Bert C Lynn

University of Kentucky, Lexington, Kentucky, United States

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Publications (57)273.93 Total impact

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    ABSTRACT: In an attempt to mimic white-rot fungi lignin degradation via in vivo Fenton chemistry, solution phase Fenton chemistry (10g biomass, 176mmol hydrogen peroxide and 1.25mmol Fe(2+) in 200mL of water) was applied to four different biomass feedstocks. An enzymatic saccharification of Fenton pretreated biomass showed an average 212% increase relative to untreated control across all four feedstocks (P<0.05, statistically significant). A microbial fermentation of the same Fenton pretreated biomass showed a threefold increase in gas production upon a sequential co-culture with Clostridium thermocellum and Clostridium beijerinckii. These results demonstrate the use of solution phase Fenton chemistry as a viable pretreatment method to make cellulose more bioavailable for microbial biofuel conversion.
    Bioresource Technology 04/2014; 162C:273-278. DOI:10.1016/j.biortech.2014.03.151 · 5.04 Impact Factor
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    ABSTRACT: In an attempt to mimic white-rot fungi lignin degradation via in vivo Fenton chemistry, solution phase Fenton chemistry (10 g biomass, 176 mmol hydrogen peroxide and 1.25 mmol Fe2+ in 200 mL of water) was applied to four different biomass feedstocks. An enzymatic saccharification of Fenton pretreated biomass showed an average 212% increase relative to untreated control across all four feedstocks (P < 0.05, statistically significant). A microbial fermentation of the same Fenton pretreated biomass showed a threefold increase in gas production upon a sequential co-culture with Clostridium thermocellum and Clostridium beijerinckii. These results demonstrate the use of solution phase Fenton chemistry as a viable pretreatment method to make cellulose more bioavailable for microbial biofuel conversion.
    Bioresource Technology 01/2014; 162:273–278. · 5.04 Impact Factor
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    ABSTRACT: Studies of oxidative damage during the progression of Alzheimer's disease (AD) suggest its central role in disease pathogenesis. To investigate levels of nucleic acid oxidation in both early and late stages of AD, levels of multiple base adducts were quantified in nuclear and mitochondrial DNA from the superior and middle temporal gyri (SMTG), inferior parietal lobule (IPL), and cerebellum (CER) of age-matched normal control subjects, subjects with mild cognitive impairment, preclinical AD, late-stage AD, and non-AD neurological disorders (diseased control; DC) using gas chromatography/mass spectrometry. Median levels of multiple DNA adducts in nuclear and mitochondrial DNA were significantly (P < 0.05) elevated in the SMTG, IPL, and CER in multiple stages of AD and in DC subjects. Elevated levels of fapyguanine and fapyadenine in mitochondrial DNA suggest a hypoxic environment early in the progression of AD and in DC subjects. Overall, these data suggest that oxidative damage is an early event not only in the pathogenesis of AD, but is also present in neurodegenerative diseases in general. This article is protected by copyright. All rights reserved.
    Journal of Neurochemistry 09/2013; DOI:10.1111/jnc.12444 · 4.24 Impact Factor
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    ABSTRACT: Aims: FUsed in sarcoma (FUS) is a multifunctional DNA/RNA-binding protein that possesses diverse roles, such as RNA splicing, RNA transport, DNA repair, translation, and transcription. The network of enzymes and processes regulated by FUS is far from being fully described. In this study, we have focused on the mechanisms of FUS-regulated MnSOD gene transcription. Results: Here, we demonstrate that FUS is a component of the transcription complex that regulates the expression of MnSOD. Overexpression of FUS increased MnSOD expression in a dose-dependent manner and knockdown of FUS by siRNA led to the inhibition of MnSOD gene transcription. Reporter analyses, ChIP assay, EMSA, affinity chromatography, and SPR analyses revealed the far upstream region of MnSOD promoter as an important target of FUS-mediated MnSOD transcription and confirmed that FUS binds to the MnSOD promoter and interacts with Sp1. Importantly, overexpression of familial amyotropic lateral sclerosis (fALS)-linked R521G mutant FUS resulted in a significantly reduced level of MnSOD expression and activity, which is consistent with the decline in MnSOD activity observed in fibroblasts from fALS patients with the R521G mutation. R521G-mutant FUS abrogates MnSOD promoter-binding activity and interaction with Sp1. Innovation and Conclusion: This study identifies FUS as playing a critical role in MnSOD gene transcription and reveals a previously unrecognized relationship between MnSOD and mutant FUS in fALS.
