Search Results (8)

During choriogenesis in insects, chorion (eggshell) is formed by surrounding follicular epithelial cells in ovarioles. However, the regulatory endocrine factor(s) activating choriogenesis and the effect of chemical components on eggshell deserve further exploration. In two representative coleopterans, a coccinellid Henosepilachna vigintioctopunctata and a chrysomelid Leptinotarsa decemlineata, genes encoding the 20-hydroxyecdysone (20E) receptor heterodimer, ecdysone receptor (EcR) and ultraspiracle (USP), and two chitin biosynthesis enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP) and chitin synthase (ChS1), were highly expressed in ovaries of the young females. RNA interference (RNAi)-aided knockdown of either HvEcR or Hvusp in H. vigintioctopunctata inhibited oviposition, suppressed the expression of HvChS1, and lessened the positive signal of Calcofluor staining on the chorions, which suggests the reduction of a chitin-like substance (CLS) deposited on eggshells. Similarly, RNAi of LdEcR or Ldusp in L. decemlineata constrained oviposition, decreased the expression of LdUAP1 and LdChS1, and reduced CLS contents in the resultant ovaries. Knockdown of LdUAP1 or LdChS1 caused similar defective phenotypes, i.e., reduced oviposition and CLS contents in the L. decemlineata ovaries. These results, for the first time, indicate that 20E signaling activates choriogenesis in two coleopteran species. Moreover, our findings suggest the deposition of a CLS on the chorions. Full article

Chondroitin is a glycosaminoglycan that has gained widespread use in nutraceuticals and pharmaceuticals, mainly for treating osteoarthritis. Traditionally, it has been extracted from animal cartilage but recently, biotechnological processes have emerged as a commercial alternative to avoid the risk of viral or prion contamination and offer a vegan-friendly source. Typically, these methods involve producing the chondroitin backbone using pathogenic bacteria and then modifying it enzymatically through the action of sulfotransferases. Despite the challenges of expressing active sulfotransferases in bacteria, the use of eukaryotic microorganisms is still limited to a few works using Pichia pastoris. To create a safer and efficient biotechnological platform, we constructed a biosynthetic pathway for chondroitin production in S. cerevisiae as a proof-of-concept. Up to 125 mg/L and 200 mg/L of intracellular and extracellular chondroitin were produced, respectively. Furthermore, as genome-scale models are valuable tools for identifying novel targets for metabolic engineering, a stoichiometric model of chondroitin-producing S. cerevisiae was developed and used in optimization algorithms. Our research yielded several novel targets, such as uridine diphosphate (UDP)-N-acetylglucosamine pyrophosphorylase (QRI1), glucosamine-6-phosphate acetyltransferase (GNA1), or N-acetylglucosamine-phosphate mutase (PCM1) overexpression, that might enhance chondroitin production and guide future experimental research to develop more efficient host organisms for the biotechnological production process. Full article

UDP-N-acetylglucosamine pyrophosphorylase (UAP) is a key enzyme in the chitin biosynthesis pathway of insects. Here, we described the gene SfUAP in the white-backed planthopper Sogatella furcifera (Horváth) with an open reading frame of 1470 bp. Quantitative real-time polymerase chain reaction (qPCR) suggested that SfUAP exhibits a different developmental expression pattern and a higher expression after molting. The highest expression of SfUAP was observed in the integument tissues of adults, whereas head tissues showed negligible expression. RNAi-based gene silencing decreased the mRNA transcript levels in S. furcifera nymphs injected with double-stranded RNA of SfUAP. Finally, SfUAP silencing led to 84% mortality and malformed phenotypes in nymphs. Thus, our results can help better understand the role of SfUAP in S. furcifera. Full article

