Publications by authors named "Ryan C Hill"

45 Publications

The effect of normal, metaplastic, and neoplastic esophageal extracellular matrix upon macrophage activation.

J Immunol Regen Med 2021 Aug 28;13. Epub 2020 Dec 28.

McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA.

Introduction: Macrophages are capable of extreme plasticity and their activation state has been strongly associated with solid tumor growth progression and regression. Although the macrophage response to extracellular matrix (ECM) isolated from normal tissue is reasonably well understood, there is a relative dearth of information regarding their response to ECM isolated from chronically inflamed tissues, pre-neoplastic tissues, and neoplastic tissues. Esophageal adenocarcinoma (EAC) is a type of neoplasia driven by chronic inflammation in the distal esophagus, and the length of the esophagus provides the opportunity to investigate macrophage behavior in the presence of ECM isolated from a range of disease states within the same organ.

Methods: Normal, metaplastic, and neoplastic ECM hydrogels were prepared from decellularized EAC tissue. The hydrogels were evaluated for their nanofibrous structure (SEM), biochemical profile (targeted and global proteomics), and direct effect upon macrophage (THP-1 cell) activation state (qPCR, ELISA, immunolabeling) and indirect effect upon epithelial cell (Het-1A) migration (Boyden chamber).

Results: Nanofibrous ECM hydrogels from the three tissue types could be formed, and normal and neoplastic ECM showed distinctive protein profiles by targeted and global mass spectroscopy. ECM proteins functionally related to cancer and tumorigenesis were identified in the neoplastic esophageal ECM including collagen alpha-1(VIII) chain (COL8A1), lumican, and elastin. Metaplastic and neoplastic esophageal ECM induce distinctive effects upon THP-1 macrophage signaling compared to normal esophageal ECM. These effects include activation of pro-inflammatory IFNγ and TNFα gene expression and anti-inflammatory IL1RN gene expression. Most notably, neoplastic ECM robustly increased macrophage TNFα protein expression. The secretome of macrophages pre-treated with metaplastic and neoplastic ECM increases the migration of normal esophageal epithelial cells, similar behavior to that shown by tumor cells. Metaplastic ECM shows similar but less pronounced effects than neoplastic ECM suggesting the abnormal signals also exist within the pre-cancerous state.

Conclusion: A progressively diseased ECM, as exists within the esophagus exposed to chronic gastric reflux, can provide insights into novel biomarkers of early disease and identify potential therapeutic targets.
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http://dx.doi.org/10.1016/j.regen.2020.100037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8136242PMC
August 2021

Injectable Myocardial Matrix Hydrogel Mitigates Negative Left Ventricular Remodeling in a Chronic Myocardial Infarction Model.

JACC Basic Transl Sci 2021 Apr 10;6(4):350-361. Epub 2021 Mar 10.

Department of Bioengineering, University of California, San Diego, La Jolla, California, USA.

A first-in-man clinical study on a myocardial-derived decellularized extracellular matrix hydrogel suggested the potential for efficacy in chronic myocardial infarction (MI) patients. However, little is understood about the mechanism of action in chronic MI. In this study, the authors investigated the efficacy and mechanism by which the myocardial matrix hydrogel can mitigate negative left ventricular (LV) remodeling in a rat chronic MI model. Assessment of cardiac function via magnetic resonance imaging demonstrated preservation of LV volumes and apical wall thickening. Differential gene expression analyses showed the matrix is able to prevent further negative LV remodeling in the chronic MI model through modulation of the immune response, down-regulation of pathways involved in heart failure progression and fibrosis, and up-regulation of genes important for cardiac muscle contraction.
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http://dx.doi.org/10.1016/j.jacbts.2021.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8093531PMC
April 2021

Bio-inspired incorporation of phenylalanine enhances ionic selectivity in layer-by-layer deposited polyelectrolyte films.

Soft Matter 2021 May 13. Epub 2021 May 13.

Sandia National Laboratories, PO Box 5800, MS 1411, Albuquerque, NM 87185, USA.

The addition of a common amino acid, phenylalanine, to a Layer-by-Layer (LbL) deposited polyelectrolyte (PE) film on a nanoporous membrane can increase its ionic selectivity over a PE film without the added amino acid. The addition of phenylalanine is inspired by detailed knowledge of the structure of the channelrhodopsins family of protein ion channels, where phenylalanine plays an instrumental role in facilitating sodium ion transport. The normally deposited and crosslinked PE films increase the cationic selectivity of a support membrane in a controllable manner where higher selectivity is achieved with thicker PE coatings, which in turn also increases the ionic resistance of the membrane. The increased ionic selectivity is desired while the increased resistance is not. We show that through incorporation of phenylalanine during the LbL deposition process, in solutions of NaCl with concentrations ranging from 0.1 to 100 mM, the ionic selectivity can be increased independently of the membrane resistance. Specifically, the addition is shown to increase the cationic transference of the PE films from 81.4% to 86.4%, an increase on par with PE films that are nearly triple the thickness while exhibiting much lower resistance compared to the thicker coatings, where the phenylalanine incorporated PE films display an area specific resistance of 1.81 Ω cm2 in 100 mM NaCl while much thicker PE membranes show a higher resistance of 2.75 Ω cm2 in the same 100 mM NaCl solution.
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http://dx.doi.org/10.1039/d1sm00134eDOI Listing
May 2021

Evaluation and Refinement of Sample Preparation Methods for Extracellular Matrix Proteome Coverage.

