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The use of RNA interference (RNAi) technology to control pests is explored by researchers globally. Even though RNA is a new class of pest control compound unlike conventional chemical pesticides, the evolution of pest resistance needs to be considered. Here, we first investigate RNAi-based biopesticide resistance of Fusarium asiaticum, which is responsible for devastating diseases of plants, for example, Fusarium head blight. Five resistant strains were isolated from 500 strains that treated with UV-mutagenesis. The mutation common to all of the five resistant mutants occurred in the gene encoding Dicer2 (point mutations at codon 1005 and 1007), which were under strong purifying selection pressure. To confirm whether the mutations in Dicer2 confer resistance to RNAi, we exchanged the Dicer2 locus between the sensitive strain and the resistant strain by homologous double exchange. The transformed mutants, Dicer2R1005D and Dicer2E1007H, exhibited resistance to dsRNA in vitro. Further study showed that mutations of R1005D and E1007H affected the intramolecular interactions of Dicer2, resulting in the dysfunction of RNase III domain of Dicer2. The amount of sRNAs produced by Dicer2R1005D and Dicer2E1007H was extremely reduced along with variation of sRNA length. Together, these findings revealed a new potential mechanism of RNAi resistance and provided insight into RNAi-related biopesticide deployment for fungal control.

Modern oncology is rapidly evolving, driven by recent advances in RNA-based therapeutics. As new emerging cutting-edge technology, mRNA vaccines hold excellent promise for encoding immunostimulatory molecules, tumor-associated antigens, neoantigens, and chimeric antigen receptors for T-cell reprogramming. RNA interference tools enable highly effective post-transcriptional gene silencing that has rapidly progressed towards more tailored antitumor treatments targeting key molecular players in tumor progression and drug resistance. The inherent challenges and limitations of RNA-based tools, such as size, low stability and surface charges hindering direct cell entry, along with the short circulatory half-life and rapid clearance, call for new and improved RNA delivery systems enabling enhanced gene delivery. Nanoplatforms, particularly certain types of lipid, polymeric nanoparticles and inorganic nanoparticles, provide designed means to address the challenges of RNA delivery and cellular uptake. This paper explores the challenges and barriers while giving insight into the future perspective of RNA-based cancer therapeutics in the context of delivery nanoplatforms and the challenges during development.

Microvitellogenin (mVg) belongs to the lepidopteran-specific lipoprotein_11 super family and is thought to be involved in insect reproduction. However, the function of mVg in reproduction has not been established. In this study, we identified and characterized MsmVg in Mythimna separata, a destructive insect pest of grain crops. MsmVg is comprised of a 1,310 bp open reading frame (ORF) encoding 437 amino acids. Structural analysis showed that MsmVg contains three conserved domains in the lipoprotein_11 super family, including the all-α-N-terminal domain (NTD), a Pseudomonas putida homologous domain (PPD), and an all-β-C-terminal domain (CTD). MsmVg was highly expressed in mature female adults and enriched in the ovaries of 3-day-old female adults. Disruption of MsmVg by RNA interference did not change the expression of MsmVg, vitellogenin (MsVg) or the vitellogenin receptor (MsVgR) in fat bodies, but it inhibited their transcription in ovaries. Phenotype analysis showed that knockdown of MsmVg did not affect yolk deposition in ovaries, but impaired eggshell formation and led to a reduction in the reproductive capacity. Furthermore, knockdown of MsmVg significantly inhibited the expression of genes associated with eggshell formation (MsChP1-3 and MsFcP3C). Taken together, these data suggest that MsmVg plays an important role in female reproduction by affecting eggshell formation in M. separata, which provides a potential target for managing this insect pest.

Tetrahydrobiopterin (BH4) expression is normally strictly controlled; however, its intracellular levels increase considerably following nerve damage. GTP cyclohydrolase I (GCH1) plays a crucial role in regulating BH4 concentration, with an upregulation observed in the dorsal root ganglion in cases of neuropathic pain. In this study, we aimed to develop and evaluate the clinical potential of an RNA interference-based adeno-associated virus (AAV) targeting GCH1 across various species to decrease BH4 levels and, consequently, alleviate neuropathic pain symptoms. We identified universal small-interfering RNA sequences effective across species and developed an AAV-u-shRNA that successfully suppressed GCH1 expression with minimal off-target effects. Male Sprague Dawley rats were divided into four groups: normal, spared nerve injury, AAV-shCON, and AAV-u-shGCH1. The rats were sacrificed on post-injection day 28 to collect blood for BH4 level assessment. The AAV-u-shGCH1 group demonstrated remarkable improvement in the mechanical withdrawal threshold by PID 28, significantly outperforming the normal, spared nerve injury, and AAV-shCON groups. Plasma BH4 levels confirmed that AAV-u-shGCH1 effectively reduced neuropathic pain by inhibiting BH4 synthesis in vivo, introducing a novel, multispecies-compatible therapeutic strategy. Our results suggest that a single application of AAV-u-shGCH1 could offer a viable solution for neuropathic pain relief.

Hepatocellular carcinoma (HCC) poses a significant clinical challenge due to high mortality and limited treatment options. Radiofrequency ablation (RFA) is commonly used but can be limited by tumor recurrence. This study explores the potential of combining RFA with an attenuated Salmonella strain carrying siRNA-PD-L1 and endostatin to enhance HCC treatment. In this study, an H22 subcutaneous tumor mouse model was used, with animals divided into five groups for treatment with a blank control, a blank Salmonella plasmid, RFA alone, siRNA-PD-L1-endostatin, or a combination of RFA and siRNA-PD-L1-endostatin. The combination therapy significantly reduced tumor growth, angiogenesis, and PD-L1/VEGF expression in tumor tissues post-RFA. Additionally, it induced tumor cell apoptosis, inhibited proliferation and migration, and increased the infiltration of T lymphocytes, granzyme B+ T cells, and CD86+macrophages within tumors. There was also a notable rise in T and NK cell populations in the spleen. In conclusion, combining RFA with siRNA-PD-L1-endostatin delivered by attenuated Salmonella synergistically enhances anti-tumor effects, boosts the anti-tumor immune response, and improves RFA efficacy for HCC.

Chitinases are pivotal enzymes for the degradation of chitin, which play a role in food digestion. However, the functional roles of chitinases in teleosts remain under-explored. In this study, a full-length cDNA of chitinase gene (namely, OpCht) was obtained from dark sleeper Odontobutis potamophila. The predicted amino acid sequence of OpCht consisted of a typical catalytic domain and a chitin-binding domain interconnected with a linker region. Phylogenetic analyses suggest that OpCht has appeared in the teleost-specific monophyletic branches (CHIO). Quantitative real-time PCR analyses showed that OpCht was mainly expressed in stomach, liver, intestine and increased significantly during the period of juvenile. The optimal gradient concentrations and intervals/efficiency of RNA interference (RNAi) in OpCht were established, the results demonstrated that RNAi significantly repressed the expression of OpCht and diminished the chitinase activity. After 8 weeks of long-term RNAi, knockdown of OpCht reduced the height of intestinal villus and the thickness of muscular layer, which led to a decline in weight gain rate (WGR), while the accompanying intestinal inflammation contributed to a decreased survival rate (SR) in O. potamophila. In conclusion, OpCht is implicated in chitin degradation and plays an important role in intestinal health and growth performance in O. potamophila.

