Validation of Non-Targeted Analysis Methods: Establishing and Communicating Confidence in Results

Presentation to NACRW on 7/25/2023 in Fort Lauderdale, FloridaScience Inventory, CCTE products: https://cfpub.epa.gov/si/si_public_search_results.cfm?advSearch=true&showCriteria=2&keyword=CCTE&TIMSType=&TIMSSubTypeID=&epaNumber=&ombCat=Any&dateBeginPublishedPresented=07/01/2017&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&DEID=&personName=&personID=&role=Any&journalName=&journalID=&publisherName=&publisherID=&sortBy=pubDate&count=25</p

Taming the wild west of NTA: advancement of tools & applications

Presentation to LC-MSMS on Sept. 25-25, 2023 in Buffalo, NYScience Inventory, CCTE products: https://cfpub.epa.gov/si/si_public_search_results.cfm?advSearch=true&showCriteria=2&keyword=CCTE&TIMSType=&TIMSSubTypeID=&epaNumber=&ombCat=Any&dateBeginPublishedPresented=07/01/2017&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&DEID=&personName=&personID=&role=Any&journalName=&journalID=&publisherName=&publisherID=&sortBy=pubDate&count=25</p

Crystallographic and Spectroscopic Insights into Heme Degradation by <i>Mycobacterium tuberculosis</i> MhuD

Mycobacterium heme utilization degrader (MhuD) is a heme-degrading protein from Mycobacterium tuberculosis responsible for extracting the essential nutrient iron from host-derived heme. MhuD has been previously shown to produce unique organic products compared to those of canonical heme oxygenases (HOs) as well as those of the IsdG/I heme-degrading enzymes from Staphylococcus aureus. Here, we report the X-ray crystal structure of cyanide-inhibited MhuD (MhuD–heme–CN) as well as detailed ¹H nuclear magnetic resonance (NMR), UV/vis absorption, and magnetic circular dichroism (MCD) spectroscopic characterization of this species. There is no evidence for an ordered network of water molecules on the distal side of the heme substrate in the X-ray crystal structure, as was previously reported for canonical HOs. The degree of heme ruffling in the crystal structure of MhuD is greater than that observed for HO and less than that observed for IsdI. As a consequence, the Fe 3d_<i>xz</i>-, 3d_<i>yz</i>-, and 3d_<i>xy</i>-based MOs are very close in energy, and the room-temperature ¹H NMR spectrum of MhuD–heme–CN is consistent with population of both a ²E_<i>g</i> electronic state with a (d_<i>xy</i>)²(d_<i>xz</i>,d_<i>yz</i>)³ electron configuration, similar to the ground state of canonical HOs, and a ²B_2<i>g</i> state with a (d_<i>xz</i>,d_<i>yz</i>)⁴(d_<i>xy</i>)¹ electron configuration, similar to the ground state of cyanide-inhibited IsdI. Variable temperature, variable field MCD saturation magnetization data establishes that MhuD–heme–CN has a ²B_2<i>g</i> electronic ground state with a low-lying ²E_<i>g</i> excited state. Our crystallographic and spectroscopic data suggest that there are both structural and electronic contributions to the α-meso regioselectivity of MhuD-catalyzed heme cleavage. The structural distortion of the heme substrate observed in the X-ray crystal structure of MhuD–heme–CN is likely to favor cleavage at the α- and γ-meso carbons, whereas the spin density distribution may favor selective oxygenation of the α-meso carbon

Annotating and Visualizing a Database of Analytical Methods

Presentation for SERMACS on October 25-28, 2023 in Durham, NCScience Inventory, CCTE products: https://cfpub.epa.gov/si/si_public_search_results.cfm?advSearch=true&showCriteria=2&keyword=CCTE&TIMSType=&TIMSSubTypeID=&epaNumber=&ombCat=Any&dateBeginPublishedPresented=07/01/2017&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&DEID=&personName=&personID=&role=Any&journalName=&journalID=&publisherName=&publisherID=&sortBy=pubDate&count=25</p

Increased Abscess Formation and Defective Chemokine Regulation in CREB Transgenic Mice

