The rise in bacterial resistance to common antibiotics has raised an increased need for alternative treatment strategies. The natural antibacterial product, 18β-glycyrrhetinic acid (GRA) has shown efficacy against community-associated methicillin-resistant Staphylococcus aureus (MRSA), although its interactions against planktonic and biofilm modes of growth remain poorly understood. This investigation utilized biochemical and metabolic approaches to further elucidate the effects of GRA on MRSA. Prolonged exposure of planktonic MRSA cell cultures to GRA resulted in increased production of staphyloxanthin, a pigment known to exhibit antioxidant and membrane-stabilizing functions. Then, 1D ¹H NMR analyses of intracellular metabolite extracts from MRSA treated with GRA revealed significant changes in intracellular polar metabolite profiles, including increased levels of succinate and citrate, and significant reductions in several amino acids, including branch chain amino acids. These changes reflect the MRSA response to GRA exposure, including potentially altering its membrane composition, which consumes branched chain amino acids and leads to significant energy expenditure. Although GRA itself had no significant effect of biofilm viability, it seems to be an effective biofilm disruptor. This may be related to interference with cell–cell aggregation, as treatment of planktonic MRSA cultures with GRA leads to a significant reduction in micro-aggregation. The dispersive nature of GRA on MRSA biofilms may prove valuable for treatment of such infections and could be used to increase susceptibility to complementary antibiotic therapeutics.

Environmental metabolomics has the potential to facilitate the establishment of a new suite of tools for assessing the physiological status of important wildlife species. A first step in developing such tools is to evaluate the impacts of various capture techniques on metabolic profiles as capture is necessary to obtain the biological samples required for assays. This study employed ¹H nuclear magnetic resonance (NMR)-based metabolite profiling of 562 blood serum samples from wild bighorn sheep to identify characteristic molecular serum makers of three capture techniques (dart, dropnet, and helicopter-based captures) to inform future sampling protocols for metabolomics studies, and to provide insights into the physiological impacts of capture. We found that different capture techniques induce distinct changes in amino acid serum profiles, the urea cycle, and glycolysis, and attribute the differences in metabolic patterns to differences in physical activity and stress caused by the different capture methods. These results suggest that when designing experiments involving the capture of wild animals, it may be prudent to employ a single capture technique to reduce confounding factors. Our results also supports administration of tranquilizers as soon as animals are restrained to mitigate short-term physiological and metabolic responses when using pursuit and physical restraint capture techniques.

Hypoglycemia may be induced by a variety of physiologic and pathologic stimuli and can result in life-threatening consequences if untreated. However, hypoglycemia may also play a role in the purported health benefits of intermittent fasting and caloric restriction. Previously, we demonstrated that systemic administration of ricin toxin induced fatal hypoglycemia in mice. Here, we examine the metabolic landscape of the hypoglycemic state induced in the liver of mice by two different stimuli: systemic ricin administration and fasting. Each stimulus produced the same decrease in blood glucose and weight loss. The polar metabolome was studied using ¹H NMR, quantifying 59 specific metabolites, and untargeted LC-MS on approximately 5000 features. Results were analyzed by multivariate analyses, using both principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), to identify global metabolic patterns, and by univariate analyses (ANOVA) to assess individual metabolites. The results demonstrated that while there were some similarities in the responses to the two stimuli including decreased glucose, ADP, and glutathione, they elicited distinct metabolic states. The metabolite showing the greatest difference was O-phosphocholine, elevated in ricin-treated animals and known to be affected by the pro-inflammatory cytokine TNF-α. Another difference was the alternative fuel source utilized, with fasting-induced hypoglycemia primarily ketotic, while the response to ricin-induced hypoglycemia involves protein and amino acid catabolism.

