23040732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6017673PMC
March 2018

The influence of REM sleep and SWS on emotional memory consolidation in participants reporting depressive symptoms.

Cortex 2018 02 15;99:281-295. Epub 2017 Dec 15.

School of Psychology, College of Social Science, University of Lincoln. Sarah Swift Building, Brayford Wharf East, Lincoln, Lincolnshire, United Kingdom. Electronic address:

Negative emotional memory bias is thought to play a causal role in the onset and maintenance of major depressive disorder. Rapid Eye Movement (REM) sleep has been shown to selectively consolidate negative emotional memories in healthy participants, and is greater in quantity and density in depressed patients. Slow-Wave Sleep (SWS) is typically associated with the consolidation of non-emotional memories. However, the effects of REM sleep and SWS on emotional memory consolidation have not been investigated in participants reporting depressive symptoms. In this study, we recruited two groups of healthy participants; one reporting mild-to-moderate depressive symptoms, and another reporting minimal depressive symptoms (assessed using the Beck Depression Inventory; BDI-II). Using a within-subjects split-night design, we measured consolidation of positive, neutral and negative images across a 3 h retention interval rich in either REM sleep or SWS. We found a significant sleep condition x image valence interaction in participants reporting depressive symptoms [F (2, 20) = 4.73, p = .021], but not participants reporting minimal depressive symptoms [F (2, 22) = .17, p = .845]. Participants reporting depressive symptoms consolidated significantly more neutral memories during SWS, and marginally more negative memories during REM sleep, than those reporting minimal depressive symptoms [t (21) = 2.44, p = .023; t (21) = 1.96, p = .064, respectively]. Our preliminary results demonstrate that REM sleep and SWS have differential effects on the consolidation of emotional and neutral images in participants reporting depressive symptoms. Further studies including larger sample sizes are required to investigate whether REM sleep alterations promote the development of negative memory bias in major depressive disorder.
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http://dx.doi.org/10.1016/j.cortex.2017.12.004DOI Listing
February 2018

Expression and purification of human and Saccharomyces cerevisiae equilibrative nucleoside transporters.

Protein Expr Purif 2018 Feb 14;142:68-74. Epub 2017 Sep 14.

Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA; Stephenson Oklahoma Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA; Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. Electronic address:

Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis.
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http://dx.doi.org/10.1016/j.pep.2017.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679208PMC
February 2018

MCT1 in Invasive Ductal Carcinoma: Monocarboxylate Metabolism and Aggressive Breast Cancer.

Front Cell Dev Biol 2017 3;5:27. Epub 2017 Apr 3.

Department of Medical Oncology, Sidney Kimmel Cancer Center, Thomas Jefferson UniversityPhiladelphia, PA, USA.

Monocarboxylate transporter 1 (MCT1) is an importer of monocarboxylates such as lactate and pyruvate and a marker of mitochondrial metabolism. MCT1 is highly expressed in a subgroup of cancer cells to allow for catabolite uptake from the tumor microenvironment to support mitochondrial metabolism. We studied the protein expression of MCT1 in a broad group of breast invasive ductal carcinoma specimens to determine its association with breast cancer subtypes and outcomes. MCT1 expression was evaluated by immunohistochemistry on tissue micro-arrays (TMA) obtained through our tumor bank. Two hundred and fifty-seven cases were analyzed: 180 cases were estrogen receptor and/or progesterone receptor positive (ER+ and/or PR+), 62 cases were human epidermal growth factor receptor 2 positive (HER2+), and 56 cases were triple negative breast cancers (TNBC). MCT1 expression was quantified by digital pathology with Aperio software. The intensity of the staining was measured on a continuous scale (0-black to 255-bright white) using a co-localization algorithm. Statistical analysis was performed using a linear mixed model. High MCT1 expression was more commonly found in TNBC compared to ER+ and/or PR+ and compared to HER-2+ ( < 0.001). Tumors with an component were less likely to stain strongly for MCT1 ( < 0.05). High nuclear grade was associated with higher MCT1 staining ( < 0.01). Higher T stage tumors were noted to have a higher expression of MCT1 ( < 0.05). High MCT1 staining in cancer cells was associated with shorter progression free survival, increased risk of recurrence, and larger size independent of TNBC status ( < 0.05). MCT1 expression, which is a marker of high catabolite uptake and mitochondrial metabolism, is associated with recurrence in breast invasive ductal carcinoma. MCT1 expression as quantified with digital image analysis may be useful as a prognostic biomarker and to design clinical trials using MCT1 inhibitors.
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http://dx.doi.org/10.3389/fcell.2017.00027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376582PMC
April 2017

Ecology, Fitness, Evolution: New Perspectives on Categorization.

