Sf9 Cell Line Research Articles

Improved Expression of SARS-CoV-2 Spike RBD Using the Insect Cell-Baculovirus System.

Insect cell-baculovirus expression vector system is one of the most established platforms to produce biological products, and it plays a fundamental role in the context of COVID-19 emergency, providing recombinant proteins for treatment, diagnosis, and prevention. SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host's cellular receptor. As RBD is required for many applications, in the context of pandemic it is important to meet the challenge of producing a high amount of recombinant RBD (rRBD). For this reason, in the present study, we developed a process based on Sf9 insect cells to improve rRBD yield. rRBD was recovered from the supernatant of infected cells and easily purified by metal ion affinity chromatography, with a yield of 82% and purity higher than 95%. Expressed under a novel chimeric promoter (polh-pSeL), the yield of rRBD after purification was 21.1 ± 3.7 mg/L, which is the highest performance described in Sf9 cell lines. Finally, rRBD was successfully used in an assay to detect specific antibodies in COVID-19 serum samples. The efficient strategy herein described has the potential to produce high-quality rRBD in Sf9 cell line for diagnostic purpose.

Syntheses of 6-(2-hydroxy-6-phenylhexyl)-5,6-dihydro-2H-pyran-2-one derivatives and their cytotoxicities

The cytotoxicities against cancer cells (HL-60, HeLa) and insect cells (Sf9) of four stereoisomers of 6-(2-hydroxy-6-phenylhexyl)− 5,6-dihydro-2 H -pyran-2-one ( 1 ) were evaluated, and then their structure-activity relationships examined. The 2′-dehydroxy derivative 5 of (6 R ,2′ R )- and (6 R ,2′ S )- 1 showed the highest activity against HeLa cells (IC 50 = 1.4 μM). To evaluate the effect of the 2′-hydroxy group of 1 , 6 R -and 6 S -oxetane derivatives were also synthesized and their activities examined. Against HeLa and HL-60 cells, the activities of the less potent stereoisomers were enhanced 3–4-fold by the introduction of the oxetane moieties at the 2′-position. Against the insect cell line (Sf9), phenyl derivative 7 showed the highest activity with an IC 50 value of 8.0 μM. • The cytotoxicities of all stereoisomers of 6-(2-hydroxy-6-phenylhexyl)− 5,6-dihydro-2 H -pyran-2-one were evaluated. • Structure-cytotoxicity relationship of 6-(2-hydroxy-6-phenylhexyl)− 5,6-dihydro-2 H -pyran-2-one was examined. • The effects of stereochemistry, oxetane structure, and secondary hydroxy group were clarified. • The derivative without secondary hydroxy was most effective.

The uncommon function and mechanism of the common enzyme glyceraldehyde-3-phosphate dehydrogenase in the metamorphosis of Helicoverpa armigera.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, is commonly used as an internal reference gene in humans, mice, and insects. However, the function of GAPDH in insect development, especially in metamorphosis, has not been reported. In the present study, Helicoverpa armigera and Spodoptera frugiperda ovarian cell lines (Sf9 cells) were used as materials to study the function and molecular mechanism of GAPDH in larval metamorphosis. The results showed that HaGAPDH was more closely related to GAPDH of S. frugiperda and Spodoptera litura. The transcript peaks of HaGAPDH in sixth instar larvae were 6L-3 (epidermal and midgut) and 6L-1 (fat body) days, and 20E and methoprene significantly upregulated the transcripts of HaGAPDH of larvae in qRT-PCR. HaGAPDH-GFP-His was specifically localized in mitochondria in Sf9 cells. Knockdown of HaGAPDH by RNA interference (RNAi) in sixth instar larvae resulted in weight loss, increased mortality, and decreases in the pupation rate and emergence rates. HaGAPDH is directly bound to soluble trehalase (HaTreh1) physically and under 20E treatment in yeast two-hybrid, coimmunoprecipitation, and colocalization experiments. In addition, knockdown of HaGAPDH increased the Treh1 activity, which in turn decreased the trehalose content but increased the glucose content in larvae. Therefore, these data demonstrated that GAPDH controlled the glucose content within the normal range to ensure glucose metabolism and metamorphosis by directly binding with HaTreh1.