Piwi proteins act in interaction with piRNAs to influence germline development in a variety of organisms. Nevertheless, to date, there is limited studies have been documented regarding the function of Piwis in crustaceans. In this study, we isolated a Piwi2 gene from the mud crab Scylla paramamosain, and further explored its possible role in spermatogenesis through RNA interference (RNAi) and transcriptome analysis. The expression profile suggested that Sp-Piwi2 was specifically expressed in the testis of S. paramamosain. It had a high expression level at stage I, and that expression significantly decreased at stage II but subsequently increased at stage III. Immunofluorescence results revealed that Sp-Piwi2 was localized in the cytoplasm of spermatogonia. RNAi showed that the expression of Sp-Piwi2 was specifically down-regulated. In response to the prolonged down-regulation of Sp-Piwi2, the number of germ cells in the testis was markedly decreased. Transcriptome analysis further suggested that Sp-Piwi2 presumably regulates spermatogenesis by promoting spermatogonia proliferation and the differentiation of spermatogonia to spermatocytes, and is also involved in apoptosis and regulation of transposons. Our findings assigned the functions of Sp-Piwi2 in the spermatogenesis of the mud crab S. paramamosain, and shed a new insight into the mechanism underlying Sp-Piwi2 involved in spermatogenesis.

Acute hepatic porphyrias (AHPs) are a family of rare, autosomal, dominantly inherited conditions characterized by abnormalities in the production of heme. Advances in molecular engineering have provided new therapeutic possibilities for modifying the heme synthetic pathway in patients with porphyria. In particular, the RNA interference therapeutic givosiran was approved for the treatment of adults and adolescents with AHP aged >12 years based on the positive results of the phase III trial ENVISION. Despite the extended characterization of the activity of givosiran in clinical trials, reports on the long-term effects and effectiveness of the treatment in clinical practice are still scant. To fill this gap, this case series describes a monocentric Italian cohort of AHP patients treated with givosiran. Overall, our real-life experience supports the clinical evidence that long-term treatment with givosiran is well tolerated and able to provide sustained and continuous benefit to patients with acute intermittent porphyria, as reflected by the reduction in the frequency of attacks. In our series, givosiran treatment was also associated with improvement in assessments of quality of life, pain and fatigue.

Given that numerous genetic disorders, driven by diverse pathogenic mechanisms, may be amenable to recombinant adeno-associated virus (rAAV)-delivered gene therapies, the sustained innovation of rAAV-based therapeutic modalities is crucial. The progression and severity of genetic diseases can be reduced by targeting the toxic transcripts of a defective gene using microRNA (miRNA)-based miQURE technology delivered within an AAV vector. By adapting the delivered cassette, it may be possible to simultaneously regulate the expression profile of multiple genes involved in the pathogenesis of complex genetic diseases. The established miQURE gene silencing strategy was expanded by concatenating several miQURE molecules in a single construct, resulting in the novel linQURE platform. Here, a proof of mechanism is established by demonstrating that linQURE technology enables the concomitant expression of two synthetic miRNAs in vitro and in vivo, allowing more efficient downregulation of their disease-causing mRNA targets. This approach supports the development of multi-targeting therapeutic strategies, enabling gene therapy products to adapt to more complex multigenic indications, thus expanding the toolbox of readily available gene therapies.

Nucleic acid nanotechnology presents an exciting approach for manufacturing tailored nanoscale biomaterials with precise structures, spatial precision, and exceptional biocompatibility. These nucleic acid-based nanomaterials, here referred as nano-carriers can have versatile applications in bio-imaging, diagnostics, and therapy. In this review, we have surveyed the progress made in the field of multifunctional nucleic acid nano-carriers, particularly in the context of delivering antisense and RNA interference (RNAi) therapeutics. RNAi technology has emerged as a potent modality for conducting functional genomic analyses and holds potential as an effective approach for crafting targeted gene-silencing treatments for viral infections, cancer, and other diseases in the future. Our focus is on exploring RNAi as a therapeutic avenue, taking into account challenges such as the expense of RNAi triggers, delivering RNAi efficiently to the target site, and addressing off-target and nontarget effects, while also recognizing promising opportunities within the field of biomedical research.

transformer is a switch gene for sex determination in many insects, which cooperates with transformer-2 that is expressed in both sexes to regulate female differentiation, particularly in dipterans. Zeugodacus cucurbitae (Coquillett) is a very destructive pest worldwide, however, its sex determination pathway remains largely uncharacterized. Here, we show that the female sex ratio is sharply reduced with knockdown of either transformer or transformer-2 by RNA interference in early embryos of Z. cucurbitae. Most of the males grown from the embryos with transient transformer and transformer-2 suppression mated with wild-type females and produced mixed sex progeny, with one exception that produced only female progeny, and all of the few remaining males failed to mate with wild-type females and produced no progeny. The exceptional male and those males with mating failure were XX pseudomales as determined by the detection of Y chromosome-linked Maleness-on-the-Y, indicating that most XX pseudomales are not viable. The phenotypes of transformer and transformer-2 suggest that they play a key role in regulating sex determination and are required for female sexual development of Z. cucurbitae. Our results will be beneficial to the understanding of sex determination in Z. cucurbitae and can facilitate the development of genetic sexing strains for its biological control.

Plant-mediated RNA interference (RNAi) has emerged as a promising technology for insect control. The green peach aphid, Myzus persicae, feeds on over 400 species of host plants. Brassica napus (rape) is the second most important oilseed crop worldwide. Myzus persicae is highly reproductive and causes severe damage to the rape plants due to its quite flexible life cycle. In this study, we tested the RNAi effects of transgenic rape plants on M. persicae. By in vitro feeding M. persicae with artificial diets containing double-stranded RNAs (dsRNAs) targeting seven aphid genes, we identified a new gene encoding the partitioning-defective protein 6 (Par6) as the most potent RNAi target. Tissue- and stage-expression analysis of Par6 suggested this gene is highly expressed in the embryo and adult stage of M. persicae. We next generated transgenic rape plants expressing dsPar6 by Agrobacterium-mediated transformation and obtained nine independent transgenic lines. Compared to wild-type control plants, transgenic rape lines expressing dsPar6 showed strong resistance to M. persicae. Feeding assays revealed that feeding transgenic rape plants to M. persicae significantly decreased MpPar6 expression and survival rate and impaired fecundity. Furthermore, we showed that the resistance levels to M. persicae are positively correlated with dsPar6 expression levels in transgenic rape plants. Our study demonstrates that transgenic rape plants expressing dsPar6 are efficiently protected from M. persicae. Interfering with the genes involved in embryo development could be the effective RNAi targets for controlling aphids and potentially other insect pests.

Therapeutic applications of small interfering RNAs (siRNAs) have recently facilitated advancements in the biopharmaceutical industry, expanding opportunities for pharmacological intervention to targets previously deemed “undruggable.” Hence, determining rational design principles to inform the selection of effective siRNA sequences and appropriate chemical modifications has been a significant undertaking in the field. To accelerate the process of empirical siRNA design, machine learning (ML) techniques have been applied to the problem of siRNA efficacy prediction. This systematic review provides a comprehensive, yet succinct overview of advancements in this ML task by examining the evolution of model architectures trained to predict siRNA efficacy, features selected to represent individual samples and inform predictions, and the challenges associated with the use of ML in the context of therapeutic siRNA discovery. Consensus and conflict throughout the literature are discussed, promoting a nuanced understanding of this problem. Finally, the vast potential for future directions is addressed, supporting further research in computational biomedicine.

