Amino Acid Uptake Research Articles

Both Enantiomers of 2-Hydroxyglutarate Modulate the Metabolism of Cultured Human Neuroblastoma Cells

Elevated levels of D-2-hydroxyglutarate (D-2HG) and L-2-hydroxyglutarate (L-2HG) in the brain are associated with various pathological conditions, potentially contributing to neurological symptoms and neurodegeneration. Previous studies on animal models have revealed their capability to interfere with several cellular processes, including mitochondrial metabolism. Both enantiomers competitively inhibit the enzymatic activity of 2-oxoglutarate-dependent dioxygenases. These enzymes also execute several signaling cascades and regulate the level of covalent modifications on nucleic acids or proteins, e.g., methylation, hydroxylation, or ubiquitination, with an effect on epigenetic regulation of gene expression, protein stability, and intracellular signaling. To investigate the potential impact of 2HG enantiomers on human neuronal cells, we utilized the SH-SY5Y human neuroblastoma cell line as a model. We employed proton nuclear magnetic resonance (1H-NMR) spectroscopy of culture media that provided high-resolution insights into the changes in the content of metabolites. Concurrently, we performed biochemical assays to complement the 1H-NMR findings and to estimate the activities of lactate and 3-hydroxybutyrate dehydrogenases. Our results reveal that both 2HG enantiomers can influence the cellular metabolism of human neuroblastoma cells on multiple levels. Specifically, both enantiomers of 2HG comparably stimulate anaerobic metabolism of glucose and inhibit the uptake of several essential amino acids from the culture media. In this respect, both 2HG enantiomers decreased the catabolism capability of cells to incorporate the leucine-derived carbon atoms into their metabolism and to generate the ketone bodies. These results provide evidence that both enantiomers of 2HG have the potential to influence the metabolic and molecular aspects of human cells. Furthermore, we may propose that increased levels of 2HG enantiomers in the brain parenchyma may alter brain metabolism features, potentially contributing to the etiology of neurological symptoms in patients.

Prolactin upregulates amino acids uptake in dairy cow mammary epithelial cells via LAT1

Prolactin upregulates amino acids uptake in dairy cow mammary epithelial cells via LAT1

Identification of Anticancer Enzymes and Biomarkers for Hepatocellular Carcinoma through Constraint-Based Modeling.

Hepatocellular carcinoma (HCC) results in the abnormal regulation of cellular metabolic pathways. Constraint-based modeling approaches can be utilized to dissect metabolic reprogramming, enabling the identification of biomarkers and anticancer targets for diagnosis and treatment. In this study, two genome-scale metabolic models (GSMMs) were reconstructed by employing RNA sequencing expression patterns of hepatocellular carcinoma (HCC) and their healthy counterparts. An anticancer target discovery (ACTD) framework was integrated with the two models to identify HCC targets for anticancer treatment. The ACTD framework encompassed four fuzzy objectives to assess both the suppression of cancer cell growth and the minimization of side effects during treatment. The composition of a nutrient may significantly affect target identification. Within the ACTD framework, ten distinct nutrient media were utilized to assess nutrient uptake for identifying potential anticancer enzymes. The findings revealed the successful identification of target enzymes within the cholesterol biosynthetic pathway using a cholesterol-free cell culture medium. Conversely, target enzymes in the cholesterol biosynthetic pathway were not identified when the nutrient uptake included a cholesterol component. Moreover, the enzymes PGS1 and CRL1 were detected in all ten nutrient media. Additionally, the ACTD framework comprises dual-group representations of target combinations, pairing a single-target enzyme with an additional nutrient uptake reaction. Additionally, the enzymes PGS1 and CRL1 were identified across the ten-nutrient media. Furthermore, the ACTD framework encompasses two-group representations of target combinations involving the pairing of a single-target enzyme with an additional nutrient uptake reaction. Computational analysis unveiled that cell viability for all dual-target combinations exceeded that of their respective single-target enzymes. Consequently, integrating a target enzyme while adjusting an additional exchange reaction could efficiently mitigate cell proliferation rates and ATP production in the treated cancer cells. Nevertheless, most dual-target combinations led to lower side effects in contrast to their single-target counterparts. Additionally, differential expression of metabolites between cancer cells and their healthy counterparts were assessed via parsimonious flux variability analysis employing the GSMMs to pinpoint potential biomarkers. The variabilities of the fluxes and metabolite flow rates in cancer and healthy cells were classified into seven categories. Accordingly, two secretions and thirteen uptakes (including eight essential amino acids and two conditionally essential amino acids) were identified as potential biomarkers. The findings of this study indicated that cancer cells exhibit a higher uptake of amino acids compared with their healthy counterparts.

