One-carbon unit supplementation fuels purine synthesis in tumor-infiltrating T cells and augments checkpoint blockade.

Nucleotides perform important metabolic functions, carrying energy and feeding nucleic acid synthesis. Here, we use isotope tracing-mass spectrometry to quantitate contributions to purine nucleotides from salvage versus de novo synthesis. We further explore the impact of augmenting a key precursor for purine synthesis, one-carbon (1C) units. We show that tumors and tumor-infiltrating T cells (relative to splenic or lymph node T cells) synthesize purines de novo. Shortage of 1C units for T cell purine synthesis is accordingly a potential bottleneck for anti-tumor immunity. Supplementing 1C units by infusing formate drives formate assimilation into purines in tumor-infiltrating T cells. Orally administered methanol functions as a formate pro-drug, with deuteration enabling kinetic control of formate production. Safe doses of methanol raise formate levels and augment anti-PD-1 checkpoint blockade in MC38 tumors, tripling durable regressions. Thus, 1C deficiency can gate antitumor immunity and this metabolic checkpoint can be overcome with pharmacological 1C supplementation.

A folate inhibitor exploits metabolic differences in Pseudomonas aeruginosa for narrow-spectrum targeting.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections for which the development of antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks enzymes required to scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa is to disrupt thymidine synthesis while providing exogenous thymine. However, known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa.Here we characterize fluorofolin, a dihydrofolate reductase (DHFR) inhibitor derived from Irresistin-16, that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. Fluorofolin is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics. Metabolomics and in vitro assays using purified folA confirm that fluorofolin inhibits P. aeruginosa DHFR. Importantly, in the presence of thymine supplementation, fluorofolin activity is selective for P. aeruginosa. Resistance to fluorofolin can emerge through overexpression of the efflux pumps MexCD-OprJ and MexEF-OprN, but these mutants also decrease pathogenesis. Our findings demonstrate how understanding species-specific genetic differences can enable selective targeting of important pathogens while revealing trade-offs between resistance and pathogenesis.

Selected Ion Monitoring for Orbitrap-Based Metabolomics.

Orbitrap mass spectrometry in full scan mode enables the simultaneous detection of hundreds of metabolites and their isotope-labeled forms. Yet, sensitivity remains limiting for many metabolites, including low-concentration species, poor ionizers, and low-fractional-abundance isotope-labeled forms in isotope-tracing studies. Here, we explore selected ion monitoring (SIM) as a means of sensitivity enhancement. The analytes of interest are enriched in the orbitrap analyzer by using the quadrupole as a mass filter to select particular ions. In tissue extracts, SIM significantly enhances the detection of ions of low intensity, as indicated by improved signal-to-noise (S/N) ratios and measurement precision. In addition, SIM improves the accuracy of isotope-ratio measurements. SIM, however, must be deployed with care, as excessive accumulation in the orbitrap of similar m/z ions can lead, via space-charge effects, to decreased performance (signal loss, mass shift, and ion coalescence). Ion accumulation can be controlled by adjusting settings including injection time and target ion quantity. Overall, we suggest using a full scan to ensure broad metabolic coverage, in tandem with SIM, for the accurate quantitation of targeted low-intensity ions, and provide methods deploying this approach to enhance metabolome coverage.

TREM1 disrupts myeloid bioenergetics and cognitive function in aging and Alzheimer disease mouse models.