Small interfering RNAs (siRNAs) are revolutionizing the treatment of liver-associated indications. Yet, robust delivery to extrahepatic tissues remains a challenge. Conjugating lipids (e.g., docosanoic acid [DCA]) to siRNA supports extrahepatic delivery, but tissue accumulation remains lower than that achieved in liver by approved siRNA therapeutics. Early evidence suggests that functionalizing DCA with a head group (e.g., phosphatidylcholine [PC]) may enhance delivery to certain tissues. Here, we report the first systematic evaluation of the effect of PC head group chemistry on the extrahepatic distribution of DCA-conjugated siRNAs. We show that functionalizing DCA with a PC head group enhances siRNA accumulation in heart, muscle, lung, pancreas, duodenum, urinary bladder, and fat. Varying the size of the linker between the phosphate and choline moiety of the PC head group altered the extrahepatic accumulation of siRNA, with the optimal linker length being different for different tissues. Increasing PC head group valency also improved extrahepatic accumulation in a tissue-specific manner. This study demonstrates the structural impact of the PC moiety on the biodistribution of lipid-conjugated siRNA and introduces multiple novel PC variants for the chemical optimization of DCA-conjugated siRNA. These chemical variants can be used in the context of other lipids to increase the repertoire of conjugates for the extrahepatic distribution of siRNAs.

Marine pearls from Pinctada fucata martensii are valuable for decoration, skin care, and health care, but their quality and yield can be threatened by diseases, pearl nuclei transplantation rejection, and other external factors. The present study investigated the immune function of the tumor necrosis factor receptor (TNFR) gene in P. f. martensii to better understand immune responses related to nuclei rejection. The PmTNFR16 cDNA sequence obtained from cloning was 1675 bp in length and encoded 406 amino acid residues. Sequence analysis revealed that PmTNFR16 comprises one signal peptide domain, four TNFR domains, one transmembrane domain, and one death domain. Expression analysis revealed PmTNFR16 was highly expressed in the mantle and significantly up-regulated after exogenous stimuli. The effects of PmTNFR16 on immune effector molecules within the NF-κB signaling pathway were further explored using RNA interference probes for PmTNFR16. Pre-treatment of pearl oysters with dsRNA-PmTNFR before nucleus-insertion resulted in decreased expression of PmTNFR16 by 53 % at 24 h (p

The insulin-like signaling pathway plays diverse biological roles in regulating metabolism, immune responses, growth, and development in invertebrates. The insulin-like peptides (ILP) receptor (ILPR) is the pivotal protein in any insulin-like signaling pathway. In our previous study, we cloned the Mn-ILP gene from the oriental river prawn Macrobrachium nipponense and elucidated its function. However, the ILPR has not yet been described in M. nipponense. In the present study, we further examined the function of Mn-ILPR in M. nipponense. The full length of the Mn-ILPR mRNA was 4790 bp, encoding 1412 amino acids. Mn-ILPR was a transmembrane receptor under a subfamily of receptor tyrosine kinases. The Mn-ILPR mRNA was predominantly expressed in muscle and hepatopancreas. To elucidate the function of Mn-ILPR, we investigated its expression in different growth stages of prawns of both sexes, in prawns of different sizes at the same time, and in prawns of the same size at different stages of the molt cycle using qRT-PCR. The expression of Mn-ILPR was markedly higher: (1) in the rapid growth stage than in other stages of male individuals; (2) in the normal growth stage than in the gonad development stage of female individuals; (3) in large male individuals than in small male individuals; and (4) in the intermolt stage than in other stages of the molt cycle. Furthermore, the long-term knockdown of Mn-ILPR by RNAi effectively delayed the growth speed of M. nipponense. Meanwhile, the short-term knockdown of Mn-ILPR revealed that the ILP-ILPR signal pathway played a part in the process of growth of M. nipponense. These findings provide a greater understanding of the molecular mechanisms underlying growth and development in crustaceans.

ABSTRACT: Pulmonary artery remodeling is a characteristic feature of broiler ascites syndrome (BAS). Pulmonary artery endothelial cells (PAECs) regulated by HIF-1α play a critical role in pulmonary artery remodeling, but the underlying mechanisms of HIF-1α in BAS remain unclear. In this experiment, primary PAECs were cultured in vitro and were identified by coagulation factor VIII. After hypoxia and RNA interference, the mRNA and protein expression levels of HIF-1α and VEGF were determined by qPCR and Western blotting. The transcriptome profiles of PAECs were obtained by RNA sequencing. Our results showed that the positive rate of PAECs was more than 90%, hypoxia-induced promoted the proliferation and apoptosis of PAECs, and RNA interference significantly downregulated the expression of HIF-1α, inhibited the proliferation of PAECs, and promoted the apoptosis of PAECs. In addition, transcriptome sequencing analysis indicated that HIF-1α may regulate broiler ascites syndrome by mediating COL4A, vitronectin, vWF, ITGα8, and MKP-5 in the ECM, CAMs and MAPK pathways in PAECs. These studies lay the foundation for further exploration of the mechanisms of pulmonary artery remodeling, and HIF-1α may be a potentially effective gene for the prevention and treatment of BAS.

Chemically modified small interfering RNAs (siRNAs) are promising therapeutics guiding sequence-specific silencing of disease genes. Identifying chemically modified siRNA sequences that effectively silence target genes remains challenging. Such determinations necessitate computational algorithms. Machine learning is a powerful predictive approach for tackling biological problems but typically requires datasets significantly larger than most available siRNA datasets. Here, we describe a framework applying machine learning to a small dataset (356 modified sequences) for siRNA efficacy prediction. To overcome noise and biological limitations in siRNA datasets, we apply a trichotomous, two-threshold, partitioning approach, producing several combinations of classification threshold pairs. We then test the effects of different thresholds on random forest machine learning model performance using a novel evaluation metric accounting for class imbalances. We identify thresholds yielding a model with high predictive power, outperforming a linear model generated from the same data, that was predictive upon experimental evaluation. Using a novel model feature extraction method, we observe target site base importances and base preferences consistent with our current understanding of the siRNA-mediated silencing mechanism, with the random forest providing higher resolution than the linear model. This framework applies to any classification challenge involving small biological datasets, providing an opportunity to develop high-performing design algorithms for oligonucleotide therapies.

Blocking androgen receptor signaling is the mainstay of therapy for advanced prostate cancer (PCa). However, acquired resistance to single agents targeting this pathway results in the development of lethal castration-resistant PCa. Combination therapy approaches represent a promising strategy for the treatment of advanced disease. Here, we explore a therapeutic strategy for PCa based on the ability of shRNAs/siRNAs to function essentially as miRNAs and, via seed sequence complementarity, induce RNA interference of numerous targets simultaneously. We developed a library that contained shRNAs with all possible seed sequence combinations to identify those ones that most potently reduce cell growth and viability when expressed in PCa cells. Validation of some of these RNAi sequences indicated that the toxic effect is associated with seed sequence complementarity to the 3′ UTR of AR coregulatory and essential genes. In fact, expression of siRNAs containing the identified toxic seed sequences led to global inhibition of AR-mediated gene expression and reduced expression of cell-cycle genes. When tested in mice, the toxic shRNAs also inhibited castration-resistant PCa and exhibited therapeutic efficacy in pre-established tumors. Our findings highlight RNAi of androgen signaling networks as a promising therapeutic strategy for PCa.

