Essential Metabolic Pathways Research Articles

Hepatocyte nuclear factor 4 located in different developmental stages in Schistosoma japonicum and involved in important metabolic pathways

Nuclear receptors (NRs) are vital for regulating gene expression un organisms. Hepatocyte nuclear factor 4 (HNF4), a class of NRs, participates in blood feeding and intestinal maintenance in schistosomes. However, there is limited research on the molecular and functional characterization of HNF4 in Schistosoma japonicum (S. japonicum). Highly specific polyclonal antibodies were generated to analyze the expression and tissue localization of S. japonicum HNF4 (SjHNF4). The potential biological functions of SjHNF4 were characterized by transcriptome and pull-down analysis. Subsequently, enrichment analysis was performed to identify the specific signaling pathways linked to SjHNF4. The SjHNF4 protein was expressed heterologously and purified successfully. High purity and high potency polyclonal antibodies were further prepared. The expression of SjHNF4 was higher in female compared to male worms at both transcriptional and protein levels. Female worms expressed SjHNF4 in their perithecium, reproductive system, and certain parts of the intestinal tissues. SjHNF4 was also detected in the perithecium of male worms, as well as in the head, body of cercaria, and eggs. Furthermore, our findings highlighted the potential role of SjHNF4 in blood feeding and its interaction with crucial pathways such as glucose metabolism, lipid metabolism, and nucleotide metabolism. This study shed light on the location of SjHNF4 in different life stages of S. japonicum, particularly associated with the female schistosomes. A strong correlation was observed between SjHNF4 and essential metabolic pathways. These findings laid a solid groundwork for the research on the relationship between NRs and schistosomes.

The Active Glucuronide Metabolite of the Brain Protectant IMM-H004 with Poor Blood-Brain Barrier Permeability Demonstrates a High Partition in the Rat Brain via Multiple Mechanisms.

Glucuronidation is an essential metabolic pathway for a variety of drugs. IMM-H004 is a novel neuroprotective agent against ischemic stroke, and its glucuronide metabolite IMM-H004G exhibits similar pharmacological activity. Despite possessing a higher molecular weight and polarity, brain exposure of IMM-H004G is much higher than that of IMM-H004. This study aimed to investigate the brain metabolism and transport mechanisms of IMM-H004 and IMM-H004G. First, the possibility of IMM-H004 glucuronidation in the brain was evaluated in several human brain cell lines and rat homogenate. Subsequently, the blood-brain barrier carrier-mediated transport mechanism of IMM-H004 and IMM-H004G was studied using overexpression cell models. In addition, intracerebroventricular injection, in situ brain perfusion model, and microdialysis/microinjection techniques were performed to study the distribution profiles of IMM-H004 and IMM-H004G. IMM-H004 could be metabolized to IMM-H004G in both rat brain and HEB cells mediated by UGT1A7. However, IMM-H004G could not be hydrolyzed back into IMM-H004. Furthermore, the entry and efflux of IMM-H004 in the brain were mediated by the pyrilamine-sensitive H+/OC antiporter and P-gp, respectively, while the transport of IMM-H004G from the blood to the brain was facilitated by OATP1A2 and OATP2B1. Ultimately, stronger concentration gradients and OATP-mediated uptake played a critical role in promoting greater brain exposure of IMM-H004G. The active glucuronide metabolite of the brain protectant IMM-H004 with poor blood-brain barrier permeability demonstrates a high partition in the rat brain via multiple mechanisms, and our findings deepen the understanding of the mechanisms underlying the blood-brain barrier metabolism and transport of active glucuronide conjugates.

Abstract C077: Elucidation and exploitation of critical nutrient scavenging regulation in ERK MAPK inhibited pancreatic cancer

