N-acetylglucosamine Research Articles

Expression and immobilization of novel N-glycan-binding protein for highly efficient purification and enrichment of N-glycans, N-glycopeptides, and N-glycoproteins.

Comprehensive and selective enrichment of N-glycans, N-glycopeptides, and N-glycoproteins prior to analysis is of great significance in N-glycomics research, reducing sample complexity, removing impurity interference, increasing sample abundance and enhancing signal intensity. However, only an Fbs1 (F-box protein that recognizes sugar chain 1) GYR variant (Fg) can enrich these N-glycomolecules solely due to its substantial binding affinity for the core pentasaccharide motif of N-glycans. Stationary phase separation is commonly used to enrich N-glycomolecules efficiently. Herein, DNA encoding the Fg was cloned into pGEX-4T-1, and the protein was expressed with a GST tag, which facilitates the convenient and efficient immobilization of recombinant GST-tagged Fg to GSH agarose resin. The yield of the GST-tagged Fg reached to 0.05 g/L after optimization of the induction condition, and the purified protein exhibited good identification ability and excellent stability for months. In particular, the immobilized GST-tagged Fg can enrich N-glycans released byPNGase Fand capture derivatized N-glycans possessing an intact terminal N-acetyl glucosamine (GlcNAc). Validation of immobilized GST-tagged Fg with standard N-glycopeptides and N-glycoproteins revealed its high loading capacity, sensitivity, and selectivity. The novel immobilized GST-tagged Fg is a convenient and efficient enrichment material specific for N-glycans, N-glycopeptides, and N-glycoproteins, suggesting excellent performance and prospects for industrial application.

Development of root rot in Zanthoxylum bungeanum is closely linked to changes in soil microbial communities, enzyme activities, and physicochemical factors

Due to the emergence of root rot (RR), it can cause harm to the health of Zanthoxylum bungeanum (Z. bungeanum) plants. However, the reason for this is not clear, and further research is needed. We selected healthy and RR-affected 6-year-old Z. bungeanum trees (infected for 1 year), collected their rhizosphere and bulk soils, and analyzed soil microbial communities, enzyme activities, and physicochemical properties. When the plants were diseased, soil water content (SWC), available potassium (AK), organic matter (OM) contents, β-1,4-glucosidase (β-G) activity, and fungal Shannon index significantly decreased, while bacterial Shannon index significantly increased in the rhizosphere soil. Soil total nitrogen (TN), SWC, AK, OM contents, and β-G and N-acetyl-glucosamine (NAG) activity significantly reduced, while fungal Simpson index significantly increased in the bulk soil. There were significant differences in the bacterial communities of bulk and rhizosphere soil. However, there were no obvious differences in the fungal communities. Pearson's correlation analysis showed that changes in soil microbial communities were significantly correlated with changes in the contents of TN, AK, OM, and SWC, as well as the activities of β-G and NAG. In summary, changes in soil AK, OM, SWC, and TN resulted in changes in microbial community composition, as well as changes in β-G and NAG activities, and these alterations may lead to RR in Z. bungeanum.

Cell-free expression and biochemical characterization of polysaccharide-synthesizing glycosyltransferases

Polysaccharides like cellulose and hyaluronan are synthesized by membrane-bound family-2 glycosyltransferases (GTs) that play critical structural, metabolic, or functional roles in cells. Though GT-2 family has the maximum number of deposited gene sequences, the biochemistry is poorly understood due to enzyme production challenges. Here, we developed a cell-free expression (CFE) protocol to produce two GT-2 family representative cellulose synthase (PttCesA8 from Populus tremula x tremuloides) and hyaluronan synthase (SeHAS from Streptococcus equisimilis). The CFE products were obtained as reconstituted proteoliposomes directly at high yields and short processing times compared to other approaches. Enzyme expression was confirmed using SDS-PAGE and immunoblotting, while integration of GTs into lipid layers was observed using cryogenic electron microscopy. Both GTs were catalytically active with Michalis-Menten kinetic constants, Km for PttCesA8, was 295.8 µM, and SeHAS was 321.51 µM (toward UDP N-Acetyl Glucosamine) and 207.88 µM (toward UDP Glucuronic Acid), respectively, and with UDP inhibiting both GTs. Mutation of conserved residues in SeHAS also confirmed the importance of lysine-139, glutamine-248, and threonine-283 residues in hyaluronan biosynthesis. In summary, CFE methods enable expression of polysaccharide-synthesizing enzymes as proteoliposomes at high yields with relative ease for rapid biochemical and structural characterization studies.

