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Reprogramming of energy metabolism is regarded as one of the hallmarks of cancer; in particular, oncogenic RAS has been shown to be a critical regulator of cancer metabolism. Recently, asparagine metabolism has been heavily investigated as a novel target for cancer treatment. For example, Knott et al. showed that asparagine bioavailability governs metastasis in a breast cancer model. Gwinn et al. reported the therapeutic vulnerability of asparagine biosynthesis in KRAS-driven non-small cell lung cancer. We previously reported that KRAS-mutated CRC cells can adapt to glutamine depletion through upregulation of asparagine synthetase (ASNS), an enzyme that synthesizes asparagine from aspartate. In our previous study, we assessed the efficacy of asparagine depletion using human cancer cell lines. In the present study, we evaluated the clinical relevance of asparagine depletion using a novel patient-derived spheroid xenograft (PDSX) mouse model. First, we examined ASNS expression in 38 spheroid lines and found that 12 lines (12/37, 32.4%) displayed high ASNS expression, whereas 26 lines (25/37, 67.6%) showed no ASNS expression. Next, to determine the role of asparagine metabolism in tumor growth, we established ASNS-knockdown spheroid lines using lentiviral short hairpin RNA constructs targeting ASNS. An in vitro cell proliferation assay demonstrated a significant decrease in cell proliferation upon asparagine depletion in the ASNS-knockdown spheroid lines, and this was not observed in the control spheroids lines. In addition, we examined asparagine inhibition with the anti-leukemia drug L-asparaginase (L-Asp) and observed a considerable reduction in cell proliferation at a low concentration (0.1 U/mL) in the ASNS-knockdown spheroid lines, whereas it exhibited limited inhibition of control spheroid lines at the same concentration. Finally, we used the PDSX model to assess the effects of asparagine depletion on tumor growth in vivo. The nude mice injected with ASNS-knockdown or control spheroid lines were administered with L-Asp once a day for 28 days. Surprisingly, in mice injected with ASNS-knockdown spheroids, the administration of L-Asp dramatically inhibited tumor engraftment. On the other hands, in mice injected with control spheroids, the administration of L-Asp had no effect on tumor growth inhibition at all. These results suggest that ASNS inhibition could be critical in targeting asparagine metabolism in cancers.

Asparagine synthetase deficiency is a rare inborn error of metabolism caused by a defect in ASNS , a gene encoding asparagine synthetase. It manifests with a severe neurological phenotype manifesting as severe developmental delay, congenital microcephaly, spasticity and refractory seizures. To date, nineteen patients from twelve unrelated families have been identified. Majority of the mutations are missense and nonsense mutations in homozygous or compound heterozygous state. We add another case from India which harbored a novel homozygous missense variation in exon 11 and compare the current case with previously reported cases.

Objective: Asparagine synthetase (ASNS) is an aminotransferase responsible for the biosynthesis of aspartate by using aspartic acid and glutamine. ASNS is highly expressed in fast-growing broilers, but few studies have reported the regulatory role of ASNS in muscle development.Methods: To explore the function of ASNS in chicken muscle development, the expression of ASNS in different chicken breeds and tissues were first performed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Then, using real-time quantitative RT-PCR, western blot, EdU assay, cell cycle assay and immunofluorescence, the effects of ASNS on the proliferation and differentiation of chicken skeletal muscle satellite cell (SMSC) were investigated. Finally, potential mechanisms by which ASNS influences chicken muscle fiber differentiation were identified through RNA-Seq.Results: The mRNA expression pattern of ASNS in muscles mirrors trends in muscle fiber cross-sectional area, average daily weight gain, and muscle weight across different breeds. ASNS knockdown inhibited SMSC proliferation, while overexpression showed the opposite. Moreover, ASNS attenuated SMSC differentiation by activating the adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) pathway. Additionally, 5-aminoimidazole4-carboxamide1-β-D-ribofuranoside (AICAR) treatment suppressed the cell differentiation induced by siRNA-ASNS. RNA-Seq identified 1,968 differentially expressed genes (DEGs) during chicken SMSC differentiation when overexpression ASNS. Gene ontology (GO) enrichment analysis revealed that these DEGs primarily participated in 8 biological processes, 8 cellular components, and 4 molecular functions. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis identified several significantly enriched signaling pathways, such as the JAK-STAT signaling pathway, tumor necrosis factor signaling pathway, toll-like receptor signaling pathway, and PI3K-Akt signaling pathway.Conclusion: ASNS promotes proliferation while inhibits the differentiation of chicken SMSCs. This study provides a theoretical basis for studying the role of ASNS in muscle development.