    Antioxidants & Redox Signaling 07/2013; 20(10). DOI:10.1089/ars.2012.4984 · 8.20 Impact Factor
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    ABSTRACT: Herein we report the fast pyrolysis of the lignin monomers sinapyl and coniferyl alcohol, as well as mixtures of the two, at 650 °C using pyrolysis–GC/MS. The total ion chromatogram area % of certain marker pyrolysates for each alcohol were summed and used to calculate sinapyl:guaiacyl (S:G) ratios in the mixtures; these ratios were then plotted against the actual molar S:G ratios of the starting material. 13 coniferyl alcohol marker pyrolysates and 9 sinapyl alcohol marker pyrolysates provided acceptable linear correlation, whereas several other marker groups chosen did not correlate to the actual S:G ratio in the starting material. Results indicated that the demethoxylation of sinapyl alcohol and/or its pyrolysates occurs during pyrolysis at 650 °C; however, the amount of demethoxylated products generated is statistically insignificant. Having obtained the pyrolysis profile of the various S:G mixtures, marker pyrolysates for the calculation of the S:G ratio in lignin can be carefully selected according to unique samples. These marker groups can then be calibrated against known S:G ratios to provide analysis of the actual S:G ratio of lignin in biomass. For example, pyrolysates chosen from the pyrolysis of peach pit lignin were calibrated in order to determine the S:G ratio in peach pit lignin. The resulting S:G ratio was similar to that obtained from capillary electrophoresis of the products from KMnO4 oxidation of the peach pit lignin.
    Journal of Analytical and Applied Pyrolysis 01/2013; 99:161–169. DOI:10.1016/j.jaap.2012.10.001 · 3.07 Impact Factor
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    ABSTRACT: Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H(2)) are also produced. The effect of hydrogenase inhibitors (H(2), carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N(2) to H(2), acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H(2) or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD(+) from NADH by H(2), lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H(2) production for the conversion of synthetic gases to chemicals.
    Applied Microbiology and Biotechnology 01/2012; 93(4):1777-84. DOI:10.1007/s00253-011-3812-3 · 3.81 Impact Factor
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    ABSTRACT: The isolation of high-purity cellular biomacromolecules and sub-cellular organelles is an essential aspect to mass spectrometry based studies. Mitochondria are sub-cellular organelles that perform a central role in cellular energy production. Mitochondria are of great interest due to their potential to generate reactive oxygen species (ROS) and susceptibility to oxidative damage and subsequent functional impairment. Current methods of mitochondria isolation are optimized for respiratory-based studies that favor viability. Whereas, proteomic and lipidomics studies of mitochondria require procedures that optimize for purity and enrichment. We describe a procedure derived from previously established methods for the isolation of mitochondria, nuclear and cytosolic fractions from a neurological tissue sample. In addition to the isolation being of significant purity for mass spectral based '-omics' analysis, mitochondrial yields were routinely 500 μg per tissue wet weight, allowing multiple studies to be conducted from a single isolation procedure.
    Journal of neuroscience methods 03/2011; 197(2):279-82. DOI:10.1016/j.jneumeth.2011.02.027 · 2.30 Impact Factor
  • Alzheimer's and Dementia 07/2010; 6(4). DOI:10.1016/j.jalz.2010.05.1781 · 17.47 Impact Factor
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    ABSTRACT: The major barrier to treating or preventing Alzheimer's disease (AD) is its unknown etiology and pathogenesis. Although increasing evidence supports a role for mitochondrial dysfunction in the pathogenesis of AD, there have been few studies that simultaneously evaluate changes in multiple mitochondrial proteins. To evaluate changes in sites of potentially interacting mitochondrial proteins, we applied 2-dimensional liquid chromatography coupled with tandem mass spectrometry and the isotope coded affinity tag method to identify and quantify proteins in mitochondrial enriched fractions isolated from short postmortem interval temporal pole specimens from subjects with mild cognitive impairment (4 subjects pooled), early AD (4 subjects pooled), late-stage AD (8 subjects pooled) and age-matched normal control (7 subjects pooled) subjects. A total of 112 unique, non-redundant proteins were identified and quantified in common to all three stages of disease progression. Overall, patterns of protein change suggest activation of mitochondrial pathways that include proteins responsible for transport and utilization of ATP. These proteins include adenine nucleotide translocase, voltage dependent anion channels, hexokinase, and creatine kinase. Comparison of protein changes throughout the progression of AD suggests the most pronounced changes occur in early AD mitochondria.