Design, synthesis and insecticidal activity and mechanism research of Chasmanthinine derivatives

Unrestricted reproduction and spread of pest had caused great damage to the quality and yield of crops in recent years. Besides the use of traditional chemical pesticides, natural products also make a huge contribution against pests. Chasmanthinine, a diterpenoid alkaloid isolated from Aconitum franchetii var. villosulum, shown extremely antifeedant activity against Spodoptera exigua. Therefore, a series of novel Chasmanthinine derivatives were synthesized and their biological activity was studied in this work. Compound 33 showed the strongest antifeedant activity (EC50 = 0.10 mg/cm2) among all the test compounds. The mechanism research of 33 revealed that its antifeedant effect was related to the inhibition of carboxylesterase (CES), and proved the thiophene acyl group could form a strong binding effect with CES by molecular docking. Moreover, compound 10 exhibited the strongest cytotoxicity (IC50 = 12.87 μM) against Sf9 cell line and moderate contact toxicity. The mechanism research indicated that compound 10 could induce Sf9 cells apoptosis. In summary, the results lay a foundation for the application of diterpene alkaloids in plant protection.

Biological, morphological, and molecular characterization of the baculovirus PlxyMNPV_LBIV-11, and its virulence towards Plutella xylostella, Trichoplusia ni, and Spodoptera frugiperda larvae.

PlxyMNPV_LBIV-11 is an alphabaculovirus strain, isolated from Plutella xylostella larvae. This work characterized this strain at a biological, morphological, and molecular level to evaluate its similarity with other baculoviruses. Its ultrastructure showed a multiple arrangement of nucleocapsids within enveloped virions, all occluded within large cubical polyhedra. PlxyMNPV_LBIV-11 showed infectivity on the Hi5 and Sf9 cell lines, despite these being from heterologous origin. This in vitro infectivity was observed using either BVs or by transfection with genomic DNA. Restriction fragment patterns of PlxyMNPV_LBIV-11, using the enzymes EcoRI, BamHI and HindIII, showed a high relationship with those patterns shown by AcMNPV, except for one or two differential bands with each enzyme. Sequences of core genes lef-8 and lef-9 and the conserved polh gene showed identities ranging from 98 to 100% when compared with those of AcMNPV. Somewhat lower was the sequence identity of the gp64 gene (94%) as compared with those of AcMNPV and PlxyMNPV_CL3, which might be related to the difference in virulence. Besides, the presence of this gene in PlxyMNPV_LBIV-11 indicates that it belongs to group 1 of alphabaculoviruses. A phylogram was estimated with the core and conserved gene sequences, corroborating its high relationship with AcMNPV and PlxyMNPV_CL3. Bioassays were performed with P. xylostella larvae reared on a meridic diet, whose LC50 values indicated lower virulence than AcMNPV when tested against P. xylostella, Spodoptera frugiperda, and Trichoplusia ni larvae. Its virulence against S. frugiperda was only seven times lower than AcMNPV. Its potential as a biological control agent is discussed.

Knockout of Dicer-2 in the Sf9 cell line enhances the replication of Spodoptera frugiperda rhabdovirus and conditionally increases baculovirus replication.

The Sf9 cell line, originally isolated from the ovarian tissue of Spodoptera frugiperda larvae, is widely used in academia and industry for the baculovirus-mediated production of recombinant proteins and virus-like particles. RNA interference (RNAi) is a conserved antiviral pathway present in eukaryotic organisms and is the primary antiviral defence mechanism in insects. Recent evidence has implicated RNAi as an antiviral response to baculovirus infection in Sf9 cells. To test this hypothesis, CRISPR/Cas9 technology was used to disable the RNAi pathway in Sf9 cells by knocking out Dicer-2, the protein responsible for cleaving viral double-stranded RNA precursors into short interfering RNAs. Infection of Dicer-2 knockout Sf9 cells with either the wild-type baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), recombinant AcMNPV (rAcMNPV) expressing β-galactosidase (β-gal), or rAcMNPV expressing a wasp venom protein (Vn50) at a multiplicity of infection (m.o.i.) of 1 resulted in a modest increase in virus replication compared to control Sf9 cells under adherent culture conditions. In contrast, Dicer-2 knockout Sf9 monolayer or suspension cultures infected by the rAcMNPV expressing β-gal at higher m.o.i.s (3.5 and 20) did not exhibit increases in either viral DNA replication or β-gal production. Intriguingly, during long-term passaging in suspension, Dicer-2 knockout Sf9 cultures underwent transient crashes in cell proliferation and viability. It was discovered that these periods of low growth and viability coincided with a dramatic increase in the RNA levels of S. frugiperda rhabdovirus, a recently identified adventitious virus that persistently infects the Sf9 cell line, suggesting a role for Dicer-2 in managing chronic viral infections in this industrially relevant insect cell line.

