Widespread changes in gene expression drive tumorigenesis, yet our knowledge of how aberrant epigenomic and transcriptome profiles arise in cancer cells is poorly understood. Here, we demonstrate ...that metabolic transformation plays an important role. Butyrate is the primary energy source of normal colonocytes and is metabolized to acetyl-CoA, which was shown to be important not only for energetics but also for HAT activity. Due to the Warburg effect, cancerous colonocytes rely on glucose as their primary energy source, so butyrate accumulated and functioned as an HDAC inhibitor. Although both mechanisms increased histone acetylation, different target genes were upregulated. Consequently, butyrate stimulated the proliferation of normal colonocytes and cancerous colonocytes when the Warburg effect was prevented from occurring, whereas it inhibited the proliferation of cancerous colonocytes undergoing the Warburg effect. These findings link a common metabolite to epigenetic mechanisms that are differentially utilized by normal and cancerous cells because of their inherent metabolic differences.
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► The Warburg effect mediates butyrate's action on cell proliferation ► Butyrate regulates histone acetylation and gene expression as an acetyl-CoA donor ► The dose of butyrate determines the utilization of epigenetic mechanisms ► Butyrate modulates different genes as a HAT cofactor compared to an HDAC inhibitor
5-methylcytosine (5mC) in DNA plays an important role in gene expression, genomic imprinting, and suppression of transposable elements. 5mC can be converted to 5-hydroxymethylcytosine (5hmC) by the ...Tet (ten eleven translocation) proteins. Here, we show that, in addition to 5hmC, the Tet proteins can generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) from 5mC in an enzymatic activity— dependent manner. Furthermore, we reveal the presence of 5fC and 5caC in genomic DNA of mouse embryonic stem cells and mouse organs. The genomic content of 5hmC, 5fC, and 5caC can be increased or reduced through overexpression or depletion of Tet proteins. Thus, we identify two previously unknown cytosine derivatives in genomic DNA as the products of Tet proteins. Our study raises the possibility that DNA demethylation may occur through Tet-catalyzed oxidation followed by decarboxylation.
Per- and polyfluorinated alkyl substances (PFASs) contaminate groundwater, surface water, and finished drinking water internationally. Their ecological persistence and adverse human health effects ...demand effective remediation approaches. Motivated by the limitations in selectivity and performance of current PFAS removal technologies, we report a platform approach for the development of ionic fluorogel resins that effectively remove a chemically diverse mixture of PFAS from water. The synthesis of a material library with systematic variation in fluorous and ionic components led to the identification of a resin that demonstrated rapid removal of PFASs with high affinity and selectivity in the presence of nonfluorous contaminants commonly found in groundwater. The material can be regenerated and reused multiple times. We demonstrate ionic fluorogels as effective adsorbents for the removal of 21 legacy and emerging PFASs from settled water collected at the Sweeney Water Treatment Plant in Wilmington, North Carolina.
DNA damage and mutations induced by oxidative stress are associated with various different human pathologies including cancer. The facts that most human tumors are characterized by large genome ...rearrangements and glutathione depletion in mice results in deletions in DNA suggest that reactive oxygen species (ROS) may cause gene and chromosome mutations through DNA double strand breaks (DSBs). However, the generation of DSBs at low levels of ROS is still controversial. In the present study, we show that H2O2 at biologically-relevant levels causes a marked increase in oxidative clustered DNA lesions (OCDLs) with a significant elevation of replication-independent DSBs. Although it is frequently reported that OCDLs are fingerprint of high-energy IR, our results indicate for the first time that H2O2, even at low levels, can also cause OCDLs leading to DSBs specifically in G1 cells. Furthermore, a reverse genetic approach revealed a significant contribution of the non-homologous end joining (NHEJ) pathway in H2O2-induced DNA repair & mutagenesis. This genomic instability induced by low levels of ROS may be involved in spontaneous mutagenesis and the etiology of a wide variety of human diseases like chronic inflammation-related disorders, carcinogenesis, neuro-degeneration and aging.