    Journal of Alzheimer's disease: JAD 01/2010; 19(1):325-39. DOI:10.3233/JAD-2010-1254 · 3.61 Impact Factor
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    ABSTRACT: The major barrier to treating or preventing Alzheimer's disease (AD) is its unknown etiology and pathogenesis. Although increasing evidence supports a role for mitochondrial dysfunction in the pathogenesis of AD, there have been few studies that simultaneously evaluate changes in multiple mitochondrial proteins. To evaluate changes in sites of potentially interacting mitochondrial proteins, we applied 2-dimensional liquid chromatography coupled with tandem mass spectrometry and the isotope coded affinity tag method to identify and quantify proteins in mitochondrial enriched fractions isolated from short postmortem interval temporal pole specimens from subjects with mild cognitive impairment (4 subjects pooled), early AD (4 subjects pooled), late-stage AD (8 subjects pooled) and age-matched normal control (7 subjects pooled) subjects. A total of 112 unique, non-redundant proteins were identified and quantified in common to all three stages of disease progression. Overall, patterns of protein change suggest activation of mitochondrial pathways that include proteins responsible for transport and utilization of ATP. These proteins include adenine nucleotide translocase, voltage dependent anion channels, hexokinase, and creatine kinase. Comparison of protein changes throughout the progression of AD suggests the most pronounced changes occur in early AD mitochondria.
    Journal of Alzheimer's disease: JAD 11/2009; DOI:10.3233/JAD-2009-1254 · 3.61 Impact Factor
  • Jinchun Sun, Bert C. Lynn
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    ABSTRACT: A hybrid LC/MS/MS and proteomics method was developed for the assessment of multiple pesticide exposure. The methodology was based on the analysis of tryptic peptides resulting from inhibited butyrylcholinesterase (BChE) after exposure to pesticides including organophosphates (OPs) and carbamates (CBs). The primary advantage of the assay was its ability to simultaneously examine multiple pesticide exposures in a single analytical experiment. Application of tandem and MS3 techniques provided identities of the inhibiting pesticide, confirmation and localization of the site of inhibition and relative quantification of phosphorylated peptides present in tryptic digests of equine BChE (eBChE).
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 11/2009; 877(29-877):3681-3685. DOI:10.1016/j.jchromb.2009.09.009 · 2.78 Impact Factor
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    ABSTRACT: Accumulating evidence suggests mitochondrial alterations are intimately associated with the pathogenesis of Alzheimer's disease (AD). In order to determine if mutations of presenilin-1 (PS-1) affect levels of mitochondrial proteins at different ages we enriched mitochondrial fractions from 3-, 6-, 12-month-old knock-in mice expressing the M146V PS-1 mutation and identified, and quantified proteins using cleavable isotope-coded affinity tag labeling and two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS). Using this approach, 165 non-redundant proteins were identified with 80 of them present in all three age groups. Specifically, at young ages (3 and 6 months), Na(+)/K(+) ATPase and several signal transduction proteins exhibited elevated levels, but dropped dramatically at 12 months. In contrast, components of the oxidative phosporylation pathway (OXPHOS), the mitochondrial permeability transition pore (MPTP), and energy metabolism proteins remained unchanged at 3 months but significantly increased with age. We propose that alterations in calcium homeostasis induced by the PS-1 mutation have a major impact in young animals by inhibiting the function of relevant proteins and inducing compensatory changes. However, in older mice combination of the PS-1 mutation and accumulated oxidative damage results in a functional suppression of OXPHOS and MPTP proteins requiring a compensatory increase in expression levels. In contrast, signal transduction proteins showed decreased levels due to a break down in the compensatory mechanisms. The dysfunction of Na(+)/K(+) ATPase and signal transduction proteins may induce impaired cognition and memory before neurodegeneration occurs.
