Comparative transcriptomics reveal universal and compound-specific mechanisms of insecticide response in the mulberry looper (Phthonandria atrilineata).

BackgroundJuvenile hormones (JH) and ecdysteroids control postembryonic development in insects. They serve as valuable targets for pest management. Hence, understanding the molecular mechanisms of their action is of crucial importance. CREB-binding protein (CBP) is a universal transcriptional co-regulator. It controls the expression of several genes including those from hormone signaling pathways through co-activation of many transcription factors. However, the role of CBP during postembryonic development in insects is not well understood. Therefore, we have studied the role of CBP in postembryonic development in Tribolium, a model coleopteran insect.ResultsCBP is ubiquitously expressed in the red flour beetle, Tribolium castaneum. RNA interference (RNAi) mediated knockdown of CBP resulted in a decrease in JH induction of Kr-h1 gene expression in Tribolium larvae and led to a block in their development. Moreover, the injection of CBP double-stranded RNA (dsRNA) showed lethal phenotypes within 8 days of injection. RNA-seq and subsequent differential gene expression analysis identified CBP target genes in Tribolium. Knockdown of CBP caused a decrease in the expression of 1306 genes coding for transcription factors and other proteins associated with growth and development. Depletion of CBP impaired the expression of several JH response genes (e.g., Kr-h1, Hairy, early trypsin) and ecdysone response genes (EcR, E74, E75, and broad complex). Further, GO enrichment analyses of the downregulated genes showed enrichment in different functions including developmental processes, pigmentation, anatomical structure development, regulation of biological and cellular processes, etc.ConclusionThese data suggest diverse but crucial roles for CBP during postembryonic development in the coleopteran model insect, Tribolium. It can serve as a target for RNAi mediated pest management of this stored product pest.

A French strain of the tobacco aphid Myzus nicotianae Blackman (Homoptera: Aphididae), strain FR, showed high tolerance to imidacloprid in short-term (48-h) oral ingestion bioassays when compared to a susceptible reference strain of Myzus persicae, strain NS. The resulting tolerance factors were >50. Measures of the contact activity of imidacloprid by the FAO dip method failed to detect these high factors of tolerance. The tolerance factor was in general <10 when using the dip method. The resulting difference between tolerance factors could be attributed to a behavioural component to fitness between strain FR and strain NS as further experiments revealed. When measuring the effect of systemically applied imidacloprid on honeydew excretion, a 50% reduction occurred in both strain FR and strain NS at nearly the same concentration of imidacloprid, providing evidence for a similar antifeedant response in both strains. Starvation experiments revealed that the French strain was able to survive approximately 24 h longer than a reference laboratory strain of M. persicae. This result coincided with the fact that systemically applied imidacloprid showed the same aphicidal potential against strain FR after three days as against strain NS after two days, i.e. 24 h later. After rearing in the laboratory for six months the French strain of M. nicotianae lost its hardiness and also its apparent ability to tolerate imidacloprid. However, strain FR was a heterogeneous field strain and it is possible that a susceptible variant out-reproduced a more hardy variant. These findings indicate that the type of bioassay is very important when assessing aphid populations for resistance against the chloronicotinyl insecticide imidacloprid, because of its distinct mode of action. It is obvious that an aphid dip test, i.e. FAO dip test, produces more reliable results than the different kinds of short-term oral ingestion bioassays, because of the reversible behavioural changes induced by imidacloprid after oral uptake. Thus a short-term oral ingestion bioassay (≤48 h) is not recommended for precise detection of possible resistance of Myzus sp. to imidacloprid, although this mode of uptake for imidacloprid might be sometimes more realistic in terms of field behaviour. The ideal test to generate most accurate data would be a slightly longer (72-h) feeding bioassay, perhaps used in conjunction with a dip test. The possible influence of the results on resistance monitoring is discussed. © 1997 SCI.

The highly conserved and ubiquitous molecular chaperone heat shock protein 70 (Hsp70) plays a critical role in protein homeostasis (proteostasis). Controlled by its ATPase activity, Hsp70 cycles between two conformations, Hsp70-ATP and Hsp70-ADP, to bind and release its substrate. Chemical tools with distinct modes of action, especially those capable of modulating the ATPase activity of Hsp70, are being actively sought after in the mechanistic dissection of this system. Here, we report a conformation-specific RNA aptamer that binds only to Hsp70-ATP but not to Hsp70-ADP. We have refined this aptamer and demonstrated its inhibitory effect on Hsp70's ATPase activity. We have also shown that this inhibitory effect on Hsp70 is independent of its interaction with the Hsp40 co-chaperone. As Hsp70 is increasingly being recognized as a drug target in a number of age related diseases such as neurodegenerative, protein misfolding diseases and cancer, this aptamer is potentially useful in therapeutic applications. Moreover, this work also demonstrates the feasibility of using aptamers to target ATPase activity as a general therapeutic strategy.