Synthesis, Characterization and Osteogenic Properties of Chitin-Polydioxanone Composite Gel.

In this study, we have developed a Chitin(Ch)-Poly(dioxanone)(PDO) gel system, which can be potentially used for tissue engineering. Hydrogel has been widely used in biomedical applications for its tuneable properties and biocompatibility. Chitin (Ch) is a natural biopolymer used for its ability to mimic the natural extracellular matrix due to N-acetyl glucosamine structural units. Poly (dioxanone) (PDO) is an FDA-approved synthetic biopolymer known for its mechanical properties, good biocompatibility, and poor inflammatory response. Based on this, we have developed Ch-PDO composite gel using simple regeneration chemistry and characterized it using FT-IR and SEM. The developed composite gel showed improved gel strength, good swelling ability,and controlled degradation behaviour. It also showed good injectability with shear thinning properties and hemocompatibility. Further, the biocompatibility and cell adhesion studies of the prepared gels were studied using dental follicle stem cells (DFSCs). The prepared Ch-PDO gel was biocompatible and showed DFSCs cell attachment. Osteogenic mineralization and RUNX2 expression of the prepared Ch and Ch-PDO gel was studied and Ch-PDO gel showed an enhanced mineralization and RUNX2 expression. Therefore, the developed chitin-PDO gel could be potentially used for bone tissue engineering.

50536 A Tranexamic Acid, Niacinamide, and Acetyl Glucosamine Facial Serum Clinically Improved PIH and Post-Acne Marks in a Diverse Population

50536 A Tranexamic Acid, Niacinamide, and Acetyl Glucosamine Facial Serum Clinically Improved PIH and Post-Acne Marks in a Diverse Population

Bioorthogonal labeling of chitin in pathogenic Candida species reveals biochemical mechanisms of hyphal growth and homeostasis.

Pathogenic fungi rely on the cell wall component, chitin, for critical structural and immunological functions. Here a chitin labeling method to visualize the hyphal pathogenic response was developed. The data show that filamentous fungi, Candida albicans , transport N -acetylglucosamine (NAG) bio-orthogonal probes and incorporate them into the cell wall, indicating the probes utility for in vivo study of the morphological, pathogenic switch. As yeast reside in complex microenvironments, The data show that the opportunistic microbe C. albicans , has developed processes to utilize surrounding bacterial cell wall fragments to initiate the morphogenic switch. The probes are utilized for visualization of growth patterns of pathogenic fungi, providing insights into novel mechanisms for the development of antifungals. Remodeling chitin in fungi using NAG derivatives will advance yeast pathogenic studies.

Sub-10 μm Soft Interlayers Integrating Patterned Multivalent Biomolecular Binding Environments.