Insecticide compounds selection based on activation of ecdysteroid receptors in Diptera (S2) and Lepidoptera (Sf9) cell lines

Nuclear receptors are important targets in the research for the discovery of brand-new insecticides, and the ecdysteroid receptor is one of the most important targets nowadays. In this study, nine organocalcogenic compounds analogs of dibenzoylhydrazine were tested in a screening system, that presented at least 80% of compatibility in at least one of the three micromolar concentrations tested. We optimized in embryonic cell lines of the order Lepidoptera (SF9) and Diptera (S2), and these cell lines were transfected with a specific reporter plasmid. The nine compounds tested did not present agonist activity in both cell lines tested. In S2 cells the DAF-2 molecule inhibited the ECR-USP luminescence in 29.91%, and the DAF-4 molecule significantly inhibited the nuclear receptor luminescence in 51 to 55% compared to 20-Hydroxyecdysone (control). In SF9 cells, DAF-2 inhibited ECR-USP luminescence in 19.62%, and DAF-4 inhibited the receptor luminescence by 35.14% compared to 20-Hydroxyecdysone (control). In silico analysis of the Drosophila melanogaster receptor with the compound, DAF-2 showed inhibition of amino acids THR467 and MET-508 and with compound DAF-4 inhibition of ARG-511. For the of Heliothis virescens receptor for dendritic compounds, DAF-2 promoted inhibition of amino acids THR-343 and ARG-387 and DAF-4 inhibited ASN-504 and ARG-387

Anti-proliferative and apoptosis-inducing effects of juvenile hormone analogue, fenoxycarb in the Sf21cell line

The apprehension regarding the possible environmental effects of synthetic pesticides has led to the discovery and production of environmental friendly pesticides. Insect hormone mimics, mainly juvenile hormone analogues, like fenoxycarb have acquired attention due to their greater specificity than conventional broad-range insecticides as pest control agents. The study explored the effects of the insecticide juvenile hormone analogue (JHA), fenoxycarb, on the Sf21 cell line of Spodoptera frugiperda to illustrate the mode of action. Cytotoxicity assay was conducted at different concentrations of fenoxycarb ranging from 0.5 nM to 10 μM. The results showed the concentration-and time-dependent anti-proliferative effect of fenoxycarb. The median inhibitory concentration (IC50) was calculated as 28 nM at 48 h of exposure, and IC50 and IC25 concentrations were used for further cytotoxicity screening assays. Furthermore, the significant morphological changes of the cells after 48 h revealed the development of apoptotic bodies, membrane blebbing, cell size reduction, and irregular cell aggregation; additionally, enlarged cell spaces and widely diffused apoptotic bodies were observed after 72 h of insecticide exposure. In the confocal microscopic analysis of fenoxycarb treated cells, the nucleus was observed to condense and collapse into many fragments by Hoechst-33,342. Assessment of the relative potential of the cell cycle at two concentrations (IC50& IC25) reported the concentration-and time-dependent reduction of cells in the G1 phase with an upsurge in apoptotic cells. The percentage of cells that underwent apoptotic changes, such as loss of mitochondrial membrane potential (MMP), was strictly dependent on the fenoxycarb concentration and duration of exposure. The findings confirm the presence of fenoxycarb-mediated cell proliferation inhibition and apoptosis in Sf21 cell lines.

