Multi-omics Investigation into the Mechanism of Action of an Anti-tubercular Fatty Acid Analogue.

The mechanism of action (MoA) of a clickable fatty acid analogue 8-(2-cyclobuten-1-yl)octanoic acid (DA-CB) has been investigated for the first time. Proteomics, metabolomics, and lipidomics were combined with a network analysis to investigate the MoA of DA-CB against Mycobacterium smegmatis (Msm). The metabolomics results showed that DA-CB has a general MoA related to that of ethionamide (ETH), a mycolic acid inhibitor that targets enoyl-ACP reductase (InhA), but DA-CB likely inhibits a step downstream from InhA. Our combined multi-omics approach showed that DA-CB appears to disrupt the pathway leading to the biosynthesis of mycolic acids, an essential mycobacterial fatty acid for both Msm and Mycobacterium tuberculosis (Mtb). DA-CB decreased keto-meromycolic acid biosynthesis. This intermediate is essential in the formation of mature mycolic acid, which is a key component of the mycobacterial cell wall in a process that is catalyzed by the essential polyketide synthase Pks13 and the associated ligase FadD32. The multi-omics analysis revealed further collateral alterations in bacterial metabolism, including the overproduction of shorter carbon chain hydroxy fatty acids and branched chain fatty acids, alterations in pyrimidine metabolism, and a predominate downregulation of proteins involved in fatty acid biosynthesis. Overall, the results with DA-CB suggest the exploration of this and related compounds as a new class of tuberculosis (TB) therapeutics. Furthermore, the clickable nature of DA-CB may be leveraged to trace the cellular fate of the modified fatty acid or any derived metabolite or biosynthetic intermediate.

Phosphorus Stress-Induced Changes in Plant Root Exudation Could Potentially Facilitate Uranium Mobilization from Stable Mineral Forms.

Apparent deficiency of soil mineral nutrients often triggers specific physio-morphological changes in plants, and some of these changes could also inadvertently increase the ability of plants to mobilize radionuclides from stable mineral forms. This work, through a series of sand-culture, hydroponics, and batch-equilibration experiments, investigated the differential ability of root exudates of Andropogon virginicus grown under conditions with variable phosphorus (P) availability (KH2PO4, FePO4, Ca3(PO4)2, and no P) to solubilize uranium (U) from the uranyl phosphate mineral Chernikovite. The mineral form of P, and hence the bioavailability of P, affected the overall composition of the root exudates. The lower bioavailable forms of P (FePO4 and Ca3(PO4)2), but not the complete absence of P, resulted in a higher abundance of root metabolites with chelating capacity at 72 hrs after treatment application. In treatments with lower P-bioavailability, the physiological amino acid concentration inside of the roots increased, whereas the concentration of organic acids in the roots decreased due to the active exudation. In batch dissolution experiments, the organic acids, but not amino acids, increase the dissolution U from Chernikovite. The root exudate matrix of plants exposed to low available forms of P induced a >60% increase in U dissolution from Chernikovite due to 5-16 times greater abundance of organic acids in these treatments. However, this was ca. 70% of the theoretical dissolution achievable by this exudate matrix. These results highlight the potential of using active management of soil P as an effective tool to alter the plant-mediated mobilization of U in contaminated soil.

A reversed phase ultra-high-performance liquid chromatography-data independent mass spectrometry method for the rapid identification of mycobacterial lipids.

A rapid reversed-phase ultra-high-performance liquid chromatography-high resolution mass spectrometry based mycobacterial lipidomics approach is described. This method enables the separation of various lipid classes including lipids specific to mycobacterial, such as methoxy mycolic acid and α-mycolic acid. Lipid separation occurs during a relatively short runtime of 14 min on a charged surface hybrid C18 column. A high-resolution quadrupole-time of flight mass spectrometer and a data independent acquisition mode allowed for the simultaneous acquisition of the full scan and collision induced dissociation fragmentation. The proposed method provides lipid detection results equivalent to or better than existing methods, but with a faster throughput and an overall higher sensitivity. The reversed-phase ultra-high-performance liquid chromatography-high resolution mass spectrometry method was shown to obtain structural information for lipids extracted from Mycobacterium smegmatis, but the method is applicable to the analysis of lipids from various bacterial and mammalian cell lines.

The non-canonical mechanism of ER stress-mediated progression of prostate cancer.