Designing surfaces that enable controlled presentation of multivalent ligand clusters (e.g., for rapid screening of biomolecular binding constants or design of artificial extracellular matrices) is a cross-cutting challenge in materials and interfacial chemistry. Existing approaches frequently rely on complex building blocks or scaffolds and are often specific to individual substrate chemistries. Thus, an interlayer chemistry that enabled efficient nanometer-scale patterning on a transferrable layer and subsequent integration with other classes of materials could substantially broaden the scope of surfaces available for sensors and wearable electronics. Recently, we have shown that it is possible to assemble nanometer-resolution chemical patterns on substrates including graphite, use diacetylene polymerization to lock the molecular pattern together, and then covalently transfer the pattern to amorphous materials (e.g., polydimethylsiloxane, PDMS), which would not natively enable high degrees of control over ligand presentation. Here, we develop a low-viscosity PDMS formulation that generates very thin films (<10 μm) with dense cross-linking, enabling high-efficiency surface functionalization with polydiacetylene arrays displaying carbohydrates and other functional groups (up to 10-fold greater than other soft materials we have used previously) on very thin films that can be integrated with other materials (e.g., glass and soft materials) to enable a highly controlled multivalent ligand display. We use swelling and other characterization methods to relate surface functionalization efficiency to the average distance between cross-links in the PDMS, developing design principles that can be used to create even thinner transfer layers. In the context of this work, we apply this approach using precision glycopolymers presenting structured arrays of N-acetyl glucosamine ligands for lectin binding assays. More broadly, this interlayer approach lays groundwork for designing surface layers for the presentation of ligand clusters on soft materials for applications including wearable electronics and artificial extracellular matrix.

Sugar-mediated non-canonical ubiquitination impairs Nrf1/NFE2L1 activation

Proteasome is essential for cell survival, and proteasome inhibition induces proteasomal gene transcription via the activated endoplasmic-reticulum-associated transcription factor nuclear factor erythroid 2-like 1(Nrf1/NFE2L1). Nrf1 activation requires proteolytic cleavage by DDI2 and N-glycan removal by NGLY1. We previously showed that Nrf1 ubiquitination by SKP1-CUL1-F-box (SCF)FBS2/FBXO6, an N-glycan-recognizing E3 ubiquitin ligase, impairs its activation, although the molecular mechanism remained elusive. Here, we show that SCFFBS2 cooperates with the RING-between-RING (RBR)-type E3 ligase ARIH1 to ubiquitinate Nrf1 through oxyester bonds in human cells. Endo-β-N-acetylglucosaminidase (ENGASE) generates asparagine-linked N-acetyl glucosamine (N-GlcNAc) residues from N-glycans, and N-GlcNAc residues on Nrf1 served as acceptor sites for SCFFBS2-ARIH1-mediated ubiquitination. We reconstituted the polyubiquitination of N-GlcNAc and serine/threonine residues on glycopeptides and found that the RBR-specific E2 enzyme UBE2L3 is required for the assembly of atypical ubiquitin chains on Nrf1. The atypical ubiquitin chains inhibited DDI2-mediated activation. The present results identify an unconventional ubiquitination pathway that inhibits Nrf1 activation.

Hyperglycemia-independent neonatal streptozotocin-induced retinopathy (NSIR) in rats.

Introduction: Chemicals, such as MNU (N-methyl-N-nitrosourea) and NaIO3 (sodium iodate), are widely used to induce retinal degeneration in rodents. Streptozotocin (STZ) is an analog of N-acetyl glucosamine in which an MNU moiety is linked to a hexose and has a special toxic effect on insulin-producing pancreatic β-cells. It is commonly used to induce hyperglycemia to model diabetes. While intracerebroventricular injection of STZ can produce Alzheimer's disease independent of hyperglycemia, most retinal studies using STZ focus on the effects of hyperglycemia on the retina, but whether STZ has any impact on retinal cells independent of hyperglycemia is unknown. We aimed to investigate the role of cytotoxicity of STZ in rat retina. Methods: Intravitreal or subcutaneous injection of STZ was performed on newborn rats. Electroretinogram (ERG) and H&E staining investigated retinal function and morphological changes. Retinal cell types, cell death, proliferation, inflammation, and angiogenesis were studied by immunostaining. RNA sequencing was performed to examine the transcriptome changes of retinal cells after intravitreal injection of STZ. Results: Intravitreal (5μg or 10μg) or subcutaneous (30mg/kg) injection of STZ at the early stage of newborn rats couldn't induce hyperglycemia but caused NSIR (Neonatal STZ-induced retinopathy), including reduced ERG amplitudes, retinal rosettes and apoptosis, cell cycle arrest, microglial activation, and delayed retinal angiogenesis. STZ did not affect the early-born retinal cell types but significantly reduced the late-born ones. Short-term and long-term hyperglycemia had no significant effects on the NSIR phenotypes. RNA sequencing revealed that STZ induces oxidative stress and activates the p53 pathway of retinal cells. Locally or systemically, STZ injection after P8 couldn't induce SINR when all retinal progenitors exit the cell cycle. Conclusion: NSIR in rats is independent of hyperglycemia but due to STZ's direct cytotoxic effects on retinal progenitor cells. NSIR is a typical reaction to STZ-induced retinal oxidative stress and DNA damage. This significant finding suggests that NSIR may be a valuable model for studying retinal progenitor DNA damage-related diseases, potentially leading to new insights and treatments.