Respiratory syncytial virus (RSV), or human orthopneumovirus, is a negative-sense RNA virus that is the causative agent of severe lower respiratory tract infections in children and is associated with exacerbations of adult lung disease. The mechanisms how severe and/or repetitive virus infections cause declines in pulmonary capacity are not fully understood. We have recently discovered that viral replication triggers epithelial plasticity and metabolic reprogramming involving the hexosamine biosynthetic pathway (HBP). In this study, we examine the relationship between viral induced innate inflammation and the activation of hexosamine biosynthesis in small airway epithelial cells. We observe that RSV induces ~2-fold accumulation of intracellular UDP-GlcNAc, the end-product of the HBP and the obligate substrate of N glycosylation. Using two different silencing approaches, we observe that RSV replication activates the HBP pathway in a manner dependent on the RELA proto-oncogene (65 kDa subunit). To better understand the effect of RSV on the cellular N glycoproteome, and its RELA dependence, we conduct affinity enriched LC-MS profiling in wild-type and RELA-silenced cells. We find that RSV induces the accumulation of 171 N glycosylated peptides in a RELA-dependent manner; these proteins are functionally enriched in integrins and basal lamina formation. To elaborate this mechanism of HBP expression, we demonstrate that RSV infection coordinately induces the HBP pathway enzymes in a manner requiring RELA; these genes include Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT)-1/2, Glucosamine-Phosphate N-Acetyltransferase (GNPNAT)-1, phosphoglucomutase (PGM)-3 and UDP-N-Acetylglucosamine Pyrophosphorylase (UAP)-1. Using small-molecule inhibitor(s) of 8-oxoguanine DNA glycosylase1 (OGG1), we observe that OGG1 is also required for the expression of HBP pathway. In proximity ligation assays, RSV induces the formation of a nuclear and mitochondrial RELA∙OGG1 complex. In co-immunoprecipitaton (IP) experiments, we discover that RSV induces Ser 536-phosphorylated RELA to complex with OGG1. Chromatin IP experiments demonstrate a major role of OGG1 in supporting the recruitment of RELA and phosphorylated RNA Pol II to the HBP pathway genes. We conclude that the RELA∙OGG1 complex is an epigenetic regulator mediating metabolic reprogramming and N glycoprotein modifications of integrins in response to RSV. These findings have implications for viral-induced adaptive epithelial responses. Full article

Thymoquinone (2-methyl-5-propan-2-ylcyclohexa-2,5-diene-1,4-dione; TQ), a principal bioactive phytoconstituent of Nigella sativa essential oil, has been reported to have high antimicrobial potential. Thus, the current study evaluated TQ’s antimicrobial potential against a range of selected human pathogens using in vitro assays, including time-kill kinetics and anti-biofilm activity. In silico molecular docking of TQ against several antimicrobial target proteins and a detailed intermolecular interaction analysis was performed, including binding energies and docking feasibility. Of the tested bacteria and fungi, S. epidermidis ATCC 12228 and Candida albicans ATCC 10231 were the most susceptible to TQ, with 50.3 ± 0.3 mm and 21.1 ± 0.1 mm zones of inhibition, respectively. Minimum inhibitory concentration (MIC) values of TQ are in the range of 12.5–50 µg/mL, while minimum biocidal concentration (MBC) values are in the range of 25–100 µg/mL against the tested organisms. Time-kill kinetics of TQ revealed that the killing time for the tested bacteria is in the range of 1–6 h with the MBC of TQ. Anti-biofilm activity results demonstrate that the minimum biofilm inhibitory concentration (MBIC) values of TQ are in the range of 25–50 µg/mL, while the minimum biofilm eradication concentration (MBEC) values are in the range of 25–100 µg/mL, for the tested bacteria. In silico molecular docking studies revealed four preferred antibacterial and antifungal target proteins for TQ: D-alanyl-D-alanine synthetase (Ddl) from Thermus thermophilus, transcriptional regulator qacR from Staphylococcus aureus, N-myristoyltransferase from Candida albicans, and NADPH-dependent D-xylose reductase from Candida tenuis. In contrast, the nitroreductase family protein from Bacillus cereus and spore coat polysaccharide biosynthesis protein from Bacillus subtilis and UDP-N-acetylglucosamine pyrophosphorylase from Aspergillus fumigatus are the least preferred antibacterial and antifungal target proteins for TQ, respectively. Molecular dynamics (MD) simulations revealed that TQ could bind to all four target proteins, with Ddl and NADPH-dependent D-xylose reductase being the most efficient. Our findings corroborate TQ’s high antimicrobial potential, suggesting it may be a promising drug candidate for multi-drug resistant (MDR) pathogens, notably Gram-positive bacteria and Candida albicans. Full article

