Asparagine Synthase Research Articles

Clinical and genetic spectrum of 6 cases with asparagine synthetase deficiency

Objective: To explore the clinical and genetic characteristics of asparagine synthase deficiency. Methods: Case series studies. Retrospective analysis and summary of the clinical data of 6 cases with asparagine synthase deficiency who were diagnosed by genetic testing and admitted to the Third Affiliated Hospital of Zhengzhou University from May 2017 to April 2023 were analyzed retrospectively. The main clinical features, laboratory and imaging examination characteristics of the 6 cases were summarized, and the gene variation sites of them were analyzed. Results: All of the 6 cases were male, with onset ages ranging from 1 month to 1 year and 4 months. All of the 6 cases had cognitive and motor developmental delay, with 3 cases starting with developmental delay, 3 cases starting with convulsions and later experiencing developmental arrest or even regression. All of 6 cases had epilepsy, in whom 2 cases with severe microcephaly developed epileptic encephalopathy in the early stages of infancy with spasms as the main form of convulsions, 4 cases with mild or no microcephaly gradually evolved into convulsions with no fever after multiple febrile convulsions with focal seizures, tonic clonic seizures and tonic seizure as the main forms of convulsions. Three cases of 4 gradually developed into stagnation or even regression of development and ataxia after multiple convulsions with no fever. There were normal cranial imaging in 2 cases, dysplasia of the brains in 1 cases, frontal lobe apex accompanied by abnormal white matter signal in the frontal lobe and thin corpus callosum in 1 case, thin corpus callosum and abnormal lateral ventricular morphology in 1 case, and normal in early stage, but gradually developing into cerebellar atrophy at the age of 5 years and 9 months in 1 case. Two cases underwent visual evoked potential tests, the results of which were both abnormal. Three cases underwent auditory evoked potential examination, with 1 being normal and 2 being abnormal. All of 6 cases had variations in the asparagine synthase gene, with 2 deletion variations and 7 missense variations. The variations of 2 cases had not been reported so far, including c.1341_1343del and c.1283A>G, c.1165_1167del and c.1075G>A. The follow-up time ranged from 3 months to 53 months. Two cases who had severe microcephaly died in infancy, while the other 4 cases with mild or no microcephaly were in survival states until the follow-up days but the control of epilepsy was poor. Conclusions: Asparagine synthase deficiency has a certain degree of heterogeneity in clinical phenotype. Children with obvious microcephaly often present as severe cases, while children with mild or no microcephaly have relatively mild clinical manifestations. The variation of asparagine synthetase gene is mainly missense variation.

FLS2-RBOHD module regulates changes in the metabolome of Arabidopsis in response to abiotic stress.

Through crosstalk, FLAGELLIN SENSITIVE 2 (FLS2) and RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) are involved in regulating the homeostasis of cellular reactive oxygen species (ROS) and are linked to the metabolic response of plants toward both biotic and abiotic stress. In the present study, we examined the metabolome of Arabidopsis seedlings under drought and salt conditions to better understand the potential role of FLS2 and RBOHD-dependent signaling in the regulation of abiotic stress response. We identified common metabolites and genes that are regulated by FLS2 and RBOHD, and are involved in the response to drought and salt stress. Under drought conditions, D-aspartic acid and the expression of associated genes, such as ASPARAGINE SYNTHASE 2 (ASN2), increased in both fls2 and robed/f double mutants. The accumulation of amino acids, carbohydrates, and hormones, such as L-proline, D-ribose, and indoleacetaldehyde increased in both fls2 and rbohd/f double mutants under salt conditions, as did the expression of related genes, such as PROLINE IMINOPEPTIDASE, PHOSPHORIBOSYL PYROPHOSPHATE SYNTHASE 5, and NITRILASE 3. Collectively, these results indicate that the FLS2-RBOHD module regulates plant response to drought and salt stress through ROS signaling by adjusting the accumulation of metabolites and expression of genes related to metabolite synthesis.

Wild soybean salt tolerance metabolic model: Assessment of storage protein mobilization in cotyledons and C/N balance in the hypocotyl/root axis.