    Cellular and Molecular Neurobiology 03/2009; 29(5):649-64. DOI:10.1007/s10571-009-9359-5 · 2.20 Impact Factor
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    ABSTRACT: Clostridium thermocellum is a candidate organism for consolidated bioprocessing of lignocellulosic biomass into ethanol. However, commercial use is limited due to growth inhibition at modest ethanol concentrations. Recently, an ethanol-adapted strain of C. thermocellum was produced. Since ethanol adaptation in microorganisms has been linked to modification of membrane lipids, we tested the hypothesis that ethanol adaptation in C. thermocellum involves lipid modification by comparing the fatty acid composition and membrane anisotropy of wild-type and ethanol-adapted strains. Derivatization to fatty acid methyl esters provided quantitative lipid analysis. Compared to wild-type, the ethanol-adapted strain had a larger percentage of fatty acids with chain lengths >16:0 and showed a significant increase in the percentage of 16:0 plasmalogens. Structural identification of fatty acids was confirmed through mass spectral fragmentation patterns of picolinyl esters. Ethanol adaptation did not involve modification at sites of methyl branching or the unsaturation index. Comparison of steady-state fluorescence anisotropy experiments, in the absence and presence of ethanol, provided evidence for the effects of ethanol on membrane fluidity. In the presence of ethanol, both strains displayed increased fluidity by approximately 12%. These data support the model that ethanol adaptation was the result of fatty acid changes that increased membrane rigidity that counter-acted the fluidizing effect of ethanol.
    Applied Microbiology and Biotechnology 02/2009; 82(5):929-39. DOI:10.1007/s00253-009-1891-1 · 3.81 Impact Factor
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    ABSTRACT: Sample preparation plays a critical role in successful proteomic applications. Features of electrospray mass spectrometry impose limits on the types of buffers, detergents and other reagents that can be used in sample preparation. Unfortunately, many of these mass spectrometry incompatible reagents significantly enhance protein recoveries from complex matrices. This problem prompted our search for a better cleanup protocol. Our data suggest that the Three-layer Sandwich Gel Electrophoresis (TSGE) protocol can solve this problem and provide near quantitative recovery of extremely low concentration proteins from harsh solutions, a feature not available from other cleanup protocols. The hallmark of the TSGE protocol is the combination of the properties of agarose gels (that serve as the matrix to immobilize the proteins of interest) with low- and high-percentage polyacrylamide gels (that serve as the concentration and sealing layers, respectively). By electrophoretically driving the proteins of interest from the agarose matrix into the concentration layer, the TSGE protocol simultaneously concentrates the sample in the concentration layer and provides an environment amenable to downstream buffer exchange and proteolytic digestion. In combination with 2D-LC-MS/MS, the TSGE protocol was evaluated in the analysis of a whole cell extract from the protozoan parasite Toxoplasma gondii. Comparison of our experimental proteomic results with in silico predictions from gene data indicated that TSGE did not bias the protein identification.
    Journal of Proteome Research 10/2008; 7(10):4256-65. DOI:10.1021/pr800182b · 5.00 Impact Factor
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    ABSTRACT: Accurate determination of extracted ion chromatographic peak areas in isotope-labeled quantitative proteomics is difficult to automate. Manual validation of identified peaks is typically required. We have integrated a peak confidence scoring algorithm into existing tools which are compatible with analysis pipelines based on the standards from the Institute for Systems Biology. This algorithm automatically excludes incorrectly identified peaks, improving the accuracy of the final protein expression ratio calculation. SOURCE AND SUPPLEMENTARY INFORMATION: http://www.chem.uky.edu/research/lynn/Nelson.pdf.
