Influence of Asparaginase on Acrylamide Content, Color, and Texture in Oat, Corn, and Rice Cookies.

The safety of cereal-based baked goods can be compromised by acrylamide, a processing contaminant and class 2A carcinogen. One method to prevent acrylamide formation is by converting asparagine to aspartic acid using asparaginases. Four different asparaginases were tested using two dough incubation temperatures and dosages for oat, corn, and rice cookies. To evaluate the impact of asparaginases on product quality, color and texture were measured. Acrylamide was reduced by up to 97, 95, and 92% for oat, corn, and rice cookies, respectively, compared to the control. Asparaginase treatment resulted in minor changes in color and texture. There was a strong correlation between acrylamide concentrations in cookies and the free asparagine content of the flour. By minimizing the formation of acrylamide while maintaining product quality, the use of asparaginases offers a promising approach to enhancing food safety standards and protecting public health, potentially influencing regulatory guidelines and consumer preferences.

Integrating machine learning and multi-omics analysis to develop an asparagine metabolism immunity index for improving clinical outcome and drug sensitivity in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a malignancy affecting the respiratory system. Most patients are diagnosed with advanced or metastatic lung cancer due to the fact that most of their clinical symptoms are insidious, resulting in a bleak prognosis. Given that abnormal reprogramming of asparagine metabolism (AM) has emerged as an emerging therapeutic target for anti-tumor therapy. However, the clinical significance of abnormal reprogramming of AM in LUAD patients is unclear. In this study, we collected 864 asparagine metabolism-related genes (AMGs) and used a machine-learning computational framework to develop an asparagine metabolism immunity index (AMII) for LUAD patients. Through the utilization of median AMII scores, LUAD patients were segregated into either a low-AMII group or a high-AMII group. We observed outstanding performance of AMII in predicting survival prognosis in LUAD patients in the TCGA-LUAD cohort and in three externally independently validated GEO cohorts (GSE72094, GSE37745, and GSE30219), and poorer prognosis for LUAD patients in the high-AMII group. The results of univariate and multivariate analyses showed that AMII can be used as an independent risk factor for LUAD patients. In addition, the results of C-index analysis and decision analysis showed that AMII-based nomograms had a robust performance in terms of accuracy of prognostic prediction and net clinical benefit in patients with LUAD. Excitingly, LUAD patients in the low-AMII group were more sensitive to commonly used chemotherapeutic drugs. Consequently, AMII is expected to be a novel diagnostic tool for clinical classification, providing valuable insights for clinical decision-making and personalized management of LUAD patients.

Identifying targetable metabolic dependencies across colorectal cancer progression.

Colorectal cancer (CRC) is a multi-stage process initiated through the formation of a benign adenoma, progressing to an invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with disease progression. As such, targeting cancer cell metabolism is a promising therapeutic avenue in CRC. However, to identify tractable nodes of metabolic vulnerability specific to CRC stage, we must understand how metabolism changes during CRC development. Here, we use a unique model system - comprising human early adenoma to late adenocarcinoma. We show that adenoma cells transition to elevated glycolysis at the early stages of tumour progression but maintain oxidative metabolism. Progressed adenocarcinoma cells rely more on glutamine-derived carbon to fuel the TCA cycle, whereas glycolysis and TCA cycle activity remain tightly coupled in early adenoma cells. Adenocarcinoma cells are more flexible with respect to fuel source, enabling them to proliferate in nutrient-poor environments. Despite this plasticity, we identify asparagine (ASN) synthesis as a node of metabolic vulnerability in late-stage adenocarcinoma cells. We show that loss of asparagine synthetase (ASNS) blocks their proliferation, whereas early adenoma cells are largely resistant to ASN deprivation. Mechanistically, we show that late-stage adenocarcinoma cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and oxidative capacity. Resistance to ASNS loss in early adenoma cells is likely due to a feedback loop, absent in late-stage cells, allowing them to sense and regulate ASN levels and supplement ASN by autophagy. Together, our study defines metabolic changes during CRC development and highlights ASN synthesis as a targetable metabolic vulnerability in later stage disease.

Serum D-asparagine concentration adjusted for eGFR could serve as a novel screening tool for urothelial carcinoma.

