Lactic Acid Permeability of Aquaporin-9 Enables Cytoplasmic Lactate Accumulation via an Ion Trap

(1) Background: Human aquaporin-9 (AQP9) conducts several small uncharged metabolites, such as glycerol, urea, and lactic acid. Certain brain tumors were shown to upregulate AQP9 expression, and the putative increase in lactic acid permeability was assigned to severity. (2) Methods: We expressed AQP9 and human monocarboxylate transporter 1 (MCT1) in yeast to determine the uptake rates and accumulation of radiolabeled l-lactate/l-lactic acid in different external pH conditions. (3) Results: The AQP9-mediated uptake of l-lactic acid was slow compared to MCT1 at neutral and slightly acidic pH, due to low concentrations of the neutral substrate species. At a pH corresponding to the pKa of l-lactic acid, uptake via AQP9 was faster than via MCT1. Substrate accumulation was fundamentally different between AQP9 and MCT1. With MCT1, an equilibrium was reached, at which the intracellular and extracellular l-lactate/H+ concentrations were balanced. Uptake via AQP9 was linear, theoretically yielding orders of magnitude of higher substrate accumulation than MCT1. (4) Conclusions: The selectivity of AQP9 for neutral l-lactic acid establishes an ion trap for l-lactate after dissociation. This may be physiologically relevant if the transmembrane proton gradient is steep, and AQP9 acts as the sole uptake path on at least one side of a polarized cell.

Metabolic Effects of Doxorubicin on the Rat Liver Assessed With Hyperpolarized MRI and Metabolomics

Doxorubicin (DOX) is a successful chemotherapeutic widely used for the treatment of a range of cancers. However, DOX can have serious side-effects, with cardiotoxicity and hepatotoxicity being the most common events. Oxidative stress and changes in metabolism and bioenergetics are thought to be at the core of these toxicities. We have previously shown in a clinically-relevant rat model that a low DOX dose of 2 mg kg–1 week–1 for 6 weeks does not lead to cardiac functional decline or changes in cardiac carbohydrate metabolism, assessed with hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopy (MRS). We now set out to assess whether there are any signs of liver damage or altered liver metabolism using this subclinical model. We found no increase in plasma alanine aminotransferase (ALT) activity, a measure of liver damage, following DOX treatment in rats at any time point. We also saw no changes in liver carbohydrate metabolism, using hyperpolarized [1-13C]pyruvate MRS. However, using metabolomic analysis of liver metabolite extracts at the final time point, we found an increase in most acyl-carnitine species as well as increases in high energy phosphates, citrate and markers of oxidative stress. This may indicate early signs of steatohepatitis, with increased and decompensated fatty acid uptake and oxidation, leading to oxidative stress.

Soils and sediments host Thermoplasmata archaea encoding novel copper membrane monooxygenases (CuMMOs)

Copper membrane monooxygenases (CuMMOs) play critical roles in the global carbon and nitrogen cycles. Organisms harboring these enzymes perform the first, and rate limiting, step in aerobic oxidation of ammonia, methane, or other simple hydrocarbons. Within archaea, only organisms in the order Nitrososphaerales (Thaumarchaeota) encode CuMMOs, which function exclusively as ammonia monooxygenases. From grassland and hillslope soils and aquifer sediments, we identified 20 genomes from distinct archaeal species encoding divergent CuMMO sequences. These archaea are phylogenetically clustered in a previously unnamed Thermoplasmatota order, herein named the Ca. Angelarchaeales. The CuMMO proteins in Ca. Angelarchaeales are more similar in structure to those in Nitrososphaerales than those of bacteria, and contain all functional residues required for general monooxygenase activity. Ca. Angelarchaeales genomes are significantly enriched in blue copper proteins (BCPs) relative to sibling lineages, including plastocyanin-like electron carriers and divergent nitrite reductase-like (nirK) 2-domain cupredoxin proteins co-located with electron transport machinery. Ca. Angelarchaeales also encode significant capacity for peptide/amino acid uptake and degradation and share numerous electron transport mechanisms with the Nitrososphaerales. Ca. Angelarchaeales are detected at high relative abundance in some of the environments where their genomes originated from. While the exact substrate specificities of the novel CuMMOs identified here have yet to be determined, activity on ammonia is possible given their metabolic and ecological context. The identification of an archaeal CuMMO outside of the Nitrososphaerales significantly expands the known diversity of CuMMO enzymes in archaea and suggests previously unaccounted organisms contribute to critical global nitrogen and/or carbon cycling functions.