Mol Cell Proteomics 2021 Jun 3;20:100079. Epub 2021 Jun 3.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado, Aurora, Colorado, USA; Cancer Center Proteomics Core, School of Medicine, University of Colorado, Aurora, Colorado, USA. Electronic address:

The extracellular matrix is a key component of tissues, yet it is underrepresented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested, and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared with a standard in-solution digest, the optimized method yielded a fourfold improvement in unique ECM peptide identifications.
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http://dx.doi.org/10.1016/j.mcpro.2021.100079DOI Listing
June 2021

Pediatric tri-tube valved conduits made from fibroblast-produced extracellular matrix evaluated over 52 weeks in growing lambs.

Sci Transl Med 2021 Mar;13(585)

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.

There is a need for replacement heart valves that can grow with children. We fabricated tubes of fibroblast-derived collagenous matrix that have been shown to regenerate and grow as a pulmonary artery replacement in lambs and implemented a design for a valved conduit consisting of three tubes sewn together. Seven lambs were implanted with tri-tube valved conduits in sequential cohorts and compared to bioprosthetic conduits. Valves implanted into the pulmonary artery of two lambs of the first cohort of four animals functioned with mild regurgitation and systolic pressure drops <10 mmHg up to 52 weeks after implantation, during which the valve diameter increased from 19 mm to a physiologically normal ~25 mm. In a second cohort, the valve design was modified to include an additional tube, creating a sleeve around the tri-tube valve to counteract faster root growth relative to the leaflets. Two valves exhibited trivial-to-mild regurgitation at 52 weeks with similar diameter increases to ~25 mm and systolic pressure drops of <5 mmHg, whereas the third valve showed similar findings until moderate regurgitation was observed at 52 weeks, correlating to hyperincrease in the valve diameter. In all explanted valves, the leaflets contained interstitial cells and an endothelium progressing from the base of the leaflets and remained thin and pliable with sparse, punctate microcalcifications. The tri-tube valves demonstrated reduced calcification and improved hemodynamic function compared to clinically used pediatric bioprosthetic valves tested in the same model. This tri-tube valved conduit has potential for long-term valve growth in children.
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http://dx.doi.org/10.1126/scitranslmed.abb7225DOI Listing
March 2021

Alterations in extracellular matrix composition during aging and photoaging of the skin.

Matrix Biol Plus 2020 Nov 17;8:100041. Epub 2020 Jun 17.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado, 12801 E 17th Ave., Aurora, CO 80045, USA.

Human skin is composed of the cell-rich epidermis, the extracellular matrix (ECM) rich dermis, and the hypodermis. Within the dermis, a dense network of ECM proteins provides structural support to the skin and regulates a wide variety of signaling pathways which govern cell proliferation and other critical processes. Both intrinsic aging, which occurs steadily over time, and extrinsic aging (photoaging), which occurs as a result of external insults such as solar radiation, cause alterations to the dermal ECM. In this study, we utilized both quantitative and global proteomics, alongside single harmonic generation (SHG) and two-photon autofluorescence (TPAF) imaging, to assess changes in dermal composition during intrinsic and extrinsic aging. We find that both intrinsic and extrinsic aging result in significant decreases in ECM-supporting proteoglycans and structural ECM integrity, evidenced by decreasing collagen abundance and increasing fibril fragmentation. Intrinsic aging also produces changes distinct from those produced by photoaging, including reductions in elastic fiber and crosslinking enzyme abundance. In contrast, photoaging is primarily defined by increases in elastic fiber-associated protein and pro-inflammatory proteases. Changes associated with photoaging are evident even in young (mid 20s) sun-exposed forearm skin, indicating that proteomic evidence of photoaging is present decades prior to clinical signs of photoaging. GO term enrichment revealed that both intrinsic aging and photoaging share common features of chronic inflammation. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD015982.
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http://dx.doi.org/10.1016/j.mbplus.2020.100041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852213PMC
November 2020

Evidence of Structural Protein Damage and Membrane Lipid Remodeling in Red Blood Cells from COVID-19 Patients.

J Proteome Res 2020 11 26;19(11):4455-4469. Epub 2020 Oct 26.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, Colorado 80045, United States.

The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, in particular, short- and medium-chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, or mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading and metabolic rewiring toward the hexose monophosphate shunt, RBCs from COVID-19 patients may be less capable of responding to environmental variations in hemoglobin oxygen saturation/oxidant stress when traveling from the lungs to peripheral capillaries and vice versa.
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http://dx.doi.org/10.1021/acs.jproteome.0c00606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640979PMC
November 2020

Lysosomal cathepsin creates chimeric epitopes for diabetogenic CD4 T cells via transpeptidation.

J Exp Med 2021 Feb;218(2)

Department of Biomedical Research, National Jewish Health, Denver, CO.

The identification of the peptide epitopes presented by major histocompatibility complex class II (MHCII) molecules that drive the CD4 T cell component of autoimmune diseases has presented a formidable challenge over several decades. In type 1 diabetes (T1D), recent insight into this problem has come from the realization that several of the important epitopes are not directly processed from a protein source, but rather pieced together by fusion of different peptide fragments of secretory granule proteins to create new chimeric epitopes. We have proposed that this fusion is performed by a reverse proteolysis reaction called transpeptidation, occurring during the catabolic turnover of pancreatic proteins when secretory granules fuse with lysosomes (crinophagy). Here, we demonstrate several highly antigenic chimeric epitopes for diabetogenic CD4 T cells that are produced by digestion of the appropriate inactive fragments of the granule proteins with the lysosomal protease cathepsin L (Cat-L). This pathway has implications for how self-tolerance can be broken peripherally in T1D and other autoimmune diseases.
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http://dx.doi.org/10.1084/jem.20192135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590512PMC
February 2021

Matrix reverses immortalization-mediated stem cell fate determination.

