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1.
Pestic Biochem Physiol ; 179: 104965, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34802515

RESUMO

The fall armyworm (FAW), Spodoptera frugiperda, is a global pest of multiple economically important row crops and the development of resistance to commercially available insecticidal classes has inhibited FAW control. Thus, there is a need to identify chemical scaffolds that can provide inspiration for the development of novel insecticides for FAW management. This study aimed to assess the sensitivity of central neurons and susceptibility of FAW to chloride channel modulators to establish a platform for repurposing existing insecticides or designing new chemicals capable of controlling FAW. Potency of select chloride channel modulators were initially studied against FAW central neuron firing rate and rank order of potency was determined to be fipronil > lindane > Z-stilbene > DIDS > GABA > E-stilbene. Toxicity bioassays identified fipronil and lindane as the two most toxic modulators studied with topical LD50's of 41 and 75 ng/mg of caterpillar, respectively. Interestingly, Z-stilbene was toxic at 300 ng/mg of caterpillar, but no toxicity was observed with DIDS or E-stilbene. The significant shift in potency between stilbene isomers indicates structure-activity relationships between stilbene chemistry and the binding site in FAW may exist. The data presented in this study defines the potency of select chloride channel modulators to FAW neural activity and survivorship to establish a platform for development of novel chemical agents to control FAW populations. Although stilbenes may hold promise for insecticide development, the low toxicity of the scaffolds tested in this study dampen enthusiasm for their development into FAW specific insecticides.


Assuntos
Inseticidas , Estilbenos , Animais , Resistência a Inseticidas , Inseticidas/toxicidade , Spodoptera , Estilbenos/toxicidade , Zea mays
2.
Pestic Biochem Physiol ; 179: 104973, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34802523

RESUMO

The fall armyworm (FAW) Spodoptera frugiperda (Lepidoptera: Noctuidae) is a severe agricultural pest, which has invaded into China in 2019 and caused heavy damage to maize. The γ-aminobutyric acid receptor (GABAR)-targeted insecticides including broflanilide, fluralaner and fipronil exhibit high toxicity towards lepidopteran pests. However, whether they could be used for control of FAW and their possible mode of action in FAW remain unclear. In this study, broflanilide, fluralaner and fipronil exhibited high oral toxicity in FAW larvae with median lethal dose (LD50) values of 0.677, 0.711, and 23.577 mg kg-1 (active ingredient/ artificial food), respectively. In the electrophysiological assay, fluralaner and fipronil could strongly inhibit GABA-induced currents of homomeric FAW resistance to dieldrin 1 (RDL1) receptor with median inhibitory concentration (IC50) values of 5.018 nM (95% confidence interval (CI) 2.864-8.789) and 8.595 nM (95% CI 5.105-14.47), respectively, whereas broflanilide could not. In addition, the cytochrome P450 (P450), glutathione-S-transferase (GST) and carboxylesterase (CarE) activities were positively response to broflanilide, P450 and GST to fluralaner, and GST and CarE to fipronil, respectively, compared with those of control. In conclusion, we firstly reported a notable insecticidal activity of three representative GABAR-targeted insecticides to FAW in vivo, and in vitro using electrophysiological assay. The GST is the primary detoxification enzyme for three tested insecticides. Our results would guide the rotational use of GABAR-targeted insecticides in field.


Assuntos
Inseticidas , Animais , Inseticidas/toxicidade , Larva , Receptores de GABA , Spodoptera , Zea mays
3.
J Agric Food Chem ; 69(45): 13425-13435, 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34748318