<div><p>Cyclic AMP-response element-binding protein (CREB) is a transcription factor implicated in growth factor-dependent cell proliferation and survival, glucose homeostasis, spermatogenesis, circadian rhythms, and synaptic plasticity associated with memory. To study the phenotype of CREB overexpression <em>in vivo</em>, we generated CREB transgenic (TG) mice in which a myeloid specific hMRP8 promoter drives CREB expression. CREB TG mice developed spontaneous skin abscesses more frequently than wild type (WT) mice. To understand the role of CREB in myeloid function and innate immunity, chemokine expression in bone marrow derived macrophages (BMDMs) from CREB TG mice were compared with BMDMs from WT mice. Our results demonstrated decreased Keratinocyte-derived cytokine (KC) in CREB TG BMDMs but not TNFα protein production in response to lipid A (LPA). In addition, mRNA expression of KC and IL-1β (Interleukin)-1β was decreased in CREB TG BMDMs; however, there was no difference in the mRNA expression of TNFα, MCP-1, IL-6 and IL-12p40. The mRNA expression of IL-1RA and IL-10 was decreased in response to LPA. Nuclear factor kappa B (NFκB) expression and a subset of its target genes were upregulated in CREB TG mouse BMDMs. Although neutrophil migration was the same in both CREB TG and WT mice, Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was significantly increased in neutrophils from CREB TG mice. Taken together, CREB overexpression in myeloid cells results in increased abscess formation <em>in vivo</em> and aberrant cytokine and chemokine response, and neutrophil function <em>in vitro</em>.</p> </div

Purification of BMDMs and expression of CREB.

<p>(A) Whole bone marrow was obtained from CREB TG and WT mice. Flow cytometry was performed after staining cells for CD11b and F4/80 cell surface markers confirming 93% CD11b+/F4/80+ BMDM population. (B) Western blot analysis was performed with CREB antisera (UBI, Upstate, NY) and normalization for Actin expression (Santa Cruz Biotechnology, Santa Cruz, CA). To quantitate the band, ImageJ software was used (<a href="http://rsb.info.nih.go/ij/" target="_blank">http://rsb.info.nih.go/ij/</a>). CREB TG mice BMDMs had 4-fold greater CREB expression than WT mice.</p

Expression of known nuclear factor kappa B (NFκB) target genes.

<p>(A-E) qRT-PCR was performed with RNA from BMDMs from CREB and WT mice. Chemokine ligand 5 (CCL5), Colony Stimulating Factor 1 or Macrophage Colony Stimulating Factor (CSF1 or M-CSF), Interferon Factor 1 (IRF1), and NFkB were upregulated in CREB BMDMs (***p<0.005). Chemokine ligand 2 (CCL2) expression was downregulated. The data represent an average ± SEM of four mice analyzed in triplicate for ELISA and two independent experiments. ***p<0.001.</p

CREB overexpression results in reduced LPA-induced KC production and differential chemokine and cytokine expression.

<p>(A) ELISA were performed to quantify KC and TNF-α production and (B) qRT-PCR was performed to compare mRNA expression in BMDMs isolated from CREB TG or WT mice exposed for 2 hours to 10 ng/ml LPA. KC and TNF-α production was measured using ELISA and mRNA levels were measured using qRT-PCR and normalized to GAPDH mRNA levels. The data represent an average ± SEM of four mice analyzed in triplicate for ELISA and three independent experiments with three mice each for qRT-PCR experiments; *<i>p<0.05, **p<0.01, ***p<0.001,</i> ns means “not significant”.</p

Quantification of Secondary Structure Elements from Circular Dichroism with FecB variants.

Quantification of Secondary Structure Elements from Circular Dichroism with FecB variants.</p

Determination of heme affinity of FecB2 ligand-binding pocket variants.

Representative fluorescent emission intensities at 335 nm after excitation at 280 nm of FecB2 variants with increasing concentrations of heme. FecB2 variants tested for heme affinity include FecB L135R, R141S, Q233S, R240S, Y242S, Y270S, E272S, D332S, Q336S, and E339S mutations, along with the double mutants R240S-E339S and Y242S-E339S. Curves were fit using the equation in the methods and Fe-cMB affinities (Kd) are included for each titration. (PDF)</p