Curr Dir Psychol Sci 2016 Aug;25(4):266-274

Department of Psychology: Georgia State University.

Categorization's great debate has weighed single-system exemplar theory against the possibility of alternative processing systems. We take an evolutionary perspective toward this debate to illuminate it in a new way. Animals are crucial behavioral ambassadors to this area. They reveal the roots of human categorization, the basic assumptions of vertebrates entering category tasks, and the surprising weakness of exemplar memory as a category-learning strategy. These results have joined neuroscience results to prompt important changes in categorization theory. Categorization's great debate is ending. Categorization is served by multiple systems of process and representation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055127PMC
http://dx.doi.org/10.1177/0963721416652393DOI Listing
August 2016

Integral Membrane Protein Expression in Saccharomyces cerevisiae.

Methods Mol Biol 2016 ;1432:163-86

Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.

Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.
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http://dx.doi.org/10.1007/978-1-4939-3637-3_11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166409PMC
December 2017

Categorization: The View from Animal Cognition.

Behav Sci (Basel) 2016 Jun 15;6(2). Epub 2016 Jun 15.

Language Research Center, Georgia State University, 3401 Panthersville Rd, Decatur, GA 30034, USA.

Exemplar, prototype, and rule theory have organized much of the enormous literature on categorization. From this theoretical foundation have arisen the two primary debates in the literature-the prototype-exemplar debate and the single system-multiple systems debate. We review these theories and debates. Then, we examine the contribution that animal-cognition studies have made to them. Animals have been crucial behavioral ambassadors to the literature on categorization. They reveal the roots of human categorization, the basic assumptions of vertebrates entering category tasks, the surprising weakness of exemplar memory as a category-learning strategy. They show that a unitary exemplar theory of categorization is insufficient to explain human and animal categorization. They show that a multiple-systems theoretical account-encompassing exemplars, prototypes, and rules-will be required for a complete explanation. They show the value of a fitness perspective in understanding categorization, and the value of giving categorization an evolutionary depth and phylogenetic breadth. They raise important questions about the internal similarity structure of natural kinds and categories. They demonstrate strong continuities with humans in categorization, but discontinuities, too. Categorization's great debates are resolving themselves, and to these resolutions animals have made crucial contributions.
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http://dx.doi.org/10.3390/bs6020012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931384PMC
June 2016

Thyroid Cancer Metabolism: A Review.

J Thyroid Disord Ther 2016 Feb 14;5(1). Epub 2016 Jan 14.

Department of Otolaryngology-Head and Neck Surgery, Thomas Jefferson University, Philadelphia, USA.

Metabolic dysregulation within the tumor microenvironment (TME) is critical to the process of tumorigenesis in various cancer types. Thyrocyte metabolism in papillary and anaplastic thyroid cancer, however, remains poorly characterized, and studies analyzing the role of multicompartment metabolism in thyrocyte oncogenesis are sparse. We present a review of the current knowledge on cellular metabolism in non-cancerous and cancerous thyroid tissues, focusing on the monocarboxylate transporters MCT1 and MCT4, and on a transporter of the outer mitochondrial membrane TOMM20. Understanding the metabolic phenotype of tumor cells and associated stromal cells in thyroid cancer can have profound implications on the use of biomarker staining in detecting subclinical cancer, imaging as it relates to expression of various transport proteins, and therapeutic interventions that manipulate this dysregulated tumor metabolism to halt tumorigenesis and eradicate the cancer. Future studies are required to confirm the prognostic significance of these biomarkers and their correlation with existing staging schemas such as the AGES, AMES, ATA and MACIS scoring systems.
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http://dx.doi.org/10.4172/2167-7948.1000200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4874252PMC
February 2016

Mitochondrial Metabolism as a Treatment Target in Anaplastic Thyroid Cancer.

Semin Oncol 2015 Dec 24;42(6):915-22. Epub 2015 Sep 24.

