RESUMO
The extensive use of Bacillus thuringiensis (Bt) in pest management has driven the evolution of pest resistance to Bt toxins, particularly Cry1Ac. Effective management of Bt resistance necessitates a good understanding of which pest proteins interact with Bt toxins. In this study, we screened a Helicoverpa armigera larval midgut cDNA library and captured 208 potential Cry1Ac-interacting proteins. Among these, we further examined the interaction between Cry1Ac and a previously unknown Cry1Ac-interacting protein, HaDALP (H. armigera death-associated LIM-only protein), as well as its role in toxicology. The results revealed that HaDALP specifically binds to both the Cry1Ac protoxin and activated toxin, significantly enhancing cell and larval tolerance to Cry1Ac. Additionally, HaDALP was overexpressed in a Cry1Ac-resistant H. armigera strain. These findings reveal a greater number of Cry1Ac-interacting proteins than previously known and demonstrate, for the first time, that HaDALP reduces Cry1Ac toxicity by sequestering both the protoxin and activated toxin.
Assuntos
Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas , Proteínas Hemolisinas , Proteínas de Insetos , Inseticidas , Larva , Mariposas , Animais , Toxinas de Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/toxicidade , Toxinas de Bacillus thuringiensis/química , Endotoxinas/metabolismo , Endotoxinas/genética , Endotoxinas/toxicidade , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Proteínas Hemolisinas/toxicidade , Proteínas Hemolisinas/genética , Mariposas/metabolismo , Mariposas/efeitos dos fármacos , Mariposas/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Larva/metabolismo , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/genética , Inseticidas/toxicidade , Inseticidas/farmacologia , Inseticidas/química , Bacillus thuringiensis/química , Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/genética , Resistência a Inseticidas/genética , Controle Biológico de Vetores , Helicoverpa armigeraRESUMO
Regenerating inflamed bone defects represents a severe clinical challenge due to the undesirable inflammatory microenvironment. The inflammatory stimulus poses a weighty threat to the regenerative capacity of endogenously derived mesenchymal stem cells (MSCs), which are mainly responsible for osteogenic differentiation, thereby resulting in compromised endogenous bone formation. Consequently, alleviating the biological characteristics of inflammatory-impaired MSCs is crucial for promoting inflamed bone regeneration. Nano-sized small extracellular vesicles (sEVs) have emerged as promising therapeutic tools to orchestrate MSCs fate due to their intrinsic biocompatibility and encapsulated bioactive contents. In the present study, we extracted sEVs from youthful and adult dental pulp MSCs and explored their ability to recover inflammation-compromised periodontal ligament stem cells (IPDLSCs). The results indicated that both types of sEVs were capable of facilitating IPDLSCs osteogenesis. However, young sEVs exhibited a more robust potential at a lower concentration compared to adult sEVs. Mechanically, young sEVs enhanced the expression of bone morphogenetic protein 4 (BMP4) via delivering the protein Biglycan, which correspondingly promoted the osteogenic capability of IPDLSCs. Collectively, our findings emphasized that young sEVs hold enormous potential to rescue the inherent function and regenerative competence of inflammation-impaired MSCs, shedding light on their promising therapeutic prospects for infected bone regeneration.
Assuntos
Biglicano , Regeneração Óssea , Diferenciação Celular , Vesículas Extracelulares , Células-Tronco Mesenquimais , Osteogênese , Ligamento Periodontal , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Regeneração Óssea/efeitos dos fármacos , Biglicano/metabolismo , Vesículas Extracelulares/metabolismo , Osteogênese/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/metabolismo , Inflamação/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Polpa Dentária/citologia , Animais , Células-Tronco/metabolismoRESUMO
Cry2Ab is one of the important alternative Bt proteins that can be used to manage insect pests resistant to Cry1A toxins and to expand the insecticidal spectrum of pyramided Bt crops. Previous studies have showed that vacuolar H+-ATPase subunits A and B (V-ATPase A and B) may be involved in Bt insecticidal activities. The present study investigated the role of V-ATPases subunit E in the toxicity of Cry2Ab in Helicoverpa amigera. RT-PCR analysis revealed that oral exposure of H. amigera larvae to Cry2Ab led to a significant reduction in the expression of H. armigera V-ATPase E (HaV-ATPase E). Ligand blot, homologous and heterologous competition experiments confirmed that HaV-ATPases E physically and specifically bound to activated Cry2Ab toxin. Heterologous expressing of HaV-ATPase E in Sf9 cells made the cell line more susceptible to Cry2Ab, whereas knockdown of the endogenous V-ATPase E in H. zea midgut cells decreased Cry2Ab's cytotoxicity against this cell line. Further in vivo bioassay showed that H. armigera larvae fed a diet overlaid with both Cry2Ab and E. coli-expressed HaV-ATPase E protein suffered significantly higher mortality than those fed Cry2Ab alone. These results support that V-ATPases E is a putative receptor of Cry2Ab and can be used to improve Cry2Ab toxicity and manage Cry2Ab resistance at least in H. armigera.
