miR164-MhNAC1 regulates apple root nitrogen uptake under low nitrogen stress.

Nitrogen is an essential nutrient for plant growth and serves as a signaling molecule to regulate gene expression inducing physiological, growth and developmental responses. An excess or deficiency of nitrogen may have adverse effects on plants. Studying nitrogen uptake will help us understand the molecular mechanisms of utilization for targeted molecular breeding. Here, we identified and functionally validated an NAC (NAM-ATAF1/2-CUC2) transcription factor based on the transcriptomes of two apple rootstocks with different nitrogen uptake efficiency. NAC1, a target gene of miR164, directly regulates the expression of the high-affinity nitrate transporter (MhNRT2.4) and citric acid transporter (MhMATE), affecting root nitrogen uptake. To examine the role of MhNAC1 in nitrogen uptake, we produced transgenic lines that overexpressed or silenced MhNAC1. Silencing MhNAC1 promoted nitrogen uptake and citric acid secretion in roots, and enhanced plant tolerance to low nitrogen conditions, while overexpression of MhNAC1 or silencing miR164 had the opposite effect. This study not only revealed the role of the miR164-MhNAC1 module in nitrogen uptake in apple rootstocks but also confirmed that citric acid secretion in roots affected nitrogen uptake, which provides a research basis for efficient nitrogen utilization and molecular breeding in apple.

Seasonal drought promotes citrate accumulation in citrus fruit through the CsABF3-activated CsAN1-CsPH8 pathway.

Plenty of rainfall but unevenly seasonal distribution happens regularly in southern China. Seasonal drought from summer to early autumn leads to citrus fruit acidification, but how seasonal drought regulates citrate accumulation remains unknown. Herein, we employed a set of physiological, biochemical, and molecular approaches to reveal that CsABF3 responds to seasonal drought stress and modulates citrate accumulation in citrus fruits by directly regulating CsAN1 and CsPH8. Here, we demonstrated that irreversible acidification of citrus fruits is caused by drought lasting for > 30 d during the fruit enlargement stage. We investigated the transcriptome characteristics of fruits affected by drought and corroborated the pivotal roles of a bHLH transcription factor (CsAN1) and a P3A-ATPase gene (CsPH8) in regulating citrate accumulation in response to drought. Abscisic acid (ABA)-responsive element binding factor 3 (CsABF3) was upregulated by drought in an ABA-dependent manner. CsABF3 activated CsAN1 and CsPH8 expression by directly and specifically binding to the ABA-responsive elements (ABREs) in the promoters and positively regulated citrate accumulation. Taken together, this study sheds new light on the regulatory module ABA-CsABF3-CsAN1-CsPH8 responsible for citrate accumulation under drought stress, which advances our understanding of quality formation of citrus fruit.

Ctp1 and Yhm2: Two Mitochondrial Citrate Transporters to Support Metabolic Flexibility of Saccharomyces cerevisiae.

Differently from higher eukaryotic cells, in the yeast Saccharomyces cerevisiae there are two mitochondrial carrier proteins involved in the transport of citrate: Ctp1 and Yhm2. Very little is known about the physiological role of these proteins. Wild-type and mutant yeast strains deleted in CTP1 and YHM2 were grown in media supplemented with a fermentable (glucose) or a nonfermentable (ethanol) carbon source. To assess changes in Ctp1 and Yhm2 mRNA expression levels, real-time PCR was performed after total RNA extraction. In the wild-type strain, the metabolic switch from the exponential to the stationary phase is associated with an increase in the expression level of the two citrate transporters. In addition, the results obtained in the mutant strains suggest that the presence of a single citrate transporter can partially compensate for the absence of the other. Ctp1 and Yhm2 differently contribute to fermentative and respiratory metabolism. Moreover, the two mitochondrial carriers represent a link between the Krebs cycle and the glyoxylate cycle, which play a key role in the metabolic adaptation strategies of S. cerevisiae.

Overexpression of the DHA1 family, ChlH and ChlK, leads to enhanced dicarboxylic acids production in koji fungi, Aspergillus luchuensis mut. kawachii and Aspergillus oryzae.

