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It is unknown whether antiretroviral (ARV) drugs in women living with HIV (WLHIV) are associated with mitochondrial toxicity and altered fat oxidation and branched-chain amino acid metabolism in the placenta and fetus. Immediately after delivery, we froze placental biopsies from 20 WLHIV and 20 matched uninfected women. We analyzed global biochemical profiles using high-performance liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry. We used t -tests, principle component analysis, hierarchical clustering, and random forest analysis (RFA) in our analysis. Twelve WLHIV were on protease inhibitors, six on non-nucleoside reverse inhibitors, and two on integrase strand inhibitors with optimized backbone. Mean birth weight of HIV-exposed neonates was significantly lower than unexposed neonates (3,075 g vs. 3,498 g, p  = .01) at similar gestational age. RFA identified 30 of 702 analytes that differentiated the placental profiles of WLHIV from uninfected women with 72.5% predictive accuracy. Placental profiles of non-nucleoside reverse transcriptase inhibitor (NNRTI)-treated WLHIV exhibited lower levels of amino acids, including essential and branched-chain amino acids, and some medium-chain acylcarnitines. Placental metabolism may be altered in WLHIV, possibly associated with ARV exposure. The lower birth weight among neonates of WLHIV suggests the need for further studies considering potential deleterious effects of altered placenta metabolism on fetal growth and development.

Here, we report a multi OMIC (transcriptome, proteome, and metabolome) approach to investigate molecular changes in lens fiber cells (FC) of mice exposed to cigarette smoke (CS). Pregnant mice were placed in a whole-body smoke chamber and a few days later pups were born, which were exposed to CS for 5 hours/day, 5 days/week for a total of 3½ months. We examined the mice exposed to CS for CS-related cataractogenesis after completion of the CS exposure but no cataracts were observed. Lenses of CS-exposed and age-matched, untreated control mice were extracted and lens FC were subjected to multi OMIC profiling. We identified 348 genes, 130 proteins, and 14 metabolites exhibiting significant (p < 0.05) differential levels in lens FC of mice exposed to CS, corresponding to 3.6%, 4.3%, and 5.0% of the total genes, protein, and metabolites, respectively identified in this study. Our multi OMIC approach confirmed that only a small fraction of the transcriptome, the proteome, and the metabolome was perturbed in the lens FC of mice exposed to CS, which suggests that exposure of CS had a minimal effect on the mouse lens. It is worth noting that while our results confirm that CS exposure does not have a substantial impact on the molecular landscape of the mouse lens FC, we cannot rule out that CS exposure for longer durations and/or in combination with other morbidities or environmental factors would have a more robust effect and/or result in cataractogenesis., (© 2021. The Author(s).)

Scrub typhus is an acute zoonosis caused by the obligate intracellular Gram-negative bacterium Orientia tsutsugamushi. To better understand the host response elicited by natural infection by chigger feeding, ICR mice were infected by Leptotrombidium chiangraiensis (Lc1) chiggers, and the metabolic profiles of their serum were examined over several time points after initiation of feeding. ICR mice were infected by either naive Lc1 chiggers (i.e. not infected by O. tsutsugamushi, NLc1) or O. tsutsugamushi -infected Lc1 chiggers (OLc1). Serum was collected from both groups of mice at 6 hours and 10 days after initiation of feeding. Metabolites were extracted from the serum and analysed by ultra performance liquid chromatography-tandem mass spectrometry. The resulting ion/chromatographic features were matched to a library of chemical standards for identification and quantification. Biochemicals that differed significantly between the experimental groups were identified using Welch's two-sample t tests; p ≤ 0.05 was considered statistically significant. A number of biochemicals linked to immune function were found to be significantly altered between mice infected by the NLc1 and OLc1 chiggers, including itaconate, kynurenine and histamine. Several metabolites linked to energy production were also found to be altered in the animals. In addition lipid and carbohydrate metabolism, bile acid and phospholipid homeostasis, and nucleotide metabolism were also found to be different in these two groups of mice. Markers of stress and food intake were also significantly altered. Global untargeted metabolomic characterization revealed significant differences in the biochemical profiles of mice infected by the NLc1 versus OLc1 chiggers. These findings provide an important platform for further investigation of the host responses associated with chigger-borne O. tsutsugamushi infections.

Hotspot mutations in the isocitrate dehydrogenase 1 ( IDH1 ) gene occur in a number of human cancers and confer a neomorphic enzyme activity that catalyzes the conversion of α-ketoglutarate (αKG) to the oncometabolite D-(2)-hydroxyglutarate (D2HG). In malignant gliomas, IDH1 R132H expression induces widespread metabolic reprogramming, possibly requiring compensatory mechanisms to sustain the normal biosynthetic requirements of actively proliferating tumor cells. We used genetically engineered mouse models of glioma and quantitative metabolomics to investigate IDH1 R132H -dependent metabolic reprogramming and its potential to induce biosynthetic liabilities that can be exploited for glioma therapy. In gliomagenic neural progenitor cells, IDH1 R132H expression increased the abundance of dipeptide metabolites, depleted key tricarboxylic acid cycle metabolites, and slowed progression of murine gliomas. Notably, expression of glutamate dehydrogenase GDH2, a hominoid-specific enzyme with relatively restricted expression to the brain, was critically involved in compensating for IDH1 R132H -induced metabolic alterations and promoting IDH1 R132H glioma growth. Indeed, we found that recently evolved amino acid substitutions in the GDH2 allosteric domain conferred its nonredundant, glioma-promoting properties in the presence of IDH1 mutation. Our results indicate that among the unique roles for GDH2 in the human forebrain is its ability to limit IDH1 R132H -mediated metabolic liabilities, thus promoting glioma growth in this context. Results from this study raise the possibility that GDH2-specific inhibition may be a viable therapeutic strategy for gliomas with IDH mutations. Significance: These findings show that the homonid-specific brain enzyme GDH2 may be essential to mitigate metabolic liabilities created by IDH1 mutations in glioma, with possible implications to leverage its therapeutic management by IDH1 inhibitors. Cancer Res; 78(1); 36-50. ©2017 AACR ., (©2017 American Association for Cancer Research.)

