Search Results (45)

Field-evolved resistance of Helicoverpa zea to crops expressing Cry insecticidal proteins from Bacillus thuringiensis (Bt) is widespread across the United States. To comparatively evaluate physiological factors associated with Bt susceptibility, we analyzed two laboratory strains (Benzon and SIMRU) and one field colony obtained from a commercial corn field near Pickens, Arkansas. Biochemical assays of larval midgut extracts showed that Pickens exhibited significantly altered activities of chymotrypsin-like proteases, aminopeptidase N (APN), and alkaline phosphatase (ALP) compared with the SIMRU or Benzon colonies, with differences varying by larval instar. In contrast, trypsin-like protease activities did not differ significantly among the three colonies. Gene expression analyses of ten serine protease genes and seven candidate Cry receptor genes (including cadherin, ATP-binding cassette family C2, ALP, and four APN genes) revealed significant transcriptional differences in the Pickens relative to the lab colonies. Collectively, these results suggest that chymotrypsin-like proteases may play an important role in the activation of Cry toxins in H. zea. Altered chymotrypsin and APN activities, together with differential gene expressions in the Pickens population, likely contribute to reduced Bt susceptibility. The biochemical and molecular differences provide insight into potential physiological factors underlying reduced Bt susceptibility and may inform future Bt resistance monitoring and management strategies. Full article

Background: β-casomorphin-10 (CM-10), a peptide fragment derived from milk casein with the sequence YPFPGPIPNS, has demonstrated notable anxiolytic activity in BALB/c mice. Yet, its cellular responses and mechanistic pathways remain largely uncharacterized. Methods: We performed RNA-seq analysis to profile gene expression changes in δ-opioid receptor-expressing HEK293 cells (DOR-HEK), comparing CM-10-treated and untreated conditions. Results: CM-10 exposure led to differential expression of 1714 genes in DOR-HEK cells, with 34 upregulated (>1.4-fold) (1.9%) and 1680 downregulated (<0.71-fold) (98.1%), based on a predicted p-value threshold of <0.05. Notably, we identified 10 clusters that were associated with reduced cyclic AMP (cAMP) in DOR-HEK cells following CM-10 treatment. These clusters particularly involved genes related to regulatory subunits of cAMP-dependent protein kinases, such as PRKAR2A, cAMP-responsive element-binding pathway, circadian rhythms, such as CLOCK, ARNT1, CRY2, PER1, and PER2, and anxiety and depression, such as NOTCH1, NOTCH2 and ANK2. A network with these selected genes was confirmed by STRING analysis. Conclusions: These findings indicate that CM-10 may activate DOR-mediated signaling by suppressing cAMP levels, implicating a distinct molecular cascade in HEK293 cells. Full article

Aedes aegypti, a crucial vector mosquito that transmits many diseases that cause millions of deaths worldwide, can be controlled with Bacillus thuringiensis subsp. israelensis (Bti). The larvicidal activity of Bti against Ae. aegypti is due primarily to Cry4Aa, Cry4Ba, and Cry11Aa, and Cyt1Aa, a protein that synergizes the activity of the Cry proteins. Interestingly, Galectins-6 and Galectins-14, members of a family of β-galactoside-binding proteins that play a role in immune responses insects, have been shown to decrease the activity of Bti toxins. The activity of other Galectins, particularly Galectin-8A, against the Cry proteins is not known. Toward this end, we cloned the gene coding for galactin-8A and expressed the recombinant protein and purified protein. The bioassay results indicated that Galectin-8A can also reduce the toxicity of Cry11Aa, but it was much stronger than Galectin-6. To investigate the interactions among Galectin-8A, Cry11Aa, and toxin receptors, Octet Red System analysis, Western blot, far-Western blot, and ELISA assay were also performed. The Octet Red System result showed that Galectin-8A could also bind to BBMVs of Ae. aegypti, with a lower kDa value than that of Galectin-6, indicating that Galectin-8A had a stronger binding affinity to BBMVs than Galectin-6. Western blot, far-Western blot, and ELISA assay analyses also demonstrated that Galectin-8A bound to Ae. aegypti receptor ALP1 and APN2, consistent with the protein docking simulation results. These findings support the conclusion that Galectin-8A blocks with ALP1 and APN2 more effectively than Galectin-6, which may subsequently reduce the toxicity of Cry11Aa in Ae. aegypti. Full article