The white koji fungus Aspergillus luchuensis mut. kawachii secretes substantial amounts of citric acid through the expression of the citric acid exporter CexA, a member of the DHA1 family. In this study, we aimed to characterize 11 CexA homologs (Chl proteins) encoded in the genome of A. luchuensis mut. kawachii to identify novel transporters useful for organic acid production. We constructed overexpression strains of chl genes using a cexA disruptant of the A. luchuensis mut. kawachii as the host strain, which prevented excessive secretion of citric acid into the culture supernatant. Subsequently, we evaluated the effects of overexpression of chl on producing organic acids by analyzing the culture supernatant. All overexpression strains did not exhibit significant citric acid accumulation in the culture supernatant, indicating that Chl proteins are not responsible for citric acid export. Furthermore, the ChlH overexpression strain displayed an accumulation of 2-oxoglutaric and fumaric acids in the culture supernatant, while the ChlK overexpression strain exhibited the accumulation of 2-oxoglutaric, malic and succinic acids. Notably, the ChlH and ChlK overexpression led to a substantial increase in the production of 2-oxoglutaric acid, reaching approximately 25 mM and 50 mM, respectively. Furthermore, ChlH and ChlK overexpression also significantly increased the secretory production of dicarboxylic acids, including 2-oxoglutaric acid, in the yellow koji fungus, Aspergillus oryzae. Our study demonstrates that overexpression of DHA1 family gene results in enhanced secretion of organic acids in koji fungi of the genus Aspergillus.

Citrate synthase lysine K215 hypoacetylation contributes to microglial citrate accumulation and pro-inflammatory functions after traumatic brain injury.

AIMS: This study aimed to investigate the relationship between microglial metabolism and neuroinflammation by examining the impact of citrate accumulation in microglia and its potential regulation through Cs K215 hypoacetylation. METHODS: Experimental approaches included assessing Cs enzyme activity through Cs K215Q mutation and investigating the inhibitory effects of hesperidin, a natural flavanone glycoside, on citrate synthase. Microglial phagocytosis and expression of pro-inflammatory cytokines were also examined in relation to Cs K215Q mutation and hesperidin treatment. RESULTS: Cs K215Q mutation and hesperidin exhibited significant inhibitory effects on Cs enzyme activity, microglial citrate accumulation, phagocytosis, and pro-inflammatory cytokine expression. Interestingly, Sirt3 knockdown aggravated microglial pro-inflammatory functions during neuroinflammation, despite its proven role in Cs deacetylation. CONCLUSION: Cs K215Q mutation and hesperidin effectively inhibited microglial pro-inflammatory functions without reversing the metabolic reprogramming. These findings suggest that targeting Cs K215 hypoacetylation and utilizing hesperidin may hold promise for modulating neuroinflammation in microglia.

Time-Series Transcriptome of Cucumis melo Reveals Extensive Transcriptomic Differences with Different Maturity.

As the most important melon cultivar grown in the north-western provinces of China, Hami melon (Cucumis melo) produces large edible fruits that serve as an important dietary component in the world. In general, as a climacteric plant, melon harvested at 60% maturity results in a product with bad quality, while the highest-quality product can be guaranteed when harvesting at 90% maturity. In order to clarify the genetic basis of their distinct profiles of metabolite accumulation, we performed systematic transcriptome analyses between 60% and 90% maturity melons. A total of 36 samples were sequenced and over 1.7 billion reads were generated. Differentially expressed genes in 60% and 90% maturity melons were detected. Hundreds of these genes were functionally enriched in the sucrose and citric acid accumulation process of C. melo. We also detected a number of distinct splicing events between 60% and 90% maturity melons. Many genes associated with sucrose and citric acid accumulation displayed as differentially expressed or differentially spliced between different degrees of maturity of Hami melons, including CmCIN2, CmSPS2, CmBGAL3, and CmSPS2. These results demonstrate that the phenotype pattern differences between 60% and 90% maturity melons may be largely resulted from the significant transcriptome regulation.

Dissolution behavior of stone wool fibers in synthetic lung fluids: Impact of iron oxidation state changes induced by heat treatment for binder removal.