Investigating the Multi-Targeted Pharmacological profile of an Exopolysaccharide from Bacillus rugosus SYG20 via In Vitro Evaluation of its Antioxidant, Anti-inflammatory, Anti-diabetic, Wound Healing, and Antimicrobial Properties

IntroductionExopolysaccharides (EPSs) derived from marine microorganisms are a newly recognized reservoir of bioactive therapeutic compoundsMaterial and methodsWe isolated a high EPS-yielding bacterial strain from the Red Sea, identified as Bacillus rugosus SYG20. Its purified EPS (EPSR9) contains 45.33% uronic acid, 9.98% sulfate groups, and 5.40% N-acetyl glucosamine. The HPLC chromatogram revealed four monosaccharides - glucose, xylose, galacturonic acid, and arabinose, in a distinct molar ratio of 2:3:1:1. EPSR9 showed a wide array of bioactivities.ResultsIt displayed antioxidant activity with an IC50 of 25.6 μg/ml in the DPPH assay and a total antioxidant capacity (TAC) of 417.77μg/ml equivalent AAE and 62.67 μg/ml equivalent AAE in Ferric reducing antioxidant power (FRAP) assays. It exhibited substantial anti-inflammatory properties. The anticoagulant effect of the EPS was demonstrated by a dose-dependent increase in prothrombin time. The scratch assay resulted in a 72.66% increase in wound closure, promoting in vitro wound healing after 48 h. Anti-obesity activity was evidenced by 83.8% lipase inhibition at 1000 μg/ml with IC50 of 107.73μg/ml. EPSR9 demonstrated inhibitory effects on α-amylase with an IC50 value of 14.37μg/ml and α-glucosidase with an IC50 value of 26.73 μg/ml, highlighting its potential as an anti-diabetic agent. Then, EPS showed bactericidal properties with MBC/MIC≤2 against both G+ve and G-ve bacteria, Staphylococcus aureus , Enterococcus faecalis (MIC=3.9μg/ml), Salmonella typhi, and Helicobacter pylori.ConclusionsThe marine EPSR9 exhibited considerable potential for pharmaceutical applications as a multi-bioactive microbial metabolite. Its in vivo potency and mechanisms of action warrant further investigation.

Enzymatic hydrolysis of chitinous wastes pretreated by deep eutectic solvents into N-acetyl glucosamine

In this study, we present an efficient and green extraction-pretreatment integrated approach for enhancing enzymatic conversion of chitinous wastes into N-acetyl-d-glucosamine (GlcNAc). Firstly, the enzyme cocktail containing a chitinase CmChi1 and a N-acetyl glucosaminase CmNAGase were constructed for hydrolyzing chitin into sole GlcNAc. Secondly, deep eutectic solvent (DES), consisting of choline chloride and glycollic acid was used to treat chitinous wastes. Under optimal conditions, chitin yield reach to 72 % with a purity of 98 %. Fourier-transform infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction analysis revealed that the crystallinity and thermal stability of the obtained chitin decreased upon DES treatment without alteration of the chemical structure or deacetylation. Finally, the concentration of GlcNAc was increased 2–6 folds by enzymatic hydrolysis of DES-treated chitinous wastes (including shrimp shell, crab shell, ganoderma spores wall, and mycelium). The process provides a promising strategy for degrading chitinous wastes to produce high valued GlcNAc.