Acid-catalyzed multiphase chemistry of isoprene epoxydiols (IEPOX) on sulfate aerosol produces substantial amounts of water-soluble secondary organic aerosol (SOA) constituents, including ...2-methyltetrols, methyltetrol sulfates, and oligomers thereof in atmospheric fine particulate matter (PM2.5). These constituents have commonly been measured by gas chromatography interfaced to electron ionization mass spectrometry (GC/EI-MS) with prior derivatization or by reverse-phase liquid chromatography interfaced to electrospray ionization high-resolution mass spectrometry (RPLC/ESI-HR-MS). However, both techniques have limitations in explicitly resolving and quantifying polar SOA constituents due either to thermal degradation or poor separation. With authentic 2-methyltetrol and methyltetrol sulfate standards synthesized in-house, we developed a hydrophilic interaction liquid chromatography (HILIC)/ESI-HR-quadrupole time-of-flight mass spectrometry (QTOFMS) protocol that can chromatographically resolve and accurately measure the major IEPOX-derived SOA constituents in both laboratory-generated SOA and atmospheric PM2.5. 2-Methyltetrols were simultaneously resolved along with 4-6 diastereomers of methyltetrol sulfate, allowing efficient quantification of both major classes of SOA constituents by a single non-thermal analytical method. The sum of 2-methyltetrols and methyltetrol sulfates accounted for approximately 92%, 62%, and 21% of the laboratory-generated β-IEPOX aerosol mass, laboratory-generated δ-IEPOX aerosol mass, and organic aerosol mass in the southeastern U.S., respectively, where the mass concentration of methyltetrol sulfates was 171-271% the mass concentration of methyltetrol. Mass concentrations of methyltetrol sulfates were 0.39 and 2.33 μg m-3 in a PM2.5 sample collected from central Amazonia and the southeastern U.S., respectively. The improved resolution clearly reveals isomeric patterns specific to methyltetrol sulfates from acid-catalyzed multiphase chemistry of β- and δ-IEPOX. We also demonstrate that conventional GC/EI-MS analyses overestimate 2-methyltetrols by up to 188%, resulting (in part) from the thermal degradation of methyltetrol sulfates. Lastly, C5-alkene triols and 3-methyltetrahydrofuran-3,4-diols are found to be largely GC/EI-MS artifacts formed from thermal degradation of 2-methyltetrol sulfates and 3-methyletrol sulfates, respectively, and are not detected with HILIC/ESI-HR-QTOFMS.
Whether dietary fiber protects against colorectal cancer is controversial because of conflicting results from human epidemiologic studies. However, these studies and mouse models of colorectal cancer ...have not controlled the composition of gut microbiota, which ferment fiber into short-chain fatty acids such as butyrate. Butyrate is noteworthy because it has energetic and epigenetic functions in colonocytes and tumor-suppressive properties in colorectal cancer cell lines. We used gnotobiotic mouse models colonized with wild-type or mutant strains of a butyrate-producing bacterium to demonstrate that fiber does have a potent tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. Furthermore, due to the Warburg effect, butyrate was metabolized less in tumors where it accumulated and functioned as a histone deacetylase (HDAC) inhibitor to stimulate histone acetylation and affect apoptosis and cell proliferation. To support the relevance of this mechanism in human cancer, we demonstrate that butyrate and histone-acetylation levels are elevated in colorectal adenocarcinomas compared with normal colonic tissues.
These results, which link diet and microbiota to a tumor-suppressive metabolite, provide insight into conflicting epidemiologic findings and suggest that probiotic/prebiotic strategies can modulate an endogenous HDAC inhibitor for anticancer chemoprevention without the adverse effects associated with synthetic HDAC inhibitors used in chemotherapy.
Inhaled formaldehyde is classified as a known human and animal carcinogen, causing nasopharyngeal cancer. Additionally, limited epidemiological evidence for leukemia in humans is available; however, ...this is inconsistent across studies. Both genotoxicity and cytotoxicity are key events in formaldehyde nasal carcinogenicity in rats, but mechanistic data for leukemia are not well established. Formation of DNA adducts is a key event in initiating carcinogenesis. Formaldehyde can induce DNA monoadducts, DNA-DNA cross-links, and DNA protein cross-links. In this study, highly sensitive liquid chromatography-tandem mass spectrometry-selected reaction monitoringmethods were developed and 13CD2-formaldehyde exposures utilized, allowing differentiation of DNA adducts and DNA-DNA cross-links originating from endogenous and inhalation-derived formaldehyde exposure. The results show that exogenous formaldehyde induced N2-hydroxymethyl-dG monoadducts and dG-dG cross-links in DNA from rat respiratory nasal mucosa but did not form 13CD2-adducts in sites remote to the portal of entry, even when five times more DNA was analyzed. Furthermore, no N6-HO13CD2-dA adducts were detected in nasal DNA. In contrast, high amounts of endogenous formaldehyde dG and dA monoadducts were present in all tissues examined. The number of exogenous N2-HO13CD2-dG in 1- and 5-day nasal DNA samples from rats exposed to 10-ppm 13CD2-formaldehyde was 1.28 ± 0.49 and 2.43 ± 0.78 adducts/107 dG, respectively, while 2.63 ± 0.73 and 2.84 ± 1.13 N2-HOCH2-dG adducts/107 dG and 3.95 ± 0.26 and 3.61 ± 0.95 N6-HOCH2-dA endogenous adducts/107 dA were present. This study provides strong evidence supporting a genotoxic and cytotoxic mode of action for the carcinogenesis of inhaled formaldehyde in respiratory nasal epithelium but does not support the biological plausibility that inhaled formaldehyde also causes leukemia.
