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Across different kingdoms of life, ATP citrate lyase (ACLY, also known as ACL) catalyses the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate, a metabolic product of the Krebs cycle, to oxaloacetate and the high-energy biosynthetic precursor acetyl-CoA 1 . The latter fuels pivotal biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine 2 , and the acetylation of histones and proteins 3 , 4 . In autotrophic prokaryotes, ACLY is a hallmark enzyme of the reverse Krebs cycle (also known as the reductive tricarboxylic acid cycle), which fixates two molecules of carbon dioxide in acetyl-CoA 5 , 6 . In humans, ACLY links carbohydrate and lipid metabolism and is strongly expressed in liver and adipose tissue 1 and in cholinergic neurons 2 , 7 . The structural basis of the function of ACLY remains unknown. Here we report high-resolution crystal structures of bacterial, archaeal and human ACLY, and use distinct substrate-bound states to link the conformational plasticity of ACLY to its multistep catalytic itinerary. Such detailed insights will provide the framework for targeting human ACLY in cancer 8 – 11 and hyperlipidaemia 12 , 13 . Our structural studies also unmask a fundamental evolutionary relationship that links citrate synthase, the first enzyme of the oxidative Krebs cycle, to an ancestral tetrameric citryl-CoA lyase module that operates in the reverse Krebs cycle. This molecular transition marked a key step in the evolution of metabolism on Earth. Crystal structures of ATP citrate lyase from bacteria, archaea and humans unravel how the enzyme directs the formation of the central metabolite acetyl-CoA, and shed light onto the evolutionary origins of the Krebs cycle.

Like citrate, the molecule hydroxycitrate is shown to inhibit growth of the crystal that is the principal component of kidney stones, suggesting that hydroxycitrate could be another treatment for kidney stone disease. Novel inhibitor action on 'kidney stone' crystal growth This paper reports that both citrate and hydroxycitrate molecules are able to inhibit growth of crystals of calcium oxalate monohydrate — the principal component of kidney stones — even in a supersaturated solution where the inhibitor concentration is far smaller than the concentration of the solutes. The mechanism for crystal growth inhibition diverges from the classical picture of such processes: atomic force microscopy images and other data point to a mechanism in which inhibitor–crystal interactions impart localized strain to the crystal lattice that are alleviated by oxalate and calcium ions. Potassium citrate is an established treatment for kidney stone disease and the authors suggest that the clinical potential of hydroxycitrate might also be worth exploring and report preliminary in vitro investigations to support this suggestion. Crystalline materials are crucial to the function of living organisms, in the shells of molluscs 1 , 2 , 3 , the matrix of bone 4 , the teeth of sea urchins 5 , and the exoskeletons of coccoliths 6 . However, pathological biomineralization can be an undesirable crystallization process associated with human diseases 7 , 8 , 9 . The crystal growth of biogenic, natural and synthetic materials may be regulated by the action of modifiers, most commonly inhibitors, which range from small ions and molecules 10 , 11 to large macromolecules 12 . Inhibitors adsorb on crystal surfaces and impede the addition of solute, thereby reducing the rate of growth 13 , 14 . Complex inhibitor–crystal interactions in biomineralization are often not well elucidated 15 . Here we show that two molecular inhibitors of calcium oxalate monohydrate crystallization—citrate and hydroxycitrate—exhibit a mechanism that differs from classical theory in that inhibitor adsorption on crystal surfaces induces dissolution of the crystal under specific conditions rather than a reduced rate of crystal growth. This phenomenon occurs even in supersaturated solutions where inhibitor concentration is three orders of magnitude less than that of the solute. The results of bulk crystallization, in situ atomic force microscopy, and density functional theory studies are qualitatively consistent with a hypothesis that inhibitor–crystal interactions impart localized strain to the crystal lattice and that oxalate and calcium ions are released into solution to alleviate this strain. Calcium oxalate monohydrate is the principal component of human kidney stones 16 , 17 , 18 , 19 and citrate is an often-used therapy 20 , but hydroxycitrate is not. For hydroxycitrate to function as a kidney stone treatment, it must be excreted in urine. We report that hydroxycitrate ingested by non-stone-forming humans at an often-recommended dose leads to substantial urinary excretion. In vitro assays using human urine reveal that the molecular modifier hydroxycitrate is as effective an inhibitor of nucleation of calcium oxalate monohydrate nucleation as is citrate. Our findings support exploration of the clinical potential of hydroxycitrate as an alternative treatment to citrate for kidney stones.