The Inhibition Effect of Linezolid With Reyanning Mixture on MRSA and its Biofilm is More Significant than That of Linezolid Alone

Methicillin-resistant Staphylococcus aureus (MRSA) is a superbacterium, and when it forms biofilms, it is difficult to treat even with the first-line of antibiotic linezolid (LNZ). Reyanning mixture (RYN), a compound-based Chinese medicine formula, has been found to have inhibitory effects on biofilms. This study aims to explore the synergistic inhibitory effect and corresponding mechanisms of their (LNZ&RYN) combination on the planktonic as well as biofilm cells of MRSA. Broth microdilution and chessboard methods were employed for the determination of minimum inhibitory concentrations (MICs) and synergistic concentration of LNZ&RYN, respectively. The effect of the combined medication on biofilm and mature biofilm of MRSA were observed by biofilm morphology and permeability experiments, respectively. To unveil the molecular mechanism of action of the synergistic combination of LNZ and RYN, RT-PCR based biofilm-related gene expression analysis and ultra-high pressure liquid chromatography-time-of-flight mass spectrometry based endogenous metabonomic analysis were deployed. The results indicated that 1/16RYN as the best combined dose reduced LNZ (4 μg/ml) to 2 μg/ml. The combined treatment inhibited living MRSA before and after biofilm formation, removed the residual structure of dead bacteria in MRSA biofilms and affected the shape and size of bacteria, resulting in the improvement of biofilm permeability. The mechanism was that biofilm-related genes such as agrC, atlA, and sarA, as well as amino acid uptake associated with the metabolism of 3-dehydrocarnitine, kynurenine, L-leucine, L-lysine and sebacic acid were inhibited. This study provides evidence for the treatment of MRSA and its biofilms with LNZ combined with RYN.

Nutritional Status Predicts Fatty Acid Uptake from Fish and Soybean Oil Supplements for Treatment of Cancer-Related Fatigue: Results from a Phase II Nationwide Study

Cancer-related fatigue is a prevalent and debilitating condition that persists for years into survivorship. Studies evaluating both fish oil supplementation on fatigue and associations between fish oil consumption and fatigue have shown mixed effects; it is unknown what factors contribute to these differential effects. Herein, we investigate whether the nutritional status of cancer survivors was associated with serum omega-3 concentration or change in serum omega-3s throughout a fish oil supplementation study, and then if any of these factors were associated with fatigue. Breast cancer survivors 4–36 months post-treatment with moderate-severe fatigue were randomized to take 6 g fish oil, 6 g soybean oil, or 3 g of each daily for 6 weeks. Baseline nutritional status was calculated using the Controlling Nutritional Status tool (serum albumin, lymphocytes, cholesterol). At baseline and post-intervention, serum fatty acids were quantified and fatigue was assessed using the Multidimensional Fatigue Symptom Inventory. Participants (n = 85) were 61.2 ± 9.7 years old with a body mass index of 31.9 ± 6.7 kg/m2; 69% had a good nutritional score and 31% had light-moderate malnutrition. Those with good nutritional status had greater total serum omega-3s at baseline (p = 0.013) and a greater increase in serum omega-3s with supplementation (p = 0.003). Among those who were supplemented with fish oil, greater increases in serum omega-3s were associated with greater improvements in fatigue. In conclusion, good nutritional status may increase uptake of fatty acid supplements, increasing their ability to improve fatigue.

Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8

Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS–PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology.

Duodenal mucosal resurfacing with GLP-1 receptor agonism increases postprandial unconjugated bile acid in patients with insulin-dependent type 2 diabetes

Introduction: Duodenal Mucosal Resurfacing (DMR) is a new endoscopic ablation technique aimed at improving glycemia and metabolic control in patients with type 2 diabetes mellitus (T2DM). DMR appears to improve insulin resistance, which is the root cause of T2DM, but its mechanism of action is largely unknown. Bile acids function as intestinal signalling molecules in glucose and energy metabolism via the activation of farnesoid X receptor and secondary signalling (e.g. via fibroblast growth factor 19[FGF19]), and are linked to metabolic health. Methods: We investigated the effect of DMR and GLP-1 on postprandial bile acid responses in 16 patients with insulin-dependent T2DM, using mixed meal tests performed at baseline and six months after the DMR procedure. Results: The combination treatment allowed discontinuation of insulin treatment in 11/16 (69%) of patients while improving glycaemic and metabolic health. We found increased postprandial unconjugated bile acid responses (all p<0.05), an overall increased secondary bile acid response (p=0.036) and a higher 12α-hydroxylated:non12α-hydroxylated ratio (p<0.001). Total bile acid concentrations were unaffected by the intervention. Postprandial FGF19 and 7-alpha-hydroxy-4-cholesten-3-one (C4) concentrations decreased post-intervention (both p<0.01). Conclusion and discussion: Our study demonstrates that DMR with GLP-1 modulates the postprandial bile acid response. The alterations in postprandial bile acid responses may be the result of changes in the microbiome, ileal bile acid uptake and improved insulin sensitivity. Controlled studies are needed to elucidate the mechanism linking the combination treatment to metabolic health and bile acids.