The sensitivity of currently available screening tools for urothelial carcinoma (UC) remains unsatisfactory particularly at early stages. Hence, we aimed to establish a novel blood-based screening tool for urothelial carcinoma. We measured serum d-amino acid levels in 108 and 192 patients with and without UC individuals in the derivation cohort, and 15 and 25 patients with and without UC in the validation cohort. Serum d-asparagine levels were significantly higher in patients with UC than in those without UC (p < 0.0001). We developed a novel screening equation for the diagnosis of urothelial carcinoma using d-asparagine in serum and estimated the glomerular filtration rate (eGFR). Serum d-asparagine levels adjusted for eGFR exhibited high performance in the diagnosis of UC (AUC-ROC, 0.869; sensitivity, 80.6 %; specificity, 82.7 %), even in early-stage UC (AUC-ROC: 0.859, sensitivity: 83.3 %, specificity: 82.3 %), which were previously misdiagnosed via urinary occult blood or urine cytology. This established strategy combined with urinary occult blood, improves diagnostic ability (sensitivity: 93.7 %, specificity: 70.1 %).

L-asparaginase-mediated pH shift and carbon dot fluorescence modulation: A sensitive ratiometric method for quantifying L-asparagine in diverse potato varieties under variable storage conditions.

This study presents a novel and selective method for the determination of l-asparagine in diverse potato varieties under various storage conditions. L-asparagine levels serve as a crucial indicator for acrylamide formation, a hazardous substance in processed potato products. The fluorometric method utilized blue-emitting CDs (B-CDs), orange-emitting CDs (O-CDs), and the enzyme L-asparaginase for ratiometric detection of L-asparagine. Upon enzymatic hydrolysis of L-asparagine by L-asparaginase, liberated ammonia induced a pH increase in the reaction medium. This pH shift enhanced the fluorescence of B-CDs while simultaneously decreasing that of O-CDs, enabling sensitive and selective L-asparagine quantification. Comprehensive characterization of the CDs was performed using various spectroscopic techniques and transmission electron microscopy. The method demonstrated excellent sensitivity (LOD = 0.31 µM) and a wide linear range (1.0-50.0 µM). When the method was applied to potato samples, high recovery values (98.00-100.33 %) with low relative standard deviations (RSDs) were achieved, confirming the accuracy and precision of the method. The approach was employed to determine L-asparagine levels in three potato varieties (Lady Rosetta, Spunta, and Nicola) under different storage temperatures and durations. This method provides a valuable tool for monitoring L-asparagine content in potatoes, potentially aiding in the mitigation of acrylamide formation during processing. The robust performance and simplicity of the proposed technique make it suitable for routine analysis in both research and industrial applications within the potato industry.

Asparagine as a Signal for Glutamine Sufficiency via Asparagine Synthetase: A Fresh Evidence-Based Framework in Physiology and Oncology.

Among the twenty proteinogenic amino acids, glutamine and asparagine represent a unique cohort in containing a terminal amide in their side chain, and share a direct metabolic relationship, with glutamine generating asparagine through the ATP-dependent asparagine synthetase (ASNS) reaction. Circulating glutamine levels and metabolic flux through cells and tissues greatly exceed those for asparagine, and "glutamine addiction" in cancer has likewise received considerable attention. However, historic and recent evidence collectively suggest that in spite of its modest presence, asparagine plays an outsized regulatory role in cellular function. Here, we present a unifying evidence-based hypothesis that the amides constitute a regulatory signaling circuit, with glutamine as a driver and asparagine as a second messenger that allosterically regulates key biochemical and physiological functions, particularly cell growth and survival. Specifically, it is proposed that ASNS serves as a sensor of substrate sufficiency for S-phase entry and progression in proliferating cells. ASNS-generated asparagine serves as a subsequent second messenger that modulates the activity of key regulatory proteins and promotes survival in the face of cellular stress, and serves as a feed-forward driver of S-phase progression in cell growth. We propose that this signaling pathway be termed the Amide Signaling Circuit (ASC) in homage to the SLC1A5-encoded ASCT2 that transports both glutamine and asparagine in a bidirectional manner, and has been implicated in the pathogenesis of a broad spectrum of human cancers. Support for the ASC model is provided by the recent discovery that glutamine is sensed in primary cilia via ASNS during metabolic stress.

Gestational Interrelationships among Gut-Metabolism-Transcriptome in Regulating Early Embryo Implantation and Placental Development in Mice.

