Response of xenobiotic biodegradation and metabolic genes in Tribolium castaneum following eugenol exposure.

Eugenol, a plant-derived component possessing small side effects, has an insecticidal activity to Tribolium castaneum; however, the underlying molecular mechanisms of eugenol acting on T. castaneum are currently unclear. Here, a nerve conduction carboxylesterase and a detoxifying glutathione S-transferase were significantly inhibited after eugenol exposure, resulting in the paralysis or death of beetles. Then, RNA-sequencing of eugenol-exposed and control samples identified 362 differentially expressed genes (DEGs), containing 206 up-regulated and 156 down-regulated genes. RNA-seq data were validated further by qRT-PCR. GO analysis revealed that DEGs were associated with 1308 GO terms of which the most enriched GO terms were catalytic activity, and integral component of membrane; KEGG pathway analysis showed that these DEGs were distributed in 151 different pathways, of which some pathways associated with metabolism of xenobiotics or drug were significantly enriched, which indicated that eugenol most likely disturbed the processes of metabolism, and detoxication. Moreover, several DEGs including Hexokinase type 2, Isocitrate dehydrogenase, and Cytochrome b-related protein, might participate in the respiratory metabolism of eugenol-exposed beetles. Some DEGs encoding CYP, UGT, GST, OBP, CSP, and ABC transporter were involved in the xenobiotic or drug metabolism pathway, which suggested that these genes of T. castaneum participated in the response to eugenol exposure. Additionally, TcOBPC11/ TcGSTs7, detected by qRT-PCR and RNA-interference against these genes, significantly increased the mortality of eugenol-treated T. castaneum, providing further evidence for the involvement of OBP/GST in eugenol metabolic detoxification in T. castaneum. These results aid eugenol insecticidal mechanisms and provide the basis of insect control.

Aldehyde oxidases mediate plant toxicant susceptibility and fecundity in the red flour beetle, Tribolium castaneum.

Aldehyde oxidases (AOXs) are a group of metabolic enzymes that play critical roles in the degradation of xenobiotics and chemicals. However, the physiological function of this enzyme in insects remains poorly understood. In this study, three TcAOX genes (TcAOX1, TcAOX2, TcAOX3) were identified and characterized from Tribolium castaneum genome. Spatiotemporal expression profiling showed that TcAOX1 expression was most highly expressed at the early pupal stage and was predominantly expressed in the antennae of adults, indicating that TcAOX1 was involved in the degradation of chemical signals; TcAOX2 expression was most highly expressed at the late pupal stage and was mainly expressed in the fat body, epidermis of larvae and adults, respectively; and TcAOX3 expression was in all stages and was primarily expressed in the head of adults. Moreover, the transcripts of TcAOX2 and TcAOX3 were significantly induced after exposure to plant oil, and RNA interference (RNAi) targeting of each of them enhanced the susceptibility of beetles to this plant toxicant, suggesting that these two genes are associated with plant toxicant detoxification. Intriguingly, knockdown of the TcAOX1 led to reductions in female egg-laying but unchanged the hatchability and the development of genital organs, suggesting that this gene may mediate fecundity by effecting the inactivation of chemical signals in T. castaneum. Overall, these results shed new light on the function of AOX genes in insects, and could facilitate the development of research on pest control management.

Odorant binding protein C12 is involved in the defense against eugenol in Tribolium castaneum.

