Bulk phase resource ratio alters carbon steel corrosion rates and endogenously produced extracellular electron transfer mediators in a sulfate-reducing biofilm.

Desulfovibrio alaskensis G20 biofilms were cultivated on 316 steel, 1018 steel, or borosilicate glass under steady-state conditions in electron-acceptor limiting (EAL) and electron-donor limiting (EDL) conditions with lactate and sulfate in a defined medium. Increased corrosion was observed on 1018 steel under EDL conditions compared to 316 steel, and biofilms on 1018 carbon steel under the EDL condition had at least twofold higher corrosion rates compared to the EAL condition. Protecting the 1018 metal coupon from biofilm colonization significantly reduced corrosion, suggesting that the corrosion mechanism was enhanced through attachment between the material and the biofilm. Metabolomic mass spectrometry analyses demonstrated an increase in a flavin-like molecule under the 1018 EDL condition and sulfonates under the 1018 EAL condition. These data indicate the importance of S-cycling under the EAL condition, and that the EDL is associated with increased biocorrosion via indirect extracellular electron transfer mediated by endogenously produced flavin-like molecules.

Exposome-Scale Investigations Guided by Global Metabolomics, Pathway Analysis, and Cognitive Computing.

Concurrent exposure to a wide variety of xenobiotics and their combined toxic effects can play a pivotal role in health and disease, yet are largely unexplored. Investigating the totality of these exposures, i.e., the "exposome", and their specific biological effects constitutes a new paradigm for environmental health but still lacks high-throughput, user-friendly technology. We demonstrate the utility of mass spectrometry-based global exposure metabolomics combined with tailored database queries and cognitive computing for comprehensive exposure assessment and the straightforward elucidation of biological effects. The METLIN Exposome database has been redesigned to help identify environmental toxicants, food contaminants and supplements, drugs, and antibiotics as well as their biotransformation products, through its expansion with over 700 000 chemical structures to now include more than 950 000 unique small molecules. More importantly, we demonstrate how the XCMS/METLIN platform now allows for the readout of the biological effect of a toxicant through metabolomic-derived pathway analysis, and further, artificial intelligence provides a means of assessing the role of a potential toxicant. The presented workflow addresses many of the methodological challenges current exposomics research is facing and will serve to gain a deeper understanding of the impact of environmental exposures and combinatory toxic effects on human health.

Thermal Degradation of Small Molecules: A Global Metabolomic Investigation.

Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization, especially in gas chromatography mass spectrometry (GC/MS). In this study the effect of heating was examined on a set of 64 small molecule standards and, separately, on human plasma metabolite extracts. The samples, either derivatized or underivatized, were heated at three different temperatures (60, 100, and 250 °C) at different exposure times (30 s, 60 s, and 300 s). All the samples were analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry (LC/MS) and the data processed by XCMS Online ( xcmsonline.scripps.edu ). The results showed that heating at an elevated temperature of 100 °C had an appreciable effect on both the underivatized and derivatized molecules, and heating at 250 °C created substantial changes in the profile. For example, over 40% of the molecular peaks were altered in the plasma metabolite analysis after heating (250 °C, 300s) with a significant formation of degradation and transformation products. The analysis of 64 small molecule standards validated the temperature-induced changes observed on the plasma metabolites, where most of the small molecules degraded at elevated temperatures even after minimal exposure times (30 s). For example, tri- and diorganophosphates (e.g., adenosine triphosphate and adenosine diphosphate) were readily degraded into a mono-organophosphate (e.g., adenosine monophosphate) during heating. Nucleosides and nucleotides (e.g., inosine and inosine monophosphate) were also found to be transformed into purine derivatives (e.g., hypoxanthine). A newly formed transformation product, oleoyl ethyl amide, was identified in both the underivatized and derivatized forms of the plasma extracts and small molecule standard mixture, and was likely generated from oleic acid. Overall these analyses show that small molecules and metabolites undergo significant time-sensitive alterations when exposed to elevated temperatures, especially those conditions that mimic sample preparation and analysis in GC/MS experiments.

