RNA Interference-mediated Gene Silencing Research Articles

Challenges and Opportunities in Plant Disease Management: A Brief Review

Plant diseases can reduce human food availability. Modern plant disease management faces problems due to climate change, fungicide resistance, pesticide residues and biodiversity loss. This review discusses problems and challenges in plant disease management and future research needs for effective management. Plant disease forecasting models can be used to predict plant diseases ahead of time. Protected cultivation combats climate change. It is necessary to find more evidence that plant pathogen diversity has a dilution effect on disease incidence. Deep learning-based disease diagnosis will help detect diseases faster. More hybrid fungicides should be developed to minimize fungicidal resistance problems. Among the molecular methods of plant disease management, RNA interference-mediated gene silencing and genome editing with CRISPR is more promising in plant disease management.

Moesin affects the plasma membrane expression and the immune checkpoint function of CD47 in human ovarian clear cell carcinoma

Among major histological subtypes of epithelial ovarian cancer, a higher incidence of ovarian clear cell carcinoma (OCCC) is observed in East Asian populations, particularly in Japan. Despite recent progress in the immune checkpoint inhibitors for a wide variety of cancer cell types, patients with OCCC exhibit considerably low response rates to these drugs. Hence, urgent efforts are needed to develop a novel immunotherapeutic approach for OCCC. CD47, a transmembrane protein, is overexpressed in almost all cancer cells and disrupts macrophage phagocytic activity in cancer cells. Ezrin-Radixin-Moesin (ERM) family member of proteins serve as scaffold proteins by crosslinking certain transmembrane proteins with the actin cytoskeleton, contributing to their plasma membrane localization. Here, we examined the role of ERM family in the plasma membrane localization and functionality of CD47 in OCCC cell lines derived from Japanese women. Confocal laser scanning microscopy analysis showed colocalization of CD47 with all three ERM in the plasma membrane of OCCC cells. RNA interference-mediated gene silencing of moesin, but not others, decreased the plasma membrane expression and immune checkpoint function of CD47, as determined by flow cytometry and in vitro phagocytosis assay using human macrophage-like cells, respectively. Interestingly, clinical database analysis indicated that moesin expression in OCCC was higher than that in other histological subtypes of ovarian cancers, and the expression of CD47 and moesin increased with the cancer stage. In conclusion, moesin is overexpressed in OCCC and may be the predominant scaffold protein responsible for CD47 plasma membrane localization and function in OCCC.

LC‒MS/MS and transcriptome analyses reveal saliva components of the seed-feeding truebug Pyrrhocoris apterus

Saliva secretion is essential for successful feeding by piercing–sucking insects. Despite extensive studies of the saliva composition of phloem- and blood-feeding insects, less is known about the oral secretions of seed-feeding insects. The firebug Pyrrhocoris apterus is a polyphagous insect that feeds primarily on dry seeds of various plant species. Here, liquid chromatography‒mass spectrometry/mass spectrometry (LC‒MS/MS) was used to identify 165 P. apterus salivary proteins, including 110 gelling-specific proteins, 24 watery-specific proteins, and 31 proteins common to both groups. Transcriptomic analysis of saliva glands identified 567 potential salivary proteins, of which 87 overlapped with those detected by LC‒MS/MS. Comparative analysis showed that 98 out of 165 (59.4%) P. apterus saliva proteins were associated with extra-oral digestion and detoxification and had homologs in ten other species of arthropods examined. In addition, 67 proteins were specific to P. apterus, of which 22 proteins were defined with unknown functions. RNA interference-mediated gene silencing assays indicated that P. apterus-specific proteins were vital for P. apterus survival. Our findings shed light on the function of salivary proteins in feeding by seed-feeding insects and the evolution of feeding habits in piercing–sucking insects.