Biomaterials 2021 01 16;265:120387. Epub 2020 Sep 16.

Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV, USA; WVU Cancer Institute, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, USA. Electronic address:

Primary cell culture in vitro suffers from cellular senescence. We hypothesized that expansion on decellularized extracellular matrix (dECM) deposited by simian virus 40 large T antigen (SV40LT) transduced autologous infrapatellar fat pad stem cells (IPFSCs) could rejuvenate high-passage IPFSCs in both proliferation and chondrogenic differentiation. In the study, we found that SV40LT transduced IPFSCs exhibited increased proliferation and adipogenic potential but decreased chondrogenic potential. Expansion on dECMs deposited by passage 5 IPFSCs yielded IPFSCs with dramatically increased proliferation and chondrogenic differentiation capacity; however, this enhanced capacity diminished if IPFSCs were grown on dECM deposited by passage 15 IPFSCs. Interestingly, expansion on dECM deposited by SV40LT transduced IPFSCs yielded IPFSCs with enhanced proliferation and chondrogenic capacity but decreased adipogenic potential, particularly for the dECM group derived from SV40LT transduced passage 15 cells. Our immunofluorescence staining and proteomics data identify matrix components such as basement membrane proteins as top candidates for matrix mediated IPFSC rejuvenation. Both cell proliferation and differentiation were endorsed by transcripts measured by RNASeq during the process. This study provides a promising model for in-depth investigation of the matrix protein influence on surrounding stem cell differentiation.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944411PMC
January 2021

Serum Proteomics in COVID-19 Patients: Altered Coagulation and Complement Status as a Function of IL-6 Level.

J Proteome Res 2020 11 14;19(11):4417-4427. Epub 2020 Aug 14.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado 80045, United States.

Over 5 million people around the world have tested positive for the beta coronavirus SARS-CoV-2 as of May 29, 2020, a third of which are in the United States alone. These infections are associated with the development of a disease known as COVID-19, which is characterized by several symptoms, including persistent dry cough, shortness of breath, chills, muscle pain, headache, loss of taste or smell, and gastrointestinal distress. COVID-19 has been characterized by elevated mortality (over 100 thousand people have already died in the US alone), mostly due to thromboinflammatory complications that impair lung perfusion and systemic oxygenation in the most severe cases. While the levels of pro-inflammatory cytokines such as interleukin-6 (IL-6) have been associated with the severity of the disease, little is known about the impact of IL-6 levels on the proteome of COVID-19 patients. The present study provides the first proteomics analysis of sera from COVID-19 patients, stratified by circulating levels of IL-6, and correlated to markers of inflammation and renal function. As a function of IL-6 levels, we identified significant dysregulation in serum levels of various coagulation factors, accompanied by increased levels of antifibrinolytic components, including several serine protease inhibitors (SERPINs). These were accompanied by up-regulation of the complement cascade and antimicrobial enzymes, especially in subjects with the highest levels of IL-6, which is consistent with an exacerbation of the acute phase response in these subjects. Although our results are observational, they highlight a clear increase in the levels of inhibitory components of the fibrinolytic cascade in severe COVID-19 disease, providing potential clues related to the etiology of coagulopathic complications in COVID-19 and paving the way for potential therapeutic interventions, such as the use of pro-fibrinolytic agents. Raw data for this study are available through ProteomeXchange with identifier PXD020601.
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http://dx.doi.org/10.1021/acs.jproteome.0c00365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640953PMC
November 2020

Esophageal extracellular matrix hydrogel mitigates metaplastic change in a dog model of Barrett's esophagus.

Sci Adv 2020 Jul 1;6(27):eaba4526. Epub 2020 Jul 1.

McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA.

Chronic inflammatory gastric reflux alters the esophageal microenvironment and induces metaplastic transformation of the epithelium, a precancerous condition termed Barrett's esophagus (BE). The microenvironmental niche, which includes the extracellular matrix (ECM), substantially influences cell phenotype. ECM harvested from normal porcine esophageal mucosa (eECM) was formulated as a mucoadhesive hydrogel, and shown to largely retain basement membrane and matrix-cell adhesion proteins. Dogs with BE were treated orally with eECM hydrogel and omeprazole ( = 6) or omeprazole alone ( = 2) for 30 days. eECM treatment resolved esophagitis, reverted metaplasia to a normal, squamous epithelium in four of six animals, and downregulated the pro-inflammatory tumor necrosis factor-α cell infiltrate compared to control animals. The metaplastic tissue in control animals ( = 2) did not regress. The results suggest that in vivo alteration of the microenvironment with a site-appropriate, mucoadhesive ECM hydrogel can mitigate the inflammatory and metaplastic response in a dog model of BE.
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http://dx.doi.org/10.1126/sciadv.aba4526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329334PMC
July 2020

Evidence for structural protein damage and membrane lipid remodeling in red blood cells from COVID-19 patients.

medRxiv 2020 Jun 30. Epub 2020 Jun 30.