The fermentation of nitrogen-containing compounds by biological nitrogen fixation is a sustainable strategy that is independent of the Haber–Bosch process. We previously reported that the nitrogen-fixing bacterium Klebsiella pasteurii (formerly K. oxytoca ) NG13 synthesized and excreted large amounts of ʟ-glutamate using gaseous nitrogen when citrate synthase (CS) and citrate transporter (CitS) were overproduced; however, the majority of carbon atoms in ʟ-glutamate were derived from citrate, not glucose, in the glucose and citrate-containing medium. To examine biased carbon flux to ʟ-glutamate, K. pasteurii overproducing CS and a 2-oxoglutarate (2-OG) transporter (KgtP) was constructed, and its carbon origin was investigated. This strain produced 2-OG-derived ʟ-glutamate in a culture medium containing glucose and 2-OG as the carbon sources. Since CS was inhibited by 2-OG competitively with oxaloacetate, a cognate substrate of CS, the deviated carbon flux from citrate/2-OG to ʟ-glutamate was attributed to the suppression of CS by 2-OG. Based on the structural model of CS from K. pasteurii (KpCS), H227 and V362 were selected as candidates to detect 2-OG binding, and KpCS variants (KpCS*) with H227L, H227Q, and V362L substitutions were confirmed to have inhibition constants that increased by 2.5- to 12.5-fold. As expected, the strains co-overproducing each of the KpCS variants and CitS generated larger amounts of ʟ-glutamate from glucose than the wild-type KpCS + CitS strain. When the KpCS(H227Q) + CitS strain was cultured under continuous glucose-fed conditions, maximum ʟ-glutamate production reached 2.35 g L −1 . These results suggest the potential of the Haber–Bosch process-independent strategy as a technological basis for the sustainable and eco-friendly utilization of nitrogen. Key points • CS was inhibited by 2-OG in K. pasteurii • CS variants with increased K i 2−OG allowed glucose-derived ʟ-glutamate production • Under glucose-fed culture, ʟ-glutamate production finally reached 2.35 g L −1

In a trial, patients with moderate to severely advanced idiopathic pulmonary fibrosis were treated with nintedanib plus sildenafil or nintedanib alone, with the goal of decreasing IPF symptoms. There were no between-group differences in any of three symptom measures.

Minimizing the duration of mechanical ventilation is one of the most important therapeutic goals during the care of preterm infants at neonatal intensive care units (NICUs). The rate of extubation failure among preterm infants is between 16% and 40% worldwide. Numerous studies have been conducted on the assessment of extubation suitability, the optimal choice of respiratory support around extubation, and the effectiveness of medical interventions. Since the Caffeine Therapy for Apnea of Prematurity (CAP) trial, caffeine has become one of the essential drugs at NICUs. However, the optimal dosage and timing for adequate effectiveness still need to be more conclusive. Previous studies suggest that higher doses of caffeine treatment increase the success rate of extubation. Therefore, we aim to determine whether using a single additional loading dose of caffeine citrate one hour prior to extubation impacts the success rate of extubation. The study is an open-label, multicenter randomized clinical trial testing the effectiveness and safety of pre-extubational loading dose of caffeine citrate. Inclusion criteria will be infants born before the 32nd gestational week, before the first extubation attempt after at least 48 hours of mechanical ventilation, and a signed parental informed consent. A total of 226 patients will be randomly allocated to either the experimental or control group. The randomization will be stratified by gestational age and antenatal steroid prophylaxis. Preterm infants in the experimental group will receive an additional intravenous (IV) loading dose (20 mg/kg) of caffeine citrate one hour before the first planned extubation, in addition to the standard dosing regimen (20 mg/kg caffeine citrate IV on the first day of life and 5 to 10 mg/kg IV or orally caffeine citrate each consecutive day). Preterm infants in the control group will receive the standard dosing regimen. The primary outcome will be reintubation within 48 hours. A pre-extubational loading dose of caffeine citrate can reduce extubation failure. Obtaining evidence on this feature has the potential to contribute to finding the optimal dosing regimen. The study protocol was approved by the Hungarian Ethics Committee for Clinical Pharmacology of the Medical Research Council and National Institute of Pharmacy and Nutrition (OGYÉI/6838-11/2023). ClinicalTrials.gov identifier NCT06401083 Registered 06. May 2024.; EudraCT number: 2022-003202-77.

Objective: To determine whether intermittent hypoxia (IH) persisting after 36 weeks postmenstrual age (PMA) can be attenuated using caffeine doses sufficient to maintain caffeine concentrations >20 μg ml −1 . Study Design: Twenty-seven infants born <32 weeks were started on caffeine citrate at 10 mg kg −1  day −1 when clinical caffeine was discontinued. At 36 weeks PMA, the dose was increased to 14 or 20 mg kg −1  day −1 divided twice a day (BID) to compensate for progressively increasing caffeine metabolism. Caffeine concentrations were measured weekly. The extent of IH derived from continuous pulse oximetry was compared to data from 53 control infants. Result: The mean (s.d.) gestational age of enrolled infants was 27.9±2 weeks. Median caffeine levels were >20 μg ml −1 on study caffeine doses. IH was significantly attenuated through 38 weeks PMA compared with the control group. Conclusion: Caffeine doses of 14 to 20 mg kg −1  day −1 were sufficient to maintain caffeine concentrations >20 μg ml −1 and reduce IH in preterm infants at 36 to 38 weeks PMA.

Short-term studies indicate that bempedoic acid, an ATP citrate lyase inhibitor, reduces LDL cholesterol levels. In a 1-year trial, bempedoic acid added to maximally tolerated statin therapy did not lead to a higher incidence of adverse events than placebo and led to significantly lower LDL cholesterol levels.