Salt stress has become one of the main factors limiting crop yield in recent years. The post-germinative growth is most sensitive to salt stress in soybean. In this study, cultivated and wild soybeans were used for an integrated metabonomics and transcriptomics analysis to determine whether wild soybean can resist salt stress by maintaining the mobilization of stored substances in cotyledons and the balance of carbon and nitrogen in the hypocotyl/root axis (HRA). Compared with wild soybean, the growth of cultivated soybean was significantly inhibited during the post-germinative growth period under salt stress. Integrating analysis found that the breakdown products of proteins, such as glutamate, glutamic acid, aspartic acid, and asparagine, increased significantly in wild soybean cotyledons. Asparagine synthase and fumarate hydratase genes and genes encoding HSP20 family proteins were specifically upregulated. In wild soybean HRA, levels of glutamic acid, aspartic acid, asparagine, citric acid, and succinic acid increased significantly, and the glutamate decarboxylase gene and the gene encoding carbonic anhydrase in nitrogen metabolism were significantly upregulated. The metabolic model indicated that wild soybean enhanced the decomposition of stored proteins and the transport of amino acids to the HRA in cotyledons and the GABA shunt to maintain carbon and nitrogen balance in the HRA to resist salt stress. This study provided a theoretical basis for cultivating salt-tolerant soybean varieties and opened opportunities for the development of sustainable agricultural practices.

Prediction of functional proteins associated with the gut microbiome of an adult population in Lagos State, Nigeria

BackgroundThe human gut microbiome is implicated in health and disease. Yet, its functions in healthy adults from a divergent population like those seen in Africa are quite unclear. We set to reveal the roles of the gut microbiome of a healthy rural and urban Yoruba population of Lagos State. MethodologySociodemographic and clinical data, and fecal samples were obtained from rural (n = 10) and urban (n = 10) participants. fecal DNA extraction was done using ZR Fecal DNA MiniPrep™ D6010 and metagenomic next generation sequencing performed on Illumina Miseq platform. Functional roles prediction and antibiotics resistance genes (ARGs) were done using the Clusters of Orthologous Groups (COG) and Comprehensive Antibiotic Resistance Database (CARD) tools, respectively. The potential link between phenotypes and COG abundance were inferred using principal component analysis (PCA). ResultsWe found 12 (urban) and 11 (rural) COG classes of proteins from the gut microbes. Class E protein which denotes amino acid transport and metabolism was unique to urban respondents and was dominated by 5-enolpyruvylshikimate-3-phosphate synthase (45%) and asparagine synthase (30%), implying a high protein-based diet. Class S was found in both groups. COG families of proteins revealed the presence of exo-beta-1,3-glucanase (31%), pyruvate-decarboxylase, and thiamine pyrophosphate-requiring enzymes (22%) among the rural respondents’ indicative of carbohydrate-rich diets. In addition, uncharacterized protein linked to hypothetical functions was found in both. The COG proteins clustered with female sex, BMI (body mass index) and BP (blood pressure) for urban while for the rural populace, it clustered with height, BMI, and BP. CARD analysis showed ARGs in both groups (urban 90%; rural 10%) and all were Escherichia coli 16S rRNA (rrsB) mutation conferring resistance to streptomycin with gene variant model A523C n/a to aminoglycoside antibiotics. ConclusionThe study defined the classes of proteins from gut microbes of urban and rural respondents and highlighted the presence of uncharacterized proteins linked to hypothetical and unknown functions. Differences seen among the predicted roles appear to be driven by diet, clinical and sociodemographic factors. The higher abundance of ARGs observed in the urban population poses a serious public health concern with regards to the spread of antibiotic resistance.

Comparative analysis reveals distinctive genomic features of Taiwan hot-spring cyanobacterium Thermosynechococcus sp. TA-1.