BACKGROUND: The development of persistent endoplasmic reticulum (ER) stress is one of the cornerstones of prostate carcinogenesis; however, the mechanism is missing. Also, alcohol is a physiological ER stress inducer, and the link between alcoholism and progression of prostate cancer (PCa) is well documented but not well characterized. According to the canonical model, the mediator of ER stress, ATF6, is cleaved sequentially in the Golgi by S1P and S2P proteases; thereafter, the genes responsible for unfolded protein response (UPR) undergo transactivation. METHODS: Cell lines used were non-malignant prostate epithelial RWPE-1 cells, androgen-responsive LNCaP, and 22RV1 cells, as well as androgen-refractory PC-3 cells. We also utilized PCa tissue sections from patients with different Gleason scores and alcohol consumption backgrounds. Several sophisticated approaches were employed, including Structured illumination superresolution microscopy, Proximity ligation assay, Atomic force microscopy, and Nuclear magnetic resonance spectroscopy. RESULTS: Herein, we identified the trans-Golgi matrix dimeric protein GCC185 as a Golgi retention partner for both S1P and S2P, and in cells lacking GCC185, these enzymes lose intra-Golgi situation. Progression of prostate cancer (PCa) is associated with overproduction of S1P and S2P but monomerization of GCC185 and its downregulation. Utilizing different ER stress models, including ethanol administration, we found that PCa cells employ an elegant mechanism that auto-activates ER stress by fragmentation of Golgi, translocation of S1P and S2P from Golgi to ER, followed by intra-ER cleavage of ATF6, accelerated UPR, and cell proliferation. The segregation of S1P and S2P from Golgi and activation of ATF6 are positively correlated with androgen receptor signaling, different disease stages, and alcohol consumption. Finally, depletion of ATF6 significantly retarded the growth of xenograft prostate tumors and blocks production of pro-metastatic metabolites. CONCLUSIONS: We found that progression of PCa associates with translocation of S1P and S2P proteases to the ER and subsequent ATF6 cleavage. This obviates the need for ATF6 transport to the Golgi and enhances UPR and cell proliferation. Thus, we provide the novel mechanistic model of ATF6 activation and ER stress implication in the progression of PCa, suggesting ATF6 is a novel promising target for prostate cancer therapy.

Comparative Metabolomic Analyses of Ipomoea lacunosa Biotypes with Contrasting Glyphosate Tolerance Captures Herbicide-Induced Differential Perturbations in Cellular Physiology.

Glyphosate-tolerant Ipomoea lacunosa is emerging as a problematic weed in the southeastern United States. Metabolomic profiling was conducted to examine the innate physiology and the glyphosate induced perturbations in two biotypes of I. lacunosa (WAS and QUI) that had contrasting glyphosate tolerance. Compared to the less tolerant QUI-biotype, the innate metabolism of the more tolerant WAS-biotype was characterized by a higher abundance of amino acids, and pyruvate; whereas the sugar profile of the QUI biotype was dominated by the transport sugar sucrose. Glyphosate application (80 g ae/ha) caused similar shikimate accumulation in both biotypes. Compared to QUI, in WAS, the content of aromatic amino acids was less affected by glyphosate treatment, and the content of Ala, Val, Ile, and Pro increased. However, the total sugars decreased by ∼75% in WAS, compared to ∼50% decrease in QUI. The innate, higher proportional abundance, of the transport-sugar sucrose in QUI coud partly explain the higher translocation and greater sensitivity of this biotype to glyphosate. The decrease in sugars, accompanied by an increase in amino acids could delay feedback regulation of upstream enzymes of the shikimate acid pathway in WAS, which could contribute to a greater glyphosate tolerance. Our study, through a metabolomics approach, provides complementary data that elucidates the cellular physiology of herbicide tolerance in Ipomoea lacunosa biotypes.

Metabolic Profiling and Enzyme Analyses Indicate a Potential Role of Antioxidant Systems in Complementing Glyphosate Resistance in an Amaranthus palmeri Biotype.

Metabolomics and biochemical assays were employed to identify physiological perturbations induced by a commercial formulation of glyphosate in susceptible (S) and resistant (R) biotypes of Amaranthus palmeri. At 8 h after treatment (HAT), compared to the respective water-treated control, cellular metabolism of both biotypes were similarly perturbed by glyphosate, resulting in abundance of most metabolites including shikimic acid, amino acids, organic acids and sugars. However, by 80 HAT the metabolite pool of glyphosate-treated R-biotype was similar to that of the control S- and R-biotypes, indicating a potential physiological recovery. Furthermore, the glyphosate-treated R-biotype had lower reactive oxygen species (ROS) damage, higher ROS scavenging activity, and higher levels of potential antioxidant compounds derived from the phenylpropanoid pathway. Thus, metabolomics, in conjunction with biochemical assays, indicate that glyphosate-induced metabolic perturbations are not limited to the shikimate pathway, and the oxidant quenching efficiency could potentially complement the glyphosate resistance in this R-biotype.