The rice leaf folder, Cnaphalocrocis medinalis is a major pest of rice and is difficult to control. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is a key enzyme in the chitin synthesis pathway in insects. In this study, the UAP gene from C. medinalis (CmUAP) was cloned and characterized. The cDNA of CmUAP is 1788 bp in length, containing an open reading frame of 1464 nucleotides that encodes 487 amino acids. Homology and phylogenetic analyses of the predicted protein indicated that CmUAP shared 91.79%, 87.89%, and 82.75% identities with UAPs of Glyphodes pyloalis, Ostrinia furnacalis, and Heortia vitessoides, respectively. Expression pattern analyses by droplet digital PCR demonstrated that CmUAP was expressed at all developmental stages and in 12 tissues of C. medinalis adults. Silencing of CmUAP by injection of double-stranded RNA specific to CmUAP caused death, slow growth, reduced feeding and excretion, and weight loss in C. medinalis larvae; meanwhile, severe developmental disorders were observed. The findings suggest that CmUAP is essential for the growth and development of C. medinalis, and that targeting the CmUAP gene through RNAi technology can be used for biological control of this insect. Full article

To minimize treatment toxicities, recent anti-cancer research efforts have switched from broad-based chemotherapy to targeted therapy, and emerging data show that altered cellular metabolism in cancerous cells can be exploited as new venues for targeted intervention. In this study, we focused on, among the altered metabolic processes in cancerous cells, altered glycosylation due to its documented roles in cancer tumorigenesis, metastasis and drug resistance. We hypothesize that the enzymes required for the biosynthesis of UDP-hexoses, glycosyl donors for glycan synthesis, could serve as therapeutic targets for cancers. Through structure-based virtual screening and kinetic assay, we identified a drug-like chemical fragment, GAL-012, that inhibit a small family of UDP-hexose pyrophosphorylases-galactose pyro-phosphorylase (GALT), UDP-glucose pyrophosphorylase (UGP2) and UDP-N-acetylglucosamine pyrophosphorylase (AGX1/UAP1) with an IC₅₀ of 30 µM. The computational docking studies supported the interaction of GAL-012 to the binding sites of GALT at Trp190 and Ser192, UGP2 at Gly116 and Lys127, and AGX1/UAP1 at Asn327 and Lys407, respectively. One of GAL-012 derivatives GAL-012-2 also demonstrated the inhibitory activity against GALT and UGP2. Moreover, we showed that GAL-012 suppressed the growth of PC3 cells in a dose-dependent manner with an EC₅₀ of 75 µM with no effects on normal skin fibroblasts at 200 µM. Western blot analysis revealed reduced expression of pAKT (Ser473), pAKT (Thr308) by 77% and 72%, respectively in the treated cells. siRNA experiments against the respective genes encoding the pyrophosphorylases were also performed and the results further validated the proposed roles in cancer growth inhibition. Finally, synergistic relationships between GAL-012 and tunicamycin, as well as bortezomib (BTZ) in killing cultured cancer cells were observed, respectively. With its unique scaffold and relatively small size, GAL-012 serves as a promising early chemotype for optimization to become a safe, effective, multi-target anti-cancer drug candidate which could be used alone or in combination with known therapeutics. Full article

β-N-acetylglucosaminidases (NAGs) are carbohydrate enzymes that degrade chitin oligosaccharides into N-acetylglucosamine monomers. This process is important for chitin degradation during insect development and metamorphosis. We identified and evaluated a β-N-acetylglucosaminidase 2 gene (LsNAG2) from the cigarette beetle, Lasioderma serricorne (Fabricius). The full-length open reading frame of LsNAG2 was 1776 bp and encoded a 591 amino acid protein. The glycoside hydrolase family 20 (GH20) catalytic domain and an additional GH20b domain of the LsNAG2 protein were highly conserved. Phylogenetic analysis revealed that LsNAG2 clustered with the group II NAGs. Quantitative real-time PCR analyses showed that LsNAG2 was expressed in all developmental stages and was most highly expressed in the late larval and late pupal stages. In the larval stage, LsNAG2 was predominantly expressed in the integument. Knockdown of LsNAG2 in fifth instar larvae disrupted larval–pupal molting and reduced the expression of four chitin synthesis genes (trehalase 1 (LsTRE1), UDP-N-acetylglucosamine pyrophosphorylase 1 and 2 (LsUAP1 and LsUAP2), and chitin synthase 1 (LsCHS1)). In late pupae, LsNAG2 depletion resulted in abnormal adult eclosion and wing deformities. The expression of five wing development-related genes (teashirt (LsTSH), vestigial (LsVG), wingless (LsWG), ventral veins lacking (LsVVL), and distal-less (LsDLL)) significantly declined in the LsNAG2-depleted beetles. These findings suggest that LsNAG2 is important for successful molting and wing development of L. serricorne. Full article