Browsing by Author "Vogel, Hans J."

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    Analysis of Metabolomics Data via Mixed Models
    (2020-08) Ren, Austin Mu Qing; de Leon, Alexander R.; Kopciuk, Karen Arlene; Vogel, Hans J.; Sajobi, Tolulope T.
    Generalized linear mixed models have been widely studied and used in many different disciplines, yet very little application of them can be found with metabolomics data analysis. Traditional methods of cancer classification used to determine disease severity, such as biopsies, can be harmful to the health of the patients. Classification based on metabolomics data analysis demonstrates a main advantage as it only requires non-invasive procedures such as the drawing of a small amount of blood from patients. However, data analysis in cancer research often requires the handling of multiple correlated measurements of disease severity. The methods that are most commonly used with metabolomics data, such as partial least squares discriminant analysis, were traditionally designed to handle univariate data only, and can be very challenging to work with when applied to data with multiple correlated outcomes. Therefore, different methods should be considered for metabolomics data analysis in cancer classification. In this thesis, we proposed bivariate generalized linear mixed models with binary outcomes using the probit link function for the analysis of metabolomics data. The models were specifically designed to handle multiple correlated outcomes via the inclusion of subject-specific random intercepts. Random slopes were not included in the models to reduce complexity. We specifically designed three settings for the random intercept models: shared, independent, and correlated between the outcomes. An extensive number of simulations were carried out to test our models' parameters, including: standard deviation and correlation of the distribution of the random intercepts, correlation between the covariates as well as correlation between the covariates and the outcomes, the proportion of data missing among the covariates, misspecified distribution of the random intercepts, and misspecified conditional correlation between the outcomes. In addition, we also incorporated the nearest neighbors algorithm as a missing values imputation method and LASSO as a feature selection method to our mixed models in order to handle the common issues of high dimensional covariates and missing values in metabolomics data. Finally, our proposed mixed models were applied to a real dataset with prostate cancer patients to evaluate our models' performance on outcome predictions.
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    Bacterial periplasmic binding proteins
    (2006) Krewulak, Karla Dawn; Vogel, Hans J.
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    Characterization of Bacterial Ferrous Iron Transport: the Feo System
    (2013-01-11) Lau, Cheryl Kar Yee; Vogel, Hans J.
    The Feo system is the main ferrous iron uptake pathway for the majority of prokaryotic organisms. The feo operon consists of two proteins, ferrous iron transport proteins A (FeoA) and B (FeoB). Many studies in relation to the system’s role in bacterial virulence and the crystal structures of the N-terminal domain of FeoB (NFeoB) have been reported, however, none have explored the solution characteristics associated with FeoA and FeoB. This dissertation presents the solution structure of Escherichia coli FeoA along with the backbone amide dynamics of this protein. Further, NMR dynamic studies of E. coli NFeoB, along with interaction studies between FeoA and NFeoB are presented. Lastly, a detailed bioinformatics analysis of this system is presented to bring together our findings and new hypotheses are suggested. Our studies provide insight to the function of FeoA from a structural perspective and its potential role in interacting with FeoB, a co-regulated gene. The solution structure of FeoA shows a resemblance to eukaryotic SH3 protein-protein interaction domains. The addition of an α-helix in FeoA for one of the conserved interaction loops in eukaryotic SH3-domains results in a greater resemblance towards SH3-like domains present in prokaryotic metalloregulators, that are involved in transcriptional regulation. Backbone dynamics on the fast NMR timescale of FeoA indicate a rigid monomeric structure. The backbone dynamics of the apo and guanine nucleotide forms of NFeoB on the fast NMR timescale suggest communication between the N-terminal cytoplasmic domains of FeoB and the transmembrane domain of FeoB. Backbone dynamics on the slow NMR timescale indicate that global conformational changes associated with guanine-nucleotide binding may be important for the association of NFeoB with the transmembrane domain. Interaction studies between FeoA and NFeoB indicate a transient interaction under the conditions tested. Furthermore, 31P NMR studies with FeoA and NFeoB suggests that FeoA does not act as an activator of the G-domain of FeoB as previously suggested. Bioinformatics analyses of the Feo system provide different perspectives towards the roles of FeoA and FeoB; along with the unique conservation of certain domains in FeoB that are important to its function.