Decidualization of the uterine endometrium is a critical process for embryo implantation in mammals, primarily occurring on gestational day 8 in pregnant mice. However, the interplay between the maternal gut microbiome, metabolism, and the uterus at this specific time point remains poorly understood. This study employed a multi-omics approach to investigate the metabolic, gut microbiome, and transcriptomic changes associated with early pregnancy (gestational day 8 (E8)) in mice. Serum metabolomics revealed a distinct metabolic profile at E8 compared to controls, with the differential metabolites primarily enriched in amino acid metabolism pathways. The gut microbial composition showed that E8 mice exhibited higher alpha-diversity and a significant shift in beta-diversity. Specifically, the E8 group displayed a decrease in pathogenic Proteobacteria and an increase in beneficial Bacteroidetes and S24-7 taxa. Transcriptomics identified myriads of distinct genes between the E8 and control mice. The differentially expressed genes were enriched in pathways involved in alanine, aspartate, and glutamate metabolism, PI3K-Akt signaling, and the PPAR signaling pathway. Integrative analysis of the multi-omics data uncovered potential mechanistic relationships among the differential metabolites, gut microbiota, and uterine gene expression changes. Notably, the gene Asns showed strong correlations with specific gut S24-7 and metabolite L-Aspartatic acid, suggesting its potential role in mediating the crosstalk between the maternal environment and embryo development during early pregnancy. These findings provide valuable insights into the complex interplay between the maternal metabolome, the gut microbiome, and the uterine transcriptome in the context of early pregnancy, which may contribute to our understanding of the underlying mechanisms of embryo implantation and development.

Methods for production and assaying catalysis of isolated recombinant human aspartate/asparagine-ß-hydroxylase.

Aspartate/asparagine-ß-hydroxylase (AspH) is a transmembrane 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of aspartate- and asparagine-residues in epidermal growth factor-like domains (EGFDs) of its substrate proteins. Upregulation of ASPH and translocation of AspH from the endoplasmic reticulum membrane to the surface membrane of cancer cells is associated with enhanced cell motility and worsened clinical prognosis. AspH is thus a potential therapeutic and diagnostic target for cancer. This chapter describes methods for the production and purification of soluble constructs of recombinant human AspH suitable for biochemical and crystallographic studies. The chapter also describes efficient methods for performing turnover and inhibition assays which monitor catalysis of isolated recombinant human AspH in vitro using solid phase extraction coupled to mass spectrometry (SPE-MS). The SPE-MS assays employ synthetic disulfide- or thioether-bridged macrocyclic oligopeptides as substrates; a macrocycle is an apparently essential requirement for productive AspH catalysis and mimics an EGFD disulfide isomer that is not typically observed in crystal and NMR structures. SPE-MS assays can be used to monitor catalysis of 2OG oxygenases other than AspH; the methods described herein are representative for 2OG oxygenase SPE-MS assays useful for performing kinetic and/or inhibition studies.

Targeting NAT10 inhibits osteosarcoma progression via ATF4/ASNS-mediated asparagine biosynthesis.

Despite advances in treatment, the prognosis of patients with osteosarcoma remains unsatisfactory, and searching for potential targets is imperative. Here, we identify N4-acetylcytidine (ac4C) acetyltransferase 10 (NAT10) as a candidate therapeutic target in osteosarcoma through functional screening. NAT10 overexpression is correlated with a poor prognosis, and NAT10 knockout inhibits osteosarcoma progression. Mechanistically, NAT10 enhances mRNA stability of activating transcription factor 4 (ATF4) through ac4C modification. ATF4 induces the transcription of asparagine synthetase (ASNS), which catalyzes asparagine (Asn) biosynthesis, facilitating osteosarcoma progression. Utilizing virtual screening, we identify paliperidone and AG-401 as potential NAT10 inhibitors, and both inhibitors are found to bind to NAT10 proteins. Inhibiting NAT10 suppresses osteosarcoma progression in vivo. Combined treatment using paliperidone and AG-401 produces synergistic inhibition for osteosarcoma in patient-derived xenograft (PDX) models. Our findings demonstrate that NAT10 facilitates osteosarcoma progression through the ATF4/ASNS/Asn axis, and pharmacological inhibition of NAT10 may be a feasible therapeutic approach for osteosarcoma.

Targeting Asparagine Metabolism in Well-Differentiated/Dedifferentiated Liposarcoma.