Neuronal activity-driven O-GlcNAcylation promotes mitochondrial plasticity

Neuronal activity is an energy-intensive process that is largely sustained by instantaneous fuel utilization and ATP synthesis. However, how neurons couple ATP synthesis rate to fuel availability is largely unknown. Here, we demonstrate that the metabolic sensor enzyme O-linked N-acetyl glucosamine (O-GlcNAc) transferase regulates neuronal activity-driven mitochondrial bioenergetics in hippocampal and cortical neurons. We show that neuronal activity upregulates O-GlcNAcylation in mitochondria. Mitochondrial O-GlcNAcylation is promoted by activity-driven glucose consumption, which allows neurons to compensate for high energy expenditure based on fuel availability. To determine the proteins that are responsible for these adjustments, we mapped the mitochondrial O-GlcNAcome of neurons. Finally, we determine that neurons fail to meet activity-driven metabolic demand when O-GlcNAcylation dynamics are prevented. Our findings suggest that O-GlcNAcylation provides a fuel-dependent feedforward control mechanism in neurons to optimize mitochondrial performance based on neuronal activity. This mechanism thereby couples neuronal metabolism to mitochondrial bioenergetics and plays a key role in sustaining energy homeostasis.

Marine Bacillus haynesii chitinase: Purification, characterization and antifungal potential for sustainable chitin bioconversion

The development of chitinase tailored for the bioconversion of chitin to chitin oligosaccharides has attracted significant attention due to its potential to alleviate environmental pollution associated with chemical conversion processes. In this present investigation, we purified extracellular chitinase derived from marine Bacillus haynesii to homogeneity and subsequently characterized it. The molecular weight of BhChi was approximately 35 kDa. BhChi displayed its peak catalytic activity at pH 6.0, with an optimal temperature of 37 °C. It exhibited stability across a pH range of 6.0–9.0. In addition, BhChi showed activation in the presence of Mn2+ with the improved activity of 105 U mL−1. Ca2+ and Fe2+ metal ions did not have any significant impact on enzyme activity. Under the optimized enzymatic conditions, there was a notable enhancement in catalytic activity on colloidal chitin with Km of 0.01 mg mL−1 and Vmax of 5.75 mmol min−1. Kcat and catalytic efficiency were measured at 1.91 s−1 and 191 mL mg−1 s−1, respectively. The product profiling of BhChi using thin layer chromatography and Mass spectrometric techniques hinted an exochitinase mode of action with chitobiose and N-Acetyl glucosamine as the products. This study represents the first report on an exochitinase from Bacillus haynesii. Furthermore, the chitinase showcased promising antifungal properties against key pathogens, Fusarium oxysporum and Penicillium chrysogenum, reinforcing its potential as a potent biocontrol agent.

Combinatorial metabolic engineering of Escherichia coli for de novo production of structurally defined and homogeneous Amino oligosaccharides