Department of Otolaryngology, Thomas Jefferson University, Philadelphia, PA. Electronic address:

Anaplastic thyroid cancer (ATC) is one of the most aggressive human cancers. Key signal transduction pathways that regulate mitochondrial metabolism are frequently altered in ATC. Our goal was to determine the mitochondrial metabolic phenotype of ATC by studying markers of mitochondrial metabolism, specifically monocarboxylate transporter 1 (MCT1) and translocase of the outer mitochondrial membrane member 20 (TOMM20). Staining patterns of MCT1 and TOMM20 in 35 human thyroid samples (15 ATC, 12 papillary thyroid cancer [PTC], and eight non-cancerous thyroid) and nine ATC mouse orthotopic xenografts were assessed by visual and Aperio digital scoring. Staining patterns of areas involved with cancer versus areas with no evidence of cancer were evaluated independently where available. MCT1 is highly expressed in human anaplastic thyroid cancer when compared to both non-cancerous thyroid tissues and papillary thyroid cancers (P<.001 for both). TOMM20 is also highly expressed in both ATC and PTC compared to non-cancerous thyroid tissue (P<.01 for both). High MCT1 and TOMM20 expression is also found in ATC mouse xenograft tumors compared to non-cancerous thyroid tissue (P<.001). These xenograft tumors have high (13)C- pyruvate uptake. ATC has metabolic features that distinguish it from PTC and non-cancerous thyroid tissue, including high expression of MCT1 and TOMM20. PTC has low expression of MCT1 and non-cancerous thyroid tissue has low expression of both MCT1 and TOMM20. This work suggests that MCT1 blockade may specifically target ATC cells presenting an opportunity for a new drug target.
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http://dx.doi.org/10.1053/j.seminoncol.2015.09.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4663018PMC
December 2015

Conformation-Dependent Human p52Shc Phosphorylation by Human c-Src.

Biochemistry 2015 Jun 26;54(22):3469-82. Epub 2015 May 26.

†Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.

Phosphorylation of the human p52Shc adaptor protein is a key determinant in modulating signaling complex assembly in response to tyrosine kinase signaling cascade activation. The underlying mechanisms that govern p52Shc phosphorylation status are unknown. In this study, p52Shc phosphorylation by human c-Src was investigated using purified proteins to define mechanisms that affect the p52Shc phosphorylation state. We conducted biophysical characterizations of both human p52Shc and human c-Src in solution as well as membrane-mimetic environments using the acidic lipid phosphatidylinositol 4-phosphate or a novel amphipathic detergent (2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside). We then identified p52Shc phosphorylation sites under various solution conditions, and the amount of phosphorylation at each identified site was quantified using mass spectrometry. These data demonstrate that the p52Shc phosphorylation level is altered by the solution environment without affecting the fraction of active c-Src. Mass spectrometry analysis of phosphorylated p52Shc implies functional linkage among phosphorylation sites. This linkage may drive preferential coupling to protein binding partners during signaling complex formation, such as during initial binding interactions with the Grb2 adaptor protein leading to activation of the Ras/MAPK signaling cascade. Remarkably, tyrosine residues involved in Grb2 binding were heavily phosphorylated in a membrane-mimetic environment. The increased phosphorylation level in Grb2 binding residues was also correlated with a decrease in the thermal stability of purified human p52Shc. A schematic for the phosphorylation-dependent interaction between p52Shc and Grb2 is proposed. The results of this study suggest another possible therapeutic strategy for altering protein phosphorylation to regulate signaling cascade activation.
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http://dx.doi.org/10.1021/acs.biochem.5b00122DOI Listing
June 2015

A case of periampullary adenocarcinoma in neurofibromatosis type 1.

J Gastrointest Oncol 2014 Dec;5(6):E96-9

1 Department of Medicine, 2 Department of Medical Oncology, 3 Department of Surgery, 4 Department of Pathology, 5 Department of Medicine, Division of Gastroenterology and Hepatology, Thomas Jefferson University Hospital, Philadelphia, PA, USA.

Neurofibromatosis type 1 (NF-1) is an autosomal dominant genetic disorder with a known predisposition to gastrointestinal neoplasms such as stromal tumors and carcinoids. Adenocarcinomas (ACs) of the gastrointestinal tract are relatively rare in patients with NF-1, especially those found in the periampullary region. We present a case report of periampullary adenocarcinoma in a 56-year-old woman with NF-1 who presented with abdominal pain and obstructive jaundice.
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http://dx.doi.org/10.3978/j.issn.2078-6891.2014.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226813PMC
December 2014

Overproduction and biophysical characterization of human HSP70 proteins.