Mixed heavy metal stress induces global iron starvation response.

Multiple heavy metal contamination is an increasingly common global problem. Heavy metals have the potential to disrupt microbially mediated biogeochemical cycling. However, systems-level studies on the effects of combinations of heavy metals on bacteria are lacking. For this study, we focused on the Oak Ridge Reservation (ORR; Oak Ridge, TN, USA) subsurface which is contaminated with several heavy metals and high concentrations of nitrate. Using a native Bacillus cereus isolate that represents a dominant species at this site, we assessed the combined impact of eight metal contaminants, all at site-relevant concentrations, on cell processes through an integrated multi-omics approach that included discovery proteomics, targeted metabolomics, and targeted gene-expression profiling. The combination of eight metals impacted cell physiology in a manner that could not have been predicted from summing phenotypic responses to the individual metals. Exposure to the metal mixture elicited a global iron starvation response not observed during individual metal exposures. This disruption of iron homeostasis resulted in decreased activity of the iron-cofactor-containing nitrate and nitrite reductases, both of which are important in biological nitrate removal at the site. We propose that the combinatorial effects of simultaneous exposure to multiple heavy metals is an underappreciated yet significant form of cell stress in the environment with the potential to disrupt global nutrient cycles and to impede bioremediation efforts at mixed waste sites. Our work underscores the need to shift from single- to multi-metal studies for assessing and predicting the impacts of complex contaminants on microbial systems.

Sulfur Metabolites Play Key System-Level Roles in Modulating Denitrification.

Competition between nitrate-reducing bacteria (NRB) and sulfate-reducing bacteria (SRB) for resources in anoxic environments is generally thought to be governed largely by thermodynamics. It is now recognized that intermediates of nitrogen and sulfur cycling (e.g., hydrogen sulfide, nitrite, etc.) can also directly impact NRB and SRB activities in freshwater, wastewater, and sediment and therefore may play important roles in competitive interactions. Here, through comparative transcriptomic and metabolomic analyses, we have uncovered mechanisms of hydrogen sulfide- and cysteine-mediated inhibition of nitrate respiratory growth for the NRB Intrasporangium calvum C5. Specifically, the systems analysis predicted that cysteine and hydrogen sulfide inhibit growth of I. calvum C5 by disrupting distinct steps across multiple pathways, including branched-chain amino acid (BCAA) biosynthesis, utilization of specific carbon sources, and cofactor metabolism. We have validated these predictions by demonstrating that complementation with BCAAs and specific carbon sources relieves the growth inhibitory effects of cysteine and hydrogen sulfide. We discuss how these mechanistic insights give new context to the interplay and stratification of NRB and SRB in diverse environments.IMPORTANCE Nitrate-reducing bacteria (NRB) and sulfate-reducing bacteria (SRB) colonize diverse anoxic environments, including soil subsurface, groundwater, and wastewater. NRB and SRB compete for resources, and their interplay has major implications on the global cycling of nitrogen and sulfur species, with undesirable outcomes in some contexts. For instance, the removal of reactive nitrogen species by NRB is desirable for wastewater treatment, but in agricultural soils, NRB can drive the conversion of nitrates from fertilizers into nitrous oxide, a potent greenhouse gas. Similarly, the hydrogen sulfide produced by SRB can help sequester and immobilize toxic heavy metals but is undesirable in oil wells where competition between SRB and NRB has been exploited to suppress hydrogen sulfide production. By characterizing how reduced sulfur compounds inhibit growth and activity of NRB, we have gained systems-level and mechanistic insight into the interplay of these two important groups of organisms and drivers of their stratification in diverse environments.

Determinants of Risk Factors for Renal Impairment among HIV-Infected Patients Treated with Tenofovir Disoproxil Fumarate-Based Antiretroviral Regimen in Southern Vietnam.