Stone wool fiber materials are commonly used for thermal and acoustic insulation, horticulture and filler purposes. Biosolubility of the stone wool fiber (SWF) materials accessed through acellular in vitro dissolution tests can potentially be used in future as an indicator of fiber biopersistence in vivo. To correlate acellular in vitro studies with in vivo and epidemiological investigations, not only a robust dissolution procedure is needed, but fundamental understanding of fiber behavior during sample preparation and dissolution is required. We investigated the influence of heat treatment procedure for binder removal on the SWF iron oxidation state as well as on the SWF dissolution behavior in simulant lung fluids (with and without complexing agents). We used heat treatments at 450 °C for 5 min and 590 °C for 1 h. Both procedures resulted in complete binder removal from the SWF. Changes of iron oxidation state were moderate if binder was removed at 450 °C for 5 min, and there were no substantial changes of SWF's dissolution behavior in all investigated fluids after this heat treatment. In contrast, if binder was removed at 590 °C for 1 h, complete Fe(II) oxidation to Fe(III) was observed and significant increase of dissolution was shown in fluids without complexing agent (citrate). PHREEQC solution speciation modeling showed that in this case, released Fe(III) may form ferrihydrite precipitate in the solution. Precipitation of ferrihydrite solid phase leads to removal of iron cations from the solution, thus shifting reaction towards the dissolution products and increasing total mass loss of fiber samples. This effect is not observed for heat treated fibers if citrate is present in the fluid, because Fe(III) binds with citrate and remains mobile in the solution. Therefore, for developing the most accurate SWF in vitro acellular biosolubility test, SWF heat treatment for binder removal is not recommended in combination with dissolution testing in fluids without citrate as a complexing agent.

Citrate cross-feeding by Pseudomonas aeruginosa supports lasR mutant fitness.

Cross-feeding of metabolites between subpopulations can affect cell phenotypes and population-level behaviors. In chronic Pseudomonas aeruginosa lung infections, subpopulations with loss-of-function (LOF) mutations in the lasR gene are common. LasR, a transcription factor often described for its role in virulence factor expression, also impacts metabolism, which, in turn, affects interactions between LasR+ and LasR- genotypes. Prior transcriptomic analyses suggested that citrate, a metabolite secreted by many cell types, induces virulence factor production when both genotypes are together. An unbiased analysis of the intracellular metabolome revealed broad differences including higher levels of citrate in lasR LOF mutants. Citrate consumption by LasR- strains required the CbrAB two-component system, which relieves carbon catabolite repression and is elevated in lasR LOF mutants. Within mixed communities, the citrate-responsive two-component system TctED and its gene targets OpdH (porin) and TctABC (citrate transporter) that are predicted to be under catabolite repression control were induced and required for enhanced RhlR/I-dependent signaling, pyocyanin production, and fitness of LasR- strains. Citrate uptake by LasR- strains markedly increased pyocyanin production in co-culture with Staphylococcus aureus, which also secretes citrate and frequently co-infects with P. aeruginosa. This citrate-induced restoration of virulence factor production by LasR- strains in communities with diverse species or genotypes may offer an explanation for the contrast observed between the markedly deficient virulence factor production of LasR- strains in monocultures and their association with the most severe forms of cystic fibrosis lung infections. These studies highlight the impact of secreted metabolites in mixed microbial communities.IMPORTANCECross-feeding of metabolites can change community composition, structure, and function. Here, we unravel a cross-feeding mechanism between frequently co-observed isolate genotypes in chronic Pseudomonas aeruginosa lung infections. We illustrate an example of how clonally derived diversity in a microbial communication system enables intra- and inter-species cross-feeding. Citrate, a metabolite released by many cells including P. aeruginosa and Staphylococcus aureus, was differentially consumed between genotypes. Since these two pathogens frequently co-occur in the most severe cystic fibrosis lung infections, the cross-feeding-induced virulence factor expression and fitness described here between diverse genotypes exemplify how co-occurrence can facilitate the development of worse disease outcomes.

Citric Acid Promotes Immune Function by Modulating the Intestinal Barrier.