Amino oligosaccharides (AOs) possess various biological activities and are valuable in the pharmaceutical, food industries, and agriculture. However, the industrial manufacturing of AOs has not been realized yet, despite reports on physical, chemical, and biological approaches. In this study, the de novo production of chitin oligosaccharides (CHOS), a type of structurally defined AOs, was achieved in Escherichia coli through combinatorial pathway engineering. The most suitable glycosyltransferase for CHOS production was found to be NodCL from Mesorhizobium Loti. Then, by knocking out the nagB gene to block the flow of N-acetyl-d-glucosamine (NAG) to the glycolytic pathway in E. coli and adjusting the copy number of NodCL-coding gene, the CHOS yield was increased by 6.56 times. Subsequently, by introducing of UDP-N-acetylglucosamine (UDP-GlcNAc) salvage pathway for and optimizing fermentation conditions, the yield of CHOS reached 207.1 and 468.6 mg/L in shake-flask cultivation and a 5-L fed-batch bioreactor, respectively. Meanwhile, the concentration of UDP-GlcNAc was 91.0 mg/L, the highest level reported in E. coli so far. This study demonstrated, for the first time, the production of CHOS with distinct structures in plasmid-free E. coli, laying the groundwork for the biosynthesis of CHOS and providing a starting point for further engineering and commercial production.

Synthesis and anti-inflammatory activities of two new N-acetyl glucosamine derivatives

N-acetyl glucosamine (NAG) is a natural amino sugar found in various human tissues with previously described anti-inflammatory effects. Various chemical modifications of NAG have been made to promote its biomedical applications. In this study, we synthesized two bi-deoxygenated NAG, BNAG1 and BNAG2 and investigated their anti-inflammatory properties, using an in vivo and in vitro inflammation mouse model induced by lipopolysaccharide (LPS). Among the parent molecule NAG, BNAG1 and BNAG2, BNAG1 showed the highest inhibition against serum levels of IL-6 and TNF α and the leukocyte migration to lungs and peritoneal cavity in LPS challenged mice, as well as IL-6 and TNF α production in LPS-stimulated primary peritoneal macrophages. BNAG2 displayed an anti-inflammatory effect which was comparable to NAG. These findings implied potential application of these novel NAG derivatives, especially BNAG1, in treatment of certain inflammation-related diseases.

Malnutrition enteropathy in Zambian and Zimbabwean children with severe acute malnutrition: A multi-arm randomized phase II trial

Malnutrition underlies almost half of all child deaths globally. Severe Acute Malnutrition (SAM) carries unacceptable mortality, particularly if accompanied by infection or medical complications, including enteropathy. We evaluated four interventions for malnutrition enteropathy in a multi-centre phase II multi-arm trial in Zambia and Zimbabwe and completed in 2021. The purpose of this trial was to identify therapies which could be taken forward into phase III trials. Children of either sex were eligible for inclusion if aged 6–59 months and hospitalised with SAM (using WHO definitions: WLZ <−3, and/or MUAC <11.5 cm, and/or bilateral pedal oedema), with written, informed consent from the primary caregiver. We randomised 125 children hospitalised with complicated SAM to 14 days treatment with (i) bovine colostrum (n = 25), (ii) N-acetyl glucosamine (n = 24), (iii) subcutaneous teduglutide (n = 26), (iv) budesonide (n = 25) or (v) standard care only (n = 25). The primary endpoint was a composite of faecal biomarkers (myeloperoxidase, neopterin, α1-antitrypsin). Laboratory assessments, but not treatments, were blinded. Per-protocol analysis used ANCOVA, adjusted for baseline biomarker value, sex, oedema, HIV status, diarrhoea, weight-for-length Z-score, and study site, with pre-specified significance of P < 0.10. Of 143 children screened, 125 were randomised. Teduglutide reduced the primary endpoint of biomarkers of mucosal damage (effect size −0.89 (90% CI: −1.69,−0.10) P = 0.07), while colostrum (−0.58 (−1.4, 0.23) P = 0.24), N-acetyl glucosamine (−0.20 (−1.01, 0.60) P = 0.67), and budesonide (−0.50 (−1.33, 0.33) P = 0.32) had no significant effect. All interventions proved safe. This work suggests that treatment of enteropathy may be beneficial in children with complicated malnutrition. The trial was registered at ClinicalTrials.gov with the identifier NCT03716115.

Tapping the biosynthetic potential of marine Bacillus licheniformis LHG166, a prolific sulphated exopolysaccharide producer: structural insights, bio-prospecting its antioxidant, antifungal, antibacterial and anti-biofilm potency as a novel anti-infective lead.