The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has become a key plant-incorporated protectant (PIP) in transgenic crops targeting lepidopteran pests, particularly as resistance increasingly compromises the efficacy of Cry protein PIPs. More than a decade after its commercial deployment, Vip3Aa performance remains efficacious but increasingly vulnerable. Field screens have detected unexpectedly high baseline frequencies of Vip3Aa-resistant alleles and have produced highly resistant strains in several major pests, including Helicoverpa spp., Spodoptera spp., and Mythimna separata. Although structure–function experiments and studies on resistance to Vip3Aa have identified altered midgut processing and impaired receptor binding as candidate resistance mechanisms, the underlying genetic determinants remain poorly understood. Moreover, resistance to Vip3Aa appears to diverge from canonical Cry protein resistance pathways, underscoring the need for dedicated mechanistic studies. This review critically examines the available experimental evidence on Vip3Aa resistance mechanisms, highlighting major knowledge gaps and proposing research priorities to inform resistance monitoring and extend the durability of Vip3Aa-based pest control. Full article

Resistance to HER2 tyrosine-kinase inhibitor Lapatinib (Lap) is one of the leading causes of cancer treatment failure in HER2+ breast cancer (BC), associated with an aggressive tumor phenotype. Cryptotanshinone (Cry) is a natural terpene molecule that could function as a chemosensitizer by disturbing estrogen receptor (ERα) signaling and inhibiting the protein translation factor-4A, eIF4A. Therefore, we evaluated Cry dual regulation on eIF4A and ERα. This study aimed to elucidate the underlying mechanisms of Lap chemoresistance and the impact of Cry on them. We generated two Lap-resistant BT474 cell HER2+ variants named BT474^LapRV1 and BT474^LapRV2 with high chemoresistance levels, with 7- and 11-fold increases in EC₅₀, respectively, compared to BT474 parental cells. We found a PDCD4-p70S6Kβ axis association with Lap chemoresistance. However, a concomitant down-regulation of the RAF-MEK-ERK cell survival pathway and NF-κB was found in the chemoresistant cell variants; this phenomenon was exacerbated by joint treatment of Cry and Lap under a Lap plasmatic reported concentration. Optimized calcium management was identified as a compensatory mechanism contributing to chemoresistance, as determined by the higher expression of calcium pumps PMCA1/4 and SERCA2. Contrary to expectations, a combination of Lap and Cry did not affect the chemoresistance despite the ERα down-regulation; Cry-eIF4A binding possibly dampens this condition. Results indicated the pro-survival eIF4A/STAT/Bcl-xl pathway and that the down-regulation of the MAPK-NF-κB might function as an adaptive mechanism; this response may be compensated by calcium homeostasis in chemoresistance, highlighting new adaptations in HER2+ cells that lead to chemoresistance. Full article

Bioinsecticides based on the bacterium Bacillus thuringiensis (Bt) are widely used as safe alternatives to chemical insecticides. The insecticidal activity of Bt is occasioned by a protein toxin contained in parasporal crystals (Cry proteins) that are synthesized and laid down alongside the endospore during sporulation. The specificity of toxin action is associated with the subspecies of Bt and the individual Cry toxins they produce. Although a number of commercial Bt formulations are available to control moths, mosquitoes and beetles, there are none that control the red imported fire ant (RIFA) Solenopsis invicta. The present report is the first to describe the insecticidal activity of the Cry3A protein toxin, produced by Bacillus thuringiensis subsp. tenebrionis (Btt), against the RIFA as well as some of its key biochemical properties. Currently available commercial formulations of Btt are designed to control beetles such as the Colorado potato beetle, not ants. The Cry3A toxin (MW ~66 kDa) is embedded in a larger polypeptide (protoxin, MW ~73 kDa) and is released from the toxin enzymatically. Once activated, it can be administered to the RIFA as a soluble protein that most likely binds to an attendant receptor in the epithelial cells that line the wall of the larval ventriculus, killing the insect. Properly customized, the Cry3A toxin is a potential candidate for fire ant control. Full article