Protein Expr Purif 2015 Feb 27;106:57-65. Epub 2014 Sep 27.

Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd, Oklahoma City, OK 73104, United States; Stephenson Oklahoma Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, United States. Electronic address:

Heat shock proteins (HSP) perform vital cellular functions and modulate cell response pathways to physical and chemical stressors. A key feature of HSP function is the ability to interact with a broad array of protein binding partners as a means to potentiate downstream response pathways or facilitate protein folding. These binding interactions are driven by ATP-dependent conformational rearrangements in HSP proteins. The HSP70 family is evolutionarily conserved and is associated with diabetes and cancer progression and the etiopathogenesis of hepatic, cardiovascular, and neurological disorders in humans. However, functional characterization of human HSP70s has been stymied by difficulties in obtaining large quantities of purified protein. Studies of purified human HSP70 proteins are essential for downstream investigations of protein-protein interactions and in the rational design of novel family-specific therapeutics. Within this work, we present optimized protocols for the heterologous overexpression and purification of either the nucleotide binding domain (NBD) or the nucleotide and substrate binding domains of human HSPA9, HSPA8, and HSPA5 in either Escherichia coli or Saccharomyces cerevisiae. We also include initial biophysical characterization of HSPA9 and HSPA8. This work provides the basis for future biochemical studies of human HSP70 protein function and structure.
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http://dx.doi.org/10.1016/j.pep.2014.09.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248018PMC
February 2015

FUN26 (function unknown now 26) protein from saccharomyces cerevisiae is a broad selectivity, high affinity, nucleoside and nucleobase transporter.

J Biol Chem 2014 Aug 17;289(35):24440-51. Epub 2014 Jul 17.

From the Department of Biochemistry and Molecular Biology and the Stephenson Oklahoma Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and

Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2')- and C(5')-positions on the ribose sugar and is not stimulated by a membrane pH differential. [(3)H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [(3)H]adenosine or [(3)H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system.
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http://dx.doi.org/10.1074/jbc.M114.553503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148870PMC
August 2014

Skp1 prolyl 4-hydroxylase of dictyostelium mediates glycosylation-independent and -dependent responses to O2 without affecting Skp1 stability.

J Biol Chem 2012 Jan 29;287(3):2006-16. Epub 2011 Nov 29.

Department of Biochemistry and Molecular Biology, 975 NE 10th St., BRC 417, OUHSC, Oklahoma City, OK 73104, USA.

Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O(2) sensing in animals via modification of the transcriptional factor subunit HIFα, resulting in its polyubiquitination by the E3(VHL)ubiquitin (Ub) ligase and degradation in the 26 S proteasome. Previously thought to be restricted to animals, a homolog (P4H1) of HIFα-type PHDs is expressed in the social amoeba Dictyostelium where it also exhibits characteristics of an O(2) sensor for development. Dictyostelium lacks HIFα, and P4H1 modifies a different protein, Skp1, an adaptor of the SCF class of E3-Ub ligases related to the E3(VHL)Ub ligase that targets animal HIFα. Normally, the HO-Skp1 product of the P4H1 reaction is capped by a GlcNAc sugar that can be subsequently extended to a pentasaccharide by novel glycosyltransferases. To analyze the role of glycosylation, the Skp1 GlcNAc-transferase locus gnt1 was modified with a missense mutation to block catalysis or a stop codon to truncate the protein. Despite the accumulation of the hydroxylated form of Skp1, Skp1 was not destabilized based on metabolic labeling. However, hydroxylation alone allowed for partial correction of the high O(2) requirement of P4H1-null cells, therefore revealing both glycosylation-independent and glycosylation-dependent roles for hydroxylation. Genetic complementation of the latter function required an enzymatically active form of Gnt1. Because the effect of the gnt1 deficiency depended on P4H1, and Skp1 was the only protein labeled when the GlcNAc-transferase was restored to mutant extracts, Skp1 apparently mediates the cellular functions of both P4H1 and Gnt1. Although Skp1 stability itself is not affected by hydroxylation, its modification may affect the stability of targets of Skp1-dependent Ub ligases.
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http://dx.doi.org/10.1074/jbc.M111.314021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3265880PMC
January 2012

Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O₂ signaling in dictyostelium.