The escalating global threat of antimicrobial resistance necessitates prospecting uncharted microbial biodiversity for novel therapeutic leads. This study mines the promising chemical richness of Bacillus licheniformis LHG166, a prolific exopolysaccharide (EPSR2-7.22 g/L). It comprised 5 different monosaccharides with 48.11% uronic acid, 17.40% sulfate groups, and 6.09% N-acetyl glucosamine residues. EPSR2 displayed potent antioxidant activity in DPPH and ABTS+, TAC and FRAP assays. Of all the fungi tested, the yeast Candida albicans displayed the highest susceptibility and antibiofilm inhibition. The fungi Aspergillus niger and Penicillium glabrum showed moderate EPSR2 susceptibility. In contrast, the fungi Mucor circinelloides and Trichoderma harzianum were resistant. Among G+ve tested bacteria, Enterococcus faecalis was the most susceptible, while Salmonella typhi was the most sensitive to G-ve pathogens. Encouragingly, EPSR2 predominantly demonstrated bactericidal effects against both bacterial classes based on MBC/MIC of either 1 or 2 superior Gentamicin. At 75% of MBC, EPSR2 displayed the highest anti-biofilm activity of 88.30% against B. subtilis, while for G-ve antibiofilm inhibition, At 75% of MBC, EPSR2 displayed the highest anti-biofilm activity of 96.63% against Escherichia coli, Even at the lowest dose of 25% MBC, EPSR2 reduced biofilm formation by 84.13% in E. coli, 61.46% in B. subtilis. The microbial metabolite EPSR2 from Bacillus licheniformis LHG166 shows promise as an eco-friendly natural antibiotic alternative for treating infections and oxidative stress.

Abstract 2874: Cancer metabolism in radiation resistance: Complementary roles of O-GlcNAc transferase and PARP1

Abstract The efficacy of image-guided radiotherapy (RT) has been linked to directing increased chromosomal double strand breaks (DSBs) to tumor cells while sparing surrounding normal tissue. DSBs can block cell cycle progression and promote mitotic catastrophe, cell death and senescence. End processing of persistent DSBs releases single strand DNA (ssDNA), driving cGAS/STING signaling and anti-tumor immune response. DSB repair, whether by non-homologous end-joining (NHEJ) or homologous recombination (HR), is a resistance mechanism, supporting cell survival, repopulation and immune evasion. Decades of effort to block DSB repair to enhance benefits of RT has yet to produce approved agents. Toxicity due to lack of cancer specificity may prevent the current generation of targeted agents from reaching the clinic. An answer may come from examining cancer metabolic reprogramming as a determinant of intrinsic radiation resistance. Along with other glucose metabolites, the Warburg effect increases N-acetyl glucosamine (GlcNAc) biosynthesis, which not only supports N-glycosylation but also modification of nucleocytoplasmic proteins by O-GlcNAc transferase (OGT). OGT-dependent O-GlcNAcylation confers radiation resistance, in part by accelerating DSB repair. OGT regulates γH2AX, 53BP1 and BRCA1 foci kinetics and suppresses ssDNA formation at DSB ends, limiting HR but favoring NHEJ for S/G2 DSB repair. Blocking OGT induces radiation sensitization in vitro and in vivo. In cells, OGT inhibition results in hyperresection, persistent RPA and RAD51 foci and accumulation of cytosolic DNA. One mediator may be the histone methyltransferase EZH2, which is rapidly recruited to deposit H3K27me3 at DSBs, promoting NHEJ repair. Irradiation of EZH2 KO cells results in hyperresection that cannot be suppressed by O-GlcNAcylation. Cancer cells may also accumulate NAD+, a substrate for PARP1 incorporated into poly-ADP-ribose to initiate DSB repair. Much like OGT inhibition, blocking PARP1 induces hyperresection and shifts repair toward HR and away from NHEJ. Importantly, increasing O-GlcNAcylation can suppress ssDNA formation even in PARP1 KO cells. In turn, inhibiting PARP1 enhances hyperesection in EZH2 KO cells, leading to a marked increase in cytosolic ssDNA. Our data assign complementary roles for EZH2 and PARP1 in DSB repair pathway choice by independently limiting 5' end resection. This directs DSBs to rapid, imprecise repair by NHEJ over slow and precise repair by HR. An implication is that PARP inhibitor resistance due to the Warburg effect may be mediated by EZH2. Driving hyperresection by targeting O-GlcNAcylation or H3K27 trimethylation along with PARP inhibition may saturate the capacity of HR and produce irreversible damage in cancer cells. Targeting impacts of metabolic reprogramming on DSB repair may not only enhance the therapeutic index of radiation but also sharpen synthetic lethality with HR deficiency. Citation Format: Elena E. Efimova, Sera Averbek, Donald J. Wolfgeher, Ding Wu, Yue Liu, Stephen J. Kron. Cancer metabolism in radiation resistance: Complementary roles of O-GlcNAc transferase and PARP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2874.