BACKGROUND: The situation of renal impairment among HIV-infected patients treated with TDF-based antiretroviral (ARV) regimen greater than 3 years is little known when TDF use has been promptly increasing in Vietnam. METHODS: We analyse demographic and clinical data from a cross-sectional survey of 400 HIV-infected patients aged ≥18 years, who were treatment-naive or switched TDF regimen within over 3 years between November 2018 and March 2019. Serological tests for serum creatinine, ALT, and AST were performed. Renal impairment was defined as an estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2. Multivariate regression analyses were used to explore the risk factors associated with renal impairment. RESULTS: At the baseline, 7.8% of respondents had estimated glomerular filtration rate (eGFR) of 30-59 mL/min/1.73 m2 and 0.8% had eGFR of 15-29 mL/min/1.73 m2, out of 34 (8.5%) of participants who had renal impairment. Multivariate analysis showed that participants who had preexposure to isoniazid (adjusted PR [aPR] = 0.35 Cl: 0.14-0.91) compared with nonexposure to isoniazid who had a BMI from 18.5 up to 25 kg/m2 (aPR = 0.31 Cl: 0.15-0.62) compared with BMI below 18.5 kg/m2 were less likely to suffer from renal impairment. Patients aged greater than 60 years (aPR = 26.75, 95% Cl: 3.38-211.62) compared with those aged 20-29 years were more likely to have increased risk of renal impairment. CONCLUSION: Our findings underscore the need for longitudinal studies to assess the influence of TDF on maintaining the low prevalence of renal impairment among HIV-infected patients in Vietnam.

Palbociclib and Fulvestrant Act in Synergy to Modulate Central Carbon Metabolism in Breast Cancer Cells.

The aims of this study were to determine whether combination chemotherapeutics exhibit a synergistic effect on breast cancer cell metabolism. Palbociclib, is a selective inhibitor of cyclin-dependent kinases 4 and 6, and when patients are treated in combination with fulvestrant, an estrogen receptor antagonist, they have improved progression-free survival. The mechanisms for this survival advantage are not known. Therefore, we analyzed metabolic and transcriptomic changes in MCF-7 cells following single and combination chemotherapy to determine whether selective metabolic pathways are targeted during these different modes of treatment. Individually, the drugs caused metabolic disruption to the same metabolic pathways, however fulvestrant additionally attenuated the pentose phosphate pathway and the production of important coenzymes. A comprehensive effect was observed when the drugs were applied together, confirming the combinatory therapy's synergism in the cell model. This study also highlights the power of merging high-dimensional datasets to unravel mechanisms involved in cancer metabolism and therapy.

Metabolomics guided pathway analysis reveals link between cancer metastasis, cholesterol sulfate, and phospholipids.

BACKGROUND: Cancer cells that enter the metastatic cascade require traits that allow them to survive within the circulation and colonize distant organ sites. As disseminating cancer cells adapt to their changing microenvironments, they also modify their metabolism and metabolite production. METHODS: A mouse xenograft model of spontaneous tumor metastasis was used to determine the metabolic rewiring that occurs between primary cancers and their metastases. An "autonomous" mass spectrometry-based untargeted metabolomic workflow with integrative metabolic pathway analysis revealed a number of differentially regulated metabolites in primary mammary fat pad (MFP) tumors compared to microdissected paired lung metastases. The study was further extended to analyze metabolites in paired normal tissues which determined the potential influence of metabolites from the microenvironment. RESULTS: Metabolomic analysis revealed that multiple metabolites were increased in metastases, including cholesterol sulfate and phospholipids (phosphatidylglycerols and phosphatidylethanolamine). Metabolite analysis of normal lung tissue in the mouse model also revealed increased levels of these metabolites compared to tissues from normal MFP and primary MFP tumors, indicating potential extracellular uptake by cancer cells in lung metastases. These results indicate a potential functional importance of cholesterol sulfate and phospholipids in propagating metastasis. In addition, metabolites involved in DNA/RNA synthesis and the TCA cycle were decreased in lung metastases compared to primary MFP tumors. CONCLUSIONS: Using an integrated metabolomic workflow, this study identified a link between cholesterol sulfate and phospholipids, metabolic characteristics of the metastatic niche, and the capacity of tumor cells to colonize distant sites.

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus.

Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C terminus of the small (S) subunit. The intact C terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed.