Thermosynechococcus is a genus of thermophilic unicellular cyanobacteria that dominates microbial mats in Asian non-acidic hot springs. These cyanobacteria are the major primary producers in their ecological niches and are promising sources of thermostable enzymes for biotechnology applications. To improve our understanding of these organisms, we conducted whole-genome sequencing of a novel strain for comparative analysis with other representatives in the same genus. This newly characterized strain, Thermosynechococcus sp. TA-1, was isolated from the Taian hot springs in Taiwan. Analyses based on average nucleotide identity (ANI) and genome-scale phylogeny suggested that TA-1 and another Taiwanese strain CL-1 belong to a novel species-level taxon. Two metagenome-assembled genomes (MAGs) originated from India represent the sister group, and Thermosynechococcus elongatus PKUAC-SCTE542 from China is the next closest lineage. All cultivated strains and MAGs from Japan form a separate monophyletic clade and could be classified into two species-level taxa. Intriguingly, although TA-1 and CL-1 share 97.0% ANI, the genome alignment identified at least 16 synteny breakpoints that are mostly associated with transposase genes, which illustrates the dynamic nature of their chromosomal evolution. Gene content comparisons identified multiple features distinct at species- or strain-level among these Thermosynechococcus representatives. Examples include genes involved in bicarbonate transportation, nitric oxide protection, urea utilization, kanamycin resistance, restriction-modification system, and chemotaxis. Moreover, we observed the insertion of type II inteins in multiple genes of the two Taiwanese strains and inferred putative horizontal transfer of an asparagine synthase gene (asnB) associated with exopolysaccharides gene cluster. Taken together, while previous work suggested that strains in this genus share a highly conserved genomic core and no clear genetic differentiation could be linked to environmental factors, we found that the overall pattern of gene content divergence is largely congruent with core genome phylogeny. However, it is difficult to distinguish between the roles of phylogenetic relatedness and geographic proximity in shaping the genetic differentiation. In conclusion, knowledge of the genomic differentiation among these strains provides valuable resources for future functional characterization.

Energy homeostasis mediated by the LcSnRK1α-LcbZIP1/3 signaling pathway modulates litchi fruit senescence.

Cellular energy status is a key factor deciding the switch-on of the senescence of horticultural crops. Despite the established significance of the conserved energy master regulator sucrose non-fermenting 1 (SNF1)-related protein kinase 1 (SnRK1) in plant development, its working mechanism and related signaling pathway in the regulation of fruit senescence remain enigmatic. Here, we demonstrate that energy deficit accelerates fruit senescence, whereas exogenous ATP treatment delays it. The transient suppression of LcSnRK1α in litchi (Litchi chinensis Sonn.) fruit inhibited the expression of energy metabolism-related genes, while its ectopic expression in tomato (Solanum lycopersicum) promoted ripening and a high energy level. Biochemical analyses revealed that LcSnRK1α interacted with and phosphorylated the transcription factors LcbZIP1 and LcbZIP3, which directly bound to the promoters to activate the expression of DARK-INDUCIBLE 10 (LcDIN10), ASPARAGINE SYNTHASE 1 (LcASN1), and ANTHOCYANIN SYNTHASE (LcANS), thereby fine-tuning the metabolic reprogramming to ensure energy and redox homeostasis. Altogether, these observations reveal a post-translational modification mechanism by which LcSnRK1α-mediated phosphorylation of LcbZIP1 and LcbZIP3 regulates the expression of metabolic reprogramming-related genes, consequently modulating litchi fruit senescence.

AsnB Mediates Amidation of Meso-Diaminopimelic Acid Residues in the Peptidoglycan of Listeria monocytogenes and Affects Bacterial Surface Properties and Host Cell Invasion

A mutant of Listeria monocytogenes ScottA with a transposon in the 5' untranslated region of the asnB gene was identified to be hypersensitive to the antimicrobial t-cinnamaldehyde. Here, we report the functional characterization of AsnB in peptidoglycan (PG) modification and intracellular infection. While AsnB of Listeria is annotated as a glutamine-dependent asparagine synthase, sequence alignment showed that this protein is closely related to a subset of homologs that catalyze the amidation of meso-diaminopimelic acid (mDAP) residues in the peptidoglycan of other bacterial species. Structural analysis of peptidoglycan from an asnB mutant, compared to that of isogenic wild-type (WT) and complemented mutant strains, confirmed that AsnB mediates mDAP amidation in L. monocytogenes. Deficiency in mDAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the asnB mutant, including formation of shorter but thicker cells, susceptibility to lysozyme, loss of flagellation and motility, and a strong reduction in biofilm formation. In addition, the mutant showed reduced invasion of human epithelial JEG-3 and Caco-2 cells. Analysis by immunofluorescence microscopy revealed that asnB inactivation abrogated the proper display at the listerial surface of the invasion protein InlA, which normally gets cross-linked to mDAP via its LPXTG motif. Together, this work shows that AsnB of L. monocytogenes, like several of its homologs in related Gram-positive bacteria, mediates the amidation of mDAP residues in the peptidoglycan and, in this way, affects several cell wall and cell surface-related properties. It also for the first time implicates the amidation of peptidoglycan mDAP residues in cell wall anchoring of InlA and in bacterial virulence.