ATP-citrate lyase (ACLY) is a central metabolic enzyme and catalyses the ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate and acetyl-CoA 1 – 5 . The acetyl-CoA product is crucial for the metabolism of fatty acids 6 , 7 , the biosynthesis of cholesterol 8 , and the acetylation and prenylation of proteins 9 , 10 . There has been considerable interest in ACLY as a target for anti-cancer drugs, because many cancer cells depend on its activity for proliferation 2 , 5 , 11 . ACLY is also a target against dyslipidaemia and hepatic steatosis, with a compound currently in phase 3 clinical trials 4 , 5 . Many inhibitors of ACLY have been reported, but most of them have weak activity 5 . Here we report the development of a series of low nanomolar, small-molecule inhibitors of human ACLY. We have also determined the structure of the full-length human ACLY homo-tetramer in complex with one of these inhibitors (NDI-091143) by cryo-electron microscopy, which reveals an unexpected mechanism of inhibition. The compound is located in an allosteric, mostly hydrophobic cavity next to the citrate-binding site, and requires extensive conformational changes in the enzyme that indirectly disrupt citrate binding. The observed binding mode is supported by and explains the structure–activity relationships of these compounds. This allosteric site greatly enhances the ‘druggability’ of ACLY and represents an attractive target for the development of new ACLY inhibitors. The structure of human ATP-citrate lyase, in complex with a newly developed small-molecule inhibitor, shows extensive conformational changes that reveal an allosteric site for the inhibitor to bind and indirectly compete with the citrate substrate.

Consumption of fructose has risen markedly in recent decades owing to the use of sucrose and high-fructose corn syrup in beverages and processed foods 1 , and this has contributed to increasing rates of obesity and non-alcoholic fatty liver disease 2 – 4 . Fructose intake triggers de novo lipogenesis in the liver 4 – 6 , in which carbon precursors of acetyl-CoA are converted into fatty acids. The ATP citrate lyase (ACLY) enzyme cleaves cytosolic citrate to generate acetyl-CoA, and is upregulated after consumption of carbohydrates 7 . Clinical trials are currently pursuing the inhibition of ACLY as a treatment for metabolic diseases 8 . However, the route from dietary fructose to hepatic acetyl-CoA and lipids remains unknown. Here, using in vivo isotope tracing, we show that liver-specific deletion of Acly in mice is unable to suppress fructose-induced lipogenesis. Dietary fructose is converted to acetate by the gut microbiota 9 , and this supplies lipogenic acetyl-CoA independently of ACLY 10 . Depletion of the microbiota or silencing of hepatic ACSS2, which generates acetyl-CoA from acetate, potently suppresses the conversion of bolus fructose into hepatic acetyl-CoA and fatty acids. When fructose is consumed more gradually to facilitate its absorption in the small intestine, both citrate cleavage in hepatocytes and microorganism-derived acetate contribute to lipogenesis. By contrast, the lipogenic transcriptional program is activated in response to fructose in a manner that is independent of acetyl-CoA metabolism. These data reveal a two-pronged mechanism that regulates hepatic lipogenesis, in which fructolysis within hepatocytes provides a signal to promote the expression of lipogenic genes, and the generation of microbial acetate feeds lipogenic pools of acetyl-CoA. A genetic mouse model is used to reveal a two-pronged mechanism of fructose-induced de novo lipogenesis in the liver, in which fructose catabolism in hepatocytes provides a signal to promote lipogenesis, whereas fructose metabolism by the gut microbiota provides acetate as a substrate to feed lipogenesis.

Regional citrate versus systemic heparin anticoagulation for continuous renal replacement in critically ill patients. We determined the effect of regional citrate versus systemic heparin anticoagulation for continuous renal replacement therapy in critically ill subjects suffering from acute renal failure who were not at high risk for hemorrhagic complications. Between April 1999 and June 2002, 30 critically ill subjects requiring continuous renal replacement therapy and using 79 hemofilters were randomly assigned to receive regional citrate or systemic heparin anticoagulation. The median hemofilter survival time was 124.5 hours (95% CI 95.3 to 157.4) in the citrate group, which was significantly longer than the 38.3 hours (95% CI 24.8 to 61.9) in the heparin group (P < 0.001). Increasing illness severity score, male gender, and decreasing antithrombin-III levels were independent predictors of an increased relative hazard of hemofilter failure. After adjustment for illness severity, antithrombin-III levels increased significantly more over the period of study in the citrate as compared to the heparin group (P = 0.038). Moreover, after adjustment for antithrombin-III levels and illness severity score, the relative risk of hemorrhage with citrate anticoagulation was significantly lower than that with heparin (relative risk of 0.14; 95% CI 0.02 to 0.96, P = 0.05). Compared with systemic heparin anticoagulation, regional citrate anticoagulation significantly increases hemofilter survival time, and significantly decreases bleeding risk in critically ill patients suffering from acute renal failure and requiring continuous renal replacement therapy.