The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly-diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, especially short and medium chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, and mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading, RBCs from COVID-19 patients may be incapable of responding to environmental variations in hemoglobin oxygen saturation when traveling from the lungs to peripheral capillaries and, as such, may have a compromised capacity to transport and deliver oxygen.
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http://dx.doi.org/10.1101/2020.06.29.20142703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340206PMC
June 2020

Site-Dependent Lineage Preference of Adipose Stem Cells.

Front Cell Dev Biol 2020 15;8:237. Epub 2020 Apr 15.

Stem Cell and Tissue Engineering Laboratory, Department of Orthopedics, West Virginia University, Morgantown, WV, United States.

Adult stem cells have unique properties in both proliferation and differentiation preference. In this study, we hypothesized that adipose stem cells have a depot-dependent lineage preference. Four rabbits were used to provide donor-matched adipose stem cells from either subcutaneous adipose tissue (ScAT) or infrapatellar fat pad (IPFP). Proliferation and multi-lineage differentiation were evaluated in adipose stem cells from donor-matched ScAT and IPFP. RNA sequencing (RNA-seq) and proteomics were conducted to uncover potential molecular discrepancy in adipose stem cells and their corresponding matrix microenvironments. We found that stem cells from ScAT exhibited significantly higher proliferation and adipogenic capacity compared to those from donor-matched IPFP while stem cells from IPFP displayed significantly higher chondrogenic potential compared to those from donor-matched ScAT. Our findings are strongly endorsed by supportive data from transcriptome and proteomics analyses, indicating a site-dependent lineage preference of adipose stem cells.
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http://dx.doi.org/10.3389/fcell.2020.00237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174673PMC
April 2020

Biologically-engineered mechanical model of a calcified artery.

Acta Biomater 2020 07 16;110:164-174. Epub 2020 Apr 16.

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, United States; Department of Chemical Engineering & Materials Science, University of Minnesota, Minneapolis, MN, United States. Electronic address:

Vascular calcification is a commonly occurring pathological process and is recognized as an independent prognostic marker for cardiovascular morbidity and mortality. Recent progress in developing novel therapies to modify vascular calcification is critically hampered due to the lack of reliable in vitro experimental models that recapitulate the structural and mechanical attributes of calcified arteries. In this study, we show the ability to model the behavior of diffuse vascular calcification in vitro using biologically-engineered grafts approximating the composition, structure, and mechanical properties of arteries. Transmural calcification was achieved by exposing the acellular grafts of collagenous ECM to complete medium containing elevated Calcium (Ca) and Phosphate (P) concentrations. It was found that increasing the serum concentration from 2% to 10% increased the extent and degree of calcification based on histochemical, ultrastructural, chemical and thermal analyses. The presence of variably-sized spherical calcific deposits within the matrix further confirmed its morphological similarity to pathologic calcification. Mechanical testing demonstrated up to a 16-fold decrease in compliance due to the calcification, consistent with prior reports for calcified arteries. The model developed thus has potential to improve the design and development of interventional devices and therapies for the diagnosis and treatment of arterial calcification. STATEMENT OF SIGNIFICANCE: The presence of extensive vascular calcification makes angiographic/interventional procedures difficult due to reduced arterial compliance. Current attempts to develop safe and effective non-surgical adjunctive techniques to treat calcified arteries are largely limited by the lack of a physiologically relevant testing platform that mimics the structural and mechanical features of vascular calcification. Herein, we developed an off-the-shelf calcified artery model, with the goal to accelerate the pre-clinical development of novel therapies for the management of arterial calcification. To the extent of our knowledge, this is the first report of an in vitro tissue-engineered model of diffuse arterial calcification.
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http://dx.doi.org/10.1016/j.actbio.2020.04.018DOI Listing
July 2020

Postpartum breast cancer progression is driven by semaphorin 7a-mediated invasion and survival.

Oncogene 2020 03 4;39(13):2772-2785. Epub 2020 Feb 4.

Division of Medical Oncology, Department of Medicine, CU Anschutz Medical Campus, Aurora, CO, 80045, USA.

Young women diagnosed with breast cancer (BC) have poor prognosis due to increased rates of metastasis. In addition, women diagnosed within 10 years of most recent childbirth are approximately three times more likely to develop metastasis than age- and stage-matched nulliparous women. We define these cases as postpartum BC (PPBC) and propose that the unique biology of the postpartum mammary gland drives tumor progression. Our published results revealed roles for SEMA7A in breast tumor cell growth, motility, invasion, and tumor-associated lymphangiogenesis, all of which are also increased in preclinical models of PPBC. However, whether SEMA7A drives progression in PPBC remains largely unexplored. Our results presented herein show that silencing of SEMA7A decreases tumor growth in a model of PPBC, while overexpression is sufficient to increase growth in nulliparous hosts. Further, we show that SEMA7A promotes multiple known drivers of PPBC progression including tumor-associated COX-2 expression and fibroblast-mediated collagen deposition in the tumor microenvironment. In addition, we show for the first time that SEMA7A-expressing cells deposit fibronectin to promote tumor cell survival. Finally, we show that co-expression of SEMA7A/COX-2/FN predicts for poor prognosis in breast cancer patient cohorts. These studies suggest SEMA7A as a key mediator of BC progression, and that targeting SEMA7A may open avenues for novel therapeutic strategies.
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http://dx.doi.org/10.1038/s41388-020-1192-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103487PMC
March 2020

Role of lineage-specific matrix in stem cell chondrogenesis.