Human genetics implicate defective myeloid responses in the development of late-onset Alzheimer disease. A decline in peripheral and brain myeloid metabolism, triggering maladaptive immune responses, is a feature of aging. The role of TREM1, a pro-inflammatory factor, in neurodegenerative diseases is unclear. Here we show that Trem1 deficiency prevents age-dependent changes in myeloid metabolism, inflammation and hippocampal memory function in mice. Trem1 deficiency rescues age-associated declines in ribose 5-phosphate. In vitro, Trem1-deficient microglia are resistant to amyloid-ß42 oligomer-induced bioenergetic changes, suggesting that amyloid-ß42 oligomer stimulation disrupts homeostatic microglial metabolism and immune function via TREM1. In the 5XFAD mouse model, Trem1 haploinsufficiency prevents spatial memory loss, preserves homeostatic microglial morphology, and reduces neuritic dystrophy and changes in the disease-associated microglial transcriptomic signature. In aging APPSwe mice, Trem1 deficiency prevents hippocampal memory decline while restoring synaptic mitochondrial function and cerebral glucose uptake. In postmortem Alzheimer disease brain, TREM1 colocalizes with Iba1+ cells around amyloid plaques and its expression is associated with Alzheimer disease clinical and neuropathological severity. Our results suggest that TREM1 promotes cognitive decline in aging and in the context of amyloid pathology.

Off-target depletion of plasma tryptophan by allosteric inhibitors of BCKDK.

The activation of branched chain amino acid (BCAA) catabolism has garnered interest as a potential therapeutic approach to improve insulin sensitivity, enhance recovery from heart failure, and blunt tumor growth. Evidence for this interest relies in part on BT2, a small molecule that promotes BCAA oxidation and is protective in mouse models of these pathologies. BT2 and other analogs allosterically inhibit branched chain ketoacid dehydrogenase kinase (BCKDK) to promote BCAA oxidation, which is presumed to underlie the salutary effects of BT2. Potential "off-target" effects of BT2 have not been considered, however. We therefore tested for metabolic off-target effects of BT2 in Bckdk-/- animals. As expected, BT2 failed to activate BCAA oxidation in these animals. Surprisingly, however, BT2 strongly reduced plasma tryptophan levels and promoted catabolism of tryptophan to kynurenine in both control and Bckdk-/- mice. Mechanistic studies revealed that none of the principal tryptophan catabolic or kynurenine-producing/consuming enzymes (TDO, IDO1, IDO2, or KATs) were required for BT2-mediated lowering of plasma tryptophan. Instead, using equilibrium dialysis assays and mice lacking albumin, we show that BT2 avidly binds plasma albumin and displaces tryptophan, releasing it for catabolism. These data confirm that BT2 activates BCAA oxidation via inhibition of BCKDK but also reveal a robust off-target effect on tryptophan metabolism via displacement from serum albumin. The data highlight a potential confounding effect for pharmaceutical compounds that compete for binding with albumin-bound tryptophan.

Mitochondrial ATP generation is more proteome efficient than glycolysis.

Metabolic efficiency profoundly influences organismal fitness. Nonphotosynthetic organisms, from yeast to mammals, derive usable energy primarily through glycolysis and respiration. Although respiration is more energy efficient, some cells favor glycolysis even when oxygen is available (aerobic glycolysis, Warburg effect). A leading explanation is that glycolysis is more efficient in terms of ATP production per unit mass of protein (that is, faster). Through quantitative flux analysis and proteomics, we find, however, that mitochondrial respiration is actually more proteome efficient than aerobic glycolysis. This is shown across yeast strains, T cells, cancer cells, and tissues and tumors in vivo. Instead of aerobic glycolysis being valuable for fast ATP production, it correlates with high glycolytic protein expression, which promotes hypoxic growth. Aerobic glycolytic yeasts do not excel at aerobic growth but outgrow respiratory cells during oxygen limitation. We accordingly propose that aerobic glycolysis emerges from cells maintaining a proteome conducive to both aerobic and hypoxic growth.

The role of URO17® in diagnosis and follow up of bladder cancer patients.