Assuntos
Bacillus thuringiensis , Inseticidas , Mariposas , Animais , Helicoverpa armigera , Endotoxinas/toxicidade , Endotoxinas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Escherichia coli , Toxinas de Bacillus thuringiensis/metabolismo , Mariposas/genética , Mariposas/metabolismo , Larva/metabolismo , Inseticidas/toxicidade , Inseticidas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Proteínas Hemolisinas/metabolismo , Bacillus thuringiensis/metabolismo , Resistência a InseticidasRESUMO
The long-term use of chemical insecticides has caused serious problems of insect resistance and environmental pollution; new insecticides are needed to solve this problem. Cyclosporin A (CsA) is a polypeptide produced by many fungi, which is used to prevent or treat immune rejection during organ transplantation. However, little is known about the utility of CsA as an insecticide. Therefore, this study evaluated the insecticidal activity of CsA using Ostrinia furnacalis as a model. The results demonstrated that CsA was toxic to O. furnacalis with LC50 values of 113.02 µg/g and 198.70 µg/g for newly hatched neonates and newly molted third-instar larvae, respectively. Furthermore, CsA treatment had sublethal effects on the development of O. furnacalis, and significantly reduced the fecundity of adults; this suggests that CsA has great potential to suppress O. furnacalis populations. Further analysis revealed that CsA suppressed calcineurin activity in larvae. CsA had independent or synergistic toxic effects on O. furnacalis when combined with ß-cypermethrin, indoxacarb, emamectin benzoate, azadirachtin, and the Bacillus thuringiensis toxin Cry1Ac, which suggests that CsA can help prevent or manage resistance. Our study provides detailed information on the potential of CsA as an insecticide for controlling lepidopterans.
RESUMO
Trehalose is the major "blood sugar" of insects and it plays a crucial role in energy supply and as a stress protectant. The hydrolysis of trehalose occurs only under the enzymatic control of trehalase (Treh), which plays important roles in growth and development, energy supply, chitin biosynthesis, and abiotic stress responses. Previous reports have revealed that the vital hormone 20-hydroxyecdysone (20E) regulates Treh, but the detailed mechanism underlying 20E regulating Treh remains unclear. In this study, we investigated the function of HaTreh1 in Helicoverpa armigera larvae. The results showed that the transcript levels and enzymatic activity of HaTreh1 were elevated during molting and metamorphosis stages in the epidermis, midgut, and fat body, and that 20E upregulated the transcript levels of HaTreh1 through the classical nuclear receptor complex EcR-B1/USP1. HaTreh1 is a mitochondria protein. We also found that knockdown of HaTreh1 in the fifth- or sixth-instar larvae resulted in weight loss and increased mortality. Yeast two-hybrid, coimmunoprecipitation, and glutathione-S-transferase (GST) pull-down experiments demonstrated that HaTreh1 bound with ATP synthase subunit alpha (HaATPs-α) and that this binding increased under 20E treatment. In addition, 20E enhanced the transcript level of HaATPs-α and ATP content. Finally, the knockdown of HaTreh1 or HaATPs-α decreased the induction effect of 20E on ATP content. Altogether, these findings demonstrate that 20E controls ATP production by up-regulating the binding of HaTreh1 to HaATPs-α in H. armigera.
Assuntos
Ecdisterona , Proteínas de Insetos , Mariposas , Trealase , Trifosfato de Adenosina/metabolismo , Animais , Ecdisterona/metabolismo , Proteínas de Insetos/metabolismo , Larva/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mariposas/enzimologia , Mariposas/genética , Trealase/metabolismo , Trealose/metabolismoRESUMO
Spodoptera litura is a serious polyphagous pest in the whole world, which has developed resistance to most conventional insecticides and even some Bacillus thuringiensis (Bt) toxins. Cry1Ca has excellent insecticide activity against S. litura with potential application to control S. litura and delay the development of insect resistance. However, the mode of action of Cry1Ca in S. litura is poorly understood. Here, Cry1Ca-binding proteins were identified from S. litura by using pull down assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that aminopeptidase-N (APN), ATP binding cassette subfamily C member 2 (ABCC2), polycalin, actin and V-type proton ATPase subunit A may bind with Cry1Ca. Further study confirmed that ABCC2 fragment expressed in vitro can bind to Cry1Ca as demonstrated by Ligand blot and homologous competition experiments. The over-expression of endogenous SlABCC2 in Sf9 cells increased Cry1Ca cytotoxicity. Correspondingly, the vivo loss of function analyses by SlABCC2 small interfering RNAs (siRNAs) in S. litura larvae decreased the toxicity of Cry1Ca to larvae. Altogether, these results show that ABCC2 of S. litura is a functional receptor that is involved in the action mode of Cry1Ca.