Production of Bivalent Subunit Vaccine for Porcine via 2A-Like Sequence in Baculovirus Expression Vector System

Classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV2) have caused severe diseases in swine populations worldwide. Here, a polycistronic baculovirus vector was developed to express a bivalent vaccine, consisting of the CSFV-E2 and PCV2-Cap protein, and an immunomodulator protein derived from the Flammulina velutipes, FVE-FIP, as well as the selection marker, green fluorescent protein. The simultaneous expression of the CSFV-E2 and PCV2-Cap protein was mediated by the 2A-like sequence derived from the Perina nuda virus (PnV), while the expression of the FVE-FIP was driven by the internal ribosome entry site (IRES) element derived from the Rhophalosipum padi virus (RhPV). The Western blot analysis result suggested that the CSFV-E2, PCV2-Cap, and FVE-FIP protein were successfully co-expressed by the infected Spodoptera frugiperda IPBL-Sf21 (Sf21) cell line. The extracted cell lysate containing all three recombinant proteins was administered to Balb/C mice with or without the supplementation of Freund’s adjuvant. The ELISA analysis of the serum collected from all the immunized groups showed detectable antibodies against CSFV-E2 and PCV2-Cap. Furthermore, the immunized group without the adjuvant supplementation demonstrated a similar level of antibodies to the group with adjuvant supplementation, suggesting the efficiency of the FVE-FIP in enhancing the immune response. These results demonstrated the polycistronic baculovirus vector could be employed to develop bivalent vaccines for pigs.

Knockout of Sf-Caspase-1 generates apoptosis-resistant Sf9 cell lines: Implications for baculovirus expression.

The Sf9 cell line, originally isolated from the insect Spodoptera frugiperda, is commonly used alongside the baculovirus expression vector system (BEVS) to produce recombinant proteins and other biologics. As more BEVS-derived vaccines and therapeutics are approved by regulators and manufactured at scale, there is increasing interest in improving the Sf9 cell line to improve bioprocess robustness and increase product yields. CRISPR-Cas9 is a powerful genome-editing tool with great potential to improve cell line characteristics. Nevertheless, reports of genome-editing in Sf9 cells are scarce, and targets for engineering are elusive. To evaluate the effectiveness of CRISPR-Cas9 to improve BEVS yields, we generated Sf9 cell lines with functional knockouts in the Sf-Caspase-1 gene, which encodes an effector caspase involved in the execution of apoptosis. Deletion of Sf-Caspase-1 abolished the hallmarks of apoptotic cell death including plasma membrane blebbing and effector caspase activity. Following infection of Sf-Caspase-1 knockout Sf9 cultures with a recombinant baculovirus expressing β-galactosidase, we did not observe any differences in cell death kinetics or increases in productivity. Similar results were obtained when Sf-Caspase-1 expression was suppressed via RNA interference. We anticipate that the CRISPR-Cas9 workflow reported here will spur future efforts to rationally engineer Sf9 cells for improved baculovirus expression.

Heterologous expression of scFv fragment against Vairimorpha (Nosema) ceranae hexokinase in Sf9 cell culture inhibits microsporidia intracellular growth

Secretion of hexokinase (HK) by microsporidia into infected cells suggests an important role for this enzyme for the intracellular development of parasites. To verify whether the expression of HK-specific antibodies in the host cell cytoplasm can suppress the growth of microsporidia, we constructed an immune library of recombinant scFv fragments against the enzyme of the honey bee pathogen Vairimorpha (Nosema) ceranae (VcHK) with a representativeness of about 5 million bacterial transformants. Two variants of VcHK-specific recombinant antibodies were selected by library panning and expressed in lepidopteran Sf9 cell line. Infecting of cells expressing two selected and control scFv fragments with V. ceranae spores was followed by their cultivation for 4 days. Analysis of parasite β-tubulin as well as spore wall protein SWP32 transcripts in infected cultures by reverse transcription PCR and real-time qPCR showed (1) V. ceranae growth in cells heterologous to bee pathogens, (2) its inhibition by one of the selected VcHK-specific recombinant antibodies. The latter result once again emphasizes an important role of microsporidia hexokinases in their relationships with infected host cells and suggests further focusing on the mechanisms of such suppression, as well as on the search for new V. ceranae - inhibiting scFv fragments.

Insight into Cellular Uptake and Transcytosis of Peptide Nanoparticles in Spodoptera frugiperda Cells and Isolated Midgut.