Western corn rootworm (WCR), Diabrotica virgifera virgifera, LeConte, is an insect pest that poses a significant threat to the productivity of modern agriculture, causing significant economic and crop losses. The development of genetically modified (GM) crops expressing one or more proteins that confer tolerance to specific insect pests, such as WCR, was a historic breakthrough in agricultural biotechnology and continues to serve as an invaluable tool in pest management. Despite this, evolving resistance to existing insect control proteins expressed in current generation GM crops requires continued identification of new proteins with distinct modes of action while retaining targeted insecticidal efficacy. GM crops expressing insecticidal proteins must undergo extensive safety assessments prior to commercialization to ensure that they pose no increased risk to the health of humans or other animals relative to their non-GM conventional counterparts. As part of these safety evaluations, a weight of evidence approach is utilized to assess the safety of the expressed insecticidal proteins to evaluate any potential risk in the context of dietary exposure. This study describes the food and feed safety assessment of Vpb4Da2, a new Bacillus thuringiensis insecticidal protein that confers in planta tolerance to WCR. Vpb4Da2 exhibits structural and functional similarities to other insect control proteins expressed in commercialized GM crops. In addition, the lack of homology to known toxins or allergens, a lack of acute toxicity in mice, inactivation by conditions commonly experienced in the human gut or during cooking/food processing, and the extremely low expected dietary exposure to Vpb4Da2 provide a substantial weight of evidence to demonstrate that the Vpb4Da2 protein poses no indication of a risk to the health of humans or other animals.

Western corn rootworm (WCR), Diabrotica virgifera virgifera, LeConte, is an insect pest that poses a significant threat to the productivity of modern agriculture, causing significant economic and crop losses. The development of genetically modified (GM) crops expressing one or more proteins that confer tolerance to specific insect pests, such as WCR, was a historic breakthrough in agricultural biotechnology and continues to serve as an invaluable tool in pest management. Despite this, evolving resistance to existing insect control proteins expressed in current generation GM crops requires continued identification of new proteins with distinct modes of action while retaining targeted insecticidal efficacy. GM crops expressing insecticidal proteins must undergo extensive safety assessments prior to commercialization to ensure that they pose no increased risk to the health of humans or other animals relative to their non-GM conventional counterparts. As part of these safety evaluations, a weight of evidence approach is utilized to assess the safety of the expressed insecticidal proteins to evaluate any potential risk in the context of dietary exposure. This study describes the food and feed safety assessment of Vpb4Da2, a new Bacillus thuringiensis insecticidal protein that confers in planta tolerance to WCR. Vpb4Da2 exhibits structural and functional similarities to other insect control proteins expressed in commercialized GM crops. In addition, the lack of homology to known toxins or allergens, a lack of acute toxicity in mice, inactivation by conditions commonly experienced in the human gut or during cooking/food processing, and the extremely low expected dietary exposure to Vpb4Da2 provide a substantial weight of evidence to demonstrate that the Vpb4Da2 protein poses no indication of a risk to the health of humans or other animals.

The plant hormone auxin regulates many aspects of plant growth and developmental processes. Auxin gradient is formed in plant as a result of polar auxin transportation by three types of auxin transporters such as OsLAX, OsPIN, and OsABCB. We report here the analysis of two rice auxin transporter gene families, OsLAX and OsABCB, using bioinformatics tools, publicly accessible microarray data, and quantitative RT-PCR. There are 5 putative OsLAXs and 22 putative OsABCBs in rice genome, which were mapped on 8 chromosomes. The exon-intron structure of OsLAX genes and properties of deduced proteins were relatively conserved within grass family, while that of OsABCB genes varied greatly. Both constitutive and organ/tissue specific expression patterns were observed in OsLAXs and OsABCBs. Analysis of evolutionarily closely related “gene pairs” together with organ/tissue specific expression revealed possible “function gaining” and “function losing” events during rice evolution. Most OsLAX and OsABCB genes were regulated by drought and salt stress, as well as hormonal stimuli [auxin and Abscisic Acid (ABA)], which suggests extensive crosstalk between abiotic stresses and hormone signaling pathways. The existence of large number of auxin and stress related cis-regulatory elements in promoter regions might account for their massive responsiveness of these genes to these environmental stimuli, indicating complexity of regulatory networks involved in various developmental and physiological processes. The comprehensive analysis of OsLAX and OsABCB auxin transporter genes in this study would be helpful for understanding the biological significance of these gene families in hormone signaling and adaptation of rice plants to unfavorable environments.