The use of ribosome-nascent chain complex-seq to reveal the translated mRNA profile and the role of ASN1 in resistance to Verticillium wilt in cotton

We combined traditional mRNA-seq and RNC-seq together to reveal post-transcriptional regulation events impacting gene expression and interactions between the serious fungal pathogen Verticillium dahliae and a susceptible host, Gossypium hirsutum TM-1. After screening the differentially expressed and translated genes, V. dahliae infection was observed to influence gene transcription and translation in its host. Interestingly, the asparagine synthase (ASN1) gene transcripts increased significantly with the increase of infection time, while the rate of ASN1 protein accumulation in host TM-1 was distinctly lower than that in resistant hosts. We knocked down the ASN1 gene in resistant plants (ZZM2), and found that Verticillium-resistance was significantly reduced upon knockdown of ASN1. Our study revealed both transcriptional and post-transcriptional regulation of gene expression in TM-1 cotton plants infected by V. dahliae, and showed that ASN1 functions in the V. dahliae resistance process. These insights support breeding of disease resistance in cotton.

A Patent Review on the Use of L-Asparaginase in the Treatment of Acute Lymphocytic Leukemia.

L-asparaginase (L-asparagine amino hydrolase, E.C.3.5.1.1) is the most important chemotherapeutic drug used in treating Acute Lymphocytic Leukemia (ALL), decreasing blood asparagine rates causing apoptosis in tumor cells. However, pharmacological drugs cause several side effects making treatments difficult. Thus, searches for new sources of L-asparaginase or enzyme modifications focus on discovering new products to use in therapy. This article reviewed published patents from 2000 to 2020 related to the treatment of ALL using L-asparaginase. Many organisms have been shown as potential viable L-asparaginase producers for use in the treatment of ALL. However, this patent review shows that few of these organisms are gaining attention to becoming bioproducts for the market. It is expected that drugs in the testing phase and patents related to the treatment of ALL and other cancers will become real products. Besides, a treatment using an amino acid depletion approach, now referring to asparagine, altogether with a compound that directly interferes with the expression of the asparagine synthase gene, is more suitable for the treatment of ALL and possibly to other cancers.

Synthesis of L-asparagine Catalyzed by a Novel Asparagine Synthase Coupled With an ATP Regeneration System

Compared with low-yield extraction from plants and environmentally unfriendly chemical synthesis, biocatalysis by asparagine synthetase (AS) for preparation of L-asparagine (L-Asn) has become a potential synthetic method. However, low enzyme activity of AS and high cost of ATP in this reaction restricts the large-scale preparation of L-Asn by biocatalysis. In this study, gene mining strategy was used to search for novel AS with high enzyme activity by expressing them in Escherichia coli BL21 (DE3) or Bacillus subtilis WB600. The obtained LsaAS-A was determined for its enzymatic properties and used for subsequent preparation of L-Asn. In order to reduce the use of ATP, a class III polyphosphate kinase 2 from Deinococcus ficus (DfiPPK2-Ⅲ) was cloned and expressed in E. coli BL21 (DE3), Rosetta (DE3) or RosettagamiB (DE3) for ATP regeneration. A coupling reaction system including whole cells expressing LsaAS-A and DfiPPK2-Ⅲ was constructed to prepare L-Asn from L-aspartic acid (L-Asp). Batch catalytic experiments showed that sodium hexametaphosphate (>60 mmol L−1) and L-Asp (>100 mmol L−1) could inhibit the synthesis of L-Asn. Under fed-batch mode, L-Asn yield reached 90.15% with twice feeding of sodium hexametaphosphate. A final concentration of 218.26 mmol L−1 L-Asn with a yield of 64.19% was obtained when L-Asp and sodium hexametaphosphate were fed simultaneously.

Early transcriptional responses in Solanum peruvianum and Solanum lycopersicum account for different acclimation processes during water scarcity events