Immunotherapy has revolutionized the landscape of cancer treatment. However, single immunotherapy only works well in a small subset of patients. Combined immunotherapy with antitumor synergism holds considerable potential to boost the therapeutic outcome. Nevertheless, the synergistic, additive or antagonistic antitumor effects of combined immunotherapies have been rarely explored. Herein, we established a novel combined cancer treatment modality by synergizing p21-activated kinase 4 (PAK4) silencing with immunogenic phototherapy in engineered extracellular vesicles (EVs) that were fabricated by coating M1 macrophage-derived EVs on the surface of the nano-complex cores assembled with siRNA against PAK4 and a photoactivatable polyethyleneimine. The engineered EVs induced potent PAK4 silencing and robust immunogenic phototherapy, thus contributing to effective antitumor effects in vitro and in vivo. Moreover, the antitumor synergism of the combined treatment was quantitatively determined by the CompuSyn method. The combination index (CI) and isobologram results confirmed that there was an antitumor synergism for the combined treatment. Furthermore, the dose reduction index (DRI) showed favorable dose reduction, revealing lower toxicity and higher biocompatibility of the engineered EVs. Collectively, the study presents a synergistically potentiated cancer treatment modality by combining PAK4 silencing with immunogenic phototherapy in engineered EVs, which is promising for boosting the therapeutic outcome of cancer immunotherapy.

Ribosomal proteins (RPs) not only play an important role in protein synthesis but also have extraribosomal functions. In this study, the entire cDNA sequence of the RPL10a gene of Macrobrachium nipponense (MnRPL10a) was cloned by RACE-PCR. The obtained complete cDNA sequence of the MnRPL10a gene was 885 bp encoding 217 amino acids (AAs). The expression pattern of the MnRPL10a gene was analysed by quantitative real-time fluorescence PCR (qPCR), and its mRNA location was determined by fluorescence in situ hybridization (FISH). The results showed that during development, the highest expression of MnRPL10a in the fertilized ovum. Among tissues, it was mostly expressed in the ovary, and its highest expression was found in stage V ovaries. MnRPL10a mRNA was mainly distributed in the cytoplasm of oogonia and oocytes. The role of the MnRPL10a gene in ovarian development was verified by RNA interference (RNAi) and ovarian explant incubation with 5-hydroxytryptamine (5-HT) and dopamine (DA) experiments. The results indicated that the expression of the genes that positively regulate ovarian development, including vitellogenin (Vg), vitellogenin receptor (Vgr), cell cycle protein B (CyclinB) and cell division cyclin 2 (cdc2), decreased, while the expression of the genes that negatively regulate ovarian development, including Wee1 kinase (Wee1) and OTU domain-containing ubiquitin aldehyde-binding protein B (OTUB), increased with the decrease in MnRPL10a expression after RNAi and DA incubation. However, 5-HT incubation showed the opposite results. In summary, the MnRPL10a gene may regulate the development of the ovary in M. nipponense by affecting vitellin synthesis, oocyte meiosis and the oestrogen signalling pathway.

Termites are global pest for man-made structures, and in some parts of the world, they infest agricultural crops as well. Termite control relies predominately on chemical insecticides, but the negative impacts of synthetic termiticides on the environment limit their applications. Entomopathogenic fungi (EPF) are considered an environmentally friendly alternative. Nevertheless, termites have developed various defensive behavioral strategies to circumvent EPF. In this review, we summarize (1) the use of EPF as a biological control agent and strategies/defenses of termites that minimize the effectiveness of EPF, (2) the potential of integrating entomopathogenic fungi with RNA interference (RNAi) as an effective termite control strategy, (3) discussed nanoparticles (silver, chitosan, titanium, etc) which are biosynthesized from different EPF. Nanoparticles have the ability to deliver RNAi triggers (dsRNA/siRNA), so we proposed the synthesis of EPF-based nanoparticles and dsRNA/siRNA complex as potential strategy to control termite pests.

PDHα1 gene encodes catalytic subunit PDHE1α of pyruvate dehydrogenase (PDH) complex. Based on previous studies, it is hypothesized that inhibition of PDH activity prevents the entry of glycolytic pyruvate into TCA cycle, while promotes fatty acid oxidation and reduces liver triglyceride (TG) level, thereby alleviating nonalcoholic fatty liver disease (NAFLD). In this study, an in vitro model for NAFLD was established with medical fat emulsion; the most effective siRNA for PDHα1 gene was screened out by qPCR technology; the alterations in metabolism of glucose and lipid, and structure & function of mitochondria in the NAFLD cells were primarily evaluated after transfecting PDHα1 siRNA. As the results showed, after inhibiting the expression of PDHα1 gene, glucose level in culture medium was time-dependently increased, and LDH activity in the cells was moderately elevated after 24 h of transfection and then returned to the normal level after 48 h; intracellular TG level was decreased while LPS activity was increased in a time-dependent manner; no significant change in mitochondrial structure was observed with or without siRNA transfection, and ATP content was obviously reduced after 24 h of transfection, followed by restoration after 48 h. It can be concluded that inhibiting PDHα1 gene in fatty liver cells enhances lipid degradation, and represses the utilization of glucose to an extent, thus reducing TG level without impacting energy generation required for cell survival.

A gene-silencing platform (miQURE) has been developed and successfully used to deliver therapeutic microRNA (miRNA) to the brain, reducing levels of neurodegenerative disease-causing proteins/RNAs via RNA interference and improving the disease phenotype in animal models. This study evaluates the use of miQURE technology to deliver therapeutic miRNA for liver-specific indications. Angiopoietin-like 3 (ANGPTL3) was selected as the target mRNA because it is produced in the liver and because loss-of-function ANGPTL3 mutations and/or pharmacological inhibition of ANGPTL3 protein lowers lipid levels and reduces cardiovascular risk. Overall, 14 candidate miRNA constructs were tested in vitro, the most potent of which (miAngE) was further evaluated in mice. rAAV5-miAngE led to dose-dependent (≤−77%) decreases in Angptl3 mRNA in WT mice with ≤−90% reductions in plasma ANGPTL3 protein. In dyslipidemic APOE∗3-Leiden.CETP mice, AAV5-miAngE significantly reduced cholesterol and triglyceride levels vs. vehicle and scrambled (miSCR) controls when administrated alone, with greater reductions when co-administered with lipid-lowering therapy (atorvastatin). A significant decrease in total atherosclerotic lesion area (−58% vs. miSCR) was observed in AAV5-miAngE-treated dyslipidemic mice, which corresponded with the maintenance of a non-diseased plaque phenotype and reduced lesion severity. These results support the development of this technology for liver-directed indications.