Abstract Alteration of essential metabolic pathways is a major mechanism by which oncogenic KRAS promotes tumor development and growth in pancreatic ductal adenocarcinoma (PDAC). KRAS-driven PDAC is dependent on nutrient scavenging pathways, including macropinocytosis and autophagy to fuel the high metabolic demand of rapid proliferation. Thus, these metabolic processes are attractive targets for the development of treatments for PDAC. KRAS loss results in downregulation of macropinocytosis in PDAC. Additionally, our lab demonstrated that KRAS loss or inhibition of ERK MAPK signaling decreased glucose consumption and glycolysis but increased autophagy, thereby enhancing dependency on autophagy for survival and growth. Accordingly, dual ERK MAPK and autophagy inhibition (via chloroquine) synergistically enhanced anti-tumor efficacy in PDAC. Early clinical data has demonstrated that resistance to this treatment arises over time through unknown mechanisms. Our preliminary data indicates that following ERK MAPK inhibition both autophagy induction and macropinocytosis downregulation is transient—with autophagic and macropinocytic activity returning to/surpassing basal levels after prolonged treatment. The underlying mechanistic and signaling crosstalk between autophagy and macropinocytosis remains poorly understood. We hypothesize that there is compensatory regulation between autophagy and macropinocytosis signaling following ERK MAPK inhibition. We propose that prolonged activation of autophagy upregulates macropinocytosis over time, consequently abrogating dependency on autophagy and therefore reducing sensitivity to autophagy inhibition. We demonstrate an inverse temporal relationship between autophagic flux and macropinocytosis following genetic loss of KRAS or pharmacological inhibition of the KRAS-ERK MAPK pathway. Furthermore, we show that chloroquine, which is commonly thought of as a lysosomotropic drug, does not prevent degradation of proteins engulfed via macropinocytosis. Together our data suggest that upregulation of macropinocytosis may mediate resistance to the combination of ERK MAPK inhibition and chloroquine. Additionally, we present the unexpected observation that prolonged pharmacological inhibition of the ERK MAPK pathway results in the upregulation of macropinocytosis. Furthermore, we demonstrate that this upregulation of macropinocytosis is accompanied by increased albumin uptake and sensitivity to albumin-bound paclitaxel (nab-paclitaxel). Given the dependence of PDAC on macropinocytic uptake for tumorigenic growth, as well as the potential to exploit macropinocytosis for drug-delivery, it is imperative to understand how ERK MAPK inhibitors regulate macropinocytosis. A better understanding of the regulation underlying these essential metabolic pathways will better inform future ERK MAPK inhibitor combinations. Citation Format: Ryan Robb, Kirsten L. Bryant. Elucidation and exploitation of critical nutrient scavenging regulation in ERK MAPK inhibited pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C077.

Insilico exploration C. koseri ATP synthase inhibitors by pharmacophore-based virtual screening, molecular docking and MD simulation.

Citrobacter koseri is a gram-negative rod that causes infections in people who have significant comorbidities and are immunocompromised. Antibiotic-resistant strains are becoming more common, which complicates infection treatment and highlights the need for innovative, effective drugs to fight these resistant strains. The enzyme complex ATP synthase participates in the adenosine triphosphate (ATP) synthesis, the fundamental energy currency of cells. This study used Computer-Aided Drug Design approaches to identify potential inhibitors of C. koseri ATP synthase. SWISS-MODEL was used to predict the 3D structure of C. koseri ATP synthase. A ligand-based pharmacophore model was developed using chemical features of ampicillin. Following ligand-based virtual screening across nine databases, the 2043 screened hits were docked to the ATP synthase active site using the standard precision mode of the glide tool. Based on their binding affinities, the top ten compounds were selected for additional investigation. The binding affinities of the chosen compounds ranged from -10.021 to -8.452 kcal/mol. The top four compounds (PubChem-25230613, PubChem-74936833, CHEMBL263035, PubChem-44208924) with the best ADMET characteristics and binding modes were chosen. Thus, the feasible binding mechanisms of the selected compounds were subjected to stability analysis using the MD Simulation study, which revealed the compounds' stability as potent inhibitors within the protein binding pocket. This computational approach provides important insights into the rational design of novel therapeutics and emphasizes the importance of targeting essential metabolic pathways when combating antibiotic-resistant pathogens. Future experimental validation and optimization of the identified inhibitors is required to determine their efficacy and safety profiles for clinical use.

Phosphorus-induced improvement of photosynthate synthesis and transport in the leaf subtending to cotton boll provided sufficient sucrose for fiber thickening during the crucial period and improved fiber bundle strength