OBJECTIVE: to evaluate the role of urinary URO17® biomarker in the detection of urothelial tumors in haematuria patients and the detection of recurrence in non-muscle invasive bladder urothelial tumors. MATERIALS AND METHODS: Our study was formed of two cohorts of patients, group I represents patients presenting with haematuria (n = 98), while group II represents patients with known non-muscle invasive bladder cancers on their scheduled follow up cystoscopic investigation (n = 51). For both groups, patients were asked to provide urine samples before cystoscopy, either primary as part of the haematuria investigation or as a scheduled follow-up. Urine samples were sent anonymously for standard urine cytology and URO17® biomarker immunostaining. Results were compared to cystoscopic findings using Chi-square analysis and Fisher's exact test (P < 0.05). RESULTS: Group I was formed of 98 patients, with an average age of 60 years. URO17® showed 100% sensitivity and 96.15% specificity with a negative predictive value (NPV) of 100 and a positive predictive value (PPV) of 95.83. The results showed statistical significance with P value < 0.001. Group II was formed of 51 patients, with an average age of 75 years. URO17® was shown to have a sensitivity of 85.71% and NPV of 95.45. Eleven patients of group II were on scheduled BacillusCalmette-Guerin (BCG) and another 5 received Mitomycin C (MMC). The overall results of both groups combined (n = 149) showed statistical significance between flexible cystoscopy results and the results of urinary URO17® and urine cytology. CONCLUSION: URO17® has a potential to be a reliable test for diagnosis and follow up of urothelial cancer patients and a screening tool adjunct to flexible cystoscopy. TRIAL REGISTRATION: Not applicable as the current study is not a clinical trial, as per according to the National Institutes of Health, "studies that involve a comparison of methods and that do not evaluate the effect of the interventions on the participant do not meet the NIH clinical trial definition."

Systematic review of the ophthalmic complications of robotic-assisted laparoscopic prostatectomy.

This study aims to review ophthalmic injuries sustained during of robotic-assisted laparoscopic prostatectomy (RALP). A search of Medline, Embase, Cochrane and grey literature was performed using methods registered a priori. Eligible studies were published 01/01/2010-01/05/2023 in English and reported ophthalmic complications in cohorts of > 100 men undergoing RALP. The primary outcome was injury incidence. Secondary outcomes were type and permanency of ophthalmic complications, treatments, risk factors and preventative measures. Nine eligible studies were identified, representing 100,872 men. Six studies reported rates of corneal abrasion and were adequately homogenous for meta-analysis, with a weighted pooled rate of 5 injuries per 1000 procedures (95% confidence interval 3-7). Three studies each reported different outcomes of xerophthalmia, retinal vascular occlusion, and ophthalmic complications unspecified in 8, 5 and 2 men per 1000 procedures respectively. Amongst identified studies, there were no reports of permanent ophthalmic complications. Injury management was poorly reported. No significant risk factors were reported, while one study found African-American ethnicity protective against corneal abrasion (0.4 vs. 3.9 per 1000). Variables proposed (but not proven) to increase risk for corneal abrasion included steep Trendelenburg position, high pneumoperitoneum pressure, prolonged operative time and surgical inexperience. Compared with standard of care, occlusive eyelid dressings (23 vs. 0 per 1000) and foam goggles (20 vs. 1.3 per 1000) were found to reduce rates of corneal abrasion. RALP carries low rates of ophthalmic injury. Urologists should counsel the patient regarding this potential complication and pro-actively implement preventative strategies.

Reprogramming neuroblastoma by diet-enhanced polyamine depletion.

Neuroblastoma is a highly lethal childhood tumor derived from differentiation-arrested neural crest cells1,2. Like all cancers, its growth is fueled by metabolites obtained from either circulation or local biosynthesis3,4. Neuroblastomas depend on local polyamine biosynthesis, with the inhibitor difluoromethylornithine showing clinical activity5. Here we show that such inhibition can be augmented by dietary restriction of upstream amino acid substrates, leading to disruption of oncogenic protein translation, tumor differentiation, and profound survival gains in the TH-MYCN mouse model. Specifically, an arginine/proline-free diet decreases the polyamine precursor ornithine and augments tumor polyamine depletion by difluoromethylornithine. This polyamine depletion causes ribosome stalling, unexpectedly specifically at adenosine-ending codons. Such codons are selectively enriched in cell cycle genes and low in neuronal differentiation genes. Thus, impaired translation of these codons, induced by the diet-drug combination, favors a pro-differentiation proteome. These results suggest that the genes of specific cellular programs have evolved hallmark codon usage preferences that enable coherent translational rewiring in response to metabolic stresses, and that this process can be targeted to activate differentiation of pediatric cancers.