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    Digestomics: elucidating protein catabolism through the quantitative mapping of in vivo, endogenous peptides to the proteome at an amino acid level of resolution.
    (2020-03-13) Bingeman, Travis Shane; Storey, Douglas G.; Lewis, Ian A.; Vogel, Hans J.; Harrison, Joe J.
    Plasmodium falciparum is the most lethal malaria-causing parasite. During the human stage of its lifecycle it invades erythrocytes and rapidly degrades 65-70% of the cytoplasmic hemoglobin. Despite extensive in vitro investigation of the proteases responsible for this impressive catabolic activity, the definitive purpose of this digestion remains unresolved. Many of the known proteases have been knocked out yet these parasites have minimal growth phenotypes. The common explanation is that proteolytic redundancy allows parasites to survive. I, however, hypothesize that the protease(s) essential for hemoglobin catabolism have yet to be identified. To test this, I developed a new approach to directly investigate in vivo protein catabolism. I analyze a panel of knockout parasites and show that in vivo proteolytic cleavage patterns are unchanged in knockouts, find evidence that published cut sites match poorly to in vivo data and find convincing proteolytic patterns suggesting the presence of previously unidentified cut sites.
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    Evaluation of yellow pea fibre supplementation on weight loss and the gut microbiota: a randomized controlled trial
    (BioMed Central, 2014-04-08) Lambert, Jennifer E.; Parnell, Jill A.; Han, Jay; Sturzenegger, Troy; Paul, Heather A.; Vogel, Hans J.; Reimer, Raylene A.
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    Immunomodulatory Role of Cathelicidin at Colonic Epithelium
    (2020-03-23) Holani, Ravi; Cobo, Eduardo R.; Proud, David; Barkema, Herman W.; Vogel, Hans J.
    Cathelicidin, a small, cationic and amphiphilic host defense peptide, is expressed by hematopoietic and non-hematopoietic cells. Initially considered an antimicrobial, cathelicidin are more than simply natural antibiotics. Due to their ability to interact with multiple receptors, cathelicidin can modulate immune responses by a variety of host cells. However, their immunomodulatory effects vary based on their micro-environment, including tissue type (hematopoietic vs. non-hematopoietic), availability of microbes or associated factor(s) and effective concentration of cathelicidin. Since most studies are performed in leukocytes, ability of cathelicidin to regulate immune responses by intestinal epithelium remains largely unknown. The objective was to elucidate immunomodulatory potential of cathelicidin in colonic epithelium, under homeostasis as well as during bacterial colitis. In the first study, we determined how cathelicidin synergized with lipopolysaccharide or Salmonella enterica Typhimurium to enhance production of neutrophil chemokine CXCL8 (human) or murine CXCL1 (functional homologue of CXCL8). Further, we identified a two-signal mechanism triggered by the complex of LPS-LL37, in which nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling was involved in CXCL8 mRNA synthesis, whereas p38 mitogen-activated protein kinase (p38MAPK) was involved in CXCL8 mRNA stabilization. In a Citrobacter rodentium model of colitis, we corroborated increased synthesis of CXCL1 chemokine, effective neutrophil recruitment and reduced bacterial burden in Camp+/+ versus Camp-/- (cathelicidin null) mice. In the second study, under physiological conditions, cathelicidin induced synthesis of Toll-like receptor (TLR) negative regulator, Toll-interacting protein (Tollip). This induced Tollip production inhibited TLR-mediated apoptosis in colonic epithelium, both in vitro by cytokines tumour necrosis factor- α and interferon- γ as well as in vivo in a C. rodentium model of colitis. In conclusion, cathelicidin regulated colonic innate immunity via reduction of unwarranted inflammation by inhibiting TLR responses through Tollip, while promoting a neutrophil response to infection.