Biomaterials 2020 02 16;231:119681. Epub 2019 Dec 16.

Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV, 26506, USA; WVU Cancer Institute, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, 26506, USA. Electronic address:

Cartilage repair in clinics is a challenge owing to the limited regenerative capacities of cartilage. Synovium-derived stem cells (SDSCs) are suggested as tissue-specific stem cells for chondrogenesis. In this study, we hypothesize that decellularized extracellular matrix (dECM) deposited by SDSCs could provide a superior tissue-specific matrix microenvironment for optimal rejuvenation of adult SDSCs for cartilage regeneration. dECMs were deposited by adult stem cells with varying chondrogenic capacities; SDSCs (strong) (SECM), adipose-derived stem cells (weak) (AECM) and dermal fibroblasts (weak) (DECM), and urine-derived stem cells (none) (UECM). Plastic flasks (Plastic) were used as a control substrate. Human SDSCs were expanded on the above substrates for one passage and examined for chondrogenic capacities. We found that each dECM consisted of unique matrix proteins and exhibited varied stiffnesses, which affected cell morphology and elasticity. Human SDSCs grown on dECMs displayed a significant increase in cell proliferation and unique surface phenotypes. Under induction media, dECM expanded cells yielded pellets with a dramatically increased number of chondrogenic markers. Interestingly, SECM expanded cells had less potential for hypertrophy compared to those grown on other dECMs, indicating that a tissue-specific matrix might provide a superior microenvironment for stem cell chondrogenic differentiation.
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http://dx.doi.org/10.1016/j.biomaterials.2019.119681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6958706PMC
February 2020

Quantification of the anti-murine PD-1 monoclonal antibody RMP1-14 in BALB/c mouse plasma by liquid chromatography-tandem mass spectrometry and application to a pharmacokinetic study.

Anal Bioanal Chem 2020 Jan 12;412(3):739-752. Epub 2019 Dec 12.

Covance Laboratories, Inc., 8211 SciCor Drive, Indianapolis, IN, 46214, USA.

RMP1-14 is a monoclonal antibody that targets the murine PD-1 protein, and has been used extensively to probe the effects of PD-1 inhibition in preclinical murine models. However, to date, no quantitative analytical methods have been published for RMP1-14. To evaluate its anti-tumor activity in BALB/c mice inoculated with CT26.WT murine colon cancer cells, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify RMP1-14 in BALB/c mouse KEDTA plasma was developed and validated. The methodology used a signature peptide (GFYPPDIYTEWK) as a surrogate for RMP1-14 quantitation and an isotopically labeled analog of the signature peptide as the internal standard. Initial method development focused on a hybrid LC-MS/MS assay involving Protein G immunoprecipitation, but this strategy was abandoned due to lack of selectivity. The final validated method consisted of dilution with Tris-buffered saline, trypsin digestion, and desalting using micro solid-phase extraction. Analytical run time was 3.50 min, and the method demonstrated linearity between 0.500 and 50.0 μg/mL of intact RMP1-14. Accuracy, precision, and robustness were all acceptable, and the method was demonstrated to be comparable to a commercially available fit-for-purpose enzyme-linked immunosorbent assay (ELISA) capable of measuring RMP1-14. The validated method was used to generate pharmacokinetic parameters from tumor-bearing BALB/c mice dosed with RMP1-14 at either 2.50 or 7.50 mg/kg. Overall, the validated method represents a novel tool that can be used to evaluate RMP1-14 activity in future immuno-oncology studies.
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http://dx.doi.org/10.1007/s00216-019-02292-1DOI Listing
January 2020

The consequences of increased 4E-BP1 in polycystic kidney disease.

Hum Mol Genet 2019 12;28(24):4132-4147

Division of Renal Diseases and Hypertension, University of Colorado at Denver, Denver, CO, USA.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease, characterized by cyst formation and growth. Hyperproliferation is a major contributor to cyst growth. At the nexus of regulating proliferation, is 4E-BP1. We demonstrate that ADPKD mouse and rat models, ADPKD patient renal biopsies and PKD1-/- cells exhibited hyperphosphorylated 4E-BP1, a biomarker of increased translation and proliferation. We hypothesized that expression of constitutively active 4E-BP1 constructs (4E-BP1F113A and 4E-BP1R13AF113A) would decrease proliferation and reduce cyst expansion. Utilizing the Pkd1RC/RC mouse, we determined the effect of 4E-BP1F113A on PKD. Unexpectedly, 4E-BP1F113A resulted in increased cyst burden and suppressed apoptosis markers, increased anti-apoptotic Bcl-2 protein and increased mitochondrial proteins. Exogenous 4E-BP1 enhanced proliferation, decreased apoptosis, increased anti-apoptotic Bcl-2 protein, impaired NADPH oxidoreductase activity, increased mitochondrial proteins and increased superoxide production in PKD patient-derived renal epithelial cells. Reduced 4E-BP1 expression suppressed proliferation, restored apoptosis and improved cellular metabolism. These findings provide insight into how cyst-lining cells respond to 4E-BP1.
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http://dx.doi.org/10.1093/hmg/ddz244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968387PMC
December 2019

Manufacturing considerations for producing and assessing decellularized extracellular matrix hydrogels.