Small interfering RNA (siRNA) and short hairpin RNA (shRNA) are widely used as RNA interference (RNAi) reagents. Recently, truncated shRNAs that trigger RNAi in a Dicer-independent manner have been developed. We generated a novel class of RNAi reagent, designated enforced strand bias (ESB) RNA, in which an siRNA duplex was chemically bridged between the 3′ terminal overhang region of the guide strand and the 5′ terminal nucleotide of the passenger strand. ESB RNA, which is chemically bridged at the 2′ positions of ribose (2′-2′ ESB RNA), functions in a Dicer-independent manner and was highly effective at triggering RNAi without the passenger strand-derived off-target effect. In addition, the 2′-2′ ESB RNA exhibited a unique target sequence preference that differs from siRNA and silenced target sequences that could not be effectively suppressed by siRNA. Our results indicate that ESB RNA has the potential to be an effective RNAi reagent even when the target sequence is not suitable for siRNA.

Exosome is an excellent vesicle for in vivo delivery of therapeutics, including RNAi and chemical drugs. The extremely high efficiency in cancer regression can partly be attributed to its fusion mechanism in delivering therapeutics to cytosol without endosome trapping. However, being composed of a lipid-bilayer membrane without specific recognition capacity for aimed-cells, the entry into nonspecific cells can lead to potential side-effects and toxicity. Applying engineering approaches for targeting-capacity to deliver therapeutics to specific cells is desirable. Techniques with chemical modification in vitro and genetic engineering in cells have been reported to decorate exosomes with targeting ligands. RNA nanoparticles have been used to harbor tumor-specific ligands displayed on exosome surface. The negative charge reduces nonspecific binding to vital cells with negatively charged lipid-membrane due to the electrostatic repulsion, thus lowering the side-effect and toxicity. In this review, we focus on the uniqueness of RNA nanoparticles for exosome surface display of chemical ligands, small peptides or RNA aptamers, for specific cancer targeting to deliver anticancer therapeutics, highlighting recent advances in targeted delivery of siRNA and miRNA that overcomes the previous RNAi delivery roadblocks. Proper understanding of exosome engineering with RNA nanotechnology promises efficient therapies for a wide range of cancer subtypes.

Platinum-based chemotherapy resistance is a key factor of poor prognosis and recurrence in hepatocellular carcinoma (HCC). Herein, RNAseq analysis revealed that elevated tubulin folding cofactor E (TBCE) expression is associated with platinum-based chemotherapy resistance. High expression of TBCE contributes to worse prognoses and earlier recurrence among liver cancer patients. Mechanistically, TBCE silencing significantly affects cytoskeleton rearrangement, which in turn increases cisplatin-induced cycle arrest and apoptosis. To develop these findings into potential therapeutic drugs, endosomal pH-responsive nanoparticles (NPs) were developed to simultaneously encapsulate TBCE siRNA and cisplatin (DDP) to reverse this phenomena. NPs (siTBCE + DDP) concurrently silenced TBCE expression, increased cell sensitivity to platinum treatment, and subsequently resulted in superior anti-tumor effects both in vitro and in vivo in orthotopic and patient-derived xenograft (PDX) models. Taken together, NP-mediated delivery and the co-treatment of siTBCE + DDP proved to be effective in reversing chemotherapy resistance of DDP in multiple tumor models.

In insects, ecdysteroids are synthesized by genes of the Halloween family and play important roles in several key developmental events, including molting and metamorphosis. However, the roles of these genes in Agasicles hygrophila are still largely unknown. In this study, the expression patterns of the two Halloween genes AhCYP307A2 and AhCYP314A1 were determined by quantitative PCR (qPCR) at different developmental stages. Moreover, the functions of these two genes were explored using RNA interference (RNAi), and ovarian development was observed by dissecting the ovaries of A. hygrophila females. The qPCR results showed that AhCYP307A2 and AhCYP314A1 were highly expressed in last instar larvae and in adult females. In addition, AhCYP307A2 was also highly expressed in eggs and pupae but was markedly lower than in third-instar larvae and females. The RNAi results showed that the injection of dsAhCYP307A2 or dsAhCYP314A1 markedly inhibited their expression and the transcription levels of three related AhVgs. Knockdown of AhCYP307A2 or AhCYP314A1 significantly inhibited larval molting, impaired last instar larva–pupa–adult transition, delayed ovarian development, and stopped egg production (i.e., no eggs were laid). These results indicate that AhCYP307A2 and AhCYP314A1 play important regulatory roles in last instar larva–pupa–adult transition and reproduction in A. hygrophila.

As intracellular fatty acid (FA) carriers, FA-binding proteins (FABPs) widely participate in the absorption, transport, and metabolism of FAs. It is a key protein in insect lipid metabolism and plays an important role in various physiological activities of insects. An FABP gene (HvFABP) was cloned from the transcriptional library of Heortia vitessoides Moore (Lepidoptera: Crambidae), and its expression patterns were determined using reverse transcription quantitative PCR (RT-qPCR). Stage- and tissue-specific expression profiles indicated that HvFABP highly expressed from prepupal to adult stages and in larval midgut and adult wings. HvFABP expression may be induced through starvation, mRNA expression was downregulated at 24 and 48 h and upregulated at 72 h after starvation. Furthermore, 20-hydroxyecdysone can induce the upregulation of its expression. RNA interference-mediated silencing of HvFABP significantly inhibited HvFABP expression, resulting in delayed development, abnormal molting or lethal phenotypes, and a significantly reduced survival rate. These results indicate that HvFABP plays a key role in the molting of H. vitessoides.

The initiation of sex differentiation in insects is regulated by primary sex determination signals. In the Medfly Ceratitis capitata and other Tephritids, Maleness-on-the-Y (MoY) is the master gene for male sex determination. However, the primary signal in Zeugodacus cucurbitae (Coquillett), a very destructive Tephritid pest across the world, remains ambiguous. In this study, we have isolated and characterized the Medfly MoY homolog in Z. cucurbitae, ZcMoY. ZcMOY protein shows high sequence conservation to its homologs in Bactrocera species. ZcMoY transcription begins and peaks at very early embryonic stages and then becomes undetectable except the testes and heads of day 1 male adults. Silencing ZcMoY in early embryos by RNAi causes abnormal external genitalia and interior reproductive organs, giving rise to intersexes and feminization of XY individuals. The expression pattern and knockdown phenotypes of ZcMoY indicate that ZcMoY plays a key role in regulating sex determination of Z. cucurbitae males. Our findings will help the understanding of sex determination in Z. cucurbitae and facilitate the development of genetic sexing strains in its biological control.

The western flower thrips (WFT; Frankliniella occidentalis) is a mesophyll cell feeder that damages many crops. Management of WFT is complex due to factors such as high fecundity, short reproduction time, ability to feed on a broad range of host plants, and broad pesticide resistance. These challenges have driven research into developing alternative pest control approaches for WFT. This study analyzed the feasibility of a biological control-based strategy to manage WFT using RNA interference (RNAi)-mediated silencing of WTF endogenous genes. For the delivery of RNAi, we developed transgenic tomato lines expressing double-stranded RNA (dsRNA) of coatomer protein subunit epsilon (CopE) and Toll-like receptor 6 (TLR6) from WFT. These genes are involved in critical biological processes of WFT, and their dsRNA can be lethal to these insects when ingested orally. Adult WFT that fed on the transgenic dsRNA-expressing tomato flower stalk showed increased mortality compared with insects that fed on wild-type samples. In addition, WFT that fed on TLR6 and CopE transgenic tomato RNAi lines showed reduced levels of endogenous CopE and TLR6 transcripts, suggesting that their mortality was likely due to RNAi-mediated silencing of these genes. Thus, our findings demonstrate that transgenic tomato plants expressing dsRNA of TLR6 and CopE can be lethal to F. occidentalis, suggesting that these genes may be deployed to control insecticide-resistant WFT.