ContextCotton fiber is an important natural textile raw material, and its quality directly affects spinning performance. Phosphorus (P) deficiency reduces fiber quality, particularly fiber strength. Fiber strength formation relies on sucrose supplied by the leaf subtending to cotton boll (LSCB), and low P may affect it by regulating sucrose supply and utilization in LSCB and fiber. ObjectiveOur study aimed to investigate how P regulates the formation of fiber bundle strength through essential carbohydrate metabolic pathways. MethodsA three-year experiment on Lu 54 (low-P sensitive) and Yuzaomian 9110 (low-P tolerant) was carried out to study the influence of P on sucrose synthesis and export in LSCB, sucrose absorption and utilization in fiber under 0 (Deficient P), 100 (Critical P), and 200 (Excessive P) kg P2O5 ha−1 in soil with 16.9 mg kg−1 available P. ResultsP application significantly increased fiber bundle strength (8.9 %−14.6 %), primarily attributed to the enhanced sucrose supply and utilization in LSCB and fiber during the crucial period for fiber bundle strength formation (17–24 days post anthesis). During this period, P application promoted the sucrose synthesis in LSCB by improving the net photosynthetic rate (9.0 %−16.8 %), and sucrose phosphate synthase (6.3 %−12.4 %) and sucrose synthase (10.0 %−17.4 %) activities. Concomitantly, P application up-regulated the expressions of sucrose transporter genes in LSCB and fiber, promoting sucrose transport and increasing the sucrose content (28.1 %−54.0 %) in fiber. Furthermore, P application markedly enhanced the sucrose utilization in fiber by increasing sucrose synthase (9.7 %−19.2 %) and invertase (14.0 %−35.7 %) activities, improving cellulose content (6.7 %−11.5 %) and fiber bundle strength. Lu 54 exhibited a greater sensitivity to P in the carbohydrate metabolism of LSCB and fiber than Yuzaomian 9110, making its cellulose content and fiber bundle strength increase more. Incremental effects of unit P on the above measurements attenuated markedly with P rates. The critical P content in LSCB (LPC) required for effective fiber bundle strength formation was 0.40 % for Lu 54 versus 0.36 % for Yuzaomain 9110, on the basis of the content of sucrose (the key substrate) in LSCB and fiber. ConclusionP application increased cotton fiber bundle strength mainly by enhancing sucrose synthesis in LSCB, sucrose transport between LSCB and fiber, and sucrose utilization in fiber during the crucial period. ImplicationThis report revealed the crucial carbohydrate metabolic path by which P regulated the fiber bundle strength formation and quantified its LPC demand during the crucial period, providing theoretical support for P diagnosis and regulation in cotton cultivation.

Antimicrobial combinations against Helicobacter pylori including benzoxadiazol-based flavodoxin inhibitors: in vitro characterization.

The antimicrobial resistance of Helicobacter pylori (Hp) currently poses a threat to available treatment regimens. Developing antimicrobial drugs targeting new bacterial targets is crucial, and one such class of drugs includes Hp-flavodoxin (Hp-fld) inhibitors that target an essential metabolic pathway in Hp. Our study demonstrated that combining these new drugs with conventional antibiotics used for Hp infection treatment prevented the regrowth observed with drugs used alone. Hp-fld inhibitors show promise as new drugs to be incorporated into the treatment of Hp infection, potentially reducing the development of resistance and shortening the treatment duration.

ACOX1-mediated peroxisomal fatty acid oxidation contributes to metabolic reprogramming and survival in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is still an incurable disease, with many patients developing resistance to conventional and targeted therapies. To better understand the physiology of CLL and facilitate the development of innovative treatment options, we examined specific metabolic features in the tumor CLL B-lymphocytes. We observed metabolic reprogramming, characterized by a high level of mitochondrial oxidative phosphorylation activity, a low glycolytic rate, and the presence of C2- to C6-carnitine end-products revealing an unexpected, essential role for peroxisomal fatty acid beta-oxidation (pFAO). Accordingly, downmodulation of ACOX1 (a rate-limiting pFAO enzyme overexpressed in CLL cells) was enough to shift the CLL cells' metabolism from lipids to a carbon- and amino-acid-based phenotype. Complete blockade of ACOX1 resulted in lipid droplet accumulation and caspase-dependent death in CLL cells, including those from individuals with poor cytogenetic and clinical prognostic factors. In a therapeutic translational approach, ACOX1 inhibition spared non-tumor blood cells from CLL patients but led to the death of circulating, BCR-stimulated CLL B-lymphocytes and CLL B-cells receiving pro-survival stromal signals. Furthermore, a combination of ACOX1 and BTK inhibitors had a synergistic killing effect. Overall, our results highlight a less-studied but essential metabolic pathway in CLL and pave the way towards the development of new, metabolism-based treatment options.

Metabolomics Analysis and Biochemical Profiling of Arsenic-Induced Metabolic Impairment and Disease Susceptibility.