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    Improving the therapeutic potential of staphylokinase, a potent thrombolytic agent
    (2023-05-26) Baharian, Azin; Vogel, Hans J.; MacDonald, Justin A.; MacCallum, Justin
    Staphylokinase (Sak) is a small bacterial-derived protein (15.5 kDa) that can hydrolyze fibrin-rich blood clots by activating the conversion of plasminogen into plasmin. Several studies have shown the effectiveness of Sak as a thrombolytic agent that could be used to treat stroke or cardiac arrest. However, its short half-life in blood circulation and immunogenicity are the main issues that prevent Sak from clinical applications. Herein, we explore two modifications of Sak to improve its therapeutic potential. Covalently attaching PEG (polyethylene glycol) polymers to therapeutic proteins has been widely applied to improve their pharmacokinetic properties, resulting in several FDA-approved protein-based drugs. Although PEGylation of Sak has previously been investigated, the effects of PEGylation on the 3D structure of Sak have not been studied in detail. In the first part of this research project, site-specific PEGylation of the truncated version of Sak was performed in the immunogenic region of the protein, and three covalent PEGylated derivatives of the protein were prepared. NMR studies showed a slight structural perturbation upon PEGylation, mostly close to the PEGylation site, and a direct relationship between the hydrodynamic radius of the PEGylated protein and the PEG size. Together, the results suggest that PEG and Sak∆10 move relatively independently from each other. As a second approach, we introduce cyclic Sak (cyc-Sak), a novel form of staphylokinase with higher stability and improved plasminogen activation activity. Using an Ssp GyrB split intein, the N- and C-terminal ends of the linear Sak were connected by a peptide bond, rendering the protein into a cyclic form (cyc-Sak). This structural modification was generated at the protein expression level in Escherichia coli, and the cyclic protein could be purified by common chromatography techniques. Successful backbone cyclization was confirmed by NMR spectroscopy of the 13C,15N-labeled cyc-Sak and by chemical cleavage assays. Our studies show that mono-PEGylation and intein-mediated backbone cyclization of Sak are powerful strategies to improve its therapeutic potential. These approaches could be combined to facilitate the design of protein-based medications when the stability of therapeutic proteins is an obstacle to their clinical application.
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    Metabolomics and Metallomics Analyses of Renal Cell Carcinoma and Prostate Cancer
    (2019-09-12) Falegan, Oluyemi Seuntitun; Vogel, Hans J.; Hyndman, Matthew Eric; Hollenberg, Morley Donald; Bathe, Oliver F.
    Prostate cancer and renal cell carcinoma of the kidney are the most frequently diagnosed and the most lethal male genitourinary cancers respectively. While prostate cancer screening and diagnosis using serum prostate-specific-antigen is flawed by its low sensitivity and inability to detect indolent disease, the histological diversity of renal cell carcinoma poses a clinical challenge for its identification in asymptomatic individuals. It is believed that applying new technologies such as metabolomics to the discovery of potential diagnostic and prognostic biomarkers, can enhance early detection of these cancers and improve overall patient survival while preserving a high quality of life. Considering this, the metabolome of biofluids associated with renal cell carcinoma and prostate cancer were analyzed and studied. Briefly, nuclear magnetic resonance spectroscopy and gas chromatography mass spectrometry techniques were used to analyze serum and urine samples obtained from renal cell carcinoma patients for isolating disease-specific metabolic profiles. Increased levels of lactate and pyruvate accompanied by reduced levels of specific tricarboxylic acid cycle metabolites were associated with renal cell carcinoma. Furthermore, benign oncocytomas of the kidney showed a differential metabolic profile from chromophobe renal cell carcinoma, with glycine and carnitine compounds at the center of the metabolic distinction. Our results showed that the prevalence of the Warburg effect, amino acid dysregulation and glutamine metabolism may distinguish renal cell carcinoma from benign renal lesions and thus presents a platform for identifying renal neoplastic transformations in asymptomatic individuals. On the other hand, the metabolome of seminal plasma measured by nuclear magnetic resonance spectroscopy distinguished prostate cancer Gleason grade 6 from 7, specifically increased lysine levels and reduced serine levels were significantly associated with low risk Gleason grade 6 disease. Also, the plasma and urinary metabolic pattern implicated amino acid dysregulation in bone metastasis of prostate tumors compared to low and high-risk disease. Our findings showed great potential for discriminating indolent from more aggressive prostate cancer, using a non-invasive method. Finally, our metallomics results indicate that metal ions may play a vital role in kidney cancer.