Nitrogen Assimilation and Nitrification in Surface Waters of the Amazon and Pará Estuaries

AbstractUnder increasing anthropogenic impacts, the hydrology, weathering, turbidity, and biogeochemistry of the Amazon and Pará river catchments are changing, potentially affecting the concentration and speciation of nitrogen (N) entering the estuary, as well as the subsequent primary productivity. Primary production and several N‐cycling rates have been studied in the northern river plume, and new studies focusing on specific processes in the river mouth emerged. Yet, a general overview is still lacking. For the first time, we provide an overview of primary production along with nitrification, ammonium (NH4+), nitrate (NO3−), and amino acid uptake rates in the Amazon and Pará river mouths which exhibit different degrees of catchment land use. Our results indicate that the Amazon supplies tenfold more NH4+ and NO3−, and its nitrification rates are four times higher than in the Pará River. The stable isotope composition of ambient water and NO3− confirmed that nitrification is a dominant pathway, with fluxes reaching 8.5 × 108 mol N d−1 in the Amazon. Nutrients are transported to regions with reduced turbidity and little nitrification, facilitating NH4+ (0.2–2.4 × 108 mol N d−1) and carbon uptake (4.6 and 14.0 × 108 mol C d−1 in the Pará and the Amazon, respectively) by phytoplankton. The lower N‐cycling rates measured in the Pará relative to the Amazon River mouth likely originate from differences in river discharge and watershed characteristics.

High Overexpression of SiAAP9 Leads to Growth Inhibition and Protein Ectopic Localization in Transgenic Arabidopsis.

Amino acid permeases (AAPs) transporters are crucial for the long-distance transport of amino acids in plants, from source to sink. While Arabidopsis and rice have been extensively studied, research on foxtail millet is limited. This study identified two transcripts of SiAAP9, both of which were induced by NO3- and showed similar expression patterns. The overexpression of SiAAP9L and SiAAP9S in Arabidopsis inhibited plant growth and seed size, although SiAAP9 was found to transport more amino acids into seeds. Furthermore, SiAAP9-OX transgenic Arabidopsis showed increased tolerance to high concentrations of glutamate (Glu) and histidine (His). The high overexpression level of SiAAP9 suggested its protein was not only located on the plasma membrane but potentially on other organelles, as well. Interestingly, sequence deletion reduced SiAAP9's sensitivity to Brefeldin A (BFA), and SiAAP9 had ectopic localization on the endoplasmic reticulum (ER). Protoplast amino acid uptake experiments indicated that SiAAP9 enhanced Glu transport into foxtail millet cells. Overall, the two transcripts of SiAAP9 have similar functions, but SiAAP9L shows a higher colocalization with BFA compartments compared to SiAAP9S. Our research identifies a potential candidate gene for enhancing the nutritional quality of foxtail millet through breeding.

Changes in Amino Acids Profile and Uptake on Maize Seedlings Treated with Protein Hydrolysates and Humic Substances

Protein hydrolysates (PH) and humic substances (HS) are biostimulants that promote crop growth. Despite the widening of knowledge and insights provided by research activities over the past thirty years, there are still uncertainties concerning the possible direct absorption by plants of organic nitrogen as amino acids (AA) delivered as PH and the role of HS. This study aimed to assess the impact of PH treatment on the amino acid profile of maize seedlings and the influence of HS on amino acid uptake. Maize seedlings were treated with either PH from the yeast of the sugarcane fermentation process or a synthetic mixture of amino acids mimicking the PH composition using dual-labeled amino acids (13C, 15N glutamic acid) combined with commercially available humic substances. The amino acid profile was analyzed using HPLC, and plant tissues were examined for 15N using nuclear magnetic resonance (NMR) and mass spectrometry. The application of PH stimulated maize growth, with a more significant effect observed in the presence of humic substances. The treatments significantly altered the plants’ total amino acid content and composition profile. Maize seedlings actively uptake amino acids, representing 21% of the total nitrogen composition. The ascertained improvement of amino acid uptake stimulated by humic substances is associated with their effect on the differential expression of amino permease transporters and plasma membrane H+-ATPase. PH and amino acids enhance maize growth by significantly increasing amino acid uptake. The addition of humic substances further improved the biostimulant effects.