Tribolium castaneum (T. castaneum) is a worldwide pest of stored grain that mainly harms flour, and not only causes serious loss of flour quality but also leads to deterioration of flour quality. Chemical detection plays a key role in insect behavior, and the role of odorant-binding proteins (OBPs) in insect chemical detection has been widely studied. However, the mechanism of OBPs in insect defense against exogenous toxic substances is still unclear. In this study, biochemical analysis showed that eugenol, the active component of A. vulgaris essential oil, significantly induced the expression of the OBP gene OBPC12 from T. castaneum (TcOBPC12). The mortality of late larvae treated with eugenol was higher than that of the control group after RNA interference (RNAi) against TcOBPC12, which indicates that the OBP gene is involved in the eugenol defense mechanism and leads to a decrease in sensitivity to eugenol. Tissue expression profiling showed that the expression of TcOBPC12 in the epidermis, hemolymph, and intestine was higher than in other larval tissues, and TcOBPC12 was expressed mainly in the epidermis, head, and fat body of adults. The developmental expression profile showed that the expression of TcOBPC12 in late eggs, early and late larval stages, and late adult stages was higher than in other developmental stages. These data suggest that TcOBPC12 may be involved in the absorption of exogenous toxic substances by the larvae from T. castaneum. Our results provide a theoretical basis for the metabolism and degradation mechanism of exogenous toxic substances and help explore more potential target genes of insect pests.

Metastasis-associated 1 localizes to the sarcomeric Z-disc and is implicated in skeletal muscle pathology.

Metastasis-associated 1 (MTA1), a subunit of the nucleosome remodeling and histone deacetylation (NuRD) corepressor complex, was reported to be expressed in the cytoplasm of skeletal muscles. However, the exact subcellular localization and the functional implications of MTA1 in skeletal muscles have not been examined. This study aims to demonstrate the subcellular localization of MTA1 in skeletal muscles and reveal its possible roles in skeletal muscle pathogenesis. Striated muscles (skeletal and cardiac) from C57BL/6 mice of 4-5 weeks were collected to examine the expression of MTA1 by Western blotting and immunohistochemistry. Immunofluorescence and immunoelectron microscopy were performed for MTA1, α-actinin (a Z-disc marker protein), and SMN (survival of motor neuron) proteins. Gene Expression Omnibus (GEO) data sets were analyzed using the GEO2R online tool to explore the functional implications of MTA1 in skeletal muscles. MTA1 expression was detected by Western blotting and immunohistochemistry in skeletal and cardiac muscles. Subcellular localization of MTA1 was found in the Z-disc of sarcomeres, where α-actinin and SMN were expressed. Data mining of GEO profiles suggested that MTA1 dysregulation is associated with multiple skeletal muscle defects, such as Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, nemaline myopathy, and dermatomyositis. The GEO analysis also showed that MTA1 expression gradually decreased with age in mouse skeletal muscle precursor cells. The subcellular localization of MTA1 in sarcomeres of skeletal muscles implies its biological roles in sarcomere structures and its possible contribution to skeletal muscle pathology.

Construction and performance evaluation of fully biomimetic hyaline cartilage matrix scaffolds for joint defect regeneration.

Due to the absence of nerves and blood vessels in articular cartilage, its regeneration and repair present a significant and complex challenge in osteoarthritis treatment. Developing a specialized physical and chemical microenvironment supporting cell growth has been difficult in cartilage grafting, especially when aiming for comprehensive biomimetic solutions. Based on previous research, we have designed a tissue-engineered decellularized living hyaline cartilage graft (dLhCG). The study developed a method to improve the hydrophilicity and stiffness of scaffolds by employing chemical grafting techniques and designed a decellularized hyaline cartilage phenotype matrix scaffold for tissue engineering. Here, we reported a method using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride /N-hydroxysuccinimide (EDC/NHS) to achieve the grafting of chondroitin sulfate (CS) onto dLhCG, ultimately producing a tissue-engineered hyaline cartilage graft with the CS (dLhCG/CS). Young's modulus measurements revealed that the cross-linked scaffolds exhibited enhanced mechanical properties. We implanted the cross-linked dLhCG/CS scaffolds into the trochlear region of rat joints and evaluated their functionality through histological analysis and biomechanical tests. After 12 weeks, the dLhCG/CS scaffolds demonstrated excellent bioinductive activity comparable to dLhCG. The regenerated tissue effectively maintained a hyaline cartilage phenotype and exhibited similar mechanical properties, playing a crucial role in cartilage regeneration.

Enhanced hyaline cartilage formation and continuous osteochondral regeneration via 3D-Printed heterogeneous hydrogel with multi-crosslinking inks.