    Bioinformatics 09/2008; 24(18):2103-4. DOI:10.1093/bioinformatics/btn385 · 4.62 Impact Factor
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    ABSTRACT: To determine if an aberrant protein complex consisting of prostaglandin-d-synthase (PDS) and transthyretin (TTR) in CSF differentiates between subjects with Alzheimer disease (AD) and normal control (NC) subjects. Western blot analysis and a unique sandwich ELISA were used to quantify levels of complexed PDS/TTR in ventricular CSF of subjects with autopsy-verified diagnoses and in lumbar CSF of living subjects with mild to moderate probable AD and age-matched NC subjects. Ventricular CSF was obtained from short postmortem interval autopsies of 7 NC subjects (4 men/3 women), 12 diseased control (DC) subjects (7 men/5 women), 4 subjects with mild cognitive impairment (MCI) (2 men/2 women), and 8 subjects with late-stage AD (LAD) (4 men/4 women). Lumbar CSF was obtained from 15 subjects with probable AD (5 men/10 women) and 14 age-matched NC subjects (10 men/4 women) and was analyzed in a double-blind fashion. A significant increase in complexed PDS/TTR in ventricular CSF was found in MCI and LAD subjects but not DC subjects compared with NC subjects. Double-blind analysis of complexed PDS/TTR in lumbar CSF showed a significant sixfold increase in levels of the PDS/TTR complex in living probable AD subjects compared with age-matched NC subjects and a 100% sensitivity and 93% specificity in the identification of subjects with AD. After further study of larger numbers of patients, quantifying prostaglandin-d-synthase/transthyretin complex in CSF may be useful in the diagnosis of Alzheimer disease, possibly in the early stages of the disease.
    Neurology 07/2008; 70(23):2212-8. DOI:10.1212/01.wnl.0000312383.39973.88 · 8.30 Impact Factor
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    ABSTRACT: The 3' end of mammalian introns is marked by the branchpoint binding protein, SF1, and the U2AF65-U2AF35 heterodimer bound at an adjacent sequence. Baker's yeast has equivalent proteins, branchpoint binding protein (BBP) (SF1) and Mud2p (U2AF65), but lacks an obvious U2AF35 homolog, leaving open the question of whether another protein substitutes during spliceosome assembly. Gel filtration, affinity selection and mass spectrometry were used to show that rather than a U2AF65/U2AF35-like heterodimer, Mud2p forms a complex with BBP without a third (U2AF35-like) factor. Using mutants of MUD2 and BBP, we show that the BBP-Mud2p complex bridges partner-specific Prp39p, Mer1p, Clf1p and Smy2p two-hybrid interactions. In addition to inhibiting Mud2p association, the bbpDelta56 mutation impairs splicing, enhances pre-mRNA release from the nucleus, and similar to a mud2::KAN knockout, suppresses a lethal sub2::KAN mutation. Unexpectedly, rather than exacerbating bbpDelta56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpDelta56. We propose that a BBP-Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly. In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association.
    Nucleic Acids Research 06/2008; 36(8):2787-98. DOI:10.1093/nar/gkn144 · 8.81 Impact Factor
    This article is viewable in ResearchGate's enriched format
  • William D. Nelson, Kert Viele, Bert C. Lynn
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    ABSTRACT: The presence of affinity reagents such as immunoglobulin in preparations for sensitive mass spectrometry analyses can preclude the identification of low-abundance proteins of interest. We report a method whereby antisera are purified and biotinylated prior to use in immunoprecipitation that allows for its efficient removal from proteomic samples via streptavidin capture. This method can similarly be extended to other affinity reagents such as recombinant fusion proteins for enhanced identification of interacting proteins.
    Journal of Proteome Research 01/2008; 6(12):4758-62. DOI:10.1021/pr070517a · 5.00 Impact Factor
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    Jinchun Sun, Bert C Lynn
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    ABSTRACT: A novel, proteomics based method was developed for the detection, quantification, and categorization of serum butyrylcholinesterase (BChE) inhibitors, including organophosphates (OPs) and carbamates (CBs). This method was based on the MALDI-TOF-MS analysis of the trypsin generated BChE active site peptide (191-SVTLFGESAGAASVSLHLLSPR-212) previously modified by reaction with an OP or CB. The ionization efficiency of OP modified active site peptides by MALDI was greatly improved by adding diammonium citrate to the MALDI matrix, which made the quantification of OP exposure feasible. Excellent linearity (r2 > 0.98) between the normalized abundance ratios (NARs) and OP concentrations or logarithm of carbaryl concentration was obtained. The accuracy of the developed assay was evaluated by comparison of IC50 and IC100 values from the assay with those determined by the Ellman method. Results from this method were comparable with those from the Ellman method. The advantage of the assay was that both the origin and the extent of pesticide exposure can be determined in one analysis. Our MALDI method can provide critical evidence for the pesticide exposure at low BChE inhibition levels even down to 3%, not available with the Ellman method.
    Journal of the American Society for Mass Spectrometry 05/2007; 18(4):698-706. DOI:10.1016/j.jasms.2006.11.009 · 3.19 Impact Factor