BACKGROUND: mTORC1 activity is dependent on the presence of micronutrients, including Asparagine (Asn), to promote anabolic cell signaling in many cancers. We hypothesized that targeting Asn metabolism would inhibit tumor growth by reducing mTORC1 activity in well-differentiated (WD)/dedifferentiated (DD) liposarcoma (LPS). METHODS: Human tumor metabolomic analysis was utilized to compare abundance of Asn in WD vs. DD LPS. Gene set enrichment analysis (GSEA) compared relative expression among metabolic pathways upregulated in DD vs. WD LPS. Proliferation assays were performed for LPS cell lines and organoid models by using the combination treatment of electron transport chain (ETC) inhibitors with Asn-free media. 13C-Glucose-labeling metabolomics evaluated the effects of combination treatment on nucleotide synthesis. Murine xenograft models were used to assess the effects of ETC inhibition combined with PEGylated L-Asparaginase (PEG-Asnase) on tumor growth and mTORC1 signaling. RESULTS: Asn was enriched in DD LPS compared to WD LPS. GSEA indicated that mTORC1 signaling was upregulated in DD LPS. Within available LPS cell lines and organoid models, the combination of ETC inhibition with Asn-free media resulted in reduced cell proliferation. Combination treatment inhibited nucleotide synthesis and promoted cell cycle arrest. In vivo, the combination of ETC inhibition with PEG-Asnase restricted tumor growth. CONCLUSIONS: Asn enrichment and mTORC1 upregulation are important factors contributing to WD/DD LPS tumor progression. Effective targeting strategies require limiting access to extracellular Asn and inhibition of de novo synthesis mechanisms. The combination of PEG-Asnase with ETC inhibition is an effective therapy to restrict tumor growth in WD/DD LPS.

Low asparagine wheat: Europe's first field trial of genome edited wheat amid rapidly changing regulations on acrylamide in food and genome editing of crops.

We review the undertaking of a field trial of low asparagine wheat lines in which the asparagine synthetase gene, TaASN2, has been knocked out using CRISPR/Cas9. The field trial was undertaken in 2021-2022 and represented the first field release of genome edited wheat in Europe. The year of the field trial and the period since have seen rapid changes in the regulations covering both the field release and commercialisation of genome edited crops in the UK. These historic developments are reviewed in detail. Free asparagine is the precursor for acrylamide formation during high-temperature cooking and processing of grains, tubers, storage roots, beans and other crop products. Consequently, work on reducing the free asparagine concentration of wheat and other cereal grains, as well as the tubers, beans and storage roots of other crops, is driven by the need for food businesses to comply with current and potential future regulations on acrylamide content of foods. The topic illustrates how strategic and applied crop research is driven by regulations and also needs a supportive regulatory environment in which to thrive.

Nitrate Inhibits Nodule Nitrogen Fixation by Accumulating Ureide in Soybean Plants.

The mechanism by which nitrate inhibits nitrogen fixation in soybean (Glycine max L.) is not fully understood. Accumulation of ureide in soybean plant tissues may regulate the nitrogen fixation capacity through a feedback pathway. In this study, unilaterally nodulated dual-root soybeans prepared by grafting were grown in sand culture. They were subjected to the removal of the nodulated side roots, and were given either nitrate supply or no supply to the non-nodulated side roots for 3 days (experiment I). Additionally, they received nitrate supply to the non-nodulated side roots for 1-14 days (experiment II). The results showed that nitrate supply increased the levels of asparagine and ureide in soybean shoots (Experiment I). In Experiment II, nodule dry weight, nodule number, nodule nitrogenase activity, and nodule urate oxidase activity decreased significantly after 3, 7, and 14 days of nitrate supply. Ureide content in the shoots and nodules increased after 1, 3, and 7 days of nitrate supply, but decreased after 14 days of nitrate supply. There was a significant positive correlation between urate oxidase activity and nitrogenase activity. Hence, we deduced that nitrate supply increased the asparagine content in soybean shoots, likely inhibiting ureide degradation, which induced the accumulation of ureide in soybean shoots and nodules, and, in turn, feedback inhibited the nodule nitrogen fixation. In addition, urate oxidase activity can be used to assess the nitrogen fixation capacity of nodules.

Differential effect of asparagine and glutamine removal on three adenocarcinoma cell lines.