RESUMO

Adaptation to phytochemicals in herbivorous insects can influence tolerance to insecticides. However, it is unclear how insects use phytochemicals as cues to activate their metabolic detoxification systems. In this study, we found that dietary exposure to xanthotoxin enhanced tolerance of Spodoptera litura larvae to λ-cyhalothrin. Xanthotoxin ingestion significantly elevated the mRNA levels of 35 detoxification genes as well as the transcription factors Cap 'n' collar isoform-C (CncC) and its binding factor small muscle aponeurosis fibromatosis isoform-K (MafK). Additionally, xanthotoxin exposure increased the levels of reactive oxygen species (ROS), while ROS inhibitor N-acetylcysteine (NAC) treatment blocked xanthotoxin-induced expression of CncC, MafK, and detoxification genes and also prevented xanthotoxin-enhanced larval tolerance to λ-cyhalothrin. The 20-hydroxyecdysone (20E) signaling pathway was effectively activated by xanthotoxin, while blocking of 20E signaling transduction prevented xanthotoxin-enhanced larval tolerance to λ-cyhalothrin. Application of 20E induced the expression of multiple xanthotoxin-induced detoxification genes and enhanced λ-cyhalothrin tolerance in S. litura. NAC treatment blocked xanthotoxin-induced 20E synthesis, while the CncC agonist curcumin activated the 20E signaling pathway. These results indicate that the ROS/CncC pathway controls the induction of metabolic detoxification upon exposure to xanthotoxin, at least in part, through its regulation of the 20E signaling pathway.


Assuntos
Ecdisterona , Inseticidas , Animais , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Larva/genética , Larva/metabolismo , Metoxaleno , Nitrilas , Piretrinas , Espécies Reativas de Oxigênio , Transdução de Sinais , Spodoptera/genética , Spodoptera/metabolismo
4.
Braz J Biol ; 83: e245273, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34669790

RESUMO

The possible interference of resistant pest's populations to insecticides in natural enemies in the action thas not been clarified yet. Thus, this study aimed to evaluate Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) performance on Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) eggs with resistance frequency to the Metaflumizone over six generations of product exposure. Egg cards (2.0 x 7.0 cm) containing eggs from two populations of S. frugiperda, (resistant to Metaflumizone and the other susceptible), were exposed to T. pretiosum females for 24 hours in free-choice and no-choice testing in three generations (G1, G4, and G6). A completely randomized experimental design was used with 25 replications, each consisting of an egg card (experimental unit) containing 20 eggs. The parameters evaluated were: parasitism (%), emergence (%), sex ratio, number of emerged parasitoids per egg and males/females longevity. ANOVA and Tukey test (P≤ 0.05) were applied on the results. Results showed a reduction in parasitism [41.0% (G1) and 28.4% (G4)], egg emergence (17.5%) and parasitoids/egg [16.2 (G4) and 17.2 (G6)] in eggs originating from the population with resistance frequency. Females emerging from G6 populations eggs without exposure to Metaflumizone had greater longevity (3.5 days more) than the resistant population. The sex ratio and male longevity were not affected. The results indicate a reduction in T. pretiosum activity if S. frugiperda populations have some frequency of resistance to Metaflumizone.


Assuntos
Himenópteros , Mariposas , Vespas , Animais , Feminino , Masculino , Semicarbazonas , Razão de Masculinidade , Spodoptera
5.
Nat Commun ; 12(1): 6103, 2021 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-34671049

RESUMO

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/virologia , Domínios e Motivos de Interação entre Proteínas/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/isolamento & purificação , Enzima de Conversão de Angiotensina 2/ultraestrutura , Animais , Linhagem Celular Tumoral , Cristalografia por Raios X , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , SARS-CoV-2/genética , Células Sf9 , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Spodoptera , Ressonância de Plasmônio de Superfície , Ligação Viral , Internalização do Vírus
6.
J Vis Exp ; (175)2021 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-34633386

RESUMO

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase). In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.


Assuntos
Elementos de DNA Transponíveis , Transposases , Animais , Elementos de DNA Transponíveis/genética , Células Germinativas/metabolismo , Plasmídeos , Spodoptera/genética , Spodoptera/metabolismo , Transposases/genética , Transposases/metabolismo
7.
Int J Mol Sci ; 22(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34638623