NQO1 [NAD(P)H quinone oxidoreductase 1; also known as DT-diaphorase] is a cytosolic enzyme that catalyses the two-electron reduction of various quinones including vitamin K. The enzyme may play a role in vitamin K metabolism by reducing vitamin K to vitamin K hydroquinone for utilization in the post-translational γ-glutamyl carboxylation reactions required by several proteins involved in blood coagulation. The aim of the present study was to assess the contribution of NQO1 to vitamin K reduction and haemostasis in an in vivo model. We examined the contribution of NQO1 to haemostasis by examining survival rates in mice poisoned with the anticoagulant warfarin. Supraphysiological amounts of vitamin K sufficiently reversed the effects of warfarin in both wild-type and NQO1-deficient mice. Additionally, vitamin K reductase activities distinct from VKOR (vitamin K epoxide reductase) and NQO1 were measured in vitro from both wild-type and NQO1-defecient mice. The results of the present study suggest that NQO1 does not play a major role in the production of vitamin K hydroquinone and supports the existence of multiple vitamin K reduction pathways. The properties of a NAD(P)H-dependent vitamin K reductase different from NQO1 are described.

The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a beta-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection.

The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a β-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The lipid A moiety of Escherichia coli lipopolysaccharide is a hexaacylated disaccharide of glucosamine that is phosphorylated at the 1 and 4' positions. Expression of the Francisella novicida lipid A 1-phosphatase FnLpxE in E. coli results in dephosphorylation of the lipid A proximal unit. Coexpression of FnLpxE and the Rhizobium leguminosarum lipid A oxidase RlLpxQ in E. coli converts much of the proximal glucosamine to 2-amino-2-deoxygluconate. Expression of the F. novicida lipid A 4'-phosphatase FnLpxF in wild-type E. coli has no effect because FnLpxF cannot dephosphorylate hexaacylated lipid A. However, expression of FnLpxF in E. coli lpxM mutants, which synthesize pentaacylated lipid A lacking the secondary 3'-myristate chain, causes extensive 4'-dephosphorylation. Coexpression of FnLpxE and FnLpxF in lpxM mutants results in massive accumulation of lipid A species lacking both phosphate groups, and introduction of RlLpxQ generates phosphate-free lipid A variants containing 2-amino-2-deoxygluconate. The proposed lipid A structures were confirmed by electrospray ionization mass spectrometry. Strains with 4'-dephosphorylated lipid A display increased polymyxin resistance. Heptose-deficient mutants of E. coli lacking both the 1- and 4'-phosphate moieties are viable on plates but sensitive to CaCl(2). Our methods for reengineering lipid A structure may be useful for generating novel vaccines and adjuvants.

The lipid A of Rhizobium etli, a nitrogen-fixing plant endosymbiont, displays significant structural differences when compared to that of Escherichia coli. An especially striking feature of R. etli lipid A is that it lacks both the 1- and 4'-phosphate groups. The 4'-phosphate moiety of the distal glucosamine unit is replaced with a galacturonic acid residue. The dephosphorylated proximal unit is present as a mixture of the glucosamine hemiacetal and an oxidized 2-aminogluconate derivative. Distinct lipid A phosphatases directed to the 1 or the 4'-positions have been identified previously in extracts of R. etli and Rhizobium leguminosarum. The corresponding structural genes, lpxE and lpxF, respectively, have also been identified. Here, we describe the isolation and characterization of R. etli deletion mutants in each of these phosphatase genes and the construction of a double phosphatase mutant. Mass spectrometry confirmed that the mutant strains completely lacked the wild-type lipid A species and accumulated the expected phosphate-containing derivatives. Moreover, radiochemical analysis revealed that phosphatase activity was absent in membranes prepared from the mutants. Our results indicate that LpxE and LpxF are solely responsible for selectively dephosphorylating the lipid A molecules of R. etli. All the mutant strains showed an increased sensitivity to polymyxin relative to the wild-type. However, despite the presence of altered lipid A species containing one or both phosphate groups, all the phosphatase mutants formed nitrogen-fixing nodules on Phaseolus vulgaris. Therefore, the dephosphorylation of lipid A molecules in R. etli is not required for nodulation but may instead play a role in protecting the bacteria from cationic antimicrobial peptides or other immune responses of plants., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The outer monolayer of the outer membrane of Gram-negative bacteria consists of the lipid A component of lipopolysaccharide (LPS), a glucosamine-based saccharolipid that is assembled on the inner surface of the inner membrane. The first six enzymes of the lipid A pathway are required for bacterial growth and are excellent targets for the development of new antibiotics. Following assembly, the ABC transporter MsbA flips nascent LPS to the periplasmic side of the inner membrane, whereupon additional transport proteins direct it to the outer surface of the outer membrane. Depending on the bacterium, various covalent modifications of the lipid A moiety may occur during the transit of LPS to the outer membrane. These extra-cytoplasmic modification enzymes are therefore useful as reporters for monitoring LPS trafficking. Because of its conserved structure in diverse Gram-negative pathogens, lipid A is recognized as foreign by the TLR4/MD2 receptor of the mammalian innate immune system, resulting in rapid macrophage activation and robust cytokine production.