Chromium and Methionine Effects on Amino Acid Uptake, Gene Expression, and Protein Abundance in Bovine Satellite Cells

The objective of the study was to evaluate the effects of chromium (Cr) and methionine on myogenic gene expression, GLUT4 and insulin receptor (IR) synthesis, and amino acid uptake in differentiated bovine satellite cells. Cells were treated with 0, 1, or 10 μM of chromium acetate (CrAc) and 0, 1, or 10 mg/mL of methionine, with cells utilized for the amino acid assay receiving 0.1 μM instead of 1 μM of CrAc. The addition of 10 μM of Cr tended to downregulate AMPKα mRNA gene expression (P < 0.1). Myogenin was impacted by methionine and CrAc with high doses and low doses upregulating expression (P < 0.01). The ratio and protein levels of AMPKα to pAMPKα, GLUT4, and IR abundance were not affected (P > 0.1). Increase in time decreased concentrations of leucine, isoleucine, threonine, methionine, phenylalanine, glutamine, ornithine, lysine, and cysteine (P < 0.01) in conditioned media. Over time, alanine increased (P < 0.01). Methionine inclusion increased alanine, proline, glutamic acid, ornithine, and tryptophan concentrations, as well as decreased leucine, isoleucine, and cysteine (P < 0.01). CrAc only affected glutamine and tryptophan, which both increased with CrAc dose, whereas alanine decreased (P < 0.01). Methionine decreased over time within the 10 mg/mL group (P < 0.01). Tryptophan was generally unaffected but decreased in the 0 mg/mL group at 48 h. Cysteine decreased with a decreased dosage of CrAc at 24 h (P < 0.01). Lysine was generally unaffected by this interaction (P > 0.1), with the exception of the 0.1 μM treatment group at 24 h being higher (P < 0.01). CrAc affected the uptake of alanine, glutamine, and tryptophan (P < 0.01). Methionine inclusion increased alanine, proline, glutamic acid, ornithine, and tryptophan, as well as decreased leucine, isoleucine, and cystine (P < 0.01). Increased time saw a significant decrease in leucine, isoleucine, threonine, methionine, phenylalanine, glutamine, ornithine, lysine, and cystine (P < 0.01) and an increase in alanine (P < 0.01).

Blocking of amino acid transporter OsAAP7 promoted tillering and yield by determining basic and neutral amino acids accumulation in rice

BackgroundAmino acids are not only the main form of N in rice, but also are vital for its growth and development. These processes are facilitated by amino acid transporters within the plant. Despite their significance, only a few AAP amino acid transporters have been reported.ResultsIn this study, we observed that there were differences in the expression of amino acid transporter OsAAP7 among 521 wild cultivated rice varieties, and it directly negatively correlated with tillering and grain yield per plant. We revealed that OsAAP7 protein was localized to the endoplasmic reticulum and had absorption and transport affinity for amino acids such as phenylalanine (Phe), lysine (Lys), leucine (Leu), and arginine (Arg) using subcellular localization, yeast substrate testing, fluorescent amino acid uptake, and amino acid content determination. Further hydroponic studies showed that exogenous application of amino acids Phe, Lys and Arg inhibited the growth of axillary buds in the overexpression lines, and promoted the elongation of axillary buds in the mutant lines. Finally, RNA-seq analysis showed that the expression patterns of genes related to nitrogen, auxin and cytokinin pathways were changed in axillary buds of OsAAP7 transgenic plants.ConclusionsThis study revealed the gene function of OsAAP7, and found that blocking of amino acid transporter OsAAP7 with CRISPR/Cas9 technology promoted tillering and yield by determining basic and neutral amino acids accumulation in rice.

Improving aromatic higher alcohol acetates in wines by co-fermentation of Pichia kluyveri and Saccharomyces cerevisiae: growth interaction and amino acid competition.