α-catenin interaction with YAP/FoxM1/TEAD-induced CEP55 supports liver cancer cell migration

BackgroundAdherens junctions (AJs) facilitate cell–cell contact and contribute to cellular communication as well as signaling under physiological and pathological conditions. Aberrant expression of AJ proteins is frequently observed in human cancers; however, how these factors contribute to tumorigenesis is poorly understood. In addition, for some factors such as α‐catenin contradicting data has been described. In this study we aim to decipher how the AJ constituent α‐catenin contributes to liver cancer formation.MethodsTCGA data was used to detect transcript changes in 23 human tumor types. For the detection of proteins, liver cancer tissue microarrays were analyzed by immunohistochemistry. Liver cancer cell lines (HLF, Hep3B, HepG2) were used for viability, proliferation, and migration analyses after RNAinterference-mediated gene silencing. To investigate the tumor initiating potential, vectors coding for α‐catenin and myristoylated AKT were injected in mice by hydrodynamic gene delivery. A BioID assay combined with mass spectrometry was performed to identify α‐catenin binding partners. Results were confirmed by proximity ligation and co-immunoprecipitation assays. Binding of transcriptional regulators at gene promoters was investigated using chromatin-immunoprecipitation.Resultsα‐catenin mRNA was significantly reduced in many human malignancies (e.g., colon adenocarcinoma). In contrast, elevated α‐catenin expression in other cancer entities was associated with poor clinical outcome (e.g., for hepatocellular carcinoma; HCC). In HCC cells, α‐catenin was detectable at the membrane as well as cytoplasm where it supported tumor cell proliferation and migration. In vivo, α‐catenin facilitated moderate oncogenic properties in conjunction with AKT overexpression. Cytokinesis regulator centrosomal protein 55 (CEP55) was identified as a novel α‐catenin-binding protein in the cytoplasm of HCC cells. The physical interaction between α‐catenin and CEP55 was associated with CEP55 stabilization. CEP55 was highly expressed in human HCC tissues and its overexpression correlated with poor overall survival and cancer recurrence. Next to the α‐catenin-dependent protein stabilization, CEP55 was transcriptionally induced by a complex consisting of TEA domain transcription factors (TEADs), forkhead box M1 (FoxM1), and yes-associated protein (YAP). Surprisingly, CEP55 did not affect HCC cell proliferation but significantly supported migration in conjunction with α‐catenin.ConclusionMigration-supporting CEP55 is induced by two independent mechanisms in HCC cells: stabilization through interaction with the AJ protein α‐catenin and transcriptional activation via the FoxM1/TEAD/YAP complex.

Abstract 1314: Molecular contribution of Annexin A2 in triple negative breast cancer metastasis via tumor-derived extracellular vesicles

Abstract Triple negative breast cancer (TNBC) is the most lethal subtype of all breast cancers leading to high mortality rates. Tumor-derived extracellular vesicles (TEV) along with their cell-type specific cargo are greatly implicated in tissue-specific metastasis. In addition to metastasis, TEV play a crucial role in interacting with the distant microenvironment & shaping a pre-metastatic niche (PMN) for tumor cells to home. Understanding the mechanisms by which TEV promote PMN development has great potential to improve the diagnosis, prognosis, & treatment of TNBC. Annexin A2 (AnxA2) is a plasma & endosomal membrane-associated protein. High levels of AnxA2 in TNBC patients have been correlated with poor distant metastasis-free survival and poor overall survival. It is abundantly present in the TEV & recruits cargo such as proteins and microRNAs. Our lab reported that in vivo education with AnxA2 depleted TNBC-derived extracellular vesicles (EVs) led to reduced metastasis in lungs & brain suggesting a key role in the formation of a PMN. While the presence of AnxA2 in TNBC-derived EVs has been reported, its molecular role in the formation & development of PMN via EVs is still unexplored. We aim to evaluate the implications of AnxA2 in EVs at distant sites to mediate tumor cell homing & elucidate mechanisms promoting TNBC metastasis. We have employed RNA interference-mediated gene silencing to stably downregulate AnxA2 in organotropic TNBC cell lines derived from the parent MDA MB 231 cells. We observed a significant effect of AnxA2 depletion on its physiological role in plasmin generation. Differential ultracentrifugation was used to isolate EVs from cell culture supernatant and the physical characterization was done using Nanoparticle Tracking Analyzer. Biological characterization was done in concordance with MISEV 2018 guidelines. Using immunoblotting we confirmed reduced levels of AnxA2 in EVs derived from AnxA2 depleted TNBC cells. We verified their purity using EV enriched markers - ESCRT, Heat shock proteins & tetraspanins such as CD81, CD9, CD63 & confirmed the absence of negative markers - GM130 & cytochrome c. Interestingly, a reduced yield of EV was observed with AnxA2 depletion indicating a potential effect on EV biogenesis and release. Additionally, the EVs will be subjected to quantitative proteomic analysis to identify differentially expressed proteins upon loss of AnxA2. We will further carry out transcriptome profiling of the AnxA2 depleted TNBC cells and -derived EVs to identify the differentially expressed genes. The role of AnxA2 in TEV biogenesis, release and selective cargo loading will lead to potential identification and understanding of the novel secretory and EV protein that may act as a functional regulator in promoting advanced metastasis in TNBC. [This research is supported by the NCI under Award Number R01CA220273 (JKV), awarded to Dr. Jamboor K. Vishwanatha.] Citation Format: Rucha Trivedi, Jamboor K. Vishwanatha. Molecular contribution of Annexin A2 in triple negative breast cancer metastasis via tumor-derived extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1314.