Bioconversion of Shrimp By-Product into Carotenoids Using Pigmented Yeast Sporidiobolus Pararoseus Q

Chitin, a natural polysaccharide, is the second most abundant biopolymer in the world after cellulose. It consists of N-acetylglucosamine (NAG) monomers and finds widespread applications in the food, cosmetics, and pharmaceutical industries. In this study, we selected pigmented yeast strains to synthesize carotenoids from NAG and optimized the conditions for carotenoid production. The results demonstrated that the selected strain Sporidiobolus pararoseus Q could accumulate β-carotene and carotenoids, reaching 518.84 µg/g dry biomass and 595.48 µg/g dry biomass, respectively, after 96 hours of fermentation with 30 g/L NAG at pH 5. The two-step fermentation first with 80 g/L of glucose and then 50 g/L of NAG increased the carotenoids and b-carotene yields by 41% and 35%, achieving 786.69 µg/g and 632.19 µg/g, respectively. The ability to use NAG as the feedstock for carotenoid production not only adds value to shrimp by-products but also contributes to controlling environmental pollution.

Seeds of nonhost species as sources of toxic compounds for the cowpea weevil Callosobruchus maculatus (F.)

Cowpea weevil, Callosobruchus maculatus, is the primary pest of stored cowpea seeds. The management of this infestation currently relies on insecticides, resulting in environmental pollution and selection of insecticide-resistant pests. Consequently, research efforts are being devoted to identify natural insecticides as sustainable and environment friendly alternatives for the control of C. maculatus. In this study, we explore the toxic effects of the nonhost seeds Parkia multijuga, Copaifera langsdorffii, Ormosia arborea, Amburana cearensis, Lonchocarpus guilleminianus, Sapindus saponaria, and Myroxylon peruiferum, on the cowpea weevil C. maculatus. Notably, all nonhost seeds led to reductions between 60 and 100% in oviposition by C. maculatus females. Additionally, the larvae were unable to penetrate the nonhost seeds. Artificial seeds containing 0.05% to 10% of cotyledon flour were toxic to C. maculatus larvae. Approximately 40% of larvae that consumed seeds containing 0.05% of O. arborea failed to develop, in contrast to control larvae. Proteomic analysis of A. cearensis and O. arborea seeds identify revealed a total of 371 proteins. From those, 237 are present in both seeds, 91 were exclusive to O. arborea seeds, and 43 were specific to A. cearensis seeds. Some of these proteins are related to defense, such as proteins containing the cupin domain and 11S seed storage protein. The in silico docking of cupin domain-containing proteins and 11S storage protein with N-acetylglucosamine (NAG)4 showed negative values of affinity energy, indicating spontaneous binding. These results showed that nonhost seeds have natural insecticide compounds with potential to control C. maculatus infestation.