Higher alcohol acetates (HAAs) are potent aroma-active esters that impart desirable fruity and floral aromas. However, the conversion of higher alcohol precursors into HAAs is extremely low in winemaking. To investigate the underlying yeast-yeast interaction on targeted improvement of aromatic HAAs, we evaluated fermentation activity, cell viability, amino acid consumption and HAA production when Pichia kluyveri and Saccharomyces cerevisiae were inoculated concurrently or sequentially. Pichia kluyveri PK-21 possessed the ability to survive and increased HAA level up to 5.2-fold in mixed fermentation. Such an increment may benefit from the efficient conversion of higher alcohol precursors into HAAs (>27-fold higher than S. cerevisiae). During mixed fermentation, the two yeasts exhibited crucial interactions regarding cell growth and amino acid competition. Saccharomyces cerevisiae dominated over the co-inoculated P. kluyveri by efficient uptake of amino acids and biomass production. However, this dominance decreased in sequential fermentation, where P. kluyveri growth increased due to the consumption of preferred amino acids prior to S. cerevisiae. Pearson correlation analysis indicated that phenylalanine and aspartic acid may act as positive amino acids in boosting P. kluyveri growth and HAA production. Laboratory-scale winemaking validated the fermentation performance of P. kluyveri in sequential inoculum, resulting in a balanced aroma profile with enhanced floral and tropical fruity characteristics in the final wines. This study proposes a microbial, non-genetically engineered approach for targeted increase of HAA production in winemaking and the findings provide new insights into yeast-yeast interactions. © 2024 Society of Chemical Industry.

BNCT pancreatic cancer treatment strategy with glucose-conjugated boron drug

Multidisciplinary therapy centered on radical surgery for resectable pancreatic cancer is expected to prolong prognosis, but relies on CA19-9 biomarker levels to determine treatment strategy. Boron neutron capture therapy (BNCT) is a chemoradiotherapy using tumor hyperaccumulator boron drugs and neutron irradiation. The purpose of this study is to investigate novel boron drug agents for BNCT for pancreatic cancer. Bioinformatics was used to evaluate the uptake of current boron amino acid (BPA) drugs for BNCT into pancreatic cancer. The expression of the amino acid transporter LAT1, a BPA uptake transporter, was low in pancreatic cancer and even lower in high CA19-9 pancreatic cancer. In contrast, the glucose transporter was high in high CA19-9 pancreatic cancers and inversely correlated with LAT1 expression. Considering the low EPR effect in pancreatic cancer, we synthesized a small molecule Glucose-BSH, which is boron BSH bound to glucose, and confirmed its specific uptake in pancreatic cancer. uptake of Glucose-BSH was confirmed in an environment compatible with the tumor microenvironment. The therapeutic efficacy and safety of Glucose-BSH by therapeutic neutron irradiation were confirmed with BNCT. We report Glucose-BSH boron drug discovery study of a Precision Medicine BNCT with application to high CA19-9 pancreatic cancer.

Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and β-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

The complete assembly of human LAT1-4F2hc complex provides insights into its regulation, function and localisation

The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino acids, hormones and drugs. Its dysfunction is associated with many cancers and immune/neurological disorders. Here, we apply native mass spectrometry (MS)-based approaches to provide evidence of super-dimer formation (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we discover four endogenous phosphatidylethanolamine (PE) molecules at the interface and C-terminus of both LAT1 subunits. We find that interfacial PE binding is regulated by 4F2hc-R183 and is critical for regulation of palmitoylation on neighbouring LAT1-C187. Combining native MS with mass photometry (MP), we reveal that super-dimerization is sensitive to pH, and modulated by complex N-glycans on the 4F2hc subunit. We further validate the dynamic assemblies of LAT1-4F2hc on plasma membrane and in the lysosome. Together our results link PTM and lipid binding with regulation and localisation of the LAT1-4F2hc super-dimer.

IGF-1 infusion increases growth in fetal sheep when euinsulinemia is maintained.