Silencing genes in insects by introducing double-stranded RNA (dsRNA) in the diet holds promise as a new pest management method. It has been demonstrated that nanoparticles (NPs) can potentiate dsRNA silencing effects by promoting cellular internalization and protecting dsRNA against early degradation. However, many mysteries of how NPs and dsRNA are internalized by gut epithelial cells and, subsequently, transported across the midgut epithelium remain to be unraveled. The sole purpose of the current study is to investigate the role of endocytosis and transcytosis in the transport of branched amphipathic peptide nanocapsules (BAPCs) associated with dsRNA through midgut epithelium cells. Spodoptera frugiperda midguts and the epithelial cell line Sf9, derived from S. frugiperda, were used to study transcytosis and endocytosis, respectively. Results suggest that clathrin-mediated endocytosis and macropinocytosis are largely responsible for cellular uptake, and once within the midgut, transcytosis is involved in shuttling BAPCs–dsRNA from the lumen to the hemolymph. In addition, BAPCs were not found to be toxic to Sf9 cells or generate damaging reactive species once internalized.

Identification of Helicoverpa armigera promoters for biotechnological applications

Helicoverpa armigera and Helicoverpa zea are highly polyphagous major agricultural pests with a global distribution. Their control is based on insecticides, however, new, effective, and environmentally friendly control tools are required to be developed and validated. In an effort to facilitate the development of advanced biotechnological tools in these species that will take advantage of new powerful molecular biology techniques like CRISPR/Cas9, we used available transcriptomic data and literature resources, in order to identify RNA polymerase II and III promoters active in RP-HzGUT-AW1(MG), a midgut derived cell line from Helicoverpa zea. Following functional analysis in insect cell lines, four RNA polymerase II promoters from the genes HaLabial, HaTsp-2A, HaPtx-I and HaCaudal were found to exhibit high transcriptional activity in vitro. The HaTsp-2A promoter did not exhibit any activity in the non-midgut derived cell lines Sf-9 and Hi-5 despite high sequence conservation among Lepidoptera, suggesting that it may function in a gut specific manner. Furthermore, considering the utility of RNA polymerase III U6 promoters in methodologies such as RNAi and CRISPR/Cas9, we identified and evaluated four different U6 promoters of H. armigera. In vitro experiments based on luciferase and GFP reporter assays, as well as in vivo experiments targeting an essential gene of Helicoverpa, indicate that these U6 promoters are functional and can be used to experimentally silence or knockout target genes through the expression of shRNAs and sgRNAs respectively. Taking our findings together, we provide a set of promoters useful for the genetic manipulation of Helicoverpa species, that can be used in various applications in the context of agricultural biotechnology.

H3K9me2 genome-wide distribution in the holocentric insect Spodoptera frugiperda (Lepidoptera: Noctuidae)

BackgroundEukaryotic genomes are packaged by Histone proteins in a structure called chromatin. There are different chromatin types. Euchromatin is typically associated with decondensed, transcriptionally active regions and heterochromatin to more condensed regions of the chromosomes. Methylation of Lysine 9 of Histone H3 (H3K9me) is a conserved biochemical marker of heterochromatin. In many organisms, heterochromatin is usually localized at telomeric as well as pericentromeric regions but can also be found at interstitial chromosomal loci. This distribution may vary in different species depending on their general chromosomal organization. Holocentric species such as Spodoptera frugiperda (Lepidoptera: Noctuidae) possess dispersed centromeres instead of a monocentric one and thus no observable pericentromeric compartment. To identify the localization of heterochromatin in such species we performed ChIP-Seq experiments and analyzed the distribution of the heterochromatin marker H3K9me2 in the Sf9 cell line and whole 4th instar larvae (L4) in relation to RNA-Seq data. ResultsIn both samples we measured an enrichment of H3K9me2 at the (sub) telomeres, rDNA loci, and satellite DNA sequences, which could represent dispersed centromeric regions. We also observed that density of H3K9me2 is positively correlated with transposable elements and protein-coding genes. But contrary to most model organisms, H3K9me2 density is not correlated with transcriptional repression. ConclusionThis is the first genome-wide ChIP-Seq analysis conducted in S. frugiperda for H3K9me2. Compared to model organisms, this mark is found in expected chromosomal compartments such as rDNA and telomeres. However, it is also localized at numerous dispersed regions, instead of the well described large pericentromeric domains, indicating that H3K9me2 might not represent a classical heterochromatin marker in Lepidoptera.(242 words)