The unique gradient structure and complex composition of osteochondral tissue pose significant challenges in defect regeneration. Restoration of tissue heterogeneity while maintaining hyaline cartilage components has been a difficulty of an osteochondral tissue graft. A novel class of multi-crosslinked polysaccharide-based three-dimensional (3D) printing inks, including decellularized natural cartilage (dNC) and nano-hydroxyapatite, was designed to create a gradient scaffold with a robust interface-binding force. Herein, we report combining a dual-nozzle cross-printing technology and a gradient crosslinking method to create the scaffolds, demonstrating stable mechanical properties and heterogeneous bilayer structures. Biofunctional assessments revealed the remarkable regenerative effects of the scaffold, manifesting three orders of magnitude of mRNA upregulation during chondrogenesis and the formation of pure hyaline cartilage. Transcriptomics of the regeneration site in vivo and scaffold cell interaction tests in vitro showed that printed porous multilayer scaffolds could form the correct tissue structure for cell migration. More importantly, polysaccharides with dNC provided a hydrophilic microenvironment. The microenvironment is crucial in osteochondral regeneration because it could guide the regenerated cartilage to ensure the hyaline phenotype.

DNMT inhibitors and HDAC inhibitors regulate E-cadherin and Bcl-2 expression in endometrial carcinoma in vitro and in vivo.

BACKGROUND: The effect of histone deacetylase inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMTIs) on proliferation of endometrial cancer (EC) cells in vitro and in vivo was investigated. METHODS: Changes in methylation of the CDH1 promoter in HDACI- and DNMTI-treated HEC-1-B and RL-952 EC cells were detected. Nude mice with xenografted implants of human EC HEC-1-B cells were treated with valproic acid (VPA) and decitabine (DAC) and evaluated for tumor growth, CDH1 and Bcl-2 mRNA levels. RESULTS: DAC, VPA and suberoylanilide hydroxamic acid (SAHA) inhibited proliferation, induced cell cycle arrest and enhanced the apoptotic index in both cell lines, DAC, VPA and SAHA upregulated E-cadherin mRNA and protein levels and downregulated Bcl-2 mRNA levels in vitro. DAC and VPA inhibited tumor growth, upregulated CDH1 mRNA and downregulated Bcl-2 mRNA levels in vivo. CONCLUSIONS: A combination of HDACIs and DNMTIs suppresses the growth of EC, which is likely mediated by upregulation of E-cadherin and downregulation of Bcl-2.

Knockdown of Uridine Diphosphate Glucosyltransferase 86Dg Enhances Susceptibility of Tribolium castaneum (Coleoptera: Tenebrionidae) to Artemisia vulgaris (Asterales: Asteraceae) Essential Oil.

Uridine diphosphate glucosyltransferases (UGTs), which are phase II detoxification enzymes, are found in various organisms. These enzymes play an important role in the detoxification mechanisms of plant allelopathy and in insects. Artemisia vulgaris L. (Asterales: Asteraceae: Artemisia) essential oil has strong contact toxicity to Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) larvae. However, the effect of A. vulgaris essential oil on UGTs is unclear. In this study, A. vulgaris essential oil was shown to significantly induce the expression of the TcUgt86Dg transcript. Furthermore, treatment of TcUgt86Dg-silenced individuals with A. vulgaris essential oil resulted in higher mortality than for the control individuals, indicating that TcUgt86Dg is involved in detoxification of A. vulgaris essential oil in T. castaneum. The developmental expression profile showed that the expression of TcUgt86Dg in late adults was higher than in other developmental stages. Furthermore, the expression profile in adult tissues revealed higher expression of TcUgt86Dg in the head, antenna, fat body, and accessory gland than in other tissues. These data show that TcUgt86Dg may be involved in the metabolism of exogenous toxins by T. castaneum; thus, our results have elucidated one possible mechanism of resistance to A. vulgaris essential oil and provide a theoretical basis for a control scheme for T. castaneum.