Indicator traits associated with disease resiliency would be useful to improve the health and welfare of feedlot cattle. A post hoc analysis of data collected previously ( Kayser et al., 2019a) was conducted to investigate differences in immunologic, physiologic, and behavioral responses of steers (N = 36, initial BW = 386 ± 24 kg) that had differential haptoglobin (HPT) responses to an experimentally induced challenge with Mannheimia haemolytica (MH). Rumen temperature, DMI, and feeding behavior data were collected continuously, and serial blood samples were collected following the MH challenge. Retrospectively, it was determined that 9 of the 18 MH-challenged steers mounted a minimal HPT response, despite having similar leukocyte and temperature responses to other MH-challenged steers with a greater HPT response. Our objective was to examine differences in behavioral and physiological responses between MH-challenged HPT responsive (RES; n = 9), MH-challenged HPT nonresponsive (NON; n = 9), and phosphate-buffered saline-inoculated controls (CON; n = 18). Additionally, ¹H NMR analysis was conducted to determine whether the HPT-responsive phenotype affected serum metabolite profiles. The RES steers had lesser (P < 0.05) cortisol concentrations than NON and CON steers. The magnitude of the increases in neutrophil concentrations and rumen temperature, and the reduction in DMI following the MH challenge were greatest (P < 0.05) in RES steers. Univariate analysis of serum metabolites indicated differences between RES, NON, and CON steers following the MH challenge; however, multivariate analysis revealed no difference between HPT-responsive phenotypes. Prior to the MH challenge, RES steers had longer (P < 0.05) head down and bunk visit durations, slower eating rates (P < 0.01) and greater (P < 0.05) daily variances in bunk visit frequency and head down duration compared with NON steers, suggesting that feeding behavior patterns were associated with the HPT-responsive phenotype. During the 28-d postchallenge period, RES steers had decreased (P < 0.05) final BW, tended (P = 0.06) to have lesser DMI, and had greater (P < 0.05) daily variances in head down and bunk visit durations compared with NON steers, which may have been attributed to their greater acute-phase protein response to the MH challenge. These results indicate that the HPT-responsive phenotype affected feeding behavior patterns and may be associated with disease resiliency in beef cattle.

Background: In the course of testing mAb-based therapies to eradicate the persistent reservoir of HIV infection, we investigated the efficacy and mode of killing of HIV-infected cells by two categories of cytotoxic immunoconjugates (CICs) targeted by the same mAb, an immunotoxin (IT) and antibody-drug conjugate (ADC). Methods: We performed metabolic and transcriptional analyses of treatment effects on the persistently-infected cell line H9/NL4-3. Cells were treated with CIC’s consisting of the anti-gp41 mAb 7B2 conjugated to either deglycosylated ricin A chain (dgA) or to the highly cytotoxic anthracycline derivative PNU-159682. At intervals up to 24 hr, intracellular metabolites were quantified by ¹ H nuclear magnetic resonance spectroscopy, and the transcriptome analyzed by RNA-Seq. Results: Six hr post treatment, 7B2-dgA elicited both metabolic and transcriptional alterations, whereas 7B2-PNU treated cells did not differ from untreated cells. 7B2-dgA treated cells exhibited elevated intracellular levels of many amino acids, and activation of gene pathways for apoptosis, intracellular signaling, and immune activation. By 24 hr, both 7B2-dgA and 7B2-PNU treated cells differed markedly from untreated. Many of the changes observed following 7B2-PNU treatment at 24 hr were similar to those observed at 6hr following 7B2-dgA, likely indicating processes involved in cell death, but a number of alterations were unique to either IT or ADC treated cells. Conclusions: An IT and ADC showed both similarities and differences in their cytotoxic effects. These results raise the question of whether the mode of cell killing could be a determinant of clinical efficacy. Although these studies were aimed at targeting the persistent reservoir of HIV infection, they have relevance for the design of CICs to treat cancer and other conditions. SUMMARY: The use of cytotoxic immunoconjugates, wherein an antibody is attached to a cellular poison, is effective in the treatment of cancer and other conditions. We seek to extend these results to treating HIV and other chronic viral infections. We analyzed the molecular mechanisms of cell killing when the same antibody was attached to different toxic structures. We report that each immunoconjugate induced both common and distinct patterns of killing. Such differences may have clinical relevance.

Background: Understanding the metabolic adaptations of wild bighorn sheep (Ovis c. canadensis) to nutritional stress is crucial for their conservation. Methods: This study employed ¹H nuclear magnetic resonance (NMR) metabolomics to investigate the biochemical responses of these animals to varying sub-maintenance nutritional states. Serum samples from 388 wild bighorn sheep collected between 2014 and 2017 from December (early sub-maintenance) through March (severe sub-maintenance) across Wyoming and Montana were analyzed. Multivariate statistics and machine learning analyses were employed to identify characteristic metabolic patterns and metabolic interactions between early and severe sub-maintenance nutritional states. Results: Significant differences were observed in the levels of 15 of the 49 quantified metabolites, including formate, thymine, glucose, choline, and others, pointing to disruptions in one-carbon, amino acid, and central carbon metabolic pathways. These metabolites may serve as indicators of critical physiological processes such as nutritional intake, immune function, energy metabolism, and protein catabolism, which are essential for understanding how wild bighorn sheep adapt to nutritional stress. Conclusions: This study has generated valuable insights into molecular networks underlying the metabolic resilience of wild bighorn sheep, highlighting the potential for using specific biochemical markers to evaluate nutritional and energetic states in free-ranging ungulates. These insights may help wildlife managers and ecologists compare populations across different times in seasonal cycles, providing information to assess the adequacy of seasonal ranges and support conservation efforts. This research strengthens our understanding of metabolic adaptations to environmental stressors in wild ruminants, offering a foundation for improving management practices to maintain healthy bighorn sheep populations.