Glycine homeostasis requires reverse SHMT flux.

The folate-dependent enzyme serine hydroxymethyltransferase (SHMT) reversibly converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Such one-carbon unit production plays a critical role in development, the immune system, and cancer. Using rodent models, here we show that the whole-body SHMT flux acts to net consume rather than produce glycine. Pharmacological inhibition of whole-body SHMT1/2 and genetic knockout of liver SHMT2 elevated circulating glycine levels up to eight-fold. Stable-isotope tracing revealed that the liver converts glycine to serine, which is then converted by serine dehydratase into pyruvate and burned in the tricarboxylic acid cycle. In response to diets deficient in serine and glycine, de novo biosynthetic flux was unaltered, but SHMT2- and serine-dehydratase-mediated catabolic flux was lower. Thus, glucose-derived serine synthesis is largely insensitive to systemic demand. Instead, circulating serine and glycine homeostasis is maintained through variable consumption, with liver SHMT2 a major glycine-consuming enzyme.

mTORC1 controls murine postprandial hepatic glycogen synthesis via Ppp1r3b.

In response to a meal, insulin drives hepatic glycogen synthesis to help regulate systemic glucose homeostasis. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-established insulin target and contributes to the postprandial control of liver lipid metabolism, autophagy, and protein synthesis. However, its role in hepatic glucose metabolism is less understood. Here, we used metabolomics, isotope tracing, and mouse genetics to define a role for liver mTORC1 signaling in the control of postprandial glycolytic intermediates and glycogen deposition. We show that mTORC1 is required for glycogen synthase activity and glycogenesis. Mechanistically, hepatic mTORC1 activity promotes the feeding-dependent induction of Ppp1r3b, a gene encoding a phosphatase important for glycogen synthase activity whose polymorphisms are linked to human diabetes. Reexpression of Ppp1r3b in livers lacking mTORC1 signaling enhances glycogen synthase activity and restores postprandial glycogen content. mTORC1-dependent transcriptional control of Ppp1r3b is facilitated by FOXO1, a well characterized transcriptional regulator involved in the hepatic response to nutrient intake. Collectively, we identify a role for mTORC1 signaling in the transcriptional regulation of Ppp1r3b and the subsequent induction of postprandial hepatic glycogen synthesis.

Systematic identification and characterization of genes in the regulation and biogenesis of photosynthetic machinery.

Photosynthesis is central to food production and the Earth's biogeochemistry, yet the molecular basis for its regulation remains poorly understood. Here, using high-throughput genetics in the model eukaryotic alga Chlamydomonas reinhardtii, we identify with high confidence (false discovery rate [FDR] < 0.11) 70 poorly characterized genes required for photosynthesis. We then enable the functional characterization of these genes by providing a resource of proteomes of mutant strains, each lacking one of these genes. The data allow assignment of 34 genes to the biogenesis or regulation of one or more specific photosynthetic complexes. Further analysis uncovers biogenesis/regulatory roles for at least seven proteins, including five photosystem I mRNA maturation factors, the chloroplast translation factor MTF1, and the master regulator PMR1, which regulates chloroplast genes via nuclear-expressed factors. Our work provides a rich resource identifying regulatory and functional genes and placing them into pathways, thereby opening the door to a system-level understanding of photosynthesis.

Metabolic pathway analysis using stable isotopes in patients with cancer.

Metabolic reprogramming is central to malignant transformation and cancer cell growth. How tumours use nutrients and the relative rates of reprogrammed pathways are areas of intense investigation. Tumour metabolism is determined by a complex and incompletely defined combination of factors intrinsic and extrinsic to cancer cells. This complexity increases the value of assessing cancer metabolism in disease-relevant microenvironments, including in patients with cancer. Stable-isotope tracing is an informative, versatile method for probing tumour metabolism in vivo. It has been used extensively in preclinical models of cancer and, with increasing frequency, in patients with cancer. In this Review, we describe approaches for using in vivo isotope tracing to define fuel preferences and pathway engagement in tumours, along with some of the principles that have emerged from this work. Stable-isotope infusions reported so far have revealed that in humans, tumours use a diverse set of nutrients to supply central metabolic pathways, including the tricarboxylic acid cycle and amino acid synthesis. Emerging data suggest that some activities detected by stable-isotope tracing correlate with poor clinical outcomes and may drive cancer progression. We also discuss current challenges in isotope tracing, including comparisons of in vivo and in vitro models, and opportunities for future discovery in tumour metabolism.