Our study aimed to conduct a comprehensive biochemical profiling and metabolomics analysis to investigate the effects of arsenic-induced metabolic disorders, with a specific focus on disruptions in lipid metabolism, amino acid metabolism, and carbohydrate metabolism. Additionally, we sought to assess the therapeutic potential of resveratrol (RSV) as a remedy for arsenic-induced diabetes, using metformin (MF) as a standard drug for comparison. We measured the total arsenic content in mouse serum by employing inductively coupled plasma mass spectrometry (ICP-MS) after administering a 50-ppm solution of sodium arsenate (50 mg/L) in purified water. Our findings revealed a substantial increase in total arsenic content in the exposed group, with a mean value of 166.80 ± 8.52 ppb (p < 0.05). Furthermore, we investigated the impact of arsenic exposure on various biomarkers using enzyme-linked immunosorbent assay (ELISA) methods. Arsenic exposed mice exhibited significant hyperglycemia (p < 0.001) and elevated levels of homeostatic model assessment of insulin resistance (HOMA-IR), hemoglobin A1c (Hb1Ac), Inflammatory biomarkers as well as liver and kidney function biomarkers (p < 0.05). Additionally, the levels of crucial enzymes linked to carbohydrate metabolism, including α-glucosidase, hexokinase, and glucose-6-phosphatase (G6PS), and oxidative stress biomarkers, such as levels of glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), catalase, and superoxide dismutase (SOD), were significantly reduced in the arsenic-exposed group compared to the control group (p < 0.05). However, the level of MDA was significantly increased. Molecular analysis of gene expression indicated significant upregulation of key enzymes involved in lipid metabolism, such as carnitine palmitoyl-transferase-I (CPT-I), carnitine palmitoyl-transferase-II (CPT-II), lecithin-cholesterol acyltransferase (LCAT), and others. Additionally, alterations in gene expression related to glucose transporter-2 (GLUT-2), glucose-6-phosphatase (G6PC), and glucokinase (GK), associated with carbohydrate metabolism, were observed. Amino acid analysis revealed significant decreases in nine amino acids in arsenic-exposed mice. Metabolomics analysis identified disruptions in lipid metabolomes, amino acids, and arsenic metabolites, highlighting their involvement in essential metabolic pathways. Histopathological observations revealed significant changes in liver architecture, hepatocyte degeneration, and increased Kupffer cells in the livers of arsenic-exposed mice. In conclusion, these findings enhance our comprehension of the impact of environmental toxins on metabolic health and offer potential avenues for remedies against such disruptions.

Identification and Verification of Candidate miRNA Biomarkers with Application to Infection with Emiliania huxleyi Virus.

The interactions of Emiliania huxleyi and its specific lytic virus (EhV) have a profound influence on marine biogeochemical carbon-sulfur cycles and play a prominent role in global climate change. MicroRNAs (miRNAs) have emerged as promising candidates with extensive diagnostic potential due to their role in virus-host interactions. However, the application of miRNA signatures as diagnostic markers in marine viral infection has made limited progress. Based on our previous small-RNA sequencing data, one host miRNA biomarker that is upregulated in early infection and seven viral miRNA biomarkers that are upregulated in late infection were identified and verified using qRT-PCR and a receiver operating characteristic curve analysis in pure culture, mixed culture, and natural seawater culture. The host ehx-miR20-5p was able to significantly differentiate infection groups from the control in the middle (24 h post-infection, hpi) and late infection (48 hpi) phases, while seven virus-derived miRNA biomarkers could diagnose the early and late stages of EhV infection. Functional enrichment analysis showed that these miRNAs participated in numerous essential metabolic pathways, including gene transcription and translation, cell division-related pathways, protein-degradation-related processes, and lipid metabolism. Additionally, a dual-luciferase reporter assay confirmed the targeted relationship between a viral ehv-miR7-5p and the host dihydroceramide desaturase gene (hDCD). This finding suggests that the virus-derived miRNA has the ability to inhibit the host sphingolipid metabolism, which is a specific characteristic of EhV infection during the late stage. Our data revealed a cluster of potential miRNA biomarkers with significant regulatory functions that could be used to diagnose EhV infection, which has implications for assessing the infectious activity of EhV in a natural marine environment.

Subcellular proteomics for determining iron-limited remodeling of plastids in the model diatom Thalassiosira pseudonana (Bacillariophyta).

Diatoms are important primary producers in the world's oceans, yet their growth is constrained in large regions by low bioavailable iron (Fe). Low-Fe stress-induced limitation of primary production is due to requirements for Fe in components of essential metabolic pathways including photosynthesis and other chloroplast plastid functions. Studies have shown that under low-Fe stress, diatoms alter plastid-specific processes, including components of electron transport. These physiological changes suggest changes of protein content and in protein abundances within the diatom plastid. While in silico predictions provide putative information on plastid-localized proteins, knowledge of diatom plastid proteins remains limited in comparison to well-studied model photosynthetic organisms. To address this, we employed shotgun proteomics to investigate the proteome of subcellular plastid-enriched fractions from Thalassiosira pseudonana to gain a better understanding of how the plastid proteome is remodeled in response to Fe limitation. Using mass spectrometry-based peptide identification and quantification, we analyzed T. pseudonana grown under Fe-replete and -limiting conditions. Through these analyses, we inferred the relative quantities of each protein, revealing that Fe limitation regulates major metabolic pathways in the plastid, including the Calvin cycle. Additionally, we observed changes in the expression of light-harvesting proteins. In silico localization predictions of proteins identified in this plastid-enriched proteome allowed for an in-depth comparison of theoretical versus observed plastid-localization, providing evidence for the potential of additional protein import pathways into the diatom plastid.