Insulin-like growth factor 1 (IGF-1) is a critical fetal anabolic hormone. IGF-1 infusion to the normally growing sheep fetus increases the weight of some organs but does not consistently increase body weight. However, IGF-1 infusion profoundly decreases fetal plasma insulin concentrations, which may limit fetal growth potential. In this study, normally growing late-gestation fetal sheep received an intravenous infusion of either: IGF-1 (IGF), IGF-1 with insulin and dextrose to maintain fetal euinsulinemia and euglycemia (IGF+INS), or vehicle control (CON) for 1 week. The fetus underwent a metabolic study immediately prior to infusion start and after 1 week of the infusion to measure uterine and umbilical uptake rates of nutrients and oxygen. IGF+INS fetuses were 23% heavier than CON (P = 0.0081) and had heavier heart, liver, and adrenal glands than IGF and CON (P < 0.01). By design, final fetal insulin concentrations in IGF were 62% and 65% lower than IGF+INS and CON, respectively. Final glucose concentrations were similar in all groups. IGF+INS had lower final oxygen content than IGF and CON (P < 0.0001) and lower final amino acid concentrations than CON (P = 0.0002). Final umbilical oxygen uptake was higher in IGF+INS compared to IGF and CON (P < 0.05). Final umbilical uptake of several essential amino acids was higher in IGF+INS compared to CON (P < 0.05). In summary, maintaining euinsulinemia and euglycemia during fetal IGF-1 infusion is necessary to maximally support body growth. We speculate that IGF-1 and insulin stimulate placental nutrient transport to support fetal growth.

Culture Shock! Investigating the Importance of Physiological Tumour-Like Conditions on Cultured Cancer Cells

The tumour microenvironment (TME) is complex and dynamic, characterized by poor vascularization, limited nutrient availability, hypoxia, and an acidic pH. This environment plays a critical role in cancer progression, driving hallmark changes in cancer cell metabolism, morphology, proliferation, and motility. Although mammalian cell culture is a foundational tool for investigating cancer cell biology in vitro, standard cell culture practices fall short of replicating the in vivo conditions of tumours. Recently, ‘physiologic’ cell culture media that closely resemble human plasma (e.g., Plasmax, HPLM) have been developed, along with more frequent adoption of physiological oxygen conditions (2-8% O2) rather than standard atmosphere (~18% O2). Although an improvement, these conditions are not representative of all in vivo cellular environments, particularly that of the TME. Considering the tight regulatory capacity of cancer cells to sense and respond to changes in metabolic availability, we hypothesized that alterations in nutrient and oxygen availability, even if within the physiological range, may subtly rewire the metabolism in cultured cancer cells. This may necessitate further refinement of cell-specific physiologic culture conditions to better maintain the in vivo biology of cells. In this study, we directly address this hypothesis by describing the development of a tumour microenvironment-like medium (TMEM) based on metabolomic profiling of murine pancreatic ductal adenocarcinoma tumour interstitial fluid from relevant literature. We found that murine pancreatic ductal adenocarcinoma (KPCY) cells cultured under TME-like conditions (TMEM, pH 7.0, 1.5% O2) exhibited increased glucose uptake and lactate production rates compared to those cultured under healthy plasma-like conditions (Mouse Plasma-like Medium, pH 7.4, 5% O2), despite lower initial glucose concentrations in TMEM (2.3 mM vs. 4.4 mM in Mouse Plasma-like Medium). Moreover, cell proliferation rates were lower in TMEM, suggesting an overall reduction in ATP consumption fueling cell division. Seahorse Extracellular Flux Analysis is being performed to determine effects on cellular bioenergetics. Metabolomic amino acid uptake using LCMS is also being performed to further assess the effect of TMEM on nutrient utilization. Using RNA-sequencing, we showed that culture in TMEM increased expression of genes associated with angiogenesis, amino acids biosynthesis, cell migration, and endothelial-to-mesenchymal transition. Migration assays confirmed increased motility in KPCY cells cultured in TMEM. Taken together, these results demonstrate that growth in a medium designed to simulate the TME alters KPCY cell biology in ways that are relevant to their pathology, which highlights the significance of maintaining TME-like conditions in culture. This research was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant from JAS and a NSERC Canadian Graduate Student (Masters) Grant awarded to GLG. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Increased hepatic glucose production with lower oxidative metabolism in the growth-restricted fetus.