Metabolic inhibition potentiates oxidant injury

Summary Extrafloral nectar (EFN) provides plants with indirect defence against herbivores by attracting predatory insects, predominantly ants. Decades of research have supported the role of EFN as an effective plant defence, dating back to Thomas Belt's description of ants on acacia in 1874. Despite this extensive body of literature, knowledge of the ecological role of EFN has rarely been applied in the field of pest management. We review the existing literature on the use of EFN in agriculture and consider the obstacles that have hindered this transition. Chief among these obstacles is the influence of ecological context on the outcome of EFN‐mediated interactions. As such, we consider the options for various agricultural systems in the light of the growth habit of EFN‐producing species, focusing first on orchard species and then on herbaceous crops. In each case, we highlight the benefits and difficulties of utilizing EFN as a pest management tool and of measuring its efficacy. Synthesis and applications. We argue that it is time for a shift in extrafloral nectar (EFN) research towards applied settings and seek to address the question: How can a context‐dependent and often inducible plant trait be utilized as a reliable tool in agricultural pest management? Breeding crops for increased EFN production, and intercropping with EFN‐producing plants, can enhance assemblages of beneficial insects in many agricultural settings. Orchard systems, in particular, provide an ecological context in which the attraction of ants can contribute to cost‐effective and sustainable pest management programmes over a broad geographic range.

Transgenic Bt crops are important tools for growers to manage insect pests, but their durability is threatened by the evolution of insect resistance. Implementing a resistance monitoring program is essential to detect and mitigate resistance. For non-high-dose Bt crops, resistance monitoring is challenging, because insect control is not complete, so targeted insects and insect damage will be present even without resistance. Given these challenges, sentinel plots have been used to monitor for insect resistance to non-high-dose crops by assessing changes in the efficacy of a Bt crop over time relative to a non-Bt control. We optimized a sentinel plot resistance monitoring approach for MON 88702 ThryvOn™ cotton, a new non-high-dose Bt product targeting two sucking pest taxa-Lygus (L. lineolaris and L. hesperus) and thrips (Frankliniella fusca and F. occidentalis)-and report here on the thrips monitoring methods and results. Quantifying thrips immatures was the best metric to characterize the impact of the trait, with at least a 40-60% average reduction of thrips immatures on ThryvOn relative to the control cotton at all sites with higher thrips densities. These data can be used within a ThryvOn resistance monitoring program and represent a case study for establishing a resistance monitoring approach for a non-high-dose trait product.

Panax ginseng C. A. Meyer is a highly valued medicinal plant. Cylindrocarpon destructans is a destructive pathogen that causes root rot and significantly reduces the quality and yield of P. ginseng. However, an efficient method to control root rot remains unavailable because of insufficient understanding of the molecular mechanism underlying C. destructans-P. ginseng interaction. In this study, C. destructans-induced transcriptomes at different time points were investigated using RNA sequencing (RNA-Seq). De novo assembly produced 73,335 unigenes for the P. ginseng transcriptome after C. destructans infection, in which 3,839 unigenes were up-regulated. Notably, the abundance of the up-regulated unigenes sharply increased at 0.5 d postinoculation to provide effector-triggered immunity. In total, 24 of 26 randomly selected unigenes can be validated using quantitative reverse transcription (qRT)-PCR. Gene ontology enrichment analysis of these unigenes showed that “defense response to fungus”, “defense response” and “response to stress” were enriched. In addition, differentially expressed transcription factors involved in the hormone signaling pathways after C. destructans infection were identified. Finally, differentially expressed unigenes involved in reactive oxygen species and ginsenoside biosynthetic pathway during C. destructans infection were indentified. To our knowledge, this study is the first to report on the dynamic transcriptome triggered by C. destructans. These results improve our understanding of disease resistance in P. ginseng and provide a useful resource for quick detection of induced markers in P. ginseng before the comprehensive outbreak of this disease caused by C. destructans.