Unravelling the genetic framework associated with grain quality and yield-related traits in maize (Zea mays L.).

Maize serves as a crucial nutrient reservoir for a significant portion of the global population. However, to effectively address the growing world population's hidden hunger, it is essential to focus on two key aspects: biofortification of maize and improving its yield potential through advanced breeding techniques. Moreover, the coordination of multiple targets within a single breeding program poses a complex challenge. This study compiled mapping studies conducted over the past decade, identifying quantitative trait loci associated with grain quality and yield related traits in maize. Meta-QTL analysis of 2,974 QTLs for 169 component traits (associated with quality and yield related traits) revealed 68 MQTLs across different genetic backgrounds and environments. Most of these MQTLs were further validated using the data from genome-wide association studies (GWAS). Further, ten MQTLs, referred to as breeding-friendly MQTLs (BF-MQTLs), with a significant phenotypic variation explained over 10% and confidence interval less than 2Mb, were shortlisted. BF-MQTLs were further used to identify potential candidate genes, including 59 genes encoding important proteins/products involved in essential metabolic pathways. Five BF-MQTLs associated with both quality and yield traits were also recommended to be utilized in future breeding programs. Synteny analysis with wheat and rice genomes revealed conserved regions across the genomes, indicating these hotspot regions as validated targets for developing biofortified, high-yielding maize varieties in future breeding programs. After validation, the identified candidate genes can also be utilized to effectively model the plant architecture and enhance desirable quality traits through various approaches such as marker-assisted breeding, genetic engineering, and genome editing.

The fitness cost of horizontally transferred and mutational antimicrobial resistance in Escherichia coli.

Antimicrobial resistance (AMR) in bacteria implies a tradeoff between the benefit of resistance under antimicrobial selection pressure and the incurred fitness cost in the absence of antimicrobials. The fitness cost of a resistance determinant is expected to depend on its genetic support, such as a chromosomal mutation or a plasmid acquisition, and on its impact on cell metabolism, such as an alteration in an essential metabolic pathway or the production of a new enzyme. To provide a global picture of the factors that influence AMR fitness cost, we conducted a systematic review and meta-analysis focused on a single species, Escherichia coli. By combining results from 46 high-quality studies in a multilevel meta-analysis framework, we find that the fitness cost of AMR is smaller when provided by horizontally transferable genes such as those encoding beta-lactamases, compared to mutations in core genes such as those involved in fluoroquinolone and rifampicin resistance. We observe that the accumulation of acquired AMR genes imposes a much smaller burden on the host cell than the accumulation of AMR mutations, and we provide quantitative estimates of the additional cost of a new gene or mutation. These findings highlight that gene acquisition is more efficient than the accumulation of mutations to evolve multidrug resistance, which can contribute to the observed dominance of horizontally transferred genes in the current AMR epidemic.

O-059 ntermetabolites of cholesterol synthesis in granulosa cell and oocyte aging