Key Clinical Message We add two novel variants to the existing mutation spectrum of ASNS gene. Loss of ASNS function should be suspected in newborns presenting with congenital microcephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. Acquisition and sequencing of stored newborn blood spot can be a valuable option when no biological samples are available from a deceased child. We add two novel variants to the existing mutation spectrum of ASNS gene. Loss of ASNS function should be suspected in newborns presenting with congenital microcephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. Acquisition and sequencing of stored newborn blood spot can be a valuable option when no biological samples are available from a deceased child.

The aim of this study was to investigate the role of ASN3-encoded asparagine synthetase (AS, EC 6.3.5.4) during vegetative growth, seed development and germination of Arabidopsis thaliana. Phenotypic analysis of knockout (asn3-1) and knockdown (asn3-2) T-DNA insertion mutants for the ASN3 gene (At5g10240) demonstrated wild-type contents of asparagine synthetase protein, chlorophyll and ammonium in green leaves at 35 days after sowing. In situ hybridization localized ASN3 mRNA to phloem companion cells of vasculature. Young siliques of the asn3-1 knockout line showed a decrease in asparagine but an increase in glutamate. The seeds of asn3-1 and asn3-2 displayed a wild-type nitrogen status expressed as total nitrogen content, indicating that the repression of ASN3 expression had only a limited effect on mature seeds. An analysis of amino acid labeling of seeds imbibed with (15N) ammonium for 24 h revealed that asn3-1 seeds contained 20% less total asparagine while 15N-labeled asparagine ((2-15N)asparagine, (4-15N)asparagine and (2,4-15N)asparagine) increased by 12% compared to wild-type seeds. The data indicate a fine regulation of asparagine synthesis and hydrolysis in Arabidopsis seeds.

Salt stress impairs nutrient metabolism in plant cells, leading to growth and yield penalties. However, the mechanism by which plants alter their nutrient metabolism processes in response to salt stress remains elusive. In this study, we identified and characterized the rice (Oryza sativa) rice salt tolerant 1 (rst1) mutant, which displayed improved salt tolerance and grain yield. Map-based cloning revealed that the gene RST1 encoded an auxin response factor (OsARF18). Molecular analyses showed that RST1 directly repressed the expression of the gene encoding asparagine synthetase 1 (OsAS1). Loss of RST1 function increased the expression of OsAS1 and improved nitrogen (N) utilization by promoting asparagine production and avoiding excess ammonium (NH₄⁺) accumulation. RST1 was undergoing directional selection during domestication. The superior haplotype RST1Hap III decreased its transcriptional repression activity and contributed to salt tolerance and grain weight. Together, our findings unravel a synergistic regulator of growth and salt tolerance associated with N metabolism and provide a new strategy for the development of tolerant cultivars.

Here, for the first time, a comprehensive transcriptomics study is presented of leaf senescence in the legume model Medicago truncatula, providing a broad overview of differentially expressed transcripts involved in this process. The cDNA-amplification fragment length polymorphism (AFLP) technique was used to identify > 500 genes, which were cloned and sorted into functional categories according to their gene ontology annotation. Comparison between the datasets of Arabidopsis and M. truncatula leaf senescence reveals common physiological events but differences in the nitrogen metabolism and in transcriptional regulation. In addition, it was observed that a minority of the genes regulated during leaf senescence were equally involved in other processes leading to programmed cell death, such as nodule senescence and nitric oxide signalling. This study provides a wide transcriptional profile for the comprehension of key events of leaf senescence in M. truncatula and highlights a possible regulative role for MADS box transcription factors in the terminal phases of the process.