RESUMO

How herbivorous insects adapt to host plants is a key question in ecological and evolutionary biology. The fall armyworm, (FAW) Spodoptera frugiperda (J.E. Smith), although polyphagous and a major pest on various crops, has been reported to have a rice and corn (maize) feeding strain in its native range in the Americas. The species is highly invasive and has recently established in China. We compared behavioral changes in larvae and adults of a corn population (Corn) when selected on rice (Rice) and the molecular basis of these adaptational changes in midgut and antennae based on a comparative transcriptome analysis. Larvae of S. frugiperda reared on rice plants continuously for 20 generations exhibited strong feeding preference for with higher larval performance and pupal weight on rice than on maize plants. Similarly, females from the rice selected population laid significantly more eggs on rice as compared to females from maize population. The most highly expressed DEGs were shown in the midgut of Rice vs. Corn. A total of 6430 DEGs were identified between the populations mostly in genes related to digestion and detoxification. These results suggest that potential adaptations for feeding on rice crops, may contribute to the current rapid spread of fall armyworm on rice crops in China and potentially elsewhere. Consistently, highly expressed DEGs were also shown in antennae; a total of 5125 differentially expressed genes (DEGs) s were identified related to the expansions of major chemosensory genes family in Rice compared to the Corn feeding population. These results not only provide valuable insight into the molecular mechanisms in host plants adaptation of S. frugiperda but may provide new gene targets for the management of this pest.


Assuntos
Spodoptera/genética , Spodoptera/fisiologia , Adaptação Fisiológica/genética , Animais , China , Produtos Agrícolas/parasitologia , Fenômenos Fisiológicos do Sistema Digestório , Comportamento Alimentar/fisiologia , Feminino , Ontologia Genética , Genes de Insetos , Herbivoria/genética , Herbivoria/fisiologia , Adaptação ao Hospedeiro/genética , Adaptação ao Hospedeiro/fisiologia , Especificidade de Hospedeiro/genética , Especificidade de Hospedeiro/fisiologia , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/fisiologia , Larva/fisiologia , Masculino , Oryza/parasitologia , Oviposição/fisiologia , Spodoptera/patogenicidade , Transcriptoma , Zea mays/parasitologia
8.
PLoS One ; 16(10): e0258235, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34679089

RESUMO

Carnivorous plants feed on animal prey, mainly insects, to get additional nutrients. This carnivorous syndrome is widely investigated and reported. In contrast, reports on herbivores feeding on carnivorous plants and related defenses of the plants under attack are rare. Here, we studied the interaction of a pitcher plant, Nepenthes x ventrata, with a generalist lepidopteran herbivore, Spodoptera littoralis, using a combination of LC/MS-based chemical analytics, choice and feeding assays. Chemical defenses in N. x ventrata leaves were analyzed upon S. littoralis feeding. A naphthoquinone, plumbagin, was identified in Nepenthes defense against herbivores and as the compound mainly responsible for the finding that S. littoralis larvae gained almost no weight when feeding on Nepenthes leaves. Plumbagin is constitutively present but further 3-fold increased upon long-term (> 1 day) feeding. Moreover, in parallel de novo induced trypsin protease inhibitor (TI) activity was identified. In contrast to TI activity, enhanced plumbagin levels were not phytohormone inducible, not even by defense-related jasmonates although upon herbivory their level increased more than 50-fold in the case of the bioactive jasmonic acid-isoleucine. We conclude that Nepenthes is efficiently protected against insect herbivores by naphthoquinones acting as phytoanticipins, which is supported by additional inducible defenses. The regulation of these defenses remains to be investigated.


Assuntos
Planta Carnívora/fisiologia , Herbivoria/fisiologia , Naftoquinonas/farmacologia , Compostos Fitoquímicos/farmacologia , Sarraceniaceae/fisiologia , Ácido Abscísico/farmacologia , Animais , Planta Carnívora/efeitos dos fármacos , Ciclopentanos/farmacologia , Dieta , Herbivoria/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Oxilipinas/farmacologia , Reguladores de Crescimento de Plantas/análise , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/fisiologia , Inibidores de Proteases/farmacologia , Ácido Salicílico/farmacologia , Sarraceniaceae/efeitos dos fármacos , Spodoptera/efeitos dos fármacos , Spodoptera/fisiologia
9.
Molecules ; 26(19)2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34641534