EIF3 subunit M regulates blood meal digestion in Rhodnius prolixus affecting ecdysis, reproduction, and survival.

In triatomines, blood-feeding triggers many physiological processes including post embryonic development and reproduction. Different feeding habits, such as hematophagy, can shape gene functions to meet the challenges of each type of diet. The gut of blood-sucking insects faces particular challenges after feeding due to the quantity and the quality of the food ingested. A comparison of transcriptomic and proteomic data indicates that post transcriptional regulation of gene expression is crucial in the triatomine gut. It was proposed that eukaryotic translation initiation factor 3 subunit m (eIF3m) and eIF3e define 2 different eIF3 complexes with a distinct affinity for the different mRNAs, thus selecting the set of mRNAs to be translated and constituting a post transcriptional mode of regulation of gene expression. Because the eIF3m is mainly expressed in the gut, we evaluated its relevance in Rhodnius prolixus physiology through RNA interference-mediated gene silencing. The knockdown of eIF3m reduced the digestion rate, affecting the processes triggered by a blood meal. Its silencing inhibited molting and caused premature death in nymphs while impaired ovary development, oviposition and increased resistance to starvation in adult females. The survival of males after feeding (resistance to starvation) was not affected by eIF3m knockdown. The information regarding the eIF3m function in insects is scarce and the phenotypes observed in R. prolixus upon eIF3m silencing are different and more severe than those previously described in Drosophila melanogaster, indicating a pleiotropic role of this gene in triatomines.

Aedes aegypti Malpighian tubules are immunologically activated following systemic Toll activation

BackgroundCanine heartworm is a widespread and potentially fatal mosquito-borne disease caused by infections with the parasitic nematode, Dirofilaria immitis. We have previously shown that systemic activation of the Toll immune pathway via silencing of the negative regulator Cactus in Aedes aegypti blocks parasite development in the Malpighian tubules (MT), the mosquito renal organ. However, it was not established whether the MT were directly responding to Toll activation or were alternatively responding to upregulated proteins or other changes to the hemolymph driven by other tissues. Distinguishing these possibilities is crucial for developing more precise strategies to block D. immitis while potentially avoiding the fitness cost to the mosquito associated with Cactus silencing.MethodsThis study defines the transcriptional response of the MT and changes to the hemolymph proteome of Ae. aegypti after systemic Toll activation via intra-thoracic injection of double-stranded Cactus (dsCactus) RNA.ResultsMalpighian tubules significantly increased expression of the Toll pathway target genes that significantly overlapped expression changes occurring in whole mosquitoes. A significant overlap between the transcriptional response of the MT and proteins upregulated in the hemolymph was also observed.ConclusionsOur data show that MT are capable of RNA interference-mediated gene silencing and directly respond to dsCactus treatment by upregulating targets of the canonical Toll pathway. Although not definitive, the strong correspondence between the MT transcriptional response and the hemolymph proteomic responses provides evidence that the MT may contribute to mosquito humoral immunity.Graphical

Stabilized Double-Stranded RNA Strategy Improves Cotton Resistance to CBW (Anthonomus grandis).

Cotton is the most important crop for fiber production worldwide. However, the cotton boll weevil (CBW) is an insect pest that causes significant economic losses in infested areas. Current control methods are costly, inefficient, and environmentally hazardous. Herein, we generated transgenic cotton lines expressing double-stranded RNA (dsRNA) molecules to trigger RNA interference-mediated gene silencing in CBW. Thus, we targeted three essential genes coding for chitin synthase 2, vitellogenin, and ecdysis-triggering hormone receptor. The stability of expressed dsRNAs was improved by designing a structured RNA based on a viroid genome architecture. We transformed cotton embryos by inserting a promoter-driven expression cassette that overexpressed the dsRNA into flower buds. The transgenic cotton plants were characterized, and positive PCR transformed events were detected with an average heritability of 80%. Expression of dsRNAs was confirmed in floral buds by RT-qPCR, and the T1 cotton plant generation was challenged with fertilized CBW females. After 30 days, data showed high mortality (around 70%) in oviposited yolks. In adult insects fed on transgenic lines, chitin synthase II and vitellogenin showed reduced expression in larvae and adults, respectively. Developmental delays and abnormalities were also observed in these individuals. Our data remark on the potential of transgenic cotton based on a viroid-structured dsRNA to control CBW.