Synergistic effects of botanical curcumin-induced programmed cell death on the management of Spodoptera litura Fabricius with avermectin

Chemical pesticides and adjuvants have caused many negative effects. Botanical compounds provide solutions for the development of environment friendly pesticides and the management of increasing pest resistance. Curcumin, a natural polyphenol, showed synergistic effects on avermectin upon the destructive agricultural pest, Spodoptera litura. However, the botanical synergist and its relevant mechanisms remain unclear. In the article, curcumin significantly enhanced the growth inhibition and midgut structural damage of avermectin on the larvae of S. litura, and the synergistic effects were confirmed with pot experiments. There were only a few influences on the gene expression of avermectin targets, while apoptotic and autophagic related genes and proteins were accumulated in the avermectin/curcumin mixed regent (0.013/0.0013 μg/mL) treated group. Moreover, the potential mechanism was explored with an in vitro model, insect Spodoptera frugiperda Sf9 cell line. Morphology observation featured the damage on cells and Hoechst33258 staining revealed the fragments of DNA after treating with the avermectin/curcumin mixed regent (10/1 μg/mL). Dansylcadaverine and LysoTracker staining, as well as the gene expressions, supposed that curcumin exhibited autophagy inducing effects and the mixed regent possessed a higher ability to induce apoptosis and autophagy. All these results suggested that the synergistic effects of curcumin on the pest management of avermectin potentially mainly derived from the enhancement of programed cell death. It provides new sights for the application of natural compounds in integrated pest management and enriches examples of synergistic mechanisms.

Diamide insecticide resistance in transgenic Drosophila and Sf9-cells expressing a full-length diamondback moth ryanodine receptor carrying an I4790M mutation.

BACKGROUNDResistance to diamide insecticides in Lepidoptera is known to be caused primarily by amino acid changes on the ryanodine receptor (RyR). Recently, two new target site mutations, G4946V and I4790M, have emerged in populations of diamondback moth, Plutella xylostella, as well as in other lepidopteran species, and both mutations have been shown empirically to decrease diamide efficacy. Here, we quantify the impact of the I4790M mutation on diamide activation of the receptor, as compared to alterations at the G4946 locus.RESULTSI4790M when introduced into P. xylostella RyR expressed in an insect‐derived Sf9 cell line was found to mediate just a ten‐fold reduction in chlorantraniliprole efficacy (compared to 104‐ and 146‐fold reductions for the G4946E and G4946V variants, respectively), whilst in the field its presence is associated with a ≥150‐fold reduction. I4790M‐mediated resistance to flubendiamide was estimated to be >24‐fold. When the entire coding sequence of P. xylostella RyR was integrated into Drosophila melanogaster, the I4790M variant conferred ~4.4‐fold resistance to chlorantraniliprole and 22‐fold resistance to flubendiamide in the 3rd instar larvae, confirming that it imparts only a moderate level of resistance to diamide insecticides. Although the I4790M substitution appears to bear no fitness costs in terms of the flies' reproductive capacity, when assessed in a noncompetitive environment, it does, however, have potentially major impacts on mobility at both the larval and adult stages.CONCLUSIONSI4790M imparts only a moderate level of resistance to diamide insecticides and potentially confers significant fitness costs to the insect.