Alterations in Spinal Cord Metabolism during Treatment of Neuropathic Pain.

Therapeutic options for neuropathic pain have improved over the last 20 years yet still only provide partial relief with numerous side effects. Recently, metabolomics revealed that the concentration of the endogenous metabolite N,N-dimethylsphingosine (DMS) is increased in the spinal cord in a model of neuropathic pain. Additionally, it was shown that introduction of DMS to the central nervous system (CNS) resulted in mechanical allodynia. Here, we have examined two compounds; pregabalin (Lyrica®), a drug used to treat neuropathic pain, and N-oleoylethanolamine (NOE), an endogenous endocannabinoid-like compound that is known to affect multiple lipid pathways. We found that the concentration of DMS in the spinal cord was not significantly altered upon pregabalin treatment of rats suffering from neuropathic pain. We further explored whether modulating lipid metabolism may impact neuropathic pain by testing NOE as a potential novel therapeutic.

An azobenzene-containing metal-organic framework as an efficient heterogeneous catalyst for direct amidation of benzoic acids: synthesis of bioactive compounds.

An azobenzene-containing zirconium metal-organic framework was demonstrated to be an effective heterogeneous catalyst for the direct amidation of benzoic acids in tetrahydrofuran at 70 °C. This finding was applied to the synthesis of several important, representative bioactive compounds.

Metabolism links bacterial biofilms and colon carcinogenesis.

Bacterial biofilms in the colon alter the host tissue microenvironment. A role for biofilms in colon cancer metabolism has been suggested but to date has not been evaluated. Using metabolomics, we investigated the metabolic influence that microbial biofilms have on colon tissues and the related occurrence of cancer. Patient-matched colon cancers and histologically normal tissues, with or without biofilms, were examined. We show the upregulation of polyamine metabolites in tissues from cancer hosts with significant enhancement of N(1), N(12)-diacetylspermine in both biofilm-positive cancer and normal tissues. Antibiotic treatment, which cleared biofilms, decreased N(1), N(12)-diacetylspermine levels to those seen in biofilm-negative tissues, indicating that host cancer and bacterial biofilm structures contribute to the polyamine metabolite pool. These results show that colonic mucosal biofilms alter the cancer metabolome to produce a regulator of cellular proliferation and colon cancer growth potentially affecting cancer development and progression.

Luciferase does not Alter Metabolism in Cancer Cells.

Luciferase transfected cell lines are used extensively for cancer models, revealing valuable biological information about disease mechanisms. However, these genetically encoded reporters, while useful for monitoring tumor response in cancer models, can impact cell metabolism. Indeed firefly luciferase and fatty acyl-CoA synthetases differ by a single amino acid, raising the possibility that luciferase activity might alter metabolism and introduce experimental artifacts. Therefore knowledge of the metabolic response to luciferase transfection is of significant importance, especially given the thousands of research studies using luciferase as an in vivo bioluminescence imaging (BLI) reporter. Untargeted metabolomics experiments were performed to examine three different types of lymphoblastic leukemia cell lines (Ramos, Raji and SUP T1) commonly used in cancer research, each were analyzed with and without vector transduction. The Raji model was also tested under perturbed starvation conditions to examine potential luciferase-mediated stress responses. The results showed that no significant metabolic differences were observed between parental and luciferase transduced cells for each cell line, and that luciferase overexpression does not alter cell metabolism under basal or perturbed conditions.

Remarkably favorable hydration of carbonyl substituents in dicationic bis(arene) ruthenium complexes.

The addition of water and methanol to the carbonyl substituents in the dicationic bis(arene)ruthenium complexes [(p-cymene)Ru(C6H5CHO)][OTf]2 (1) and [(p-cymene)Ru(C6H5C(O)CH3)][OTf]2 (2) is remarkably favorable-the equilibrium constants for these reactions are 1 million times greater than those for uncomplexed benzaldehyde and acetophenone. The addition of methanol to the carbonyl group in the o-anisaldehyde complex, [(p-cymene)Ru(C6H4{OCH3}CHO)][OTf]2 (6), occurs with a high degree of kinetic selectivity (94% de).