Osteosarcoma is the most common primary malignant bone tumor with a high risk of metastasis and recurrence. Metabolic reprogramming is a hallmark of osteosarcoma and other cancers and is associated with genetic and epigenetic alterations. RUNX2 is an important transcription factor for osteoblastic differentiation, and aberrant expression of the gene contributes to the development and progression of osteosarcoma. To identify the effects of RUNX2 silencing on transcriptomic and metabolomic profiles in osteosarcomas, we generated SJSA-1 osteosarcoma cells stably expressing RUNX2 shRNA and SJSA-1 cells stably expressing scramble shRNA and analyzed transcriptome and metabolome profiles in the two cell types using Illumina NovaSeq 6000 and ultrahigh-performance liquid chromatography coupled with time-of-flight mass spectrometry, respectively. The datasets can be used by researchers to identify novel targets of RUNX2 and elucidate the role and underlying mechanism of RUNX2 in osteosarcoma pathogenesis and metabolic reprogramming.

Abstract The spread of HIV is on the rise and has become a global issue, especially for underdeveloped and developing countries. This is due to the fact that HIV majorly occurs asymptomatically and is implausible for early diagnosis. Recent advances in research and science have enabled the investigation of a new potential treatment involving gene-based therapy, known as RNA interference (RNAi) that will direct gene silencing and further compensate for natural variants and viral mutants. Several types of small regulatory RNA are discussed in this present study, including microRNA (miRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA). This paper examines the mechanism of RNAi as a viable HIV therapy, using a minimum of four shRNAs to target both dispensable host components (CCR5) and viral genes (Gag, Env, Tat, Pol I, Pol II and Vif). Moreover, a multiplexed mechanism of shRNAs and miRNA is known to be effective in preventing viral escape due to mutation as the miRNA develops a general polycistronic platform for the expression of a large amount of shRNA-miRs. Several administration methods as well as the advantages of this RNAi treatment are also discussed in this study. The administration methods include (1) ex vivo delivery with the help of viral vectors, nanoparticles, and electroporation, (2) nonspecific in vivo delivery using non-viral carriers including liposomes, dendrimers and aptamers, as well as (3) targeted delivery that uses antibodies, modified nanoparticles, nucleic acid aptamers, and tissue-specific serotypes of AAV. Moreover, the advantages of this treatment are related to the effectiveness in silencing the HIV gene, which is more compatible compared to other gene therapy treatments, such as ZFN, TALEN, and CRISPR/Cas9.

Effective systemic delivery of small interfering RNAs (siRNAs) to tissues other than liver remains a challenge. siRNAs are small (∼15 kDa) and therefore rapidly cleared by the kidneys, resulting in limited blood residence times and tissue exposure. Current strategies to improve the unfavorable pharmacokinetic (PK) properties of siRNAs rely on enhancing binding to serum proteins through extensive phosphorothioate modifications or by conjugation of targeting ligands. Here, we describe an alternative strategy for enhancing blood and tissue PK based on dynamic modulation of the overall size of the siRNA. We engineered a high-affinity universal oligonucleotide anchor conjugated to a high-molecular-weight moiety, which binds to the 3′ end of the guide strand of an asymmetric siRNA. Data showed a strong correlation between the size of the PK-modifying anchor and clearance kinetics. Large 40-kDa PK-modifying anchors reduced renal clearance by ∼23-fold and improved tissue exposure area under the curve (AUC) by ∼26-fold, resulting in increased extrahepatic tissue retention (∼3- to 5-fold). Furthermore, PK-modifying oligonucleotide anchors allowed for straightforward and versatile modulation of blood residence times and biodistribution of a panel of chemically distinct ligands. The effects were more pronounced for conjugates with low lipophilicity (e.g., N-Acetylgalactosamine [GalNAc]), where significant improvement in uptake by hepatocytes and dose-dependent silencing in the liver was observed.

Dominant centronuclear myopathy (CNM) is a rare form of congenital myopathy associated with a wide clinical spectrum, from severe neonatal to milder adult forms. There is no available treatment for this disease due to heterozygous mutations in the DNM2 gene encoding Dynamin 2 (DNM2). Dominant DNM2 mutations also cause rare forms of Charcot-Marie-Tooth disease and hereditary spastic paraplegia, and deleterious DNM2 overexpression was noticed in several diseases. The proof of concept for therapy by allele-specific RNA interference devoted to silence the mutated mRNA without affecting the normal allele was previously achieved in a mouse model and patient-derived cells, both expressing the most frequent DNM2 mutation in CNM. In order to have versatile small interfering RNAs (siRNAs) usable regardless of the mutation, we have developed allele-specific siRNAs against two non-pathogenic single-nucleotide polymorphisms (SNPs) frequently heterozygous in the population. In addition, allele-specific siRNAs against the p.S619L DNM2 mutation, a mutation frequently associated with severe neonatal cases, were developed. The beneficial effects of these new siRNAs are reported for a panel of defects occurring in patient-derived cell lines. The development of these new molecules allows targeting the large majority of the patients harboring DNM2 mutations or overexpression by only a few siRNAs.

Retinal gene therapy using RNA interference (RNAi) to silence targeted genes requires both efficacy and safety. Short hairpin RNAs (shRNAs) are useful for RNAi, but high expression levels and activity from the co-delivered passenger strand may cause undesirable cellular responses. Ago2-dependent shRNAs (agshRNAs) produce no passenger strand activity. To enhance efficacy and to investigate improvements in safety, we have generated VEGFA-targeting agshRNAs and microRNA (miRNA)-embedded agshRNAs (miR-agshRNAs) and inserted these RNAi effectors in Pol II/III-driven expression cassettes and lentiviral vectors (LVs). Compared with corresponding shRNAs, agshRNAs and miR-agshRNAs increased specificity and safety, while retaining a high knockdown efficacy and abolishing passenger strand activity. The agshRNAs also caused significantly smaller reductions in cell viability and reduced competition with the processing of endogenous miR21 compared with their shRNA counterparts. RNA sequencing (RNA-seq) analysis of LV-transduced ARPE19 cells revealed that expression of shRNAs in general leads to more changes in gene expression levels compared with their agshRNA counterparts and activation of immune-related pathways. In mice, subretinal delivery of LVs encoding tissue-specific miR-agshRNAs resulted in retinal pigment epithelium (RPE)-restricted expression and significant knockdown of Vegfa in transduced RPE cells. Collectively, our data suggest that agshRNAs and miR-agshRNA possess important advantages over shRNAs, thereby posing a clinically relevant approach with respect to efficacy, specificity, and safety.