Methods 2020 01 20;171:20-27. Epub 2019 Sep 20.

Department of Bioengineering, Sanford Consortium for Regenerative Medicine, University of California, San Diego, La Jolla, CA, USA. Electronic address:

Although several decellularized extracellular matrix (ECM) sheets or patches have been commercialized for use in the clinic, only one injectable decellularized ECM hydrogel, a decellularized myocardial matrix, has reached clinical trials. Consequently, very little information is available for established manufacturing standards or assessments of these materials. Here we present detailed methodology for investigating three parameters related to manufacturing optimization for a porcine derived skeletal muscle ECM hydrogel - animal-to-animal variability, bioburden reduction, and harvesting conditions. Results from characterization assays, including residual dsDNA content and sulfated glycosaminoglycan content, did not yield noteworthy differences amongst individual animals or following the addition of a bioburden reducing agent. However, the tissue collected under different harvesting conditions contained varying amounts of fat, and the protein compositions of the decellularized products differed, which could ultimately impact subsequent efficacy in vitro or in vivo. As decellularized ECM hydrogels continue to be evaluated for various applications, the differences between laboratory-scale and manufacturing-scale material batches should be thoroughly considered to avoid costly and timely optimization during scale-up.
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http://dx.doi.org/10.1016/j.ymeth.2019.09.015DOI Listing
January 2020

A unified mechanism for intron and exon definition and back-splicing.

Nature 2019 09 4;573(7774):375-380. Epub 2019 Sep 4.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

The molecular mechanisms of exon definition and back-splicing are fundamental unanswered questions in pre-mRNA splicing. Here we report cryo-electron microscopy structures of the yeast spliceosomal E complex assembled on introns, providing a view of the earliest event in the splicing cycle that commits pre-mRNAs to splicing. The E complex architecture suggests that the same spliceosome can assemble across an exon, and that it either remodels to span an intron for canonical linear splicing (typically on short exons) or catalyses back-splicing to generate circular RNA (on long exons). The model is supported by our experiments, which show that an E complex assembled on the middle exon of yeast EFM5 or HMRA1 can be chased into circular RNA when the exon is sufficiently long. This simple model unifies intron definition, exon definition, and back-splicing through the same spliceosome in all eukaryotes and should inspire experiments in many other systems to understand the mechanism and regulation of these processes.
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http://dx.doi.org/10.1038/s41586-019-1523-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939996PMC
September 2019

Reversible MOF-Based Sensors for the Electrical Detection of Iodine Gas.

ACS Appl Mater Interfaces 2019 Aug 29;11(31):27982-27988. Epub 2019 Jul 29.

Sandia National Laboratories , Albuquerque 87185 , New Mexico , United States.

Iodine detection is crucial for nuclear waste clean-up and first responder activities. For ease of use and durability of response, robust active materials that enable the direct electrical detection of I are needed. Herein, a large reversible electrical response is demonstrated as I is controllably and repeatedly adsorbed and desorbed from a series of metal-organic frameworks (MOFs) MFM-300(X), each possessing a different metal center (X = Al, Fe, In, or Sc) bridged by biphenyl-3,3',5,5'-tetracarboxylate linkers. Impedance spectroscopy is used to evaluate how the different metal centers influence the electrical response upon cycling of I gas, ranging from 10× to 10× decrease in resistance upon I adsorption in air. This large variation in electrical response is attributed not only to the differing structural characteristics of the MOFs but also to the differing MOF morphologies and how this influences the degree of reversibility of I adsorption. Interestingly, MFM-300(Al) and MFM-300(In) displayed the largest changes in resistance (up to 10×) yet lost much of their adsorption capacity after five I adsorption cycles in air. On the other hand, MFM-300(Fe) and MFM-300(Sc) revealed more moderate changes in resistance (10-100×), maintaining most of their original adsorption capacity after five cycles. This work demonstrates how changes in MOFs can profoundly affect the magnitude and reversibility of the electrical response of sensor materials. Tuning both the intrinsic (resistivity and adsorption capacity) and extrinsic (surface area and particle morphology) properties is necessary to develop highly reversible, large signal-generating MOF materials for direct electrical readout for I sensing.
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http://dx.doi.org/10.1021/acsami.9b09938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814244PMC
August 2019

Functional Insights from the Proteomic Inventory of Ovine Forestomach Matrix.

J Proteome Res 2019 04 25;18(4):1657-1668. Epub 2019 Mar 25.

Aroa Biosurgery Limited , Airport Oaks , Auckland 2022 , New Zealand.

Ovine forestomach matrix (OFM) is a decellularized extracellular matrix (dECM) biomaterial that serves as a scaffold for remodeling damaged soft tissue. dECM biomaterials are used in a variety of clinical applications, and their regenerative capacity is encoded not only in their biophysical properties but also in their molecular diversity. In this study, the proteome of OFM was characterized via both targeted and global mass spectrometry (MS) with the use of heavy isotope labeled (SIL) internal standards. Proteins were identified following either chemical digestion or extraction using saline or guanidine hydrochloride, followed by high resolution size exclusion chromatography. Identified proteins were annotated using the matrisome database and molecular function using the gene ontology database. The characterization identified 153 unique matrisome proteins, including 25 collagens, 58 glycoproteins, 12 proteoglycans, 13 ECM affiliated proteins, 20 ECM regulators, and 23 secreted factors. This inventory represents a comprehensive array of matrix proteins that are retained in OFM after processing. The diversity of proteins identified may contribute to OFM's remodeling capacity in clinical applications.
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http://dx.doi.org/10.1021/acs.jproteome.8b00908DOI Listing
April 2019

Methylation of protein aspartates and deamidated asparagines as a function of blood bank storage and oxidative stress in human red blood cells.