Amino Acid Sensing and Assimilation by the Fungal Pathogen Candida albicans in the Human Host

Nutrient uptake is essential for cellular life and the capacity to perceive extracellular nutrients is critical for coordinating their uptake and metabolism. Commensal fungal pathogens, e.g., Candida albicans, have evolved in close association with human hosts and are well-adapted to using diverse nutrients found in discrete host niches. Human cells that cannot synthesize all amino acids require the uptake of the “essential amino acids” to remain viable. Consistently, high levels of amino acids circulate in the blood. Host proteins are rich sources of amino acids but their use depends on proteases to cleave them into smaller peptides and free amino acids. C. albicans responds to extracellular amino acids by pleiotropically enhancing their uptake and derive energy from their catabolism to power opportunistic virulent growth. Studies using Saccharomyces cerevisiae have established paradigms to understand metabolic processes in C. albicans; however, fundamental differences exist. The advent of CRISPR/Cas9-based methods facilitate genetic analysis in C. albicans, and state-of-the-art molecular biological techniques are being applied to directly examine growth requirements in vivo and in situ in infected hosts. The combination of divergent approaches can illuminate the biological roles of individual cellular components. Here we discuss recent findings regarding nutrient sensing with a focus on amino acid uptake and metabolism, processes that underlie the virulence of C. albicans.

Transcriptomes and metabolism define mouse and human MAIT cell heterogeneity

Mucosal-associated invariant T (MAIT) cells are a subpopulation of T lymphocytes that respond to microbial metabolites. We performed single-cell RNA sequencing and metabolic analyses of MAIT cell subsets in thymus and peripheral tissues from mice and humans to define the heterogeneity and developmental pathway of these innate-like lymphocytes. We show that the predominant mouse subset, which produces IL-17 (MAIT17), and the subset that produces IFNγ (MAIT1), have greatly different transcriptomes and metabolic states in the thymus and periphery. A splenic MAIT subset has a transcriptome similar to circulating lymphocytes, and in mice these also are found in recent thymic emigrants, suggesting partially mature cells emigrate from the thymus. Human MAIT cells are predominantly MAIT1 cells, but have a different metabolism from their mouse counterparts with increased fatty acid uptake and storage. Although mouse and human subsets are similar in thymus, in the periphery they diverge, likely reflecting environmental influences.

A systemic analysis of amino acid transporters identifies Gnp2 as the main proline permease in Candida albicans

The tight association of Candida albicans with the human host has driven the evolution of mechanisms that permit metabolic flexibility. Amino acids, present in free form or peptide bound, are an abundant carbon and nitrogen source in many host niches. Further,the capacity to sense and utilize certain amino acids, like proline, is directly linked to virulence. The C. albicans genome encodes for at least 24 amino acid permeases (AAPs), highlighting the importance of flexible amino acid uptake for fungal growth and virulence. Although the substrate specificity and role of certain AAPs has been investigated, a comprehensive characterization was missing. Therefore, we assembled a library of AAP deletion strains, which was tested for resistance to toxic amino acid analogs. Most striking was the specific resistance of gnp2Δ to the proline analog 3,4-dehydroproline. Subsequent tests validated that Gnp2 is a specific proline permease in C. albicans, which is contrary to the model yeast Saccharomyces cerevisiae where proline transport is mediated by four permeases. Furthermore, the induction of GNP2 appears to be independent of the SPS (Ssy1-Ptr3-Ssy5) regulatory pathway that controls proline utilization in the model yeast, pointing towards rewired proline uptake in C. albicans. Additionally, strains lacking GNP2were unable to respond to proline-induced filamentation, displayed decreased cytotoxicity to macrophages and showed increased sensitivity to oxidative stress, underlining the importance of proline uptake for fungal virulence. Taken together, the role of Gnp2-mediated proline uptake illustrates the importance of metabolism-driven virulence in C. albicans.