The Notch signaling pathway plays important roles in cell proliferation, differentiation and apoptosis. To explore the regulatory mechanism of the Notch signaling pathway in the growth and development of crustaceans, notch1 was cloned from Exopalaemon carinicauda by 5′/3′ RACE and its roles in growth and development were studied by qPCR, RNA interference and in situ hybridization. The results showed that the full-length cDNA of notch1 in E. carinicauda was 7868 bp in length, encoding a total of 2504 amino acids, with a predicted molecular weight of 650.7 kDa and a theoretical isoelectric point of 5.38. Notch1 was expressed in all tested tissues, with the highest expression in the ovary. The expression of notch1 in the RNA interference group was significantly lower than that in the control group between 12 and 48 h post-notch1 interference. The expression levels of key genes in the Notch signaling pathway, including jagged, delta and strawberry notch1 (sbno1); genes related to growth and development including chh, gih and mih; and genes related to ovary development including vitellogenin and cathepsin L, decreased significantly between 12 and 24 h post-notch1 interference and were significantly lower than that in the control. In situ hybridization showed that notch1 was highly expressed on both follicles and the cell membrane. The results showed that notch1 was involved in the growth and development of E. carinicauda, especially in the development of ovaries. This study elucidates growth and development mechanism of crustaceans and provides a theoretical basis for the molecular breeding of E. carinicauda.

This systematic review aimed to shed light on the trend of current clinical trials of non-coding RNA (ncRNA)-based therapeutics for malignant diseases. We conducted a database search for published literature and ongoing clinical trials using PubMed, clinicaltrials.gov, and University Medical Information Network (UMIN) clinical trial registry. To ensure that our review was based on up-to-date clinical trials, we limited our search to literature published within the last five years (January 2017–September 2022). Furthermore, due to the “clinical” nature of our review, we focused only on studies involving human participants. Among ncRNAs, microRNAs have been extensively explored in observational studies of malignant diseases as potential diagnostic markers and prognostic predictors, as well as for their therapeutic monitoring and profiling capabilities. As therapeutic agents, microRNA or siRNA were estimated in interventional human clinical trials and showed promising outcomes; however, the number of trials was small. Evidence and ongoing clinical trials in which ncRNAs other than microRNA or siRNA have been evaluated for their potential as therapeutic agents are limited. Here, we summarized microRNA as a potential therapeutic agent in malignant diseases, but most of the current evidence suggests that it is useful as a potential biomarker. siRNA is also a promising ncRNA technique in cancer, however more data from clinical trials are warranted for clinical use.

Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins (PGs), which are involved in ovarian development and ovulation in crustaceans. In this study, complementary DNA (cDNA) of COX in Macrobrachium nipponense (MnCOX) was identified, and its function in ovarian development and ovulation was investigated by quantitative real-time PCR (qPCR), fluorescence in situ hybridization (FISH), RNA interference (RNAi) and arachidonic acid (AA), dexamethasone (DEX) and DEX+ prostaglandin E2 (PGE2) administration. The results showed that the complete cDNA of MnCOX is 3238 bp, including an 1848 bp open reading frame (ORF) that encodes 615 amino acids. The MnCOX protein conservatively possesses a membrane-binding domain, an epidermal growth factor-like (EGF-like) domain, an animal haem peroxide domain, three N-glycosylation sites and seven essential catalytic residues. The richest MnCOX transcript of nine tissues was found in the gill, followed by the ovary and intestine. In six ovarian development stages, the MnCOX transcript was the richest in the stage Ⅴ ovary. FISH showed that the MnCOX transcript was present in the cytoplasm of oocytes. MnCOX knockdown led to decreases in PGE2 content, expression levels of the vitellogenin (Vg) and vitellogenin receptor (Vgr) genes and the gonadsomatic index (GSI) of prawns. Moreover, oocyte development was delayed, and the oviposition rate and the number of eggs ovulated were reduced after MnCOX knockdown. AA administration increased the expression of MnCOX, PGE2 content and the oviposition rate of prawns. However, DEX administration decreased the expression of MnCOX, PGE2 content, oviposition rate, and the number of eggs ovulated by prawns. The addition of PGE2 to DEX injection rescued the negative effect of DEX on the ovulation of M. nipponense. This investigation illustrates that MnCOX plays an important role in regulating ovarian development and ovulation in M. nipponense by affecting the synthesis of PGs.

Ridgetail white prawn, Exopalaemon carinicauda, with high reproductive capacity can reproduce up to three or four generations a year. In exploring the genetic factors affecting the gonadal development of E. carinicauda, we cloned and studied the function of the ubiquitin-like, containing PHD and RING finger domains 1 (uhrf1) gene. The open reading frame of uhrf1 gene was 2286 bp, encoding a total of 761 amino acids. The Uhrf1 has UBL, TTD, PhD, SRA, and ring finger domains that are associated with genome methylation, histone recognition and chromatin modification. According to the phylogenetic analysis, the amino acid sequence of Ec-uhrf1 has the highest similarity with Litopenaeus vannamei uhrf1, and it is a branch of Penaeus monodon and L. vannamei. The real-time quantitative PCR showed that the expression level of uhrf1 was highest in ridgetail white prawn ovarian tissue, which was significantly higher than that of other tissues (P

The diversity of host plants is an important reason for the global spread of Hyphantria cunea. However, no studies have explored the role of the antioxidant defense system with catalase (CAT) as the core at the molecular level in the adaptation of the H. cunea to host plant secondary metabolites. Herein, the purpose is to explore how HcCAT2, highly expressed in cytisine- or coumarin-treated H. cunea larvae, mediates the adaptation of H. cunea to cytisine and coumarin, and to develop nucleic acid pesticides targeting HcCAT2. Findings revealed that H. cunea larvae treated with dsHcCAT2 alongside cytisine or coumarin exhibited significantly reduced body weight, survival rate, and expression levels of growth-related genes, energy metabolism genes, and oxidative damage regulatory genes compared to treated with cytisine or coumarin alone. HcCAT2 overexpression enhanced cell viability, lowered apoptosis rates, Ca 2+ concentrations, ROS levels, and MPTP opening, and increased mitochondrial membrane potential in cytisine or coumarin-treated SF9 cells. Encapsulation of dsHcCAT2 in chitosan (CS) improved stability and gene silencing efficacy. CS-dsHcCAT2 treatment did not significantly affect SF9 cell, Lymantria dispar larvae, and Arma chinensis nymphs. However, H. cunea larvae treated with CS-dsHcCAT2 combined with cytisine or coumarin showed significantly reduced body weight and survival compared to those receiving secondary metabolites alone. Therefore, HcCAT2 is a critical antioxidant defense gene for adaptation of H. cunea larvae to cytisine and coumarin stress, with the ability of maintaining energy metabolism homeostasis and antioxidant defense level. The constructed CS-dsHcCAT2 can be developed as a synergistic agent for plant-derived pesticides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