Fetal growth restriction (FGR) is accompanied by early activation of hepatic glucose production (HGP), a hallmark of type 2 diabetes (T2D). Here, we used fetal hepatic catheterization to directly measure HGP and substrate flux in a sheep FGR model. We hypothesized that FGR fetuses would have increased hepatic lactate and amino acid uptake to support increased HGP. Indeed, FGR fetuses compared with normal (CON) fetuses had increased HGP and activation of gluconeogenic genes. Unexpectedly, hepatic pyruvate output was increased, while hepatic lactate and gluconeogenic amino acid uptake rates were decreased in FGR liver. Hepatic oxygen consumption and total substrate uptake rates were lower. In FGR liver tissue, metabolite abundance, 13C-metabolite labeling, enzymatic activity, and gene expression supported decreased pyruvate oxidation and increased lactate production. Isolated hepatocytes from FGR fetuses had greater intrinsic capacity for lactate-fueled glucose production. FGR livers also had lower energy (ATP) and redox state (NADH/NAD+ ratio). Thus, reduced hepatic oxidative metabolism may make carbons available for increased HGP, but also produces nutrient and energetic stress in FGR liver. Intrinsic programming of these pathways regulating HGP in the FGR fetus may underlie increased HGP and T2D risk postnatally.

Electrogenic transport of amino acids and glucose differs between jejunum and ileum of female and castrated male finishing pigs

It has been shown that the jejunum represents the most important site for the nutrient’s absorption in several species. However, in pigs, this information seems to be controversial and limited information are available about differences in intestinal physiology between female and castrated male pigs. The trans-epithelial electrical resistance (TEER) and the active uptake of L-glutamate (L-Glu), L-arginine (L-Arg), L-methionine (L-Met) and D-glucose (D-Glu) in the jejunum and ileum of female (n = 5) and castrated male (n = 7) pigs fed a protein-restricted grower and finisher diet (128 and 112 g of crude protein/kg dry matter) were investigated. The intestine segments were collected at slaughter and mounted in Ussing chambers. Results were further investigated by protein expression analysis of the D-Glu transporter Sodium-dependent Glucose Transporter 1 (SGLT1) and using serum creatinine, non-esterified fatty acids (NEFAs) and serum glucose concentrations measurements as indicators of muscle mass deposition and metabolic status of the animals. A linear mixed-effects regression (Lme4) model was used for data analysis. Independent of sex, the uptake of both L-Met and D-Glu was higher (p < 0.001) in the ileum than in the jejunum (2.1- and 3.6-fold increases, respectively). The L-Arg uptake was higher (p = 0.001) in females compared to castrates (1.9-fold increase). No significant differences were observed between the segments in the SGLT1 protein expression, regardless of sex. Serum measurements were also not significantly different between the female and castrates. This study suggests that the ileum has an important role in the active uptake of amino acids and D-Glu uptake and differences exist between female and castrated finishing pigs.

Intestinal carbapenem-resistant Klebsiella pneumoniae undergoes complex transcriptional reprogramming following immune activation

ABSTRACT Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is a significant threat to public health worldwide. The primary reservoir for CR-Kp is the intestinal tract. There, the bacterium is usually present at low density but can bloom following antibiotic treatment, mostly in hospital settings. The impact of disturbances in the intestinal environment on the fitness, survival, expansion, and drug susceptibility of this pathogen is not well-understood, yet it may be relevant to devise strategies to tackle CR-Kp colonization and infection. Here, we adopted an in vivo model to examine the transcriptional adaptation of a CR-Kp clinical isolate to immune activation in the intestine. We report that as early as 6 hours following host treatment with anti-CD3 antibody, CR-Kp underwent rapid transcriptional changes including downregulation of genes involved in sugar utilization and amino acid biosynthesis and upregulation of genes involved in amino acid uptake and catabolism, antibiotic resistance, and stress response. In agreement with these findings, treatment increased the concentration of oxidative species and amino acids in the mouse intestine. Genes encoding for proteins containing the domain of unknown function (DUF) 1471 were strongly upregulated, however their deletion did not impair CR-Kp fitness in vivo upon immune activation. Transcription factor enrichment analysis identified the global regulator cAMP-Receptor Protein, CRP, as a potential orchestrator of the observed transcriptional signature. In keeping with the recognized role of CRP in regulating utilization of alternative carbon sources, crp deletion in CR-Kp resulted in strongly impaired gut colonization, although this effect was not amplified by immune activation. Thus, following intestinal colonization, which occurs in a CRP-dependent manner, CR-Kp can rapidly respond to immune cues by implementing a well-defined and complex transcriptional program whose direct relevance toward bacterial fitness warrants further investigation. Additional analyses utilizing this model may identify key factors to tackle CR-Kp colonization of the intestine.