Cultivated tomato Solanum lycopersicum (Slyc) is sensitive to water shortages, while its wild relative Solanum peruvianum L. (Sper), an herbaceous perennial small shrub, can grow under water scarcity and soil salinity environments. Plastic Sper modifies the plant architecture when suffering from drought, which is mediated by the replacement of leaf organs, among other changes. The early events that trigger acclimation and improve these morphological traits are unknown. In this study, a physiological and transcriptomic approach was used to understand the processes that differentiate the response in Slyc and Sper in the context of acclimation to stress and future consequences for plant architecture. In this regard, moderate (MD) and severe drought (SD) were imposed, mediating PEG treatments. The results showed a reduction in water and osmotic potential during stress, which correlated with the upregulation of sugar and proline metabolism-related genes. Additionally, the senescence-related genes FTSH6 protease and asparagine synthase were highly induced in both species. However, GO categories such as “protein ubiquitination” or “endopeptidase inhibitor activity” were differentially enriched in Sper and Slyc, respectively. Genes related to polyamine biosynthesis were induced, while several cyclins and kinetin were downregulated in Sper under drought treatments. Repression of photosynthesis-related genes was correlated with a higher reduction in the electron transport rate in Slyc than in Sper. Additionally, transcription factors from the ERF, WRKY and NAC families were commonly induced in Sper. Although some similar responses were induced in both species under drought stress, many important changes were detected to be differentially induced. This suggests that different pathways dictate the strategies to address the early response to drought and the consequent episodes in the acclimation process in both tomato species.

Selected Articles from This Issue

Glioblastomas (GBM) are highly invasive brain tumors that can spread rapidly throughout the brain. GBM are marked by a high rate of disease recurrence after therap. by virtue of the self-renewal capacity of glioma stem cells (GSCs), which are highly heterogeneous. Here, Thomas and colleagues identify a subset of GSCs bearing copy number alterations of the ASNS gene, leading to elevated expression of asparagine synthase. ASNS-high GSCs bore distinct metabolic features from other GSCs, including increased capacity for both glycolysis and oxidative phosphorylation and the ability to switch between them as needed in response to environmental cues. This metabolic plasticity allowed for rapid dissemination of ASNS-high GSCs throughout the brain tissue and a marked resistance to oxidative stress, rendering these cells de novo resistant to radiation. These findings nominate ASNS as a potential therapeutic target for GBM, which could restrict the pathogenicity of GSCs and enhance the efficacy of standard treatment modalities.Inhibitors of poly (ADP-ribose) polymerase (PARPi) are known to be effective in treating a range of tumor types, particularly as a synthetic-lethal approach for tumors with defects in the homologous recombination DNA repair pathway. However, dose-limiting toxicities, particularly hematological toxicities, can cause early cessation of treatment and loss of therapeutic benefit. Here, using a CRISPR/Cas9 screen, Xu and colleagues identify the CHK2-p53 pathway as a key mediator of hematological toxicity in response to the clinically approved PARPi agent, olaparib. CHK2 was required for efficient activation of p53 in response to PARPi in blood cells, and genetic approaches that blocked this connection allowed cells to better tolerate olaparib treatment. Concurrent inhibition of CHK2 alongside olaparib emulated this protective effect, but importantly did not affect PARPi-mediated killing of ovarian cancer cell lines. If this finding extends to people, then these data nominate CHK2 inhibition as a potential target to spare patients from hematological adverse effects in response to PARPi, which could broaden the window of therapeutic benefit for these agents.Analogous to epigenetic control of gene expression through histone methylation, epitranscriptomic modifications such as N6-methyladenosine (m6A) function to modulate RNA stability, processing, and translation. In this study, Cotter and colleagues profile the activity of METTL3, a key constituent of the methyltransferase complex that catalyzes m6A modifications, and its role in establishing the pathologic transcriptome observed in metastatic castration-resistant prostate cancer (mCRPC). The authors observe that ablation of METTL3—and by extension, m6A modifications—is associated with resistance to androgen receptor signaling inhibitors, a key therapeutic modality in the clinical management of prostate cancer. This effect was shown to be mediated independently of the androgen signaling pathway, and instead relied on activation of a gastrointestinal-specific transcriptional signature driven by the expression of the nuclear receptor NR5A2/LRH-1. Taken together, these observations elucidate a novel mechanism by which epitranscriptomic m6A modifications may play a role in establishing the mCRp. transcriptome and in promoting disease progression.Pancreatic ductal adenocarcinoma (PDAC) patients often present with metastatic disease and face a dismal prognosis. Because of the high rate of metastasis observed in PDAC tumors, systemic therapies are frequently the only viable option for clinical management; however, PDAC lacks an effective armamentarium of targeted interventions, and tumors quickly develop resistance to standard chemotherapeutic regimens. Using single-cell sequencing of mouse PDAC tumors, Du and colleagues identify the receptor tyrosine kinase AXL as a mediator of PDAC progression and metastatic spread. The authors demonstrate that AXL is overexpressed in PDAC tumor cells with a mesenchymal phenotype, and that its activity promotes and is required for epithelial-mesenchymal transition and metastasis in vivo. Concordantly, PDAC tumors that developed in AXL-deficient mice were less aggressive, better differentiated, and more sensitive to gemcitabine chemotherapy than PDAC in mice bearing wild-type AXL. These data suggest that targeting AXL expression and/or activity may present an opportunity to suppress or treat PDAC progression.