Transfusion 2018 12 12;58(12):2978-2991. Epub 2018 Oct 12.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado.

Background: Being devoid of de novo protein synthesis capacity, red blood cells (RBCs) have evolved to recycle oxidatively damaged proteins via mechanisms that involve methylation of dehydrated and deamidated aspartate and asparagine residues. Here we hypothesize that such mechanisms are relevant to routine storage in the blood bank.

Study Design And Methods: Within the framework of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, packed RBC units (n = 599) were stored under blood bank conditions for 10, 23, and 42 days and profiled for oxidative hemolysis and time-dependent metabolic dysregulation of the trans-sulfuration pathway.

Results: In these units, methionine consumption positively correlated with storage age and oxidative hemolysis. Mechanistic studies show that this phenomenon is favored by oxidative stress or hyperoxic storage (sulfur dioxide >95%), and prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and formation of methyl-Asp on key structural proteins and enzymes, including Band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase, biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of glyceraldehyde 3-phosphate dehydrogenase) and/or residues interacting with the N-terminal cytosolic domain of Band 3.

Conclusion: While methylation of basic amino acid residues serves as an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated asparagines is a nonepigenetic posttranslational sensor of oxidative stress and refrigerated storage in anucleated human RBCs.
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http://dx.doi.org/10.1111/trf.14936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357231PMC
December 2018

Targeted matrisome analysis identifies thrombospondin-2 and tenascin-C in aligned collagen stroma from invasive breast carcinoma.

Sci Rep 2018 08 28;8(1):12941. Epub 2018 Aug 28.

Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1111 Highland Ave., WIMR II Rm. 4528, Madison, WI, 53705, United States.

Increasing evidence demonstrates an important role for the extracellular matrix (ECM) in breast cancer progression. Collagen type I, a core constituent of the fibrous ECM, undergoes a significant set of changes that accompany tumor progression, termed Tumor Associated Collagen Signatures (TACS). Late stages of this progression are characterized by the presence of bundled, straight collagen (TACS-2) that become oriented perpendicular to the tumor-stromal boundary (TACS-3). Importantly, the presence of TACS-3 collagen is an independent predictor of poor patient outcome. At present, it remains unclear whether reorganization of the collagen matrix is the consequence of mechanical or compositional tissue remodeling. Here, we identify compositional changes in ECM correlating to collagen fiber reorganization from nineteen normal and invasive ductal carcinoma (IDC) patient biopsies using matrisome-targeted proteomics. Twenty-seven ECM proteins were significantly altered in IDC samples compared to normal tissue. Further, a set of nineteen matrisome proteins positively correlate and five proteins inversely correlate with IDC tissues containing straightened collagen fibers. Tenascin-C and thrombospondin-2 significantly co-localized with aligned collagen fibers in IDC tissues. This study highlights the compositional change in matrisome proteins accompanying collagen re-organization during breast cancer progression and provides candidate proteins for investigation into cellular and structural influences on collagen alignment.
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http://dx.doi.org/10.1038/s41598-018-31126-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113240PMC
August 2018

Author Correction: CryoEM structure of Saccharomyces cerevisiae U1 snRNP offers insight into alternative splicing.

Nat Commun 2018 04 11;9(1):1495. Epub 2018 Apr 11.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver Anschutz Medical Campus, Aurora, CO, 80045, USA.

The originally published version of this Article contained several errors in Figure 2, panel a: the basepair register in SL3-4 of yeast U1 snRNA was depicted incorrectly; the basepair for A287-U295 in yeast U1 snRNA was erroneously present; basepairs for U84-G119, G309-U532, A288-U295 and U289-A294 in yeast U1 snRNA were missing; the bulging nucleotide in SL3 of human U1 snRNA was depicted as G instead of C; and the dashed boxes defining the 5' ss binding site and Sm site in both human and yeast snRNAs were not drawn accurately. These have now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-018-03708-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895711PMC
April 2018

Structure of the yeast spliceosomal postcatalytic P complex.

Science 2017 12 16;358(6368):1278-1283. Epub 2017 Nov 16.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver (UCD), Anschutz Medical Campus, Aurora, CO 80045, USA.

The spliceosome undergoes dramatic changes in a splicing cycle. Structures of B, B, C, C*, and intron lariat spliceosome complexes revealed mechanisms of 5'-splice site (ss) recognition, branching, and intron release, but lacked information on 3'-ss recognition, exon ligation, and exon release. Here we report a cryo-electron microscopy structure of the postcatalytic P complex at 3.3-angstrom resolution, revealing that the 3' ss is mainly recognized through non-Watson-Crick base pairing with the 5' ss and branch point. Furthermore, one or more unidentified proteins become stably associated with the P complex, securing the 3' exon and potentially regulating activity of the helicase Prp22. Prp22 binds nucleotides 15 to 21 in the 3' exon, enabling it to pull the intron-exon or ligated exons in a 3' to 5' direction to achieve 3'-ss proofreading or exon release, respectively.
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http://dx.doi.org/10.1126/science.aar3462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828012PMC
December 2017

CryoEM structure of Saccharomyces cerevisiae U1 snRNP offers insight into alternative splicing.