RESUMO

Spodoptera litura Fab. is a polyphagous pest causing damage to many agriculture crops leading to yield loss. Recurrent usage of synthetic pesticides to control this pest has resulted in resistance development. Plant-derived diterpenoid compound andrographolide was isolated from the leaves of Andrographis paniculata. It was analysed by gas chromatography-mass spectroscopy and quantified by HPLC. Nutritional indices and digestive enzymatic profile were evaluated. Third, fourth and fifth instar larvae were treated with different concentrations of andrographolide. At 3, 6 and 9 ppm-treated concentrations the larvae showed decreased RGR, RCR, ECI, ECD values with adverse increase in AD. The digestive enzymes were significantly inhibited when compared with control. Conspicuously, andrographolide showed pronounced mortality of S. litura by inhibition of enzyme secretion and intake of food. The binding ability of andrographolide with CYTP450 showed high affinity with low binding energy. Andrographolide has the potential to be exploited as a biocontrol agent against S. litura as an eco-friendly pesticide.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Diterpenos/farmacologia , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Spodoptera/efeitos dos fármacos , Amilases/metabolismo , Andrographis/química , Animais , Diterpenos/isolamento & purificação , Diterpenos/metabolismo , Diterpenos/toxicidade , Relação Dose-Resposta a Droga , Inativação Metabólica/efeitos dos fármacos , Inseticidas/isolamento & purificação , Inseticidas/metabolismo , Inseticidas/toxicidade , Larva/efeitos dos fármacos , Lipase/metabolismo , Simulação de Acoplamento Molecular , Peptídeo Hidrolases/metabolismo
10.
Toxicon ; 203: 22-29, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34600912

RESUMO

Phenolics are the most abundant secondary metabolites of plants that are widely distributed in the plant kingdom. In the last few years, the development and identification of phenolic compounds from various plants have become a major area of the environment and health-related research. In the present study, different phenolic compounds were purified from the bark of the medicinally important plant Acacia nilotica which is rich in polyphenols and were evaluated for their insecticidal potential against a polyphagous pest, Spodoptera litura (Fab.). The compounds viz. Catechin, Chlorogenic acid, and Umbelliferone were purified from ethyl acetate-acetone (E-AE) fraction using Semi-preparative HPLC and were identified using melting point determination, Nuclear Magnetic Resonance (NMR), and Mass Spectroscopy (MS). These phenolic compounds recorded detrimental effects on the growth and development of second instar larvae (6 days old) of S. litura. The larval growth, survival, adult emergence, pupal weight, and different nutritional indices were adversely affected by the various concentrations of these purified compounds. The findings revealed the insecticidal potential of polyphenols obtained from the bark of A. nilotica, which can provide an alternative for resistance management, as these plant phytochemicals are highly effective against insecticide-resistant insect pests.


Assuntos
Acacia , Inseticidas , Animais , Inseticidas/toxicidade , Larva , Fenóis/toxicidade , Spodoptera
11.
J Insect Sci ; 21(5)2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34581410

RESUMO

RNAi is an effective tool for gene function analysis and a promising strategy to provide environmentally friendly control approaches for pathogens and pests. Recent studies support the utility of bacterium-mediated RNAi as a cost-effective method for gene function study and a suitable externally applied delivery mechanism for pest control. Here, we developed a bacterium-mediated RNAi system in Spodoptera frugiperda based on four target genes, specifically, Chitinase (Sf-CHI), Chitin synthase B (Sf-CHSB), Sugar transporter SWEET1 (Sf-ST), and Hemolin (Sf-HEM). RNAi conducted by feeding larvae with bacteria expressing dsRNAs of target genes or injecting pupae and adults with bacterially synthesized dsRNA induced silencing of target genes and resulted in significant negative effects on growth and survival of S. frugiperda. However, RNAi efficiency and effects were variable among different target genes and dsRNA delivery methods. Injection of pupae with dsCHI and dsCHSB induced a significant increase in wing malformation in adults, suggesting that precise regulation of chitin digestion and synthesis is crucial during wing formation. Injection of female moths with dsHEM resulted in lower mating, fecundity, and egg hatching, signifying a critical role of Sf-HEM in the process of egg production and/or embryo development. Our collective results demonstrate that bacterium-mediated RNAi presents an alternative technique for gene function study in S. frugiperda and a potentially effective strategy for control of this pest, and that Sf-CHI, Sf-CHSB, Sf-ST, and Sf-HEM encoding genes can be potent targets.