Extracellular domains of CARs reprogramme T cell metabolism without antigen stimulation.

Metabolism is an indispensable part of T cell proliferation, activation and exhaustion, yet the metabolism of chimeric antigen receptor (CAR)-T cells remains incompletely understood. CARs are composed of extracellular domains-often single-chain variable fragments (scFvs)-that determine ligand specificity and intracellular domains that trigger signalling following antigen binding. Here, we show that CARs differing only in the scFv variously reprogramme T cell metabolism. Even without exposure to antigens, some CARs increase proliferation and nutrient uptake in T cells. Using stable isotope tracers and mass spectrometry, we observed basal metabolic fluxes through glycolysis doubling and amino acid uptake overtaking anaplerosis in CAR-T cells harbouring a rituximab scFv, unlike other similar anti-CD20 scFvs. Disparate rituximab and 14G2a-based anti-GD2 CAR-T cells are similarly hypermetabolic and channel excess nutrients to nitrogen overflow metabolism. Modest overflow metabolism of CAR-T cells and metabolic compatibility between cancer cells and CAR-T cells are identified as features of efficacious CAR-T cell therapy.

TNF-α signals through ITK-Akt-mTOR to drive CD4+ T cell metabolic reprogramming, which is dysregulated in rheumatoid arthritis.

Upon activation, T cells undergo metabolic reprogramming to meet the bioenergetic demands of clonal expansion and effector function. Because dysregulated T cell cytokine production and metabolic phenotypes coexist in chronic inflammatory disease, including rheumatoid arthritis (RA), we investigated whether inflammatory cytokines released by differentiating T cells amplified their metabolic changes. We found that tumor necrosis factor-α (TNF-α) released by human naïve CD4+ T cells upon activation stimulated the expression of a metabolic transcriptome and increased glycolysis, amino acid uptake, mitochondrial oxidation of glutamine, and mitochondrial biogenesis. The effects of TNF-α were mediated by activation of Akt-mTOR signaling by the kinase ITK and did not require the NF-κB pathway. TNF-α stimulated the differentiation of naïve cells into proinflammatory T helper 1 (TH1) and TH17 cells, but not that of regulatory T cells. CD4+ T cells from patients with RA showed increased TNF-α production and consequent Akt phosphorylation upon activation. These cells also exhibited increased mitochondrial mass, particularly within proinflammatory T cell subsets implicated in disease. Together, these findings suggest that T cell-derived TNF-α drives their metabolic reprogramming by promoting signaling through ITK, Akt, and mTOR, which is dysregulated in autoinflammatory disease.

Amino acid preference and fermentation performance of Pichia kluyveri strains in a synthetic wort

Pichia kluyveri is a maltose-negative yeast applied for non- and low-alcoholic beer (NABLAB) production due to its fruity aroma attributes, however, related literature is still limited. Thus, this study aims to investigate basic fermentation parameters, such as growth, sugar consumption, and production of primary fermentation metabolites, but also the amino acid preference of three different P. kluyveri strains in an agitated model brewers’ wort substrate. Furthermore, the effect of agitation on these parameters was studied for one of the strains. The three strains assessed, PKCH_01, PKCH_02 and PKCH_03, shared a glucophilic nature, a cell growth from 4E+07 to 6E+07 E+07 CFU/mL, similar amino acid preference, and equivalent ethanol yield factor (Yse) from 0.45 to 0.5 g ethanol/g glucose. In addition, agitation increased the cell growth yield factor by 78% and favored the uptake of amino acids, minimizing their release to the medium. Interestingly, a strong preference for methionine (above 80% consumption) by P. kluyveri in a beer-related medium was reported for the first time, whereas glutamine and glutamate were within the low-preference group (below 15% consumption). This study provides a better understanding about fermentation and physiological attributes of P. kluyveri, thereby revealing its potential for brewing applications.