Abstract With the development of society, there has been a significant delay in female fertility and an increasing desire for childbearing among females of advanced maternal age in recent decades. Nevertheless, women over 35 years old have more risk of infertility and miscarriage compared to young women, due to decreased oocyte quality. These changes are mainly caused by meiotic defects, especially aneuploidy. Therefore, exploring the mechanisms of aging-related meiotic defects and aneuploidy in oocytes is of great significant for improving the pregnancy outcomes of aged women. Oocytes are enveloped by granulosa cells (GCs) to form follicles, which constitute the reproductive units in ovaries. GCs form a metabolic community with oocytes for oocyte growth and maturation. Numerous studies have reported that metabolic coupling between GCs and oocytes could affect the process of oocyte meiosis resumption, spindle assembly, and chromatin arrangement. Maternal aging is related to disruption of oocyte-GC interactions that leads to disorders of the oocyte meiotic process. Therefore, abnormal metabolic coupling between GCs and oocytes is an important reason for decreased quality in aged oocytes. To further clarify the metabolic coupling changes between GCs and oocytes during ovarian aging, we systematically characterized the dynamic changes in the overall transcriptomic landscapes of oocytes and GCs from young and aged mice throughout oocyte meiosis (during the growing oocyte [GO], full-grown oocyte [FGO], metaphase I [MI] oocyte [MIO], and metaphase II [MII] oocyte [MIIO] phases). Data revealed that the mevalonate (MVA) pathway, an essential metabolic pathway that uses acetyl-CoA to produce cholesterol and isoprenoids, was specifically highly expressed in GCs with the resumption of oocyte meiosis, and the expression of MVA pathway in GCs decreased with age. Atorvastatin-mediated inhibition of MVA metabolism in GCs decreased the first polar body extrusion (PBE) rate, and increased oocyte meiotic defects and aneuploidy in young cumulus-oocyte complexes (COCs). Further studies showed that the effect of the MVA pathway on oocyte meiosis was mainly regulated by protein isoprenoylation mediated by the inter-metabolites of cholesterol synthesis. Importantly, upregulation of the MVA pathway in aged GCs could ameliorate the depletion of ovarian reserve, decrease oocyte meiotic defects, and improve oocyte quality. In conclusion, metabolite in granulosa cell is an important factor determining the oocyte quality; MVA pathway in GCs is a critical regulator of meiotic maturation and euploidy in oocytes, and age-associated MVA pathway abnormalities contribute to oocyte meiotic defects and aneuploidy; Supplementation of inter-metabolites of cholesterol synthesis is a new therapeutic target for clinical intervention to improve oocyte quality. Trial registration number XXXX

Integrated transcriptomics and metabolomics analyses reveal key genes and essential metabolic pathways for the acquisition of cold tolerance during dormancy in apple

Low temperature is a primary abiotic stress that significantly affects plant growth and development in the cool regions of Northeast China. It is the primary limiting factor in the development of the global apple industry. Numerous studies have investigated the alterations in transcriptional metabolism under low-temperature stress in various plants. However, research on the role of dormancy in regulating differences in cold tolerance among apple varieties is limited. Therefore, this study conducted a comprehensive analysis of the physiological and biochemical indices, transcriptome, and metabolome of the cold-tolerant variety ‘Hanfu’ (HF) and cold-sensitive variety ‘Naganofuji 2′ (CF) during endodormancy and ecodormancy. Under the low-temperature treatment, the cross-section of a 1-year-old branch of CF exhibited more severe browning than HF did, and the relative electrolyte leakage value of CF was higher than that of HF. Transcriptomics and metabolomics analysis revealed the key pathways of apples in response to low-temperature stress. Functional enrichment analysis showed that hormone signal transduction pathways and amino acid metabolism-related pathways were significantly enriched during endodormancy and ecodormancy, and these pathways were considered an important way for apples to respond to low-temperature freezing injury. The galactose metabolism pathway was significantly enriched only during endodormancy, while the membrane lipid metabolism pathway was significantly enriched only during ecodormancy. A gene co-expression network was constructed using weighted gene correlation network analysis, and six modules with biological significance were selected from the network. Among them, genes encoding transcription factors such as DREB1C/CBF2, JUB1, CCCH, and VIP1 are hub genes in different modules, which can be used as candidate genes to explore the network regulation pathway of apple response to low-temperature stress. Overall, these findings help to elucidate the molecular mechanism underlying freeze-tolerance in apples and provide novel insights into the regulatory network involved in the response of apples to low-temperature stress during dormancy.

Alpha‐ketoglutarate Suppresses Tumor Growth of Diffuse Large B‐cell Lymphoma by Inducing Ferroptosis