Abstract 2857: Asparagine synthetase regulate β-catenin dependent lung cancer metastasis

Abstract Objective: Lung cancer was a malignant tumor with high morbidity and mortality. The lead cause of lung cancer-related death was metastasis. Asparagine synthetase (ASNS) that was a key enzyme to convert glutamine to asparagine played important role in cell metabolism. Based on previous metabolomics data, the high level of asparagine synthase was significantly correlated with tumor metastasis. The purpose of this study was to investigate the role of asparagine synthase in lung cancer metastasis, furthermore for anti-cancer target therapy. Method: For pursuing high ASNS correlated to metastasis, a cohort were collected from Xiangya hospital. And selected paired lung cancer cell lines were used for discovering potential molecular interactions. Importantly, some key molecules or pathways were found by cell biology study and bioinformatics analysis. In the part of cellular and molecule study, Transwell, scratch test, Western blot, immunofluorescence, immunohistochemistry and co-immunoprecipitation experiments were used. The results of the inquiry were validated by using a mouse metastasis model. Results: With provided from Xiangya hospital and TCGA cohort, we could validate high ASNS had relations to patient's metastasis and survival. In part of cell biology study, high wild-type ASNS could promote lung cancer cell metastasis and proliferation in vivo and vitro. Overexpression of amidotransferase-dysfunction ASNS had partially potential to drive metastasis but have nothing to increase cell proliferation. Furthermore, Wnt/β-catenin cascades were responsible for tumor mestasis among wild-type and amidotransferase-mutated type groups. The accumulation and nuclear location of β-catenin definitely depend on high ASNS expression even with amidotransferase mutation. With processing, ASNS possible interacted with key proteins located in Wnt/β-catenin signaling pathway to mediate signal conduction. But the activation of ASNS amidotransferase was necessary to promoted tumor proliferation. Conclusion: ASNS promoted metastasis by mediating Wnt/β-catenin signaling conduction even without ASNS amidotransferase activity, while cell proliferation was required for amidotransferase function. Maybe dual inhibition of ASNS and Wnt/β-catenin would be an effective target for anti-tumor metastasis and proliferation. Key Words: ASNS, metastasis, lung cancer, β-catenin Citation Format: Dong-Jing Cai, Cheng-Ping Hu, Li li, Zi-Yu Zhang, Yuan-Mao Li, Min Li. Asparagine synthetase regulate β-catenin dependent lung cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2857.

Amino Acid Metabolomic Profiles in Bovine Mammary Epithelial Cells under Essential Amino Acid Restriction.

Simple SummaryCells of the mammary gland obtain their necessary nutrients from the blood to produce milk components, such as casein. To achieve higher productivity, cows are excessively supplemented, thus generating a higher cost of production and affecting the environment. Therefore, this triggers the need for a reduction in the supplementation of essential amino acids without affecting the milk composition. The present in vitro study shows that, through homeostatic and homeorhetic processes, cells have the ability to maintain stable casein levels despite decreasing the percentage of essential amino acids (EAAs) supplied. These findings could contribute to the proposal of more efficient nutritional strategies at lower environmental and economic costs.Mammary epithelial cells (MECs) in culture are a useful model for elucidating mammary gland metabolism and changes that occur under different nutrient disponibility. MECs were exposed to different treatments: 100% EAA for 8 h and 24 h restriction (R); 2% EAA for 8 h and 24 h R; 2% EAA for 8 h and 24 h + 100% EAA for 8 h and 24 h restriction + re-feeding (R + RF). Western blotting and protein quantification was performed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) software identified the amino acids (AAs) and signaling pathways. The chi-squared test, multiple classification analysis, and analysis of variance were used for the purification and identification of data. Intracellular casein levels were not affected. The KEGG analysis revealed that the important pathways of metabolism of AAs, which were involved in processes related to metabolism and biosynthesis of phenylalanine, tyrosine, and tryptophan (fumarate, acetyl-CoA, and tricarboxylic acid (TCA) cycle), were affected by both R and R + RF treatments, mainly through the glutamic-oxaloacetic transaminase-2 enzyme. Additionally, metabolic processes mediated by the mitochondrial malate dehydrogenase, S-adenosylmethionine synthetase, and asparagine synthase proteins positively regulated the carbohydrate pathway, pyruvate, and TCA cycles, as well as the metabolism of alanine, aspartate, and glutamate metabolism (carbohydrate and TCA cycle). We hypothesized that MECs have the capacity to utilize alternative pathways that ensure the availability of substrates for composing milk proteins.