Amidst increasing concern about antibiotic resistance resulting from the overuse of antibiotics, there is a growing interest in exploring alternative agents. One such agent is citric acid, an organic compound commonly used for various applications. Our research findings indicate that the inclusion of citric acid can have several beneficial effects on the tight junctions found in the mouse intestine. Firstly, the study suggests that citric acid may contribute to weight gain by stimulating the growth of intestinal epithelial cells (IE-6). Citric acid enhances the small intestinal villus-crypt ratio in mice, thereby promoting intestinal structural morphology. Additionally, citric acid has been found to increase the population of beneficial intestinal microorganisms, including Bifidobacterium and Lactobacillus. It also promotes the expression of important protein genes such as occludin, ZO-1, and claudin-1, which play crucial roles in maintaining the integrity of the tight junction barrier in the intestines. Furthermore, in infected IEC-6 cells with H9N2 avian influenza virus, citric acid augmented the expression of genes closely associated with the influenza virus infection. Moreover, it reduces the inflammatory response caused by the viral infection and thwarted influenza virus replication. These findings suggest that citric acid fortifies the intestinal tight junction barrier, inhibits the replication of influenza viruses targeting the intestinal tract, and boosts intestinal immune function.

Citric Acid Conditioning as an Alternative to EDTA for Growth Factors Release and Stem Cell Response in Regenerative Endodontics: A Systematic Review of In Vitro Studies.

INTRODUCTION: Citric acid (CA) conditioning may be a promising alternative to ethylenediaminetetraacetic acid (EDTA) in regenerative endodontic procedures, as reported to improve growth factors' release from dentin. This review systematically investigated the effect of CA conditioning on the growth factors release from dentin and cell behavior compared to EDTA conditioning. METHODS: Searches were conducted (PubMed/MEDLINE, Scopus, Web of Science, Embase, SciELO, Cochrane Library, and grey literature) until May-2023. Only in vitro studies that evaluated the effects of CA on growth factors' release from dentin and cell behavior outcomes compared to EDTA were included. The studies were critically appraised using a modified Joanna Briggs Institute's checklist. Meta-analysis was unfeasible. RESULTS: Out of the 335 articles screened, nine were included. Among these, three studies used dentin discs/roots from permanent human teeth; the rest combined them with stem cells. 10% CA for 5 or 10 minute was the most used protocol. Meanwhile, EDTA concentrations ranged from 10% to 17%. In eight studies examining the release of growth factors, five reported a significant release of transforming growth factor-ß after dentin conditioning with 10% CA compared to 17% EDTA. Regarding cell behavior (6 studies), three studies assessed cell viability. The findings revealed that 10% CA conditioning showed cell viability similar to those of 17% EDTA. Additionally, in two out of three studies, it was observed that 10% CA conditioning did not affect cell morphology. The studies had a low risk of bias. CONCLUSIONS: The use of 10% CA to condition dentin for 5-10 minutes resulted in a notable transforming growth factor -ß1 release, but its cell responses were similar to those of EDTA.

CcNFYB3-CcMATE35 and LncRNA CcLTCS-CcCS modules jointly regulate the efflux and synthesis of citrate to enhance aluminium tolerance in pigeon pea.

Aluminium (Al) toxicity decreases crop production in acid soils in general, but many crops have evolved complex mechanisms to resist it. However, our current understanding of how plants cope with Al stress and perform Al resistance is still at the initial stage. In this study, the citrate transporter CcMATE35 was identified to be involved in Al stress response. The release of citrate was increased substantially in CcMATE35 over-expression (OE) lines under Al stress, indicating enhanced Al resistance. It was demonstrated that transcription factor CcNFYB3 regulated the expression of CcMATE35, promoting the release of citrate from roots to increase Al resistance in pigeon pea. We also found that a Long noncoding RNA Targeting Citrate Synthase (CcLTCS) is involved in Al resistance in pigeon pea. Compared with controls, overexpression of CcLTCS elevated the expression level of the Citrate Synthase gene (CcCS), leading to increases in root citrate level and citrate release, which forms another module to regulate Al resistance in pigeon pea. Simultaneous overexpression of CcNFYB3 and CcLTCS further increased Al resistance. Taken together, these findings suggest that the two modules, CcNFYB3-CcMATE35 and CcLTCS-CcCS, jointly regulate the efflux and synthesis of citrate and may play an important role in enhancing the resistance of pigeon pea under Al stress.