Summary Free asparagine is the precursor for acrylamide, which forms during the baking, toasting and high‐temperature processing of foods made from wheat. In this study, CRISPR/Cas9 was used to knock out the asparagine synthetase gene, TaASN2, of wheat (Triticum aestivum) cv. Cadenza. A 4‐gRNA polycistronic gene was introduced into wheat embryos by particle bombardment and plants were regenerated. T1 plants derived from 11 of 14 T0 plants were shown to carry edits. Most edits were deletions (up to 173 base pairs), but there were also some single base pair insertions and substitutions. Editing continued beyond the T1 generation. Free asparagine concentrations in the grain of plants carrying edits in all six TaASN2 alleles (both alleles in each genome) were substantially reduced compared with wildtype, with one plant showing a more than 90 % reduction in the T2 seeds. A plant containing edits only in the A genome alleles showed a smaller reduction in free asparagine concentration in the grain, but the concentration was still lower than in wildtype. Free asparagine concentration in the edited plants was also reduced as a proportion of the free amino acid pool. Free asparagine concentration in the T3 seeds remained substantially lower in the edited lines than wildtype, although it was higher than in the T2 seeds, possibly due to stress. In contrast, the concentrations of free glutamine, glutamate and aspartate were all higher in the edited lines than wildtype. Low asparagine seeds showed poor germination but this could be overcome by exogenous application of asparagine.

The accumulation of senescent cells (SNCs) contributes to tissue dysfunction and age‐related diseases, creating an urgent need for effective senolytic strategies. We identified a metabolic vulnerability in SNCs characterized by marked downregulation of asparagine synthetase (ASNS), rendering them uniquely dependent on exogenous asparagine (Asn). This vulnerability was exploited through combined treatment with L‐asparaginase (ASNase) and autophagy inhibitors, which synergistically deplete Asn via complementary mechanisms: ASNase degrades extracellular Asn pools, while autophagy inhibition blocks intracellular protein recycling as an alternative Asn source. This dual approach induced selective synthetic lethality across multiple SNC types in vitro. In aged mice, the combination therapy significantly reduced SNC burden in diverse tissues, improved physiological function, and attenuated progression of age‐related conditions including osteoporosis, atherosclerosis, and non‐alcoholic fatty liver disease. Our findings establish concurrent targeting of extracellular and intracellular Asn supplies as a potent, selective senolytic strategy with broad therapeutic potential for age‐related disorders. A model of cellular asparagine source and AH treatment kills senescent cells effectively and selectively. In senescent cells, there is a defect in ASNS. Treatment with ASNase to deplete extracellular Asn, combined with HCQ to inhibit autophagy‐derived Asn, results in a severe depletion of Asn and subsequent senescent cell death.

Glutamine synthetase (GS) is central for ammonium assimilation and consists of cytosolic (GS1) and chloroplastic (GS2) isoenzymes. During plant ageing, GS2 protein decreases due to chloroplast degradation, and GS1 activity increases to support glutamine biosynthesis and N remobilization from senescing leaves. The role of the different Arabidopsis GS1 isoforms in nitrogen remobilization was examined using 15N tracing experiments. Only the gln1;1-gln1; 2-gln1; 3 triple-mutation affecting the three GLN1;1, GLN1;2, and GLN1; 3 genes significantly reduced N remobilization, total seed yield, individual seed weight, harvest index, and vegetative biomass. The triple-mutant accumulated a large amount of ammonium that could not be assimilated by GS1. Alternative ammonium assimilation through asparagine biosynthesis was increased and was related to higher ASN2 asparagine synthetase transcript levels. The GS2 transcript, protein, and activity levels were also increased to compensate for the lack of GS1-related glutamine biosynthesis. Localization of the different GLN1 genes showed that they were all expressed in the phloem companion cells but in veins of different order. Our results demonstrate that glutamine biosynthesis for N-remobilization occurs in veins of all orders (major and minor) in leaves, it is mainly catalysed by the three major GS1 isoforms (GLN1; 1, GLN1; 2, and GLN1; 3), and it is alternatively supported by AS2 in the veins and GS2 in the mesophyll cells.