Nat Commun 2017 10 19;8(1):1035. Epub 2017 Oct 19.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver Anschutz Medical Campus, Aurora, CO, 80045, USA.

U1 snRNP plays a critical role in 5'-splice site recognition and is a frequent target of alternative splicing factors. These factors transiently associate with human U1 snRNP and are not amenable for structural studies, while their Saccharomyces cerevisiae (yeast) homologs are stable components of U1 snRNP. Here, we report the cryoEM structure of yeast U1 snRNP at 3.6 Å resolution with atomic models for ten core proteins, nearly all essential domains of its RNA, and five stably associated auxiliary proteins. The foot-shaped yeast U1 snRNP contains a core in the "ball-and-toes" region architecturally similar to the human U1 snRNP. All auxiliary proteins are in the "arch-and-heel" region and connected to the core through the Prp42/Prp39 paralogs. Our demonstration that homodimeric human PrpF39 directly interacts with U1C-CTD, mirroring yeast Prp42/Prp39, supports yeast U1 snRNP as a model for understanding how transiently associated auxiliary proteins recruit human U1 snRNP in alternative splicing.
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http://dx.doi.org/10.1038/s41467-017-01241-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648754PMC
October 2017

Hydroxylamine Chemical Digestion for Insoluble Extracellular Matrix Characterization.

J Proteome Res 2017 11;16(11):4177-4184

Department of Biochemistry and Molecular Genetics, and ‡Biological Mass Spectrometry Facility, University of Colorado Denver , Aurora, Colorado 80045, United States.

The extracellular matrix (ECM) is readily enriched by decellularizing tissues with nondenaturing detergents to solubilize and deplete the vast majority of cellular components. This approach has been used extensively to generate ECM scaffolds for regenerative medicine technologies and in 3D cell culture to model how the ECM contributes to disease progression. A highly enriched ECM fraction can then be generated using a strong chaotrope buffer that is compatible with downstream bottom-up proteomic analysis or 3D cell culture experiments after extensive dialysis. With most tissues, an insoluble pellet remains after chaotrope extraction that is rich in structural ECM components. Previously, we showed that this understudied fraction represented approximately 80% of total fibrillar collagen from the lung and other ECM fiber components that are known to be covalently cross-linked. Here, we present a hydroxylamine digestion approach for chaotrope-insoluble ECM analysis with comparison to an established CNBr method for matrisome characterization. Because ECM characteristics vary widely among tissues, we chose five tissues that represent unique and diverse ECM abundances, composition, and biomechanical properties. Hydroxylamine digestion is compatible with downstream proteomic workflows, yields high levels of ECM peptides from the insoluble ECM fraction, and reduces analytical variability when compared to CNBr digestion. Data are available via ProteomeXchange with identifier PXD006428.
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http://dx.doi.org/10.1021/acs.jproteome.7b00527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802359PMC
November 2017

Measurement of lipid transfer proteins in genetically engineered maize using liquid chromatography with tandem mass spectrometry (LC-MS/MS).

GM Crops Food 2017 Oct 28;8(4):239-252. Epub 2017 Aug 28.

a Dow AgroSciences LLC , Indianapolis , IN , USA.

Endogenous allergenicity evaluation is a required part of the risk assessment for genetically engineered (GE) crops. Although maize is not considered a major allergenic food, a lipid transfer protein (Zea m 14) in maize grain has been identified as a potential IgE-mediated food allergen. Currently, the relationship between allergen exposure and risk of sensitization is not well understood. Hence, reliable quantitative methods are useful for determining the natural range and variability of allergen levels across multiple geographies and genetic backgrounds. A LC-MS/MS analytical method was developed and validated in our laboratory to quantify Zea m 14 in grain from 2 GE maize hybrids and 20 non-GE maize hybrids. The measured Zea m 14 levels in GE maize grain and conventional non-GE maize grain ranged from 146.87 to 574.93 ng/mg across 16 field sites located in the United States and Argentina. The method accurately quantified endogenous Zea m 14 from maize grain and results show Zea m 14 levels in the GE maize varieties were within the natural variation observed in traditionally bred non-GE maize.
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http://dx.doi.org/10.1080/21645698.2017.1349602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836814PMC
October 2017

Transgenesis affects endogenous soybean allergen levels less than traditional breeding.

Regul Toxicol Pharmacol 2017 Oct 15;89:70-73. Epub 2017 Jul 15.

Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, United States.

The regulatory body that oversees the safety assessment of genetically modified (GM) crops in the European Union, the European Food Safety Authority (EFSA), uniquely requires that endogenous allergen levels be quantified as part of the compositional characterization of GM versions of crops, such as soybean, that are considered to be major allergenic foods. The value of this requirement for assessing food safety has been challenged for multiple reasons including negligible risk of altering allergen levels compared with traditional non-GM breeding. Scatter plots comparing the mean endogenous allergen levels in non-GM soybean isoline grain with the respective levels in GM grain or concurrently grown non-GM commercial reference varieties clearly show that transgenesis causes less change compared with traditional breeding. This visual assessment is confirmed by the quantitative fit of the line of identity (y = x) to the datasets. The current science on allergy does not support the requirement for quantifying allergen levels in GM crops to support safety assessment.
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http://dx.doi.org/10.1016/j.yrtph.2017.07.013DOI Listing
October 2017