Asparagine and glutamine depletion operated by the drug Asparaginase (ASNase) has revolutionized therapy in pediatric patients affected by Acute Lymphoblastic Leukemia (ALL), bringing remissions to a remarkable 90 % of cases. However, the knowledge of the proproliferative role of asparagine in adult and solid tumors is still limited. We have here analyzed the effect of ASNase on three adenocarcinoma cell lines (A549, lung adenocarcinoma, MCF-7, breast cancer, and 786-O, kidney cancer). In contrast to MCF-7 cells, 786-O and A549 cells proved to be a relevant target for cell cycle perturbation by asparagine and glutamine shortage. Indeed, when the cell-cycle was analyzed by flow cytometry, A549 showed a canonical response to asparaginase, 786-O cells, instead, showed a reduction of the percentage of cells in the G1 phase and an increase of those in the S-phase. Despite an increased number of PCNA and RPA70 positive nuclear foci, BrdU and EdU incorporation was absent or strongly delayed in treated 786-O cells, thus indicating a readiness of replication forks unmatched by DNA synthesis. In 786-O asparagine synthetase was reduced following treatment and glutamine synthetase was totally absent. Interestingly, DNA synthesis could be recovered by adding Gln to the medium. MCF-7 cells showed no significant changes in the cell cycle phases, in DNA-bound PCNA and in total PCNA, but a significant increase in ASNS and GS mRNA and protein expression. The collected data suggest that the effect observed on 786-O cells following ASNase treatment could rely on mechanisms which differ from those well-known and described for leukemic blasts, consisting of a complete block in the G1/S transition in proliferating cells and on an increase on non-proliferative (G0) blasts.

Characterisation of Tenebrio molitor Reared on Substrates Supplemented with Chestnut Shell.

Tenebrio molitor larvae represent a sustainable protein source for food and feed. The aim of this study was to evaluate the supplementation of chestnut shell, a by-product of the agro-industrial chain, in growth substrates for T. molitor larvae rearing. Seven-week-old larvae were reared on three different growth substrates: the control group (CTRL) was fed wheat bran, treatment group one was fed wheat bran supplemented with 12.5% w/w chestnut shell (TRT1), and treatment group two was fed wheat bran supplemented with 25% w/w chestnut shell (TRT2). Larval weight, substrate consumption, and mortality were recorded weekly. After 14 days, insect meals were produced for bromatological and colorimetric analysis, and bacterial inhibition activity assay using a microdilution method. The amino acid profile of insects was determined using quantitative nuclear magnetic resonance spectroscopy. Our results showed a lower feed conversion ratio and higher larval survival rate % in TRT2 compared to CTRL (p < 0.05). Proteins and lipids of TRT2 were higher than other groups (p < 0.05). Important differences were observed in the amino acid profile of TRT1 and TRT2 compared to CTRL (p < 0.05). TRT1 and TRT2 showed higher E. coli inhibitory activity than CTRL (p < 0.05). In conclusion, chestnut shell supplementation improved the survival and functional characteristics of larvae and likely impacted the insects' metabolism.

Safety evaluation of the food enzyme asparaginase from the genetically modified Aspergillus niger strain ASP.

The food enzyme asparaginase (l-asparagine amidohydrolase; EC 3.5.1.1) is produced with the genetically modified Aspergillus niger strain ASP by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in the prevention of acrylamide formation in foods and in the processing of yeast and yeast products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.792 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level at the highest dose tested of 1038 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1311. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Long-acting Erwinia chrysanthemi, Pegcrisantaspase, induces alternate amino acid biosynthetic pathways in a preclinical model of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease without meaningful therapeutic options beyond the first salvage therapy. Targeting PDAC metabolism through amino acid restriction has emerged as a promising new strategy, with asparaginases, enzymes that deplete plasma glutamine and asparagine, reaching clinical trials. In this study, we investigated the anti-PDAC activity of the asparaginase formulation Pegcrisantaspase (PegC) alone and in combination with standard-of-care chemotherapeutics. METHODS: Using mouse and human PDAC cell lines, we assessed the impact of PegC on cell proliferation, cell death, and cell cycle progression. We further characterized the in vitro effect of PegC on protein synthesis as well as the generation of reactive oxygen species and levels of glutathione, a major cellular antioxidant. Additional cell line studies examined the effect of the combination of PegC with standard-of-care chemotherapeutics. In vivo, the tolerability and efficacy of PegC, as well as the impact on plasma amino acid levels, was assessed using the C57BL/6-derived KPC syngeneic mouse model. RESULTS: Here we report that PegC demonstrated potent anti-proliferative activity in a panel of human and murine PDAC cell lines. This decrease in proliferation was accompanied by inhibited protein synthesis and decreased levels of glutathione. In vivo, PegC was tolerable and effectively reduced plasma levels of glutamine and asparagine, leading to a statistically significant inhibition of tumor growth in a syngeneic mouse model of PDAC. There was no observable in vitro or in vivo benefit to combining PegC with standard-of-care chemotherapeutics, including oxaliplatin, irinotecan, 5-fluorouracil, paclitaxel, and gemcitabine. Notably, PegC treatment increased tumor expression of asparagine and serine biosynthetic enzymes. CONCLUSIONS: Taken together, our results demonstrate the potential therapeutic use of PegC in PDAC and highlight the importance of identifying candidates for combination regimens that could improve cytotoxicity and/or reduce the induction of resistance pathways.