The effect of caffeine on the behavior and sleep patterns of zebrafish larvae, as well as its underlying mechanisms, has been a topic of great interest. This study aimed to investigate the impact of caffeine on zebrafish larval sleep/wake behavior and the expression of key regulatory genes such as cAMP-response element binding protein (CREB) and adenosine (ADA) in the sleep pathway. To begin, the study determined the optimal dose and duration of caffeine exposure, with the optimal doses found to be 31.25 μM, 62.5 μM, and 120 μM. Similarly, the optimal exposure time was established as no more than 120 h, ensuring a mortality rate of less than 10%. The confirmation of these conditions was achieved through the assessment of angiogenesis and the inflammatory reaction. As a result, the treatment time point of 24 h post-fertilization (hpf) was selected to examine the effects of caffeine on zebrafish larval sleep rhythm (48 h, with a light cycle of 14:10). Furthermore, the study analyzed the expression of clock genes (bmal1a, per1b, per2, per3, cry2), adenosine receptor genes (adora1a, adora1b, adora2aa, adora2ab, adora2b), and key regulatory factors (CREB and ADA). The research confirmed that caffeine could induce sleep pattern disorders, significantly upregulate adenosine receptor genes (adora1a, adora1b, adora2a, adora2ab, adora2b) (p < 0.05), and markedly decrease the total sleep time and sleep efficiency of the larvae. Additionally, the activity of ADA significantly increased during the exposure (p < 0.001), and the tissue-specific expression of CREB was also significantly increased, as assessed by immunofluorescence. Caffeine may regulate circadian clock genes through the ADA/ADORA/CREB pathway. These findings not only enhance our understanding of the effects of caffeine on zebrafish larvae but also provide valuable insights into the potential impact of caffeine on human behavior and sleep. Full article

Cry2Ab is a significant alternative Bacillus thuringiensis (Bt) protein utilized for managing insect resistance to Cry1 toxins and broadening the insecticidal spectrum of crops containing two or more Bt genes. Unfortunately, the identified receptors fail to fully elucidate the mechanism of action underlying Cry2Ab. Previous studies have demonstrated the involvement of vacuolar H⁺-ATPase subunits A, B, and E (V-ATPase A, B, and E) in Bt insecticidal activities. The present study aims to investigate the contribution of V-ATPase C to the toxicities of Cry2Ab against Helicoverpa armigera. The feeding of Cry2Ab in H. armigera larvae resulted in a significant decrease in the expression of V-ATPase C. Further investigations confirmed the interaction between V-ATPase C and activated Cry2Ab protein according to Ligand blot and homologous and heterologous competition assays. Expressing endogenous HaV-ATPase C in Sf9 cells resulted in an increase in Cry2Ab cytotoxicity, while the knockdown of V-ATPase C by double-stranded RNAs (dsRNA) in midgut cells decreased Cry2Ab cytotoxicity. Importantly, a higher toxicity of the mixture containing Cry2Ab and V-ATPase C against insects was also observed. These findings demonstrate that V-ATPase C acts as a binding receptor for Cry2Ab and is involved in its toxicity to H. armigera. Furthermore, the synergy between V-ATPase C protein and Cry2Ab protoxins provides a potential strategy for enhancing Cry2Ab toxicity or managing insect resistance. Full article