Study on the mechanism of sodium ion inhibiting citric acid fermentation in Aspergillus niger.

Excessive sodium significantly inhibits citric acid fermentation by Aspergillus niger during the recycling of citric acid wastewater. This study aimed to elucidate the inhibition mechanism at the interface of physiology and transcriptomics. The results showed that excessive sodium caused a 22.3 % increase in oxalic acid secretion and a 147.6 % increase in H+-ATPase activity at the 4 h fermentation compared to the control. Meanwhile, a 13.1 % reduction in energy charge level and a 15.2 % decline in NADH content were found, which implied the effects on carbon metabolism and redox balance. In addition, transcriptomic analysis revealed that excessive sodium altered the gene expression profiles related to ATPase, hydrolase, and oxidoreductase, as well as pathways like glyoxylate metabolism, and transmembrane transport. These findings gained insights into the metabolic regulation of A. niger response to environmental stress and provided theoretical guidance for the construction of sodium-tolerant A. niger for industrial application.

Enhancement of phycocyanin productivity and thermostability from Arthrospira platensis using organic acids.

Intracellular hyperaccumulation of phycocyanin (PC) and its high susceptibility to degradation at higher temperatures are major challenging problems associated with its production from cyanobacteria. The present study evaluated different concentrations of organic acids (1, 2, and 3 mM) (citric acid, acetic acid, succinic acid, fumaric acid, and oxalic acid) under fed-batch mode on the biomass and phycobiliproteins' production from Arthrospira platensis. Besides they were evaluated at 2.5-7.5 mM as preservative to stabilize PC at high temperatures. The incorporation of 3 mM of succinic acid into the cultivation medium enhanced the biomass and PC productivity to 164.05 and 26.70 mg L-1 day-1, which was ~ 2- and threefold higher than control, respectively. The produced PC in this treatment was food-grade with a 2.2 purity ratio. The use of organic acids also enhanced the thermal stability of PC. Citric acid (7.5 mM) markedly promoted the half-life values of PC to 189.44 min compared to 71.84 min in the control. The thermodynamic analysis confirmed higher thermostability of PC in the presence of organic acids and indicated the endothermic and non-spontaneity of the thermal denaturation process. The findings of the present study confirmed that organic acids could be utilized as cost effective and sustainable compounds for promoting not only phycobiliproteins' production but also the thermostability of PC for potential application in food industry.

Transcriptome and Metabolome Analyses Reveal Sugar and Acid Accumulation during Apricot Fruit Development.

The apricot (Prunus armeniaca L.) is a fruit that belongs to the Rosaceae family; it has a unique flavor and is of important economic and nutritional value. The composition and content of soluble sugars and organic acids in fruit are key factors in determining the flavor quality. However, the molecular mechanism of sugar and acid accumulation in apricots remains unclear. We measured sucrose, fructose, glucose, sorbitol, starch, malate, citric acid, titratable acid, and pH, and investigated the transcriptome profiles of three apricots (the high-sugar cultivar 'Shushanggan', common-sugar cultivar 'Sungold', and low-sugar cultivar 'F43') at three distinct developmental phases. The findings indicated that 'Shushanggan' accumulates a greater amount of sucrose, glucose, fructose, and sorbitol, and less citric acid and titratable acid, resulting in a better flavor; 'Sungold' mainly accumulates more sucrose and less citric acid and starch for the second flavor; and 'F43' mainly accumulates more titratable acid, citric acid, and starch for a lesser degree of sweetness. We investigated the DEGs associated with the starch and sucrose metabolism pathways, citrate cycle pathway, glycolysis pathway, and a handful of sugar transporter proteins, which were considered to be important regulators of sugar and acid accumulation. Additionally, an analysis of the co-expression network of weighted genes unveiled a robust correlation between the brown module and sucrose, glucose, and fructose, with VIP being identified as a hub gene that interacted with four sugar transporter proteins (SLC35B3, SLC32A, SLC2A8, and SLC2A13), as well as three structural genes for sugar and acid metabolism (MUR3, E3.2.1.67, and CSLD). Furthermore, we found some lncRNAs and miRNAs that regulate these genes. Our findings provide clues to the functional genes related to sugar metabolism, and lay the foundation for the selection and cultivation of high-sugar apricots in the future.