One-carbon unit supplementation fuels tumor-infiltrating T cells and augments checkpoint blockade.

Nucleotides perform important metabolic functions, carrying energy and feeding nucleic acid synthesis. Here, we use isotope tracing-mass spectrometry to quantitate the contributions to purine nucleotides of salvage versus de novo synthesis. We further explore the impact of augmenting a key precursor for purine synthesis, one-carbon (1C) units. We show that tumors and tumor-infiltrating T cells (relative to splenic T cells) synthesize purines de novo. Purine synthesis requires two 1C units, which come from serine catabolism and circulating formate. Shortage of 1C units is a potential bottleneck for anti-tumor immunity. Elevating circulating formate drives its usage by tumor-infiltrating T cells. Orally administered methanol functions as a formate pro-drug, with deuteration enabling control of formate-production kinetics. In MC38 tumors, safe doses of methanol raise formate levels and augment anti-PD-1 checkpoint blockade, tripling durable regressions. Thus, 1C deficiency can gate antitumor immunity and this metabolic checkpoint can be overcome with pharmacological 1C supplementation.

Activation of the integrated stress response rewires cardiac metabolism in Barth syndrome.

Barth Syndrome (BTHS) is an inherited cardiomyopathy caused by defects in the mitochondrial transacylase TAFAZZIN (Taz), required for the synthesis of the phospholipid cardiolipin. BTHS is characterized by heart failure, increased propensity for arrhythmias and a blunted inotropic reserve. Defects in Ca2+-induced Krebs cycle activation contribute to these functional defects, but despite oxidation of pyridine nucleotides, no oxidative stress developed in the heart. Here, we investigated how retrograde signaling pathways orchestrate metabolic rewiring to compensate for mitochondrial defects. In mice with an inducible knockdown (KD) of TAFAZZIN, and in induced pluripotent stem cell-derived cardiac myocytes, mitochondrial uptake and oxidation of fatty acids was strongly decreased, while glucose uptake was increased. Unbiased transcriptomic analyses revealed that the activation of the eIF2α/ATF4 axis of the integrated stress response upregulates one-carbon metabolism, which diverts glycolytic intermediates towards the biosynthesis of serine and fuels the biosynthesis of glutathione. In addition, strong upregulation of the glutamate/cystine antiporter xCT increases cardiac cystine import required for glutathione synthesis. Increased glutamate uptake facilitates anaplerotic replenishment of the Krebs cycle, sustaining energy production and antioxidative pathways. These data indicate that ATF4-driven rewiring of metabolism compensates for defects in mitochondrial uptake of fatty acids to sustain energy production and antioxidation.

An end-to-end pipeline for succinic acid production at an industrially relevant scale using Issatchenkia orientalis.

Microbial production of succinic acid (SA) at an industrially relevant scale has been hindered by high downstream processing costs arising from neutral pH fermentation for over three decades. Here, we metabolically engineer the acid-tolerant yeast Issatchenkia orientalis for SA production, attaining the highest titers in sugar-based media at low pH (pH 3) in fed-batch fermentations, i.e. 109.5 g/L in minimal medium and 104.6 g/L in sugarcane juice medium. We further perform batch fermentation using sugarcane juice medium in a pilot-scale fermenter (300×) and achieve 63.1 g/L of SA, which can be directly crystallized with a yield of 64.0%. Finally, we simulate an end-to-end low-pH SA production pipeline, and techno-economic analysis and life cycle assessment indicate our process is financially viable and can reduce greenhouse gas emissions by 34-90% relative to fossil-based production processes. We expect I. orientalis can serve as a general industrial platform for production of organic acids.