Dioryctria abietella Denis Schiffermuller (Lepidoptera: Pyralidae) is an oligophagous pest that mainly damages Pinaceae plants. Here, we investigated the effects of the Bacillus thuringiensis 2913 strain (Bt 2913), which carries the Cry1Ac, Cry2Ab, and Vip3Aa genes, on the D. abietella midgut transcriptome at 6, 12, and 24 h after infection. In total, 7497 differentially expressed genes (DEGs) were identified from the midgut transcriptome of D. abietella larvae infected with Bt 2913. Among these DEGs, we identified genes possibly involved in Bt 2913-induced perforation of the larval midgut. For example, the DEGs included 67 genes encoding midgut proteases involved in Cry/Vip toxin activation, 74 genes encoding potential receptor proteins that bind to insecticidal proteins, and 19 genes encoding receptor NADH dehydrogenases that may bind to Cry1Ac. Among the three transcriptomes, 88 genes related to metabolic detoxification and 98 genes related to immune defense against Bt 2913 infection were identified. Interestingly, 145 genes related to the 60S ribosomal protein were among the DEGs identified in the three transcriptomes. Furthermore, we performed bioinformatic analysis of zonadhesin, GST, CYP450, and CarE in the D. abietella midgut to determine their possible associations with Bt 2913. On the basis of the results of this analysis, we speculated that trypsin and other serine proteases in the D. abietella larval midgut began to activate Cry/Vip prototoxin at 6 h to 12 h after Bt 2913 ingestion. At 12 h after Bt 2913 ingestion, chymotrypsin was potentially involved in degrading the active core fragment of Vip3Aa toxin, and the detoxification enzymes in the larvae contributed to the metabolic detoxification of the Bt toxin. The ABC transporter and several other receptor-protein-related genes were also downregulated to increase resistance to Bt 2913. However, the upregulation of 60S ribosomal protein and heat shock protein expression weakened the resistance of larvae to Bt 2913, thereby enhancing the expression of NADH dehydrogenase and other receptor proteins that are highly expressed in the larval midgut and bind to activating toxins, including Cry1Ac. At 24 h after Bt 2913 ingestion, many activated toxins were bound to receptor proteins such as APN in the larval midgut, resulting in membrane perforation. Here, we clarified the mechanism of Bt 2913 infection in D. abietella larvae, as well as the larval immune defense response to Bt 2913, which provides a theoretical basis for the subsequent control of D. abietella using B. thuringiensis. Full article

By 2013, it had been shown that the genes cadherin-like receptor (Cad) and ATP-binding cassette transporter subfamily C2 (ABCC2) were responsible for insect resistance to several Cry1A toxins, acting as susceptibility-determining receptors, and many review articles have been published. Therefore, this review focuses on information about receptors and receptor-binding sites that have been revealed since 2014. Since 2014, studies have revealed that the receptors involved in determining susceptibility vary depending on the Cry toxin subfamily, and that binding affinity between Cry toxins and receptors plays a crucial role. Consequently, models have demonstrated that ABCC2, ABCC3, and Cad interact with Cry1Aa; ABCC2 and Cad with Cry1Ab and Cry1Ac; ABCC2 and ABCC3 with Cry1Fa; ABCB1 with Cry1Ba, Cry1Ia, Cry9Da, and Cry3Aa; and ABCA2 with Cry2Aa and Cry2Ba, primarily in the silkworm, Bombyx mori. Furthermore, since 2017, it has been suggested that the binding sites of BmCad and BmABCC2 on Cry1Aa toxin overlap in the loop region of domain II, indicating that Cry toxins use various molecules as receptors due to their ability to bind promiscuously in this region. Additionally, since 2017, several ABC transporters have been identified as low-efficiency receptors that poorly induce cell swelling in heterologously expressing cultured cells. In 2024, research suggested that multiple molecules from the ABC transporter subfamily, including ABCC1, ABCC2, ABCC3, ABCC4, ABCC10, and ABCC11, act as low-efficiency receptors for a single Cry toxin in the midgut of silkworm larvae. This observation led to the hypothesis that the presence of such low-efficiency receptors contributes to the evolution of Cry toxins towards the generation of highly functional receptors that determine the susceptibility of individual insects. Moreover, this evolutionary process is considered to offer valuable insights for the engineering of Cry toxins to overcome resistance and develop countermeasures against resistance. Full article