Assuntos
Interferência de RNA , RNA de Cadeia Dupla/farmacologia , Spodoptera , Animais , Bactérias/genética , Quitinases/efeitos dos fármacos , Quitinases/genética , Fertilidade/efeitos dos fármacos , Genes de Insetos/efeitos dos fármacos , Imunoglobulinas/efeitos dos fármacos , Imunoglobulinas/genética , Proteínas de Insetos/efeitos dos fármacos , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/fisiologia , Controle Biológico de Vetores , Pupa/efeitos dos fármacos , Pupa/genética , Pupa/fisiologia , Reprodução/efeitos dos fármacos , Spodoptera/efeitos dos fármacos , Spodoptera/genética , Spodoptera/fisiologia
12.
Nat Commun ; 12(1): 5610, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34584079

RESUMO

Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.


Assuntos
Doenças Cerebelares/metabolismo , Endonucleases/metabolismo , Mutação , Precursores de RNA/metabolismo , Splicing de RNA , RNA de Transferência/metabolismo , Animais , Doenças Cerebelares/genética , Cristalografia por Raios X , Endonucleases/química , Endonucleases/genética , Endorribonucleases/química , Endorribonucleases/genética , Endorribonucleases/metabolismo , Células HEK293 , Humanos , Íntrons/genética , Conformação Proteica , Multimerização Proteica , Precursores de RNA/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Células Sf9 , Spodoptera
13.
Arch Insect Biochem Physiol ; 108(3): e21844, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34519097

RESUMO

Animals maintain homeostasis of cell numbers, constantly creating new cells and eliminating others. Programmed cell death, apoptosis, is a mechanism of cell elimination and it acts in many aspects of animal biology. Drawing on the biomedical background, several signals launch the apoptosis mechanisms, including prostaglandins (PGs). Based on this information, we posed the hypothesis that PGs similarly induce apoptosis in insect cell lines. We used three Spodoptera frugiperda cell lines, including two newly established, BCIRL-SfNS-0518B-YL derived from the central nervous system and BCIRL-Sf4FB-0614-SGS derived from fat body, and the commercially available Sf9 cells. Using a kinetic apoptosis kit, we found treating SfNS cells for 18 h with 15 or 20 µM PGA2 led to decreases in cell numbers, coupled with increased numbers of apoptotic and dead cells. Similar exposures to 10 µM PGA2 (24 h) led to substantial increases in apoptotic cells, confirmed by a terminal deoxynucleotidyl transferase dUTP nick end labeling assay on a flow cytometer. The influence of PGA2 treatments increased with dosage, as we recorded about 20% apoptosis at 24 h post-PGA2 treatments (10 µM) and about 34% apoptosis at 24 h post-30 µM treatments. PGA2 treatments led to 10- to 30-fold increases in messenger RNAs (mRNAs) encoding apoptosis-specific caspases-1, -2, -3, and -5 at 12 h and 40- to 60-fold increases in mRNAs encoding caspases-1 and -2, 10-fold increases for caspases-3 and -5 at 24 h. These findings strongly support our hypothesis that PGs induce apoptosis in an insect cell line and confirm an additional PG action in insect biology.


Assuntos
Caspases , Prostaglandinas A/farmacologia , Células Sf9/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Caspases/efeitos dos fármacos , Caspases/metabolismo , Spodoptera/metabolismo
14.
Nat Commun ; 12(1): 5297, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489430

RESUMO

The protein kinase Akt plays a pivotal role in cellular processes. However, its isoforms' distinct functions have not been resolved to date, mainly due to the lack of suitable biochemical and cellular tools. Against this background, we present the development of an isoform-dependent Ba/F3 model system to translate biochemical results on isoform specificity to the cellular level. Our cellular model system complemented by protein X-ray crystallography and structure-based ligand design results in covalent-allosteric Akt inhibitors with unique selectivity profiles. In a first proof-of-concept, the developed molecules allow studies on isoform-selective effects of Akt inhibition in cancer cells. Thus, this study will pave the way to resolve isoform-selective roles in health and disease and foster the development of next-generation therapeutics with superior on-target properties.