G6PD Maintains Redox Homeostasis and Biosynthesis in LKB1-Deficient KRAS-Driven Lung Cancer.

Cancer cells depend on nicotinamide adenine dinucleotide phosphate (NADPH) to combat oxidative stress and support reductive biosynthesis. One major NAPDH production route is the oxidative pentose phosphate pathway (committed step: glucose-6-phosphate dehydrogenase, G6PD). Alternatives exist and can compensate in some tumors. Here, using genetically-engineered lung cancer model, we show that ablation of G6PD significantly suppresses KrasG12D/+;Lkb1-/- (KL) but not KrasG12D/+;p53-/- (KP) lung tumorigenesis. In vivo isotope tracing and metabolomics revealed that G6PD ablation significantly impaired NADPH generation, redox balance and de novo lipogenesis in KL but not KP lung tumors. Mechanistically, in KL tumors, G6PD ablation caused p53 activation that suppressed tumor growth. As tumor progressed, G6PD-deficient KL tumors increased an alternative NADPH source, serine-driven one carbon metabolism, rendering associated tumor-derived cell lines sensitive to serine/glycine depletion. Thus, oncogenic driver mutations determine lung cancer dependence on G6PD, whose targeting is a potential therapeutic strategy for tumors harboring KRAS and LKB1 co-mutations.

Comprehensive quantification of metabolic flux during acute cold stress in mice.

Cold-induced thermogenesis (CIT) is widely studied as a potential avenue to treat obesity, but a thorough understanding of the metabolic changes driving CIT is lacking. Here, we present a comprehensive and quantitative analysis of the metabolic response to acute cold exposure, leveraging metabolomic profiling and minimally perturbative isotope tracing studies in unanesthetized mice. During cold exposure, brown adipose tissue (BAT) primarily fueled the tricarboxylic acid (TCA) cycle with fat in fasted mice and glucose in fed mice, underscoring BAT's metabolic flexibility. BAT minimally used branched-chain amino acids or ketones, which were instead avidly consumed by muscle during cold exposure. Surprisingly, isotopic labeling analyses revealed that BAT uses glucose largely for TCA anaplerosis via pyruvate carboxylation. Finally, we find that cold-induced hepatic gluconeogenesis is critical for CIT during fasting, demonstrating a key functional role for glucose metabolism. Together, these findings provide a detailed map of the metabolic rewiring driving acute CIT.

Acidic Methanol Treatment Facilitates Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging of Energy Metabolism.

Detection of small molecule metabolites (SMM), particularly those involved in energy metabolism using MALDI-mass spectrometry imaging (MSI), is challenging due to factors including ion suppression from other analytes present (e.g., proteins and lipids). One potential solution to enhance SMM detection is to remove analytes that cause ion suppression from tissue sections before matrix deposition through solvent washes. Here, we systematically investigated solvent treatment conditions to improve SMM signal and preserve metabolite localization. Washing with acidic methanol significantly enhances the detection of phosphate-containing metabolites involved in energy metabolism. The improved detection is due to removing lipids and highly polar metabolites that cause ion suppression and denaturing proteins that release bound phosphate-containing metabolites. Stable isotope infusions of [13C6]nicotinamide coupled to MALDI-MSI ("Iso-imaging") in the kidney reveal patterns that indicate blood vessels, medulla, outer stripe, and cortex. We also observed different ATP:ADP raw signals across mouse kidney regions, consistent with regional differences in glucose metabolism favoring either gluconeogenesis or glycolysis. In mouse muscle, Iso-imaging using [13C6]glucose shows high glycolytic flux from infused circulating glucose in type 1 and 2a fibers (soleus) and relatively lower glycolytic flux in type 2b fiber type (gastrocnemius). Thus, improved detection of phosphate-containing metabolites due to acidic methanol treatment combined with isotope tracing provides an improved way to probe energy metabolism with spatial resolution in vivo.