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens, only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens, and identified two new ABC transporter genes: HvABCC3 and HvABCC4. Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis. Full article

Recent studies have suggested that ABC transporters are the main receptors of Cry toxins. However, the receptors of many Cry toxins have not been identified. In this study, we used a heterologous cell expression system to identify Bombyx mori ABC transporter subfamily C members (BmABCCs) that function as receptors for five Cry toxins active in Lepidopteran insects: Cry1Aa, Cry1Ca, Cry1Da, Cry8Ca, and Cry9Aa. All five Cry toxins can use multiple ABCCs as low-efficiency receptors, which induce cytotoxicity only at high concentrations. Surface plasmon resonance analysis revealed that the K_D values between the toxins and BmABCC1 and BmABCC4 were 10⁻⁵ to 10⁻⁹ M, suggesting binding affinities 8- to 10,000-fold lower than those between Cry1Aa and BmABCC2, which are susceptibility-determining receptors for Cry1Aa. Bioassays in BmABCC-knockout silkworm strains showed that these low-efficiency receptors are not involved in sensitivity to Cry toxins. The findings suggest that each family of Cry toxins uses multiple BmABCCs as low-efficiency receptors in the insect midgut based on the promiscuous binding of their receptor-binding regions. Each Cry toxin seems to have evolved to utilize one or several ABC transporters as susceptibility-determining receptors. Full article

Physiology disorders of the liver, as it is an important tissue in lipid metabolism, can cause fatty liver disease. The mechanism might be regulated by 17 circadian clock genes and 18 fat metabolism genes, together with a high-fat diet (HFD). Due to their rich nutritional and medicinal value, Chinese soft-shelled turtles (Trionyx sinensis) are very popular among the Chinese people. In the study, we aimed to investigate the influence of an HFD on the daily expression of both the core clock genes and the lipid metabolism genes in the liver tissue of the turtles. The two diets were formulated with 7.98% lipid (the CON group) and 13.86% lipid (the HFD group) to feed 180 juvenile turtles, which were randomly divided into two groups with three replicates per group and 30 turtles in each replicate for six weeks, and the diet experiment was administrated with a photophase regimen of a 24 h light/dark (12L:12D) cycle. At the end of the experiment, the liver tissue samples were collected from nine turtles per group every 3 h (zeitgeber time: ZT 0, 3, 6, 9, 12, 15, 18, 21 and 24) for 24 h to investigate the daily expression and correlation analysis of these genes. The results showed that 11 core clock genes [i.e., circadian locomotor output cycles kaput (Clock), brain and muscle arnt-like protein 1 and 2 (Bmal1/2), timeless (Tim), cryptochrome 1 (Cry2), period2 (Per2), nuclear factor IL-3 gene (Nfil3), nuclear receptor subfamily 1, treatment D, member 1 and 2 (Nr1d1/2) and retinoic acid related orphan receptor α/β/γ β and γ (Rorβ/γ)] exhibited circadian oscillation, but 6 genes did not, including neuronal PAS domain protein 2 (Npas2), Per1, Cry1, basic helix-loop-helix family, member E40 (Bhlhe40), Rorα and D-binding protein (Dbp), and 16 lipid metabolism genes including fatty acid synthase (Fas), diacylglycerol acyltransferase 1 (Dgat1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), Low-density lipoprotein receptor-related protein 1-like (Ldlr1), Lipin 1 (Lipin1), Carnitine palmitoyltransferase 1A (Cpt1a), Peroxisome proliferator activation receptor α, β and γ (Pparα/β/γ), Sirtuin 1 (Sirt1), Apoa (Apoa1), Apolipoprotein B (Apob), Pyruvate Dehydrogenase kinase 4 (Pdk4), Acyl-CoA synthase long-chain1 (Acsl1), Liver X receptors α (Lxrα) and Retinoid X receptor, α (Rxra) also demonstrated circadian oscillations, but 2 genes did not, Scd and Acaca, in the liver tissues of the CON group. However, in the HFD group, the circadian rhythms’ expressional patterns were disrupted for the eight core clock genes, Clock, Cry2, Per2, Nfil3, Nr1d1/2 and Rorβ/γ, and the peak expression of Bmal1/2 and Tim showed delayed or advanced phases. Furthermore, four genes (Cry1, Per1, Dbp and Rorα) displayed no diurnal rhythm in the CON group; instead, significant circadian rhythms appeared in the HFD group. Meanwhile, the HFD disrupted the circadian rhythm expressions of seven fat metabolism genes (Fas, Cpt1a, Sirt1, Apoa1, Apob, Pdk4 and Acsl1). Meanwhile, the other nine genes in the HFD group also showed advanced or delayed expression peaks compared to the CON group. Most importantly of all, there were remarkably positive or negative correlations between the core clock genes and the lipid metabolism genes, and their correlation relationships were altered by the HFD. To sum up, circadian rhythm alterations of the core clock genes and the lipid metabolism genes were induced by the high-fat diet (HFD) in the liver tissues of T. sinensis. This result provides experimental and theoretical data for the mass breeding and production of T. sinensis in our country. Full article