RNA interference (RNAi)-based pest control has emerged as a cutting-edge and highly promising approach in pest control, especially for insect pests, due to its advantages of reduced environmental risk, degradability, and good selectivity. This study provides a bibliometric analysis of RNAi-based pest control, evaluating the global scientific output in this field from the Web of Science Core Collection (WoSCC) and PubMed. From 2007, when the first RNAi-based Arthropod pest control strategy suited for field application was published, to August 2024, 722 English research articles were identified, focusing only on dsRNA delivery modes including feeding, soaking, and spraying, which hold high potential for field application. Articles examining gene function and potential targets by dsRNA injection were excluded. The 722 eligible articles were published in 132 journals by 3112 authors from 563 institutions in fifty countries. Over these 17 years, the number of publications on RNAi-based pest control has shown a trend of accelerating growth. PEST MANAGEMENT SCIENCE published the most articles, followed by PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY, SCIENTIFIC REPORTS. China produced most articles, followed by the United States. However, China is significantly behind the United States in developing commercial products in this field. Hot target insects in RNAi-based pest control research included Bemisia tabaci, Helicoverpa armigera, Aphis gossypii Glover, Leptinotarsa decemlineata, and Diabrotica virgifera virgifera. Frequently studied target genes included vATPaseA, CHS1, SNF7, EcR and β-actin, ect. In recent years, various advanced technologies for dsRNA delivery have been developed and utilized in RNAi-based pest control system, including nanoparticle-enabled, symbiont-mediated, and plant-mediated deliveries. This study represents the first comprehensive analysis based on bibliometric methods, aiming to investigate the forefront hotspots and research trends of RNAi-based pest control, providing valuable references for researchers and developers in this field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Nemo-like kinases (NLKs) integrate multiple signaling pathways and exhibit functional diversity in developmental processes, including the bone morphogenetic protein (BMP) pathway. However, their roles in insect wing development, particularly in hemimetabolous insects like the brown planthopper (Nilaparvata lugens), remain poorly understood. Here, we investigated the role of Nlnemo (Nlnmo), an NLK, in the wing development of N. lugens. We cloned and characterized Nlnmo and found it highly conserved across insect species. Expression analysis revealed higher Nlnmo levels in brachypterous compared to macropterous strains, particularly in wing buds. RNA interference (RNAi) of Nlnmo led to enlarged wing and thickened veins, indicating its inhibitory role in wing development. Further analysis revealed that Nlnmo suppresses BMP signaling by downregulating Nlmad1 and Nldpp. Dual knockdown of Nlmad1 and Nlnmo demonstrated that Nlnmo mitigates Nlmad1-mediated effects on wing development. These findings establish Nlnmo function as a key suppressor of wing development in N. lugens via BMP signaling inhibition through Nlmad1. This study deepens our understanding of the molecular mechanisms underlying wing development in in N. lugens and highlights potential pest management strategies by targeting migration-related traits., Competing Interests: Declaration of competing interest All authors have read and approved the contents of the manuscript. The authors declare no conflicts of interest, including no relevant financial interests, relationships, or affiliations pertaining to the subject matter of the manuscript., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Double-stranded RNA (dsRNA) mediated RNA interference (RNAi) is a tool in functional gene study and pest control. However, RNAi efficiency in Lepidoptera is low compared to the RNAi sensitive Coleoptera. Previous studies on RNAi in the silkworm Bombyx mori, the lepidopteran model insect, were performed by injection only. Successful oral RNAi in the silkworm has never been reported yet. This study aims to develop a successful oral dsRNA delivery method to the silkworm larvae. Chitosan is an economical and biodegradable polymer. Chitosan/dsRNA nanoparticles were prepared by self-assembly. These nanoparticles were found to be stable when incubated in the midgut juice of the silkworm larvae, whereas naked dsRNA underwent complete degradation. Chitosan/dsRNA nanoparticles targeting various immune genes in oral administration to the silkworm larvae mediated significant knockdown of gene transcript. This silencing effect resulted in smaller larvae and cocoons when the silkworms were fed with Chitosan/dsRNA nanoparticles targeting BmToll9-2 gene, indicating that immune genes might be used as targets in pest control. Optimization of the chitosan/dsRNA nanoparticles maintained an RNAi effect from 3-5 days. The efficient RNAi was due to the persistence of nanoparticle-shielded dsRNA in the larvae. The above findings contribute to the first oral RNAi report in the silkworm, which facilitates the application of RNAi in insects and oral RNAi in pest control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Rheumatoid arthritis (RA) is an autoimmune disease that has a complex pathogenesis and remains tough to treat. The clinical treatments with e.g. methotrexate (MTX) and TNF-α antibodies show fractional responses and lessen the symptoms only to a certain extent. Here, we developed inflammation-targeted vesicles codelivering methotrexate and TNF-α small interfering RNA (siTNFα) (ITV-MT) for effective ablation of collagen-induced arthritis (CIA) in mice. ITV-MT with tetra-mannose ligand and high loading of MTX (17.1 wt%) and siTNFα (9.0 wt%) displayed a small and uniform size (53 nm) and augmented uptake by inflammatory macrophages leading to superior regulation of macrophage phenotype from M1 to M2 in vitro compared to monotherapies. The intravenous injection of ITV-MT revealed clearly enhanced accretion in the inflamed joints. Interestingly, ITV-MT effectively repolarized M1 macrophages to M2 type, markedly reduced proinflammatory cytokine levels, and significantly attenuated symptoms including joint swelling, arthritis scores and bone damage in the CIA mouse models, by concurrently downregulating both adenosine and TNF-α pathways. This study highlights inflammation-targeted vesicles codelivering methotrexate and TNFα siRNA as a potential strategy to improved RA treatment. STATEMENT OF SIGNIFICANCE: Rheumatoid arthritis (RA) is regarded as an incurable disease, often referred to as an "incurable cancer". Current therapies, such as methotrexate (MTX) and anti-TNFα monoclonal antibodies, exhibit limited efficacy and severe adverse effects. The distinct physiochemical properties of MTX and siTNFα hinder their codelivery to RA joints and inflammatory cells. Here, we engineered inflammation-targeted vesicles (ITV-MT) for the codelivery of MTX and siTNFα to enhance therapeutic outcomes. Our findings reveal that ITV-MT significantly improves the drug uptake by macrophages, facilitating repolarization from M1 to M2 phenotypes. In CIA models, ITV-MT effectively downregulated proinflammatory cytokines while upregulating anti-inflammatory cytokines in RA joints, inhibited inflammatory cell infiltration in the synovium and protected against bone erosion. This study highlights that inflammation-targeted co-delivery of small molecular anti-RA agents and RNAi therapeutics may offer a compelling alternative to existing RA treatments, representing a promising strategy for RA treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Acta Materialia Inc. Published by Elsevier Inc. All rights reserved.)

Amphitetranychus viennensis, a destructive pest mite of fruit plants in Europe and Asia, poses a serious challenge due to its adaptability and resistance to multiple acaricides. RNA interference (RNAi)-based technologies offer a promising alternative to address this emerging issue. In this study, we screened for candidate genes that can be targeted for spray-induced gene silencing (SIGS). Suppression of AvSrp54k, AveIF4A-1, AvHel31B, AvCOPB2 and AvProsbeta5 led to a significantly higher mortality and caused minor damages to leaf discs in comparison to the controls. Among them, AvCOPB2 and AvProsbeta5 were the best candidates with the highest mortality (>95 %) and minimal leaf damages (<13 %). Given that LdProsbeta5 is the active ingredient of the first sprayable dsRNA-based biopesticide, Ledprona, against the Colorado Potato Beetle, Leptinotarsa decemlineata, we examined the suitability of AvProsbeta5 in managing A. viennensis. In comparison to the control, A. viennensis population was suppressed by >95 % at day-17, and the plant defoliation rate decreased to 0 at day-24. Our combined results not only provide two viable molecular targets for sprayable dsRNA-based biopesticides, but also confirm the practical implications of SIGS in managing A. viennensis, one of the most destructive arthropod pests in orchards and ornamental plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Delivering double-stranded RNA (dsRNA) in shrimp is challenging due to the lack of an effective carrier system. This study optimized chitosan nanoparticles (CNs) from two sources-α-chitosan from shrimp and β-chitosan from squid-to encapsulate antiviral dsRNA for oral administration via shrimp feed. Using response