Assuntos
Antineoplásicos/farmacologia , Linfócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Alostérica , Sítio Alostérico , Animais , Antineoplásicos/síntese química , Linhagem Celular , Desenho de Fármacos , Expressão Gênica , Células HEK293 , Humanos , Concentração Inibidora 50 , Linfócitos/citologia , Linfócitos/enzimologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Bibliotecas de Moléculas Pequenas/síntese química , Spodoptera , Relação Estrutura-Atividade
15.
Neuron ; 109(17): 2707-2716.e6, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34473954

RESUMO

The strychnine-sensitive pentameric glycine receptor (GlyR) mediates fast inhibitory neurotransmission in the mammalian nervous system. Only heteromeric GlyRs mediate synaptic transmission, as they contain the ß subunit that permits clustering at the synapse through its interaction with scaffolding proteins. Here, we show that α2 and ß subunits assemble with an unexpected 4:1 stoichiometry to produce GlyR with native electrophysiological properties. We determined structures in multiple functional states at 3.6-3.8 Å resolutions and show how 4:1 stoichiometry is consistent with the structural features of α2ß GlyR. Furthermore, we show that one single ß subunit in each GlyR gives rise to the characteristic electrophysiological properties of heteromeric GlyR, while more ß subunits render GlyR non-conductive. A single ß subunit ensures a univalent GlyR-scaffold linkage, which means the scaffold alone regulates the cluster properties.


Assuntos
Simulação de Dinâmica Molecular , Multimerização Proteica , Receptores de Glicina/química , Potenciais de Ação , Animais , Células HEK293 , Humanos , Bicamadas Lipídicas/metabolismo , Subunidades Proteicas , Receptores de Glicina/metabolismo , Células Sf9 , Spodoptera
16.
J Econ Entomol ; 114(5): 1934-1949, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34505143

RESUMO

The recent invasion of Africa by fall armyworm, Spodoptera frugiperda, a lepidopteran pest of maize and other crops, has heightened concerns about food security for millions of smallholder farmers. Maize genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) is a potentially useful tool for controlling fall armyworm and other lepidopteran pests of maize in Africa. In the Americas, however, fall armyworm rapidly evolved practical resistance to maize producing one Bt toxin (Cry1Ab or Cry1Fa). Also, aside from South Africa, Bt maize has not been approved for cultivation in Africa, where stakeholders in each nation will make decisions about its deployment. In the context of Africa, we address maize production and use; fall armyworm distribution, host range, and impact; fall armyworm control tactics other than Bt maize; and strategies to make Bt maize more sustainable and accessible to smallholders. We recommend mandated refuges of non-Bt maize or other non-Bt host plants of at least 50% of total maize hectares for single-toxin Bt maize and 20% for Bt maize producing two or more distinct toxins that are each highly effective against fall armyworm. The smallholder practices of planting more than one maize cultivar and intercropping maize with other fall armyworm host plants could facilitate compliance. We also propose creating and providing smallholder farmers access to Bt maize that produces four distinct Bt toxins encoded by linked genes in a single transgene cassette. Using this novel Bt maize as one component of integrated pest management could sustainably improve control of lepidopteran pests including fall armyworm.


Assuntos
Bacillus thuringiensis , Animais , Bacillus thuringiensis/genética , Endotoxinas , Proteínas Hemolisinas/genética , Plantas Geneticamente Modificadas/genética , África do Sul , Spodoptera , Estados Unidos , Zea mays/genética
17.
J Biol Chem ; 297(4): 101206, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34543623

RESUMO

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA-independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.