Obesity is a known risk factor for metabolic diseases and is often associated with chronic inflammation in adipose tissue. We previously identified the polyethoxylated flavonoid Nobiletin (NOB) as a circadian clock modulator that directly binds to and activates the ROR receptors in the core oscillator, markedly improving metabolic fitness in obese mice. Here, we show that NOB enhanced the oscillation of core clock genes in differentiated 3T3-L1 adipocytes, including ROR target genes such as Bmal1, Cry1, Dec1, and Dec2. NOB inhibited lipid accumulation in 3T3-L1 and SVF cells, concomitant with the dysregulated circadian expression of adipogenic differentiation-related genes including Cebpb, Pparg, Lpl, Scd1, and Fas. Importantly, RORα/RORγ double knockdown in 3T3-L1 cells (Ror DKD) significantly attenuated the effects of NOB on circadian gene expression and lipid accumulation. Furthermore, whereas NOB upregulated the expression of IκBα, a target of RORs, to inhibit NF-κB activation and proinflammatory cytokine expression, Ror DKD cells exhibited a heightened activation of the NF-κB pathway, further indicating a requisite role of RORs for NOB efficacy in adipocytes. Together, these results highlight a significant regulatory function of the NOB–ROR axis in the circadian expression of clock and clock-controlled genes in adipocytes, thereby governing adipogenic differentiation, lipogenesis, and inflammation. Full article

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith; Lepidoptera: Noctuidae) is an invasive agricultural pest with a global distribution, causing major crop losses annually. Its control strategies largely rely on chemical insecticides and transgenic crops expressing Bacillus thuringiensis insecticidal proteins (Cry and Vip toxins); however, the development of high resistance poses a significant issue. The ATP-binding cassette transporter C2 (ABCC2) has been linked to Cry toxin pore formation, acting as a receptor of some Cry toxins. Recently detected mutations in the SfABCC2 gene in extracellular loop 4 (ECL4) have been associated with Bt toxin resistance in FAW. In the present study, we expressed the SfABCC2 gene in Drosophila melanogaster, a species normally unaffected by the Bt toxins. We demonstrate that susceptibility can be introduced by the ectopic and tissue-specific expression of wildtype SfABCC2. Next, we introduced mutations into ECL4—both individually and in combination—that have been recently described in Brazilian FAW and functionally validated by toxicity bioassays against the foliar Bt product Xentari. Our results provide an efficient demonstration of the suitability of transgenic Drosophila for validating FAW ABCC2 resistance mutations in ECL4 against Bt toxins, and potential cross-resistance issues between closely related proteins that use ABCC2. Full article