Evaluation of insecticidal potential of organochemicals on SF9 cell line

BackgroundOrganophosphates and Pyrethroids are the most widely used pesticides worldwide and are known to have significant toxicity on the nervous system of the target pest. Assessment for combined toxicity of Organophosphate and Pyrethroid on Sf9 (Spodoptera frugiperda) cells is less explored. The present study demonstrates and compares the two organochemicals whose trade names are Ammo and Profex, for its cytotoxic potential on the insect Sf9 cells. Ammo and Profex were selected as the test chemicals as toxicity of these insecticides at molecular and cellular level is poorly understood.ResultsThe results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay demonstrated that Ammo and Profex exhibited significant cytotoxicity to Sf9 cells in a time- and dose-dependent manner. In our study, the IC50 value was obtained by MTT assay and the sub-lethal concentrations (IC50/20-17.5 µg/ml, IC50/10-35 µg/ml, and IC50/5–70 µg/ml for Ammo and IC50/20-20 µg/ml, IC50/10-40 µg/ml, and IC50/5-80 µg/ml for Profex) were selected for further tests. Acridine orange/ethidium bromide staining proved the apoptotic cell death on exposure of both the insecticides confirming its toxic potential. Furthermore, antioxidant status was assessed using DCF-DA staining and both the insecticides resulted into an increased reactive oxygen species (ROS) generation. A dose- and time-dependent significant (p < 0.05) alterations in lipid peroxidase (LPO), glutathione (GSH) and catalase (CAT) activity were observed.ConclusionThe results showed that both Ammo and Profex triggered apoptosis in Sf9 cells through an intrinsic mitochondrial pathway via the generation of ROS. Of the two insecticides, Ammo was found to be more toxic compared to Profex. The present study is important to evaluate the environmental safety and risk factors of Organochemicals’ exposure to crops and livestock.

Entomotoxic Activity of the Extracts from the Fungus, Alternaria tenuissima and Its Major Metabolite, Tenuazonic Acid.

The study of fungal antibiotics in their competitive interactions with arthropods may lead to the development of novel biorational insecticides. Extracts of Alternaria tenuissima MFP253011 obtained using various methods showed a wide range of biological activities, including entomotoxic properties. Analysis of their composition and bioactivity allowed us to reveal several known mycotoxins and unidentified compounds that may be involved in the entomotoxic activity of the extracts. Among them, tenuazonic acid (TeA), which was the major component of the A. tenuissima extracts, was found the most likely to have larvicidal activity against Galleria mellonella. In the intrahaemocoel injection bioassay, TeA was toxic to G. mellonella and of Zophobas morio with an LT50 of 6 and 2 days, respectively, at the level of 50 µg/larva. Administered orally, TeA inhibited the growth of G. mellonella larvae and caused mortality of Acheta domesticus adults (LT50 7 days) at a concentration of 250 µg/g of feed. TeA showed weak contact intestinal activity against the two phytophages, Tetranychus urticae and Schizaphis graminum, causing 15% and 27% mortality at a concentration of 1 mg/mL, respectively. TeA was cytotoxic to the Sf9 cell line (IC50 25 µg/mL). Thus, model insects such as G. mellonella could be used for further toxicological characterization of TeA.

Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro

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.

OdTx12/GNA, a chimeric variant of a β excitatory toxin from Odontobuthus doriae, reveals oral toxicity towards Locusta migratoria and Tenebrio molitor

OdTx12, a beta excitatory toxin from Odontobothus doriae had previously been identified and characterized. It had been shown that OdTx12 causes significant lethal effects on insects by injection but does not show any toxicity on mice. Due to the natural ineffectiveness of scorpion toxins to act as oral toxins, OdTx12 was fused to Galanthus nivalis agglutinin (GNA), a protein with the potential to cross the insect gut. The sequence of OdTx12/GNA gene was chemically synthesized, cloned in Escherichia coli, and expressed. The effect of the purified fusion protein (OdTx12/GNA) was assessed on the insect and mammalian cell lines, insect larvae and mice. Toxicity assay on insect cell culture (SF9 cell line) showed comparable toxicity between OdTx12 and OdTx12/GNA (LD50 of 0.0030 and 0.0048 μM, respectively). Also very similar mortality rates were observed by injecting OdTx12 and OdTx12/GNA to Locusta migratoria and Tenebrio molitor. Oral administration of OdTx12/GNA, after five days of feeding, resulted in 96.6% and 98.3% mortality of L. migratoria and T. molitor larvae with an LC50 of 0.69 and 0.43 nmol/g of insect food, respectively, while OdTx12 alone did not cause any toxic effects on the larvae orally, suggesting the role of GNA in delivering the toxin to the insect's haemolymph. No toxicity or mortality was observed after toxicity testing of OdTx12/GNA on a mammalian cell line (HEP-2) or any mortality in vivo, by testing the protein in the laboratory mouse.Herein, we demonstrated that the fusion protein OdTx12/GNA could be considered an effective toxin for the biological control of insects.