Assuntos
Bacillus thuringiensis/fisiologia , Interações Hospedeiro-Patógeno , Proteína 2 Associada à Farmacorresistência Múltipla/metabolismo , Spodoptera/metabolismo , Spodoptera/microbiologia , Animais , Toxinas de Bacillus thuringiensis/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Células Sf9 , Spodoptera/genéticaRESUMO
Cell-free based therapy is an effective strategy in regenerative medicine as it avoids controversial issues, such as immunomodulation and stability. Recently, exosomes have been explored as a favorable substitution for stem cell therapy as they exhibit multiple advantages, such as the ability to be endocytosed and innate biocompatibility. This study aimed to investigate the effects of stem cells from human exfoliated deciduous teeth (SHED)-derived exosomes (SHED-Exo) on bone marrow stromal cells (BMSCs) osteogenesis and bone recovery. SHED-Exo were isolated, characterized, and applied to the bone loss area caused by periodontitis in a mouse model. We found that the injection of SHED-Exo restored bone loss to the same extent as original stem cells. Without affecting BMSCs proliferation, SHED-Exo mildly inhibited apoptosis. Moreover, SHED-Exo specifically promoted BMSCs osteogenesis and inhibited adipogenesis compared with SHED-derived conditioned medium. The expression of osteogenic marker genes, alkaline phosphatase activity, and Alizarin Red S staining of BMSCs was significantly increased by co-culturing with SHED-Exo. Moreover, Western blot analysis showed that Runx2, a key transcriptional factor in osteogenic differentiation, and p-Smad5 were upregulated upon SHED-Exo stimulation. Expression of the adipogenic marker PPARγ and the amount of lipid droplets decreased when exosomes were present. Low doses of exosomes inhibited the expression of the inflammatory cytokines IL-6 and TNF-α. In conclusion, SHED-Exo directly promoted BMSCs osteogenesis, differentiation, and bone formation. Therefore, exosomes have the potential to be utilized in the treatment of periodontitis and other bone diseases.
Assuntos
Reabsorção Óssea/terapia , Exossomos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Dente Decíduo/fisiologia , Adipogenia/fisiologia , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/metabolismo , Reabsorção Óssea/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Exossomos/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Células-Tronco/metabolismo , Células-Tronco/fisiologia , Dente Decíduo/metabolismoRESUMO
MicroRNA-27a/b are small non-coding RNAs which are reported to regulate inflammatory response and cell proliferation. Although some studies have demonstrated that miR-27b is down-regulated in the oral specimens of patients suffering with oral lichen planus (OLP), the molecular mechanism of miR-27b decrease remains a large mystery, and the expression of miR-27a in OLP is not well explored. Here, we demonstrated both miR-27a and miR-27b, compared with healthy controls, were reduced in the oral biopsies, serum and saliva samples derived from OLP patients. The reductions of miR-27a/b were also confirmed in the lipopolysaccharide (LPS)- or activated CD4+ T cell-treated human oral keratinocytes (HOKs). Furthermore, we found vitamin D receptor (VDR) binding sites in the promoters of miR-27a/b genes and verified this finding. We also tested miR-27a/b levels in the oral epithelium from paricalcitol-treated, vitamin D deficient or VDR knockout mice. In the rescue experiments, we confirmed vitamin D and VDR inhibited LPS- or activated CD4+ T cell-induced miR-27a/b reductions in HOKs. In sum, our results show that vitamin D/VDR signaling induces miR-27a/b in oral lichen planus.
Assuntos
MicroRNAs/metabolismo , Receptores de Calcitriol/metabolismo , Transdução de Sinais , Vitamina D/metabolismo , Animais , Sítios de Ligação , Estudos de Casos e Controles , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Ergocalciferóis/farmacologia , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Líquen Plano Bucal/metabolismo , Líquen Plano Bucal/patologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/sangue , MicroRNAs/genética , Regiões Promotoras Genéticas , Receptores de Calcitriol/deficiência , Receptores de Calcitriol/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
While Cry1Ac has been known to bind with larval midgut proteins cadherin, APN (amino peptidase N), ALP (alkaline phosphatase) and ABCC2 (adenosine triphosphate-binding cassette transporter subfamily C2), little is known about the receptors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baseline cytotoxicity of activated Cry1Ac and Cry2Ab against the midgut and fat body cell lines of Helicoverpa zea and the ovary cell line of Spodoptera frugiperda (SF9). As expected, the descending order of cytotoxicity of Cry1Ac against the three cell lines in terms of 50% lethal concetration (LC50 ) was midgut (31.0 µg/mL) > fat body (59.0 µg/mL) and SF9 cell (99.6 µg/mL). By contrast, the fat body cell line (LC50 = 7.55 µg/mL) was about twice more susceptible to Cry2Ab than the midgut cell line (16.0 µg/mL), the susceptibility of which was not significantly greater than that of SF9 cells (27.0 µg/mL). Further, ligand blot showed the binding differences between Cry1Ac and Cry2Ab in the three cell lines. These results indicated that the receptors of Cry2Ab were enriched in fat body cells and thus largely different from the receptors of Cry1Ac, which were enriched in midgut cells.