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    Metabolomics Study of ARDS Diagnosis, Heterogeneity, and Mortality
    (2020-08-06) Mohamed Metwaly, Sayed Ahmed Osman; Winston, Brent W.; Vogel, Hans J.; Niven, Daniel J.; Lewis, Ian A.
    Background: Acute Respiratory Distress Syndrome (ARDS) represents 10.4% of all Intensive Care Unit (ICU) admissions with an overall mortality rate of 35.3%. Early diagnosis of ARDS is an important requirement for the timely institution of proper supportive care but it is hampered by the unreliable tissue diagnosis, lack of an early diagnostic biomarker and lack of specific therapy. Metabolomics is a systems biology approach to examine changes in metabolites in response to physiological and pathological stimuli. It is more closely related to observed phenotypes and more responsive to real-time pathophysiological changes, thus constitutes an attractive platform for studying ARDS.Hypothesis: We hypothesize that the diverse inflammatory, pathobiological and adaptive processes involved in ARDS would manifest as unique metabolic fingerprint that sets ARDS apart from other ICU conditions. Biomarker identification may help early diagnosis of ARDS, explore its mechanism, differentiate ARDS subclasses and predict ARDS mortality. Methodology: Serum samples from 108 ARDS patients and 27 ICU ventilated age- and sex-matched controls were included in this study. Samples were analyzed by proton nuclear magnetic resonance spectroscopy (1H-NMR) and gas chromatography mass spectrometry (GC-MS). The proteins/cytokines IL-6, IL-8, IL-10, TNF-?, Ang-2, RAGE, vWF, TNF-RI, ICAM-1, PAI-1, SP-D and Protein-C were also measured. Multivariate statistical models that distinguish ARDS from ICU controls (diagnose ARDS), subclassify ARDS and predict ARDS non-survival were built. Results were externally validated in an independent cohort of 188 patients comprising 118 ARDS patients and 70 ICU ventilated controls.Results: Our data indicates that NMR and GC-MS metabolomics are more accurate than proteins/cytokines in differentiating ARDS from ICU controls and ARDS subgroups. Pathway analysis of ARDS versus controls identified a dominant involvement of serine-glycine metabolism. In external validation, ARDS patients were correctly identified in 93% using NMR and 96% using GC-MS. Metabolomics was not able to predict mortality in the ARDS population studied.Conclusion: ARDS subgroups are metabolically distinct. Further research is needed to explore the role of dysfunctional folate metabolism in the development of ARDS. Metabolomics provides a novel technology to not only understand but intervene on the pathophysiology of ARDS.
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    Metal ion NMR studies of metalloproteins
    (1994) Aramini, James Mario; Vogel, Hans J.
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    Molecular dynamics simulations of peptides
    (2002) Shepherd, Craig McLean; Vogel, Hans J.
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    NMR, spectroscopic, and biochemical studies of calmodulin variants
    (2000) Brokx, Richard D.; Vogel, Hans J.
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    Novel Electrostatic Mechanisms Controlling the Conformational Switching of L-plastin
    (2018-06-12) Fanning, John Keenan; Noskov, Sergei Yu.; Vogel, Hans J.; Maccallum, Justin L.; Prenner, Elmar J.; Ng, Kenneth Kai Sing
    L-plastin is an actin-bundling protein that promotes the motility of both hematopoietic and metastatic cancer cells. The high definition structure of the calcium-binding regulatory domain of human L-plastin was recently determined, allowing computational research on this portion of the protein. The Drude polarizable force field was used to provide accurate computational simulations of the calcium-binding domain in conjunction with experimental validation to shown that L-plastin can regulate calcium-binding, and thus actin-bundling, through internal electrostatic interactions. Through this work the Drude force field was also benchmarked, to show that it provides comparable results to classical force fields with the added ability to simulate polarizability. Overall, a novel mechanism which allows L-plastin to self-regulate its calcium-binding affinity was developed.