The MarR-Type Regulator PA3458 Is Involved in Osmoadaptation Control in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a facultative human pathogen, causing acute and chronic infections that are especially dangerous for immunocompromised patients. The eradication of P. aeruginosa is difficult due to its intrinsic antibiotic resistance mechanisms, high adaptability, and genetic plasticity. The bacterium possesses multilevel regulatory systems engaging a huge repertoire of transcriptional regulators (TRs). Among these, the MarR family encompasses a number of proteins, mainly acting as repressors, which are involved in response to various environmental signals. In this work, we aimed to decipher the role of PA3458, a putative MarR-type TR from P. aeruginosa. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3458 showed changes in the mRNA level of 133 genes; among them, 100 were down-regulated, suggesting the repressor function of PA3458. Concomitantly, ChIP-seq analysis identified more than 300 PA3458 binding sites in P. aeruginosa. The PA3458 regulon encompasses genes involved in stress response, including the PA3459–PA3461 operon, which is divergent to PA3458. This operon encodes an asparagine synthase, a GNAT-family acetyltransferase, and a glutamyl aminopeptidase engaged in the production of N-acetylglutaminylglutamine amide (NAGGN), which is a potent bacterial osmoprotectant. We showed that PA3458-mediated control of PA3459–PA3461 expression is required for the adaptation of P. aeruginosa growth in high osmolarity. Overall, our data indicate that PA3458 plays a role in osmoadaptation control in P. aeruginosa.

Effects of Rhizophagus intraradices and Rhizobium trifolii on growth and N assimilation of white clover

The inoculation response of single arbuscular mycorrhiza fungi (AMF) or rhizobia (Rh) in relation to nitrogen (N) acquisition of plants is well established, while the combined effect of both AMF and Rh is poorly known with regard to N assimilation for changes in amino acids of white clover (Trifolium repens). A pot study was carried out to evaluate the effect of single versus dual inoculation of AMF (Rhizophagus intraradices) and rhizobium (Rhizobium trifolii) on plant growth, leaf and root N contents, root amino acid contents, and root N-related enzyme activities in white clover. One hundred days after inoculations, Rh inoculation significantly stimulated the root colonization by R. intraradices. A single inoculation of AMF or Rh improved the plant growth (biomass production, root projected area, and root volume), root N acquisition, and dual inoculation of AMF and Rh further expanded some of these positive effects on root projected area and root N contents than single inoculation. All the inoculations notably increased activities of root asparagines synthase, nitrate reductase, and glutamate synthase, whilst dual inoculation displayed a much stronger effect in asparagine synthase activity than single inoculation. Single Rh treatment increased root glutamate and proline content, single AMF inoculation induced an increase in glutamate, aspartate, arginine, and ornithine content, while dual inoculation stimulated the accumulation of aspartate and proline. These results suggested the cooperation between the AMF and Rh inoculations, which magnified the positive effect on partly N metabolites and N-assimilation relevant enzymes of white clover.

Acceleration of biofilm formation in start-up of sequencing batch biofilm reactor using carriers immobilized with Pseudomonas stutzeri strain XL-2

P. stutzeri strain XL-2 initially immobilized on polypropylene carriers accelerated the biofilm formation in start-up of sequencing batch biofilm reactor (SBBR) (denoted R1). The biofilm formation in R1 was approximately completed in 36 days, which was shorter than that of 48 days in an identical SBBR (denoted R2) without strain XL-2. Meanwhile, R1 presented a rapid stabilization of NH4+-N and TN removal to 81.7% and 72.4% respectively. Surface plasmon resonance demonstrated that strain XL-2 enhanced the initial adhesion of carrier surface due to the production of extracellular polymeric substances (EPS), which made it easier for other EPS-producing strains, such as Thauera and Flavobacterium, to adhere to the carriers. PICRUSt revealed that biofilm in R1 presented relatively higher activity of EPS biosynthesis enzymes (glycosyltransferase and asparagine synthase). Thus, high EPS content was obtained due to the application of carriers immobilized with strain XL-2 and finally promoted the biofilm formation.