Analysis of Maillard reaction precursors and secondary metabolites in Chilean potatoes and neoformed contaminants during frying.

Southern Chile native potatoes are an interesting raw material to produce novel snacks like colored potato chips. These novel products should be comprehensively evaluated for the presence of undesirable compounds such as acrylamide, 5-hydroxymethylfurfural and furan, the main neoformed contaminants in starchy rich fried foods. This study evaluated the neoformed contaminant levels and oil content on chips made from eleven Chilean potato accessions and compared them with commercial samples. The neoformed contaminant contents were related to Maillard reaction precursor levels (reducing sugars and asparagine) and secondary metabolites (phenolic compounds and carotenoids). Neoformed contaminants correlated well among them and were weakly correlated with reducing sugars and asparagine. Acrylamide level in native potato chips ranged from 738.2 to 1998.6 µg kg-1 while from 592.6 to 2390.5 µg kg-1 in commercial samples. Thus, there is need to implement neoformed contaminant mitigation strategies at different steps of the production chain of colored potato chips.

Biochemical characterization of l-asparagine synthetase from Streptococcus thermophilus and its application in the enzymatic synthesis of ß-aspartyl compounds.

ß-Aspartyl compounds, such as ß-aspartyl hydroxamate (serine racemase inhibitor), ß-aspartyl-l-lysine (moisture retention), and ß-aspartyl-l-tryptophan (immunomodulator) are physiologically active compounds. There is limited literature on the development of effective methods of production of ß-aspartyl compounds. In this study, we describe the biochemical characterization of asparagine synthetase (AS) from Streptococcus thermophilus NBRC 13957 (StAS) and the enzymatic synthesis of ß-aspartyl compounds using StAS. Recombinant StAS was expressed in Escherichia coli BL21(DE3) and it displayed activity towards hydroxylamine, methylamine, ethylamine, and ammonia, as acceptors of the ß-aspartyl moiety. StAS exhibited higher activity toward hydroxylamine and ethylamine as acceptor substrates compared with the enzymes from Lactobacillus delbrueckii NBRC 13953, Lactobacillus reuteri NBRC 15892, and E. coli. The coupling of the synthesis of ß-aspartyl compounds by StAS with an ATP-regeneration system using polyphosphate kinase from Deinococcus proteoliticus NBRC 101906 displayed an approximately 2.5-fold increase in the production of ß-aspartylhydroxamate from 1.06 mM to 2.53 mM after a 76-h reaction.

Unlocking the potential health improving properties of sprouted wheat.

Sprouting can enhance the bioavailability and stimulate the production of health-promoting compounds. This research explored the potential health benefits of wheat sprouting, focusing on underexplored areas in existing literature such as alterations in phenylalanine ammonia-lyase (PAL) activity and glutathione levels during wheat sprouting. Furthermore, special attention was directed toward asparagine (Asn), the main precursor of acrylamide formation, as regulatory agencies are actively seeking to impose limitations on the presence of acrylamide in baked products. The results demonstrate elevated levels of PAL (4.5-fold at 48 h of sprouting), antioxidants, and total phenolics (1.32 mg gallic acid equivalent/g dry matter at 72 h of sprouting), coupled with a reduction in Asn (i.e. 11-fold at 48 h of sprouting) and glutathione concentrations, after wheat sprouting. These findings suggest that sprouting can unlock health-promoting properties in wheat. Optimizing the sprouting process to harness these benefits, however, may have implications for the techno-functionality of wheat flour in food processing.

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa / CD16a structure.

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa / CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.