Background: Metabolic reprogramming is one of the vital characteristics of cancers. Glutamine metabolism is an essential metabolic pathway in tumorigenesis, which provides a ready carbon and nitrogen source to support tumor biosynthesis, energy metabolism, and intracellular homeostasis. As a type of lymphoma with heterogeneity, diffuse large B-cell lymphoma (DLBCL) is characterized by severe metabolic vulnerability represents. Given the importance of glutamine metabolism in human cancer, revealing the characteristics of glutamine metabolism in DLBCL is expected to provide new pathogenesis and effective treatment strategies for patients. Methods: Peripheral blood serum of 120 DLBCL patients and 60 healthy donors were collected for untargeted metabolomics sequencing, followed by metabolic characteristics analysis. Subsequently, the biological functions and mechanisms of α-ketoglutarate (α-KG) were explored by RNA sequencing. Finally, ROS detection, ATP detection, and lipid peroxidation were used to verify the mechanism of α-KG-induced ferroptosis. Results: Untargeted metabolomics profiling revealed that the metabolic characteristics of DLBCL patients were significantly different from healthy controls. Among the differentially expressed metabolic pathways, glutamine metabolism accounted for the highest weight, suggesting the importance of glutamine metabolism in the tumorigenesis of DLBCL (Figure 1A). Notably, glutamate, glutamine, and α-KG were the critical metabolites in glutamine metabolism. Clinical data analysis identified that high glutamine concentrations and low decreased α-KG were associated with poor prognosis in DLBCL patients. Subsequently, dimethyl α-ketoglutarate (DM-αKG) was used to reverse glutamine metabolism and increase α-KG concentration. In vitro studies showed that DM-αKG treatment significantly inhibited cell proliferation of DLBCL cells both in vitro and in vivo (Figure 1B). In addition, DM-αKG induced non-apoptotic cell death phenotypes, represented by cell membrane swelling and LDH release. To further explore the functional mechanisms of α-KG in DLBCL, we performed RNA-sequencing in DM-αKG-treated DLBCL cells. As shown in enrichment analysis, DM-αKG treatment showed apparent dysfunction in the hypoxia-inducible factor pathway, oxidative stress response, and ferroptosis (Figure 1C). In particular, DM-αKG treatment promoted ROS release and lipid peroxidation, followed by impaired ATP production and decreased mitochondrial pathway expression. Moreover, differentially expressed genes analysis identified an increased expression of TP53 in the ferroptosis pathway, indicating the importance of TP53 in α-KG-induced ferroptosis (Figure 1D). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Metabolism No conflicts of interests pertinent to the abstract.

Dynamic protein deacetylation is a limited carbon source for acetyl-CoA–dependent metabolism

The ability of cells to store and rapidly mobilize energy reserves in response to nutrient availability is essential for survival. Breakdown of carbon stores produces acetyl-CoA (AcCoA), which fuels essential metabolic pathways and is also the acyl donor for protein lysine acetylation. Histones are abundant and highly acetylated proteins, accounting for 40% to 75% of cellular protein acetylation. Notably, histone acetylation is sensitive to AcCoA availability, and nutrient replete conditions induce a substantial accumulation of acetylation on histones. Deacetylation releases acetate, which can be recycled to AcCoA, suggesting that deacetylation could be mobilized as an AcCoA source to feed downstream metabolic processes under nutrient depletion. While the notion of histones as a metabolic reservoir has been frequently proposed, experimental evidence has been lacking. Therefore, to test this concept directly, we used acetate-dependent, ATP citrate lyase-deficient mouse embryonic fibroblasts (Acly-/- MEFs), and designed a pulse-chase experimental system to trace deacetylation-derived acetate and its incorporation into AcCoA. We found that dynamic protein deacetylation in Acly-/- MEFs contributed carbons to AcCoA and proximal downstream metabolites. However, deacetylation had no significant effect on acyl-CoA pool sizes, and even at maximal acetylation, deacetylation transiently supplied less than 10% of cellular AcCoA. Together, our data reveal that although histone acetylation is dynamic and nutrient-sensitive, its potential for maintaining cellular AcCoA-dependent metabolic pathways is limited compared to cellular demand.

NQO1/CPT1A promotes the progression of pancreatic adenocarcinoma via fatty acid oxidation.

NQO1, a cytosolic enzyme, is closely related to the progression of cancers and poor outcome of cancer patients. However, the molecular biological mechanism of NQO1 tumorigenicity in pancreatic adenocarcinoma (PAAD) has not been clearly understood. In this study, we demonstrate the molecular mechanism of NQO1 in PAAD proliferation, metastasis and fatty acid oxidation (FAO). Multiple databases and western blot analysis show that NQO1 is overexpressed in PAAD and associated with lymph node metastasis and shorter survival. Furthermore, in vitro and in vivo experiments reveal that overexpression of NQO1 improves tumor growth, metastasis and FAO in PAAD. Mechanistically, NQO1 is able to bind to carnitine palmitoyltransferase 1A (CPT1A), a key enzyme controlling FAO. Therefore, Co-IP and a series of rescue experiments demonstrate that NQO1 promotes PAAD progression via CPT1A-mediated FAO. Our findings identify CPT1A-dependent FAO as an essential metabolic pathway for NQO1 to promote the PAAD process. Targeting the NQO1/CPT1A/FAO axis in PAAD to attenuate proliferation and dissemination is a potential approach to promote a better antitumour effect and improve patient outcomes.

Transcriptional Mechanisms of Thermal Acclimation in Prochlorococcus.