Hydrogen Sulfide Promotes Nodulation and Nitrogen Fixation in Soybean–Rhizobia Symbiotic System

The rhizobium-legume symbiotic system is crucial for nitrogen cycle balance in agriculture. Hydrogen sulfide (H2S), a gaseous signaling molecule, may regulate various physiological processes in plants. However, whether H2S has regulatory effect in this symbiotic system remains unknown. Herein, we investigated the possible role of H2S in the symbiosis between soybean (Glycine max) and rhizobium (Sinorhizobium fredii). Our results demonstrated that an exogenous H2S donor (sodium hydrosulfide [NaHS]) treatment promoted soybean growth, nodulation, and nitrogenase (Nase) activity. Western blotting analysis revealed that the abundance of Nase component nifH was increased by NaHS treatment in nodules. Quantitative real-time polymerase chain reaction data showed that NaHS treatment upregulated the expressions of symbiosis-related genes nodA, nodC, and nodD of S. fredii. In addition, expression of soybean nodulation marker genes, including early nodulin 40 (GmENOD40), ERF required for nodulation (GmERN), nodulation signaling pathway 2b (GmNSP2b), and nodulation inception genes (GmNIN1a, GmNIN2a, and GmNIN2b), were upregulated. Moreover, the expressions of glutamate synthase (GmGOGAT), asparagine synthase (GmAS), nitrite reductase (GmNiR), ammonia transporter (GmSAT1), leghemoglobin (GmLb), and nifH involved in nitrogen metabolism were upregulated in NaHS-treated soybean roots and nodules. Together, our results suggested that H2S may act as a positive signaling molecule in the soybean-rhizobia symbiotic system and enhance the system's nitrogen fixation ability.

Nitrogen utilization of potato genotypes and expression analysis of genes controlling nitrogen assimilation

IntroductionSignificant differences in nitrogen use efficiency (NUE) were detected previously among potato cultivars. Exploration of the genetic background may facilitate the breeding of cultivars with highly effective nitrogen use.MethodsExpression of NUE genes was analyzed at three different N-supply levels in five potato genotypes. Correlations of NUE gene expressions and agronomical parameters with such indices as the nitrogen uptake efficiency, nitrogen utilization efficiency, NUE, and harvest indices were analyzed.ResultsThe correlations between expression level of the nitrate-reductase, nitrite-reductase, ammonium transporter, and asparagine synthase genes and different agronomically important parameters were detected.DiscussionOur results contribute to more rational, genotype-dependent nitrogen use in potato production and have relevance in breeding of new cultivars with better nitrogen utilization, as well as in production of seed potato.

Increased biomass accumulation in maize grown in mixed nitrogen supply is mediated by auxin synthesis.

The use of mixed nitrate and ammonium as a nitrogen source can improve plant growth. Here, we used metabolomics and transcriptomics to study the underlying mechanisms. Maize plants were grown hydroponically in the presence of three forms of nitrogen (nitrate alone, 75%/25% nitrate/ammonium, and ammonium alone). Plants grown with mixed nitrogen had a higher photosynthetic rate than those supplied only with nitrate, and had the highest leaf area and shoot and root biomass among the three nitrogen treatments. In shoot and root, the concentration of nitrogenous compounds (ammonium, glutamine, and asparagine) and carbohydrates (sucrose, glucose, and fructose) in plants with a mixed nitrogen supply was higher than that with nitrate supply, but lower than that with ammonium supply. The activity of the related enzymes (glutamate synthase, asparagine synthase, phosphoenolpyruvate carboxylase, invertase, and ADP-glucose pyrophosphorylase) changed accordingly. Specifically, the mixed nitrogen source enhanced auxin synthesis via the shikimic acid pathway, as indicated by the higher levels of phosphoenolpyruvate and tryptophan compared with the other two treatments. The expression of corresponding genes involving auxin synthesis and response was up-regulated. Supply of only ammonium resulted in high levels of glutamine and asparagine, starch, and trehalose hexaphosphate. We conclude that, in addition to increased photosynthesis, mixed nitrogen supply enhances leaf growth via increasing auxin synthesis to build a large sink for carbon and nitrogen utilization, which, in turn, facilitates further carbon assimilation and nitrogen uptake.