Tomato POLLEN DEFICIENT 2 encodes a G-type lectin receptor kinase required for viable pollen grain formation.

Pollen development is a crucial biological process indispensable for seed set in flowering plants and for successful crop breeding. However, little is known about the molecular mechanisms regulating pollen development in crop species. This study reports a novel male-sterile tomato mutant, pollen deficient 2 (pod2), characterized by the production of non-viable pollen grains and resulting in the development of small parthenocarpic fruits. A combined strategy of mapping-by-sequencing and RNA interference-mediated gene silencing was used to prove that the pod2 phenotype is caused by the loss of Solanum lycopersicum G-type lectin receptor kinase II.9 (SlG-LecRK-II.9) activity. In situ hybridization of floral buds showed that POD2/SlG-LecRK-II.9 is specifically expressed in tapetal cells and microspores at the late tetrad stage. Accordingly, abnormalities in meiosis and tapetum programmed cell death in pod2 occurred during microsporogenesis, resulting in the formation of four dysfunctional microspores leading to an aberrant microgametogenesis process. RNA-seq analyses supported the existence of alterations at the final stage of microsporogenesis, since we found tomato deregulated genes whose counterparts in Arabidopsis are essential for the normal progression of male meiosis and cytokinesis. Collectively, our results revealed the essential role of POD2/SlG-LecRK-II.9 in regulating tomato pollen development.

Spatial Distribution and Retention in Loblolly Pine Seedlings of Exogenous dsRNAs Applied through Roots.

Exogenously applied double-stranded RNA (dsRNA) can induce potent host specific gene knockdown and mortality in insects. The deployment of RNA-interference (RNAi) technologies for pest suppression is gaining traction in both agriculture and horticulture, but its implementation in forest systems is lagging. While numerous forest pests have demonstrated susceptibility to RNAi mediated gene silencing, including the southern pine beetle (SPB), Dendroctonus frontalis, multiple barriers stand between laboratory screening and real-world deployment. One such barrier is dsRNA delivery. One possible delivery method is through host plants, but an understanding of exogenous dsRNA movement through plant tissues is essential. Therefore, we sought to understand the translocation and persistence of dsRNAs designed for SPB throughout woody plant tissues after hydroponic exposure. Loblolly pine, Pinus taeda, seedlings were exposed to dsRNAs as a root soak, followed by destructive sampling. Total RNA was extracted from different tissue types including root, stem, crown, needle, and meristem, after which gel electrophoresis confirmed the recovery of the exogenous dsRNAs, which were further verified using Sanger sequencing. Both techniques confirmed the presence of the exogenously applied target dsRNAs in each tissue type after 1, 3, 5, and 7 d of dsRNA exposure. These findings suggest that root drench applications of exogenous dsRNAs could provide a viable delivery route for RNAi technology designed to combat tree feeding pests.

Ezrin Contributes to the Plasma Membrane Expression of PD-L1 in A2780 Cells.

Programmed death ligand–1 (PD–L1) is one of the immune checkpoint molecule localized on the plasma membrane of numerous cancer cells that negatively regulates T-cell-mediated immunosurveillance. Despite the remarkable efficacy and safety profile of immune checkpoint inhibitors (ICIs), such as anti-PD–L1 antibodies, restricted poor therapeutic responses to ICIs are often observed in patients with ovarian cancer. Because higher expression of PD–L1 in advanced ovarian cancer is associated with a decreased survival rate, identifying the potential molecules to regulate the plasma membrane expression of PD–L1 may provide a novel therapeutic strategy to improve the efficacy of ICIs against ovarian cancers. Here, we reveal the involvement of the ezrin/radixin/moesin (ERM) family, which crosslinks transmembrane proteins with the actin cytoskeleton by serving as a scaffold protein, in the plasma membrane expression of PD–L1 in the human epithelial ovarian cancer cell line A2780. Our results demonstrate that PD–L1 and all three ERMs were expressed at the mRNA and protein levels in A2780 cells, and that PD–L1 was highly colocalized with ezrin and moesin, but moderately with radixin, in the plasma membrane. Interestingly, RNA interference-mediated gene silencing of ezrin, but not of radixin or moesin, substantially reduced the plasma membrane expression of PD–L1 without altering its mRNA expression. In conclusion, our results indicate that ezrin may be responsible for the plasma membrane expression of PD–L1, possibly by serving as a scaffold protein in A2780 cells. Ezrin is a potential therapeutic target for improving the efficacy of ICIs against ovarian cancers.