Ecologists have observed declines in the biodiversity of sensitive freshwater organisms in response to increasing concentrations of major ions (salinization). Yet, how changing salinities ...physiologically challenge aquatic organisms, such as mayflies, remains remarkably understudied. Moreover, it is not well understood the degree to which species respond and acclimate to salinity changes. Our lab is developing the Baetid mayfly, N. triangulifer, as a model organism for physiological research. We have previously described acclimatory changes in both ion flux rates and altered mRNA transcript levels in response to chronic exposures to elevated major ion concentrations at the whole animal level. In the present study, we use shotgun proteomics to identify the specific proteins associated with apical ion transport and how their abundance changes in response to chronic salinity exposures in gills. Gills were isolated from the penultimate nymphal stage of N. triangulifer reared under control culture conditions, elevated NaCl (157 mg L−1 Na), elevated CaCl2 (121 mg L−1 Ca), elevated Ca/MgSO4 (735 mg L−1 SO4). These conditions mirrored those from previously published physiological work. We also acutely exposed nymphs to dilute (50% dilution of culture water with deionized water) to explore proteomic changes in the gills in response to dilute conditions. We report 710 unique peptide sequences among treatment groups, including important apical ion transporters such as Ca-ATPase, Na/K ATPase, and V-ATPase. Treatment with elevated NaCl and Ca/MgSO4 appeared to cause more significant differential protein expression (452 and 345, respectively) compared to CaCl2 and dilute groups (134 and 17, respectively). Finally, we demonstrated the breadth of physiological functions in gills by exploring non-transport related pathways found in our dataset, including ATP synthesis, calcium signaling, and oxidative stress response. We discuss our results in the context of freshwater salinization and the challenges of working with non-model species without fully sequenced and annotated genomes.
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•Morphologically distinct ionocytes occur on the abdomen and gills.•The proteome of the gill on the mayfly, N. triangulifer, is described.•V-ATPase, Na/K ATPase, and Ca ATPase were identified as primary ion transporters.•Exposure to NaCl or Ca/MgSO4 induced major changes in peptide abundances.
Activated CD8
T cells are elevated in Nonalcoholic steatohepatitis (NASH) and are important for driving fibrosis and inflammation. Despite this, mechanisms of CD8
T cell activation in NASH are ...largely limited. Specific CD8
T cell subsets may become activated through metabolic signals or cytokines. However, studies in NASH have not evaluated the impact of antigen presentation or the involvement of specific antigens. Therefore, we determined if activated CD8
T cells are dependent on MHC class I expression in NASH to regulate fibrosis and inflammation.
We used H2Kb and H2Db deficient (MHC I KO
, Kb transgenic mice, and myeloid cell Kb deficient mice (LysM Kb KO) to investigate how MHC class I impacts CD8
T cell function and NASH. Flow cytometry, gene expression, and histology were used to examine hepatic inflammation and fibrosis. The hepatic class I immunopeptidome was evaluated by mass spectrometry.
In NASH, MHC class I isoform H2Kb was upregulated in myeloid cells. MHC I KO demonstrated protective effects against NASH-induced inflammation and fibrosis. Kb mice exhibited increased fibrosis in the absence of H2Db while LysM Kb KO mice showed protection against fibrosis but not inflammation. H2Kb restricted peptides identified a unique NASH peptide Ncf2 capable of CD8
T cell activation
. The Ncf2 peptide was not detected during fibrosis resolution.
These results suggest that activated hepatic CD8
T cells are dependent on myeloid cell MHC class I expression in diet induced NASH to promote inflammation and fibrosis. Additionally, our studies suggest a role of NADPH oxidase in the production of Ncf2 peptide generation.
The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb's innate therapeutic properties, resistance to phage and antibiotics, and ...high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb's ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae's secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.
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