We constructed small RNA and degradome sequencing libraries to identify miRNAs and targets involved in tomato pedicel abscission, and confirmed their roles via quantitative real-time PCR. MicroRNAs (miRNAs) are endogenous small RNAs which play crucial negatively regulatory roles at both the transcriptional and post-transcriptional levels in plants; however, limited knowledge is available on the expression profiles of miRNAs and their target genes during tomato pedicel abscission. Taking advantage of small RNA (sRNA) and degradome sequencing technology, a total of 56 known and 11 novel candidate miRNAs targeting 223 mRNA genes were confirmed during pedicel abscission. Gene ontology annotation and KEGG pathway analysis showed that these target genes were significantly enriched in intracellular, membrane-bounded organelle-related biological processes as well as in metabolic, plant-pathogen interaction and hormone signaling pathways. We screened 17 miRNA/target pairs for further analysis and performed quantitative real-time PCR to identify the roles. Cluster analysis of selected miRNAs revealed that the expression profiles of miRNAs varied in different stages of abscission and could be impacted by ethylene treatment. In the present study, the correlations between miRNAs and targets suggested a complex regulatory network of miRNA-mediated target interaction during pedicel abscission. Additionally, the expression profiles of miRNAs and their targets changed by ethylene might be a considerable reason why ethylene promotes pedicel abscission. Our study provides new insights into the expression and regulatory profiles of miRNAs during tomato pedicel abscission.

Monocarboxylate transporters (MCTs) are key mediators of lactate transport that have emerged as promising targets for anti-cancer therapy. The MCT1 inhibitor AZD3965 (AstraZeneca) has shown promising activity in various pre-clinical models and is currently in phase I/II clinical testing. Understanding the impact of this drug on tumour cell metabolism may unravel dependencies that could be exploited for combination therapy. Here we investigate changes in glucose metabolism induced by AZD3965 treatment using 13C NMR isotopomer analysis, and examine their significance for cell survival using mitochondrial metabolism inhibitors. Exposure of Raji human lymphoma cells to AZD3965 in media supplemented with [1-13C]glucose led to a marked reduction in glucose uptake and lactate production in the cellular growth media alongside a build-up in intracellular [3- 13C]lactate and [1-13C]glucose levels, indicative of blockade of lactate excretion and inhibition of overall glycolytic activity. These effects were concomitant with increased [4-13C]glutamate levels, consistent with re-routing of pyruvate towards mitochondrial metabolism and enhanced flux through oxidative pyruvate dehydrogenase. Further, AZD3965 treatment was paralleled with a significant increase in levels of steady state Krebs cycle-related metabolites (including succinate, fumarate, acetate and NAD+/NADH) and cellular ATP, as revealed by bioluminescent and 1H NMR analyses, indicating improved mitochondrial metabolism and cellular re-energization. Co-administration of the mitochondrial complex I inhibitor metformin or the mitochondrial pyruvate carrier inhibitor UK5099 markedly potentiated the anti-proliferative effects of AZD3965 and led to significantly increased cell death, indicating that the observed upregulation in mitochondrial metabolism was necessary to maintain cell survival under MCT1 inhibitor-induced metabolic stress. Similar effects were observed with a second human lymphoma cell line, Hut78. Our findings show that MCT1 inhibition leads to a shift in cellular metabolism towards mitochondrial bioenergetic metabolism, creating a metabolic vulnerability that could be exploited for combinatorial therapy to increase the efficacy of AZD3965. Citation Format: Mounia Beloueche-Babari, Teresa Casals Galobart, Slawomir Wantuch, Paul D. Smith, Martin O. Leach. Monocarboxylate transporter 1 inhibition with AZD3965 increases cancer cell dependence on bioenergetic metabolism predicating combination therapy with mitochondrial inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 444. doi:10.1158/1538-7445.AM2017-444

Background. Infants and toddlers often present with disseminated and lymph node tuberculosis, in which Mycobacterium tuberculosis (Mtb) is predominantly intracellular. Linezolid, used to treat tuberculosis in adults, has not been formally studied in infants. Infants clear linezolid 5 times faster than adults and achieve lower 0- to 24-hour area under the concentration-time curves (AUC0–24).Methods. To mimic intracellular disease, we infected human-derived THP-1 macrophages with Mtb and inoculated hollow fiber systems. We performed dose-effect and dose-scheduling studies in which we recapitulated the linezolid half-life of 3 hours encountered in infants. Repetitive sampling for linezolid pharmacokinetics, Mtb intracellular burden, viable monocyte count, and RNA sequencing reads were performed up to 28 days.Results. The linezolid extracellular half-life was 2.64 ± 0.38 hours, whereas intracellular half-life was 8.93 ± 1.30 hours (r2 = 0.89). Linezolid efficacy was linked to the AUC0–24 to minimum inhibitory concentration (MIC) ratio (r2 = 0.98). The exposure associated with maximal Mtb kill was an AUC0–24/MIC of 23.37 ± 1.16. We identified a 414-gene transcript on exposure to toxic linezolid doses. The largest number of genes mapped to ribosomal proteins, a signature hitherto not associated with linezolid toxicity. The second-largest number of differentially expressed genes mapped to mitochondrial enzyme inhibition. Linezolid AUC0–24 best explained the mitochondrial gene inhibition, with 50% inhibition at 94 mg × hour/L (highest r2 = 0.98).Conclusions. We identified the linezolid AUC0–24/MIC target for optimal efficacy against pediatric intracellular tuberculosis, and an AUC0–24 threshold associated with mitochondrial inhibition. These constitute a therapeutic window to be targeted for optimal linezolid doses in children with tuberculosis.