RNAi silencing of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene inhibits vitellogenesis in Chinese mitten crab Eriocheir sinensis

The sesquiterpenoid methyl farnesoate (MF), a de-epoxide form of insect juvenile hormone III (JH III), plays an essential role in regulating many crucial physiological processes in crustaceans including vitellogenesis and reproduction. 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is an important rate-limiting enzyme in the mevalonate pathway, which is critical for the synthesis of JH III and MF. In the present study, a full-length cDNA encoding HMGR (EsHMGR) in Eriocheir sinensis was isolated and characterised. Sequence analysis of EsHMGR revealed that it belongs to Class I HMGR family proteins with HMG-CoA-binding and NADPH-binding domains, both important for HMGR activity. In addition to its ubiquitous tissue expression, expression of EsHMGR was highly specific to the ovary, the main site of Vg synthesis. During ovarian development, EsHMGR expression in ovary displayed a stage-specific pattern, and was correlated with expression of vitellogenin (EsVg) in hepatopancreas, which suggests that EsHMGR possibly involved in vitellogenesis. To further investigate the functional role of EsHMGR in vitellogenin biosynthesis in E. sinensis, RNA interference-mediated gene silencing was carried out both in vitro and in vivo. Quantitative PCR results showed that injection of EsHMGR double-stranded RNA (dsRNA) led to a significant decrease in EsVg expression levels in ovary and hepatopancreas both in vitro and in vivo. Taken together, the results suggest that EsHMGR is involved in vitellogenin biosynthesis in female E. sinensis, which may provide a new resource for HMGR enzymes participating in reproduction in crustaceans.

Protein Phosphatase 4 Negatively Regulates the Immune Deficiency-NF-κB Pathway during the Drosophila Immune Response.

The evolutionarily conserved immune deficiency (IMD) signaling pathway shields Drosophila against bacterial infections. It regulates the expression of antimicrobial peptides encoding genes through the activation of the NF-κB transcription factor Relish. Tight regulation of the signaling cascade ensures a balanced immune response, which is otherwise highly harmful. Several phosphorylation events mediate intracellular progression of the IMD pathway. However, signal termination by dephosphorylation remains largely elusive. Here, we identify the highly conserved protein phosphatase 4 (PP4) complex as a bona fide negative regulator of the IMD pathway. RNA interference-mediated gene silencing of PP4-19c, PP4R2, and Falafel, which encode the catalytic and regulatory subunits of the phosphatase complex, respectively, caused a marked upregulation of bacterial-induced antimicrobial peptide gene expression in both Drosophila melanogaster S2 cells and adult flies. Deregulated IMD signaling is associated with reduced lifespan of PP4-deficient flies in the absence of any infection. In contrast, flies overexpressing this phosphatase are highly sensitive to bacterial infections. Altogether, our results highlight an evolutionarily conserved function of PP4c in the regulation of NF-κB signaling from Drosophila to mammals.

Silencing of CYP6AS160 in Solenopsis invicta Buren by RNA interference enhances worker susceptibility to fipronil.