Low temperature limits the growth and the distribution of the key oceanic primary producer Prochlorococcus, which does not proliferate above a latitude of ca. 40°. Yet, the molecular basis of thermal acclimation in this cyanobacterium remains unexplored. We analyzed the transcriptional response of the Prochlorococcus marinus strain MIT9301 in long-term acclimations and in natural Prochlorococcus populations along a temperature range enabling its growth (17 to 30°C). MIT9301 upregulated mechanisms of the global stress response at the temperature minimum (17°C) but maintained the expression levels of genes involved in essential metabolic pathways (e.g., ATP synthesis and carbon fixation) along the whole thermal niche. Notably, the declining growth of MIT9301 from the optimum to the minimum temperature was coincident with a transcriptional suppression of the photosynthetic apparatus and a dampening of its circadian expression patterns, indicating a loss in their regulatory capacity under cold conditions. Under warm conditions, the cellular transcript inventory of MIT9301 was strongly streamlined, which may also induce regulatory imbalances due to stochasticity in gene expression. The daytime transcriptional suppression of photosynthetic genes at low temperature was also observed in metatranscriptomic reads mapping to MIT9301 across the global ocean, implying that this molecular mechanism may be associated with the restricted distribution of Prochlorococcus to temperate zones. IMPORTANCE Prochlorococcus is a major marine primary producer with a global impact on atmospheric CO2 fixation. This cyanobacterium is widely distributed across the temperate ocean, but virtually absent at latitudes above 40° for yet unknown reasons. Temperature has been suggested as a major limiting factor, but the exact mechanisms behind Prochlorococcus thermal growth restriction remain unexplored. This study brings us closer to understanding how Prochlorococcus functions under challenging temperature conditions, by focusing on its transcriptional response after long-term acclimation from its optimum to its thermal thresholds. Our results show that the drop in Prochlorococcus growth rate under cold conditions was paralleled by a transcriptional suppression of the photosynthetic machinery during daytime and a loss in the organism's regulatory capacity to maintain circadian expression patterns. Notably, warm temperature induced a marked shrinkage of the organism's cellular transcript inventory, which may also induce regulatory imbalances in the future functioning of this cyanobacterium.

Decoding the crosstalk between mevalonate metabolism and T cell function.

The mevalonate pathway is an essential metabolic pathway in T cells regulating development, proliferation, survival, differentiation, and effector functions. The mevalonate pathway is a complex, branched pathway composed of many enzymes that ultimately generate cholesterol and nonsterol isoprenoids. T cells must tightly control metabolic flux through the branches of the mevalonate pathway to ensure sufficient isoprenoids and cholesterol are available to meet cellular demands. Unbalanced metabolite flux through the sterol or the nonsterol isoprenoid branch is metabolically inefficient and can have deleterious consequences for T cell fate and function. Accordingly, there is tight regulatory control over metabolic flux through the branches of this essential lipid synthetic pathway. In this review we provide an overview of how the branches of the mevalonate pathway are regulated in T cells and discuss our current understanding of the relationship between mevalonate metabolism, cholesterol homeostasis and T cell function.

Computer-aided genomic data analysis of drug-resistant Neisseria gonorrhoeae for the Identification of alternative therapeutic targets.

Neisseria gonorrhoeae is an emerging multidrug resistance pathogen that causes sexually transmitted infections in men and women. The N. gonorrhoeae has demonstrated an emerging antimicrobial resistance against reported antibiotics, hence fetching the attention of researchers to address this problem. The present in-silico study aimed to find putative novel drug and vaccine targets against N. gonorrhoeae infection by the application of bioinformatics approaches. Core genes set of 69N. gonorrhoeae strains was acquired from complete genome sequences. The essential and non-homologous metabolic pathway proteins of N. gonorrhoeae were identified. Moreover, different bioinformatics databases were used for the downstream analysis. The DrugBank database scanning identified 12 novel drug targets in the prioritized list. They were preferred as drug targets against this bacterium. A viable vaccine is unavailable so far against N. gonorrhoeae infection. In the current study, two outer-membrane proteins were prioritized as vaccine candidates via reverse vaccinology approach. The top lead B and T-cells overlapped epitopes were utilized to generate a chimeric vaccine construct combined with immune-modulating adjuvants, linkers, and PADRE sequences. The top ranked prioritized vaccine construct (V7) showed stable molecular interaction with human immune cell receptors as inferred during the molecular docking and MD simulation analyses. Considerable response for immune cells was interpreted by in-silico immune studies. Additional tentative validation is required to ensure the effectiveness of the prioritized vaccine construct against N. gonorrhoeae infection. The identified proteins can be used for further rational drug and vaccine designing to develop potential therapeutic entities against the multi-drug resistant N. gonorrhoeae.