Human Pancreatic Islets React to Glucolipotoxicity by Secreting Pyruvate and Citrate.

Progressive decline in pancreatic beta-cell function is central to the pathogenesis of type 2 diabetes (T2D). Here, we explore the relationship between the beta cell and its nutritional environment, asking how an excess of energy substrate leads to altered energy production and subsequent insulin secretion. Alterations in intracellular metabolic homeostasis are key markers of islets with T2D, but changes in cellular metabolite exchanges with their environment remain unknown. We answered this question using nuclear magnetic resonance-based quantitative metabolomics and evaluated the consumption or secretion of 31 extracellular metabolites from healthy and T2D human islets. Islets were also cultured under high levels of glucose and/or palmitate to induce gluco-, lipo-, and glucolipotoxicity. Biochemical analyses revealed drastic alterations in the pyruvate and citrate pathways, which appear to be associated with mitochondrial oxoglutarate dehydrogenase (OGDH) downregulation. We repeated these manipulations on the rat insulinoma-derived beta-pancreatic cell line (INS-1E). Our results highlight an OGDH downregulation with a clear effect on the pyruvate and citrate pathways. However, citrate is directed to lipogenesis in the INS-1E cells instead of being secreted as in human islets. Our results demonstrate the ability of metabolomic approaches performed on culture media to easily discriminate T2D from healthy and functional islets.

Restricted O2 consumption in pea roots induced by hexanoic acid is linked to depletion of Krebs cycle substrates.

Plant roots are exposed to hypoxia in waterlogged soils, and they are further challenged by specific phytotoxins produced by microorganisms in such conditions. One such toxin is hexanoic acid (HxA), which, at toxic levels, causes a strong decline in root O2 consumption. However, the mechanism underlying this process is still unknown. We treated pea (Pisum sativum L.) roots with 20 mM HxA at pH 5.0 and 6.0 for a short time (1 h) and measured leakage of key electrolytes such as metal cations, malate, citrate and nonstructural carbohydrates (NSC). After treatment, mitochondria were isolated to assess their functionality evaluated as electrical potential and O2 consumption rate. HxA treatment resulted in root tissue extrusion of K+ , malate, citrate and NSC, but only the leakage of the organic acids and NSC increased at pH 5.0, concomitantly with the inhibition of O2 consumption. The activity of mitochondria isolated from treated roots was almost unaffected, showing just a slight decrease in oxygen consumption after treatment at pH 5.0. Similar results were obtained by treating the pea roots with another organic acid with a short carbon chain, that is, butyric acid. Based on these results, we propose a model in which HxA, in its undissociated form prevalent at acidic pH, stimulates the efflux of citrate, malate and NSC, which would, in turn, cause starvation of mitochondrial respiratory substrates of the Krebs cycle and a consequent decline in O2 consumption. Cation extrusion would be a compensatory mechanism in order to restore plasma membrane potential.

Proteome insights of citric acid-mediated cadmium toxicity tolerance in Brassica napus L.

Cadmium (Cd) is a toxic substance that is uptake by plants from soils, Cd easily transfers into the food chain. Considering global food security, eco-friendly, cost-effective, and metal detoxification strategies are highly demandable for sustainable food crop production. The purpose of this study was to investigate how citric acid (CA) alleviates or tolerates Cd toxicity in Brassica using a proteome approach. In this study, the global proteome level was significantly altered under Cd toxicity with or without CA supplementation in Brassica. A total of 4947 proteins were identified using the gel-free proteome approach. Out of these, 476 proteins showed differential abundance between the treatment groups, wherein 316 were upregulated and 160 were downregulated. The gene ontology analysis reveals that differentially abundant proteins were involved in different biological processes including energy and carbohydrate metabolism, CO2 assimilation and photosynthesis, signal transduction and protein metabolism, antioxidant defense, heavy metal detoxification, plant development, and cytoskeleton and cell wall structure in Brassica leaves. Interestingly, several candidate proteins such as superoxide dismutase (A0A078GZ68) L-ascorbate peroxidase 3 (A0A078HSG4), glutamine synthetase (A0A078HLB2), glutathione S-transferase DHAR1 (A0A078HPN8), glutamine synthetase (A0A078HLB2), cysteine synthase (A0A078GAD3), S-adenosylmethionine synthase 2 (A0A078JDL6), and thiosulfate/3-mercaptopyruvate sulfur transferase 2 (A0A078H905) were involved in antioxidant defense system and sulfur assimilation-involving Cd-detoxification process in Brassica. These findings provide new proteome insights into CA-mediated Cd-toxicity alleviation in Brassica, which might be useful to oilseed crop breeders for enhancing heavy metal tolerance in Brassica using the breeding program, with sustainable and smart Brassica production in a metal-toxic environment.