Cytochrome P450 monooxygenases play a key role in pest resistance to insecticides by detoxification. Four new P450 genes, CYP6AS160, CYP6AS161, CYP4AB73 and CYP4G232 were identified from Solenopsis invicta. CYP6AS160 was highly expressed in the abdomen and its expression could be induced significantly with exposure to fipronil, whereas CYP4AB73 was not highly expressed in the abdomen and its expression could not be significantly induced following exposure to fipronil. Expression levels of CYP6AS160 and CYP4AB73 in workers were significantly higher than that in queens. RNA interference-mediated gene silencing by feeding on double-stranded RNA (dsRNA) found that the levels of this transcript decreased (by maximum to 64.6%) when they fed on CYP6AS160-specific dsRNA. Workers fed dsCYP6AS160 had significantly higher mortality after 24 h of exposure to fipronil compared to controls. Workers fed dsCYP6AS160 had reduced total P450 activity of microsomal preparations toward model substrates p-nitroanisole. However, the knockdown of a non-overexpressed P450 gene, CYP4AB73 did not lead to an increase of mortality or a decrease of total P450 activity. The knockdown effects of CYP6AS160 on worker susceptibility to fipronil, combined with our other findings, indicate that CYP6AS160 is responsible for detoxification of fipronil. Feeding insects dsRNA may be a general strategy to trigger RNA interference and may find applications in entomological research and in the control of insect pests in the field.

PAP1 activates the prophenoloxidase system against bacterial infection in Musca domestica

We previously identified three putative prophenoloxidase-activating proteinase (mdPAP1, mdPAP2, and mdPAP3) genes from housefly Musca domestica by transcriptomic analysis. In this study, mdPAP1 cDNA was cloned, and the function of its encoded protein was analyzed. The cDNA of mdPAP1 was 1358 bp, and it contained a single open reading frame of 1122 bp encoding a predicted MdPAP1 protein of 373 amino acids. The estimated molecular weight of MdPAP1 was 41267.08 Da with an isoelectric point of 6.25. The deduced amino acid sequence of MdPAP1 exhibited high similarity to known PAPs of insects. mdPAP1 was detected in larvae, pupae, and adult housefly, and the expression level of mdPAP1 was upregulated in bacterial challenged larvae. The recombinant protein of MdPAP1 expressed in Escherichia coli could cleave the prophenoloxidase into phenoloxidase in M. domestica hemolymph infected by bacteria and result in a significant increase of the total phenoloxidase activity. In addition, RNA interference-mediated gene silencing of mdPAP1 significantly increased the mortality of M. domestica larvae. Results indicated that mdPAP1 was involved in the activation of the prophenoloxidase against bacterial infection in M. domestica.

RNAi-Based Gene Silencing in Sugarcane for Production of Biofuel.

RNA interference (RNAi) is an innate cellular mechanism triggered by a double-stranded RNA (dsRNA) molecule causing selective inhibition of gene expression. Here, we demonstrated the RNAi technology for gene silencing in sugarcane for biofuel production. This chapter describes an efficient model system that established to target the caffeic acid O-methyltransferase (COMT) gene and the RNAi construct is designed and delivered through Agrobacterium mediated stable sugarcane transformation. Also, the approach for an analysis of resulting putative transgenic plants for a targeted RNAi mediated gene silencing is described.

Hepatic stellate cells specific liposomes with the Toll-like receptor 4 shRNA attenuates liver fibrosis.

The hepatic stellate cells (HSCs) play a significant role in the onset of liver fibrosis, which can be treated by the inhibition and reversal of HSC activation. The RNA interference‐mediated TLR4 gene silencing might be a potential therapeutic approach for liver fibrosis. The crucial challenge in this method is the absence of an efficient delivery system for the RNAi introduction in the target cells. HSCs have an enhanced capacity of vitamin A intake as they contain retinoic acid receptors (RARs). In the current study, we developed cationic liposomes modified with vitamin A to improve the specificity of delivery vehicles for HSCs. The outcome of this study revealed that the VitA‐coupled cationic liposomes delivered the TLR4 shRNA to aHSCs more efficiently, as compared to the uncoupled cationic liposomes, both in the in vitro and in vivo conditions. Besides, as evident from the outcome of this study, the TLR4 gene silencing inhibited the HSCs activation and attenuated the liver fibrosis via the NF‐κB transcriptional inactivation, pro‐inflammatory cytokines secretion and reactive oxygen species (ROS) synthesis. Thus, the VitA‐coupled liposomes encapsulated with the TLR4‐shRNA might prove as an efficient therapeutic agent for liver fibrosis.

Transcription factor CncC potentially regulates the expression of multiple detoxification genes that mediate indoxacarb resistance in Spodoptera litura.