Assuntos
Expressão Gênica , Lipase , Acilação , Animais , Células HEK293 , Humanos , Hidrólise , Lipase/biossíntese , Lipase/química , Lipase/genética , Lipase/isolamento & purificação , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Células Sf9 , Spodoptera
18.
Ecotoxicology ; 30(10): 2056-2070, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34546441

RESUMO

Aluminium, a substance found in large amounts in nature, has been widely used for various purposes, especially food additives. The effects of long-term and excessive exposure to aluminium on human health are receiving increasing attention. The extensive human use of aluminium food additives can also cause aluminium to enter the ecosystem, where it has significant impacts on insects. This study explored the cytotoxicity and changes in gene expression under aluminium potassium sulfate toward Spodoptera frugiperda 9 cells. We found that high concentrations of aluminium resulted in cell enlargement and cell membrane breakage, decreased cell vitality, and apoptosis. Through RNA-Seq transcriptomics, we found that aluminium ions may inhibit the expression of regulatory-associated protein of mTOR, tdIns-dependent protein kinase-1, and small heat shock proteins (heat shock 70 kDa protein and crystallin alpha B), leading to changes in mTOR-related pathways (such as the longevity regulation pathway and PI3K-Akt signalling pathway), and promoting cell apoptosis. On the other hand, aluminium ions lead to the overexpression of GSH S-transferase, prostaglandin-H2 D-isomerase and pyrimidodiazepine synthase, and induce intracellular oxidative damage, which ultimately affects cell growth and apoptosis through a series of cascade reactions.


Assuntos
Alumínio , Fosfatidilinositol 3-Quinases , Alumínio/toxicidade , Animais , Apoptose , Ecossistema , Expressão Gênica , Humanos , Células Sf9 , Spodoptera/genética , Sulfatos
19.
J Agric Food Chem ; 69(34): 9743-9753, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34465092

RESUMO

Indirect defense is an important strategy employed by sessile plants to defend against herbivorous insects by recruiting the natural enemies of herbivores mediated by herbivore-induced plant volatiles (HIPVs). We aimed to determine whether indirect defense occurs in Compositae with Chrysanthemum morifolium as the model and elucidate the mechanisms underlying the biosynthesis of HIPVs. Using two-choice olfactometer bioassays, we showed that C. morifolium plants following infestation by larvae of the tobacco cutworm (Spodoptera litura, TCW) were significantly more attractive to two natural enemies of TCW larvae than control plants, indicating that indirect defense is an active defense strategy of C. morifolium. The chemical cue responsible for indirect defense in C. morifolium was identified as a complex blend of volatiles predominated by sesquiterpenes and monoterpenes. A total of 11 candidate terpene synthase (TPS) genes were identified by comparing the transcriptomes of healthy and TCW-infested plants. Gene expression analysis confirmed that up-regulated CmjTPS genes are consistent with the elevated emission of terpenes after herbivory treatment. Our study showed that the herbivore-induced emission of JA-dependent volatile terpenes attracted both predatory and parasitic enemies of herbivores. Generally, our findings indicate that indirect defense might be an important defense mechanism against insects in C. morifolium.


Assuntos
Chrysanthemum , Herbivoria , Animais , Chrysanthemum/genética , Larva , Spodoptera/genética , Terpenos
20.
Molecules ; 26(18)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34577058

RESUMO

The strategies for controlling the insect pest Spodoptera frugiperda have been developing over the past four decades; however, the insecticide resistance and the remarkable adaptability of this insect have hindered its success. This review first analyzes the different chemical compounds currently available and the most promising options to control S. frugiperda. Then, we analyze the metabolites obtained from plant extracts with antifeedant, repellent, insecticide, or ovicide effects that could be environmentally friendly options for developing botanical S. frugiperda insecticides. Subsequently, we analyze the biological control based on the use of bacteria, viruses, fungi, and parasitoids against this pest. Finally, the use of sex pheromones to monitor this pest is analyzed. The advances reviewed could provide a wide panorama to guide the search for new pesticidal strategies but focused on environmental sustainability against S. frugiperda.


Assuntos
Agentes de Controle Biológico/toxicidade , Inseticidas/toxicidade , Controle Biológico de Vetores/métodos , Extratos Vegetais/toxicidade , Spodoptera/efeitos dos fármacos , Animais
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