Pangenome analysis provides insight into the evolution of the orange subfamily and a key gene for citric acid accumulation in citrus fruits.

The orange subfamily (Aurantioideae) contains several Citrus species cultivated worldwide, such as sweet orange and lemon. The origin of Citrus species has long been debated and less is known about the Aurantioideae. Here, we compiled the genome sequences of 314 accessions, de novo assembled the genomes of 12 species and constructed a graph-based pangenome for Aurantioideae. Our analysis indicates that the ancient Indian Plate is the ancestral area for Citrus-related genera and that South Central China is the primary center of origin of the Citrus genus. We found substantial variations in the sequence and expression of the PH4 gene in Citrus relative to Citrus-related genera. Gene editing and biochemical experiments demonstrate a central role for PH4 in the accumulation of citric acid in citrus fruits. This study provides insights into the origin and evolution of the orange subfamily and a regulatory mechanism underpinning the evolution of fruit taste.

Regulatory-systemic approach in Aspergillus niger for bioleaching improvement by controlling precipitation.

In the context of e-waste recycling by fungal bioleaching, nickel and cobalt precipitate as toxic metals by oxalic acid, whereas organic acids, such as citric, act as a high-performance chelating agent in dissolving these metals. Oxalic acid elimination requires an excess and uneconomical carbon source concentration in culture media. To resolve this issue, a novel and straightforward systems metabolic engineering method was devised to switch metabolic flux from oxalic acid to citric acid. In this technique, the genome-scale metabolic model of Aspergillus niger was applied to predicting flux variability and key reactions through the calculation of multiple optimal solutions for cellular regulation. Accordingly, BRENDA regulators and a novel molecular docking-oriented approach were defined a regulatory medium for this end. Then, ligands were evaluated in fungal culture to assess their impact on organic acid production for bioleaching of copper and nickel from waste telecommunication printed circuit boards. The protein structure of oxaloacetate hydrolase was modeled based on homology modeling for molecular docking. Metformin, glutathione, and sodium fluoride were found to be effective as inhibitors of oxalic acid production, enabling the production of 8100 ppm citric acid by controlling cellular metabolism. Indirect bioleaching demonstrated that nickel did not precipitate, and the bioleaching efficiency of copper and nickel increased from 40% and 24% to 61% and 100%, respectively. Bioleaching efficiency was evaluated qualitatively by FE-SEM, EDX, mapping, and XRD analysis. KEY POINTS: • A regulatory-systemic procedure for controlling cellular metabolism was introduced • Metformin inhibited oxalic acid, leading to 8100 ppm citric acid production • Bioleaching of copper and nickel in TPCBs improved by 21% and 76.

The dual role of citrate in cancer.

Citrate is a key metabolite of the Krebs cycle that can also be exported in the cytosol, where it performs several functions. In normal cells, citrate sustains protein acetylation, lipid synthesis, gluconeogenesis, insulin secretion, bone tissues formation, spermatozoid mobility, and immune response. Dysregulation of citrate metabolism is implicated in several pathologies, including cancer. Here we discuss how cancer cells use citrate to sustain their proliferation, survival, and metastatic progression. Also, we propose two paradoxically opposite strategies to reduce tumour growth by targeting citrate metabolism in preclinical models. In the first strategy, we propose to administer in the tumor microenvironment a high amount of citrate, which can then act as a glycolysis inhibitor and apoptosis inducer, whereas the other strategy targets citrate transporters to starve cancer cells from citrate. These strategies, effective in several preclinical in vitro and in vivo cancer models, could be exploited in clinics, particularly to increase sensibility to current anti-cancer agents.