Biochemistry 2011 Mar 9;50(10):1700-13. Epub 2011 Feb 9.

Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States.

The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα) type prolyl 4-hydroxylase (P4H1) and an α-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein) class of E3 ubiquitin ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O(2) regulation of development, suggesting the existence of an ancient O(2)-sensing mechanism related to modification of the transcription factor HIFα by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O(2) signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. (1)H NMR demonstrated formation of 4(trans)-hydroxyproline as previously found for HIFα, and highly purified P4H1 was inhibited by Krebs cycle intermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFα by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3(SCF)ubiquitin ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily related E3(VBC)ubiquitin ligases.
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http://dx.doi.org/10.1021/bi101977wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192012PMC
March 2011

Nucleotide excision repair deficiency is intrinsic in sporadic stage I breast cancer.

Proc Natl Acad Sci U S A 2010 Dec 30;107(50):21725-30. Epub 2010 Nov 30.

Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Hospital, University of Pittsburgh Medical Center, Pittsburgh, PA 15213-3180, USA.

The molecular etiology of breast cancer has proven to be remarkably complex. Most individual oncogenes are disregulated in only approximately 30% of breast tumors, indicating that either very few molecular alterations are common to the majority of breast cancers, or that they have not yet been identified. In striking contrast, we now show that 19 of 19 stage I breast tumors tested with the functional unscheduled DNA synthesis assay exhibited a significant deficiency of DNA nucleotide excision repair (NER) capacity relative to normal epithelial tissue from disease-free controls (n = 23). Loss of DNA repair capacity, including the complex, damage-comprehensive NER pathway, results in genomic instability, a hallmark of carcinogenesis. By microarray analysis, mRNA expression levels for 20 canonical NER genes were reduced in representative tumor samples versus normal. Significant reductions were observed in 19 of these genes analyzed by the more sensitive method of RNase protection. These results were confirmed at the protein level for five NER gene products. Taken together, these data suggest that NER deficiency may play an important role in the etiology of sporadic breast cancer, and that early-stage breast cancer may be intrinsically susceptible to genotoxic chemotherapeutic agents, such as cis-platinum, whose damage is remediated by NER. In addition, reduced NER capacity, or reduced expression of NER genes, could provide a basis for the development of biomarkers for the identification of tumorigenic breast epithelium.
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http://dx.doi.org/10.1073/pnas.0914772107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003008PMC
December 2010

A practical approach to detect unique metabolic patterns for personalized medicine.

Analyst 2010 Nov 13;135(11):2864-70. Epub 2010 Sep 13.

Department of Medicine, Division of Pulmonary, Allergy and Critical Care, Emory University, Atlanta, GA 30322, USA.

Information-rich technologies have advanced personalized medicine, yet obstacles limit measurement of large numbers of chemicals in human samples. Current laboratory tests measure hundreds of chemicals based upon existing knowledge of exposures, metabolism and disease mechanisms. Practical issues of cost and throughput preclude measurement of thousands of chemicals. Additionally, individuals are genetically diverse and have different exposures and response characteristics; some have disease mechanisms that have not yet been elucidated. Consequently, methods are needed to detect unique metabolic characteristics without presumption of known pathways, exposures or disease mechanisms, i.e., using a top-down approach. In this report, we describe profiling of human plasma with liquid chromatography (LC) coupled to Fourier-transform mass spectrometry (FTMS). FTMS is a high-resolution mass spectrometer providing mass accuracy and resolution to discriminate thousands of m/z features, which are peaks defined by m/z, retention time and intensity. We demonstrate that LC-FTMS detects 2000 m/z features in 10 min. These features include known and unidentified chemicals with m/z between 85 and 850, most with <10% coefficient of variation. Comparison of metabolic profiles for 4 healthy individuals showed that 62% of the m/z features were common while 10% were unique and 770 discriminated the individuals. Because the simple one-step extraction and automated analysis is rapid and cost-effective, the approach is practical for personalized medicine. This provides a basis to rapidly characterize novel metabolic patterns which can be linked to genetics, environment and/or lifestyle.
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http://dx.doi.org/10.1039/c0an00333fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3069708PMC
November 2010