The xenobiotic transcription factor cap 'n' collar isoform C (CncC) is considered the central regulator of antioxidant and detoxification genes. Previous research indicated that CncC regulates three-phase enzymes responsible for insecticide resistance. In this study, the SlituCncC gene from Spodoptera litura was obtained and characterized. Quantitative polymerase chain reaction (qPCR) analysis showed that SlituCncC was expressed in all developmental stages and tissues, but was highly expressed in 3rd- and 4th-instar larvae, and in the Malpighian tubule, fat body, and midgut. In addition, SlituCncC was up-regulated and more highly induced with indoxacarb treatment in the indoxacarb-resistant strains compared with the susceptible strain. RNA interference-mediated gene silencing of SlituCncC significantly increased mortality of S.litura when exposed to indoxacarb. Furthermore, comparative transcriptome analysis showed that 842 genes were down-regulated and 127 genes were up-regulated in SlituCncC knockdown S. litura. Further analysis indicated that 18 three-phase enzymes were identified in the down-regulated genes, of which seven were associated with indoxacarb resistance in S. litura. qPCR analysis confirmed that expression of six of these seven genes was consistent with RNA sequencing data. All six detoxification genes were induced by indoxacarb, and the expression patterns were similar to that of SlituCncC. Finally, the CncC-Maf binding site was predicted in all six gene promoters. This study indicates that the transcription factor SlituCncC may regulate multiple detoxification genes that mediate indoxacarb resistance in S. litura.

Nanoparticulate RNA delivery systems in cancer

Drug delivery system is a common practice in cancer treatment. RNA interference-mediated post-transcriptional gene silencing holds promise as an approach to knockdown in the expression of target genes responsible for cancer cell growth and metastasis. RNA interference (RNAi) can be achieved by delivering small interfering RNA (siRNA) and short hairpin RNA (shRNA) to target cells. Since neither interfering RNAs can be delivered in naked form due to poor stability, an efficient delivery system is required that protects, guides, and delivers the siRNA and shRNA to target cells as part of cancer therapy (chemotherapy). In this review, a discussion is presented about the different types of drug delivery system used to deliver siRNA and shRNA, together with an overview of the potential benefits associated with this sophisticated biomolecular therapy. Improved understanding of the different approaches used in nanoparticle (NP) fabrication, along with an enhanced appreciation of the biochemical properties of siRNA/shRNA, will assist in developing improved drug delivery strategies in basic and clinical research. These novel delivery techniques are able to solve the problems that form an inevitable part of delivering genes in more efficient manner and as part of more effective treatment protocols. The present review concludes that the nanoparticulate RNA delivery system has great possibility for cancer treatment along with several other proposed methods. Several NPs or nanocarriers are already in use, but the methods proposed here could fulfill the missing gap in cancer research. It is the future technology, which unravels the mystery of resolving genomic diseases that is, especially genomic instability and its signaling cascades.

Both LmCYP4G genes function in decreasing cuticular penetration of insecticides in Locusta migratoria.

Cuticular hydrocarbons (CHCs) have a critical role in preventing desiccation and penetration of xenobiotics in insects. Previous studies have shown that cytochrome P450 subfamily 4G (CYP4G) enzymes are oxidative decarbonylases, essential for CHC biosynthesis. However, it is unclear whether there are functional differences between the two CYP4G genes in most insects. In Locusta migratoria, we identified two CYP4G genes (LmCYP4G62 and LmCYP4G102). LmCYP4G102 plays a critical role in the synthesis of CHCs, but the function of LmCYP4G62 is unknown. We identified, characterized, and compared two LmCYP4G genes, based on L. migratoria transcriptomic and genomic databases. RT-qPCR showed that both were highly expressed in tissues with which oenocytes are associated, the integument and fat body. Immunostaining indicated that LmCYP4G62 and LmCYP4G102 were highly abundant in oenocytes in these tissues. However, the two enzymes had a different subcellular distribution, with LmCYP4G62 localized on the plasma membrane and LmCYP4G102 dispersed throughout the oenocyte cytoplasm, presumably on the endoplasmic reticulum. RNA interference-mediated gene silencing against each of the two genes resulted in reduced CHC contents, in all classes for LmCYP4G102, but mostly shorter chain CHCs for LmCYP4G62. Silencing of both genes resulted in increased insecticide penetration through the cuticle, and increased locust susceptibility to desiccation and insecticides. Our studies suggest that both LmCYP4G62 and LmCYP4G102 contribute to hydrocarbon biosynthesis and play key roles in protecting locusts from water loss and insecticide penetration, but they are not fully redundant. Further, the two LmCYP4G genes might be used as new targets for insect pest management. © 2020 Society of Chemical Industry.