Hypoxic injury was evaluated morphologically in the proximal tubule and in the medullary thick ascending limb of isolated rat kidneys perfused for 90 min without O2 or with various metabolic inhibitors. Inhibition of mitochondrial respiration (with rotenone, antimycin, oligomycin) or of intermediary metabolism (with monofluoroacetate, malonate, 2-deoxyglucose) caused reduction in renal oxygen consumption, renal function, and ATP content comparable with those elicited by oxygen deprivation. Metabolic inhibition produced hypoxiclike injury in the first portions of the proximal tubule, S1 and S2 ("clubbing" of microvilli, mitochondrial swelling), and the extent of damage was correlated with the degree of ATP depletion. In the third portion of the proximal tubule, S3, hypoxiclike damage (cytoplasmic edema or fragmentation) occurred most consistently when both aerobic and anaerobic metabolism were inhibited simultaneously. In the medullary thick ascending limb, none of the metabolic or mitochondrial inhibitors used could reproduce the injury of oxygen deprivation. Thus, the proximal tubule and the thick ascending limb have markedly different responses to cellular energy depletion, suggesting disparate mechanisms for hypoxic injury along the nephron.

Background: Diffuse Midline Glioma (DMG), a severe pediatric brain tumor, is characterized by its aggressive nature and poor prognosis. Common in brain midline structures like the thalamus and brainstem, DMGs often harbor H3K27M mutations, leading to reduced histone trimethylation at lysine 27 and loss of serine 31 phosphorylation. Building on these findings, our laboratory has undertaken a novel approach by engineering a knockout of the Polycomb Repressive Complex 2 (PRC2) in selected cell lines. This was achieved using CRISPR-Cas technology to target the EZH1 and EZH2 methyltransferases (PRC2KO), alongside the introduction or reversal of histone H3.3 mutations. The goal of this study is to investigate the effects of K27 Methylation loss and H3.3 mutations on gene expression in DMG to better understand DMG pathogenesis and potential therapeutic avenues. Methods: RNA and ATAC sequencing were performed on DMG cell lines including H3.3WT, H3.3 S31A, H3.3K27M, and PRC2KO. Utilizing 150 bp paired-end reads, 25 to 45 million unique reads were mapped to the human genome (hg38) using STAR. Differential gene expression was analyzed using DESeq2 in R, complemented by ATAC-sequencing to explore potential epigenetic changes. Results: The analysis identified 2,564; 2,946; 4,618; and 5,490 differentially expressed genes (DEGs) in the K27M, S31A, K27M-PRC2KO, and WT-PRC2KO cell lines, respectively, when compared to WT. A four-way Venn diagram analysis identified unique and shared gene cohorts among the different mutations. H3K27M cells showed 414 downregulated and 385 upregulated genes, mainly affecting cell migration. In contrast, PRC2KO cells, characterized by a complete loss of histone methylation, showed extensive changes with 1,302 genes downregulated and 1,779 upregulated, primarily impacting cell cycle and developmental processes. The S31A cells displayed a unique profile with 548 genes uniquely downregulated and 547 genes upregulated. The lack of enrichment in specific pathways for these genes suggests their involvement in a broader range of affected pathways. A significant discovery was the identification of a set of genes common to both S31 and K27M cells, but absent in PRC2 knockouts. This cohort, consisting of 350 downregulated and 389 upregulated genes, may represent a molecular link between serine phosphorylation and the K27M mutation. Additionally, ATAC-sequencing analysis was conducted to determine if the expression changes were a direct result of epigenetic dysregulation or an indirect effect. Conclusion: This study delineates the intricate gene regulation in DMG resulting from H3K27M mutations beyond loss of loss of K27 Methylation. It highlights unique and overlapping pathways influenced by, providing valuable insights into the molecular basis of DMG and guiding future therapeutic strategies. Citation Format: Suraj Bhattarai, Faruck L. Hakkim, Charles Day, Florina Grigore, Alyssa Langfald, Edward Hinchcliffe, James Robinson. Elucidating the oncogenic mechanisms in diffuse midline glioma: Comparative analysis of h3k27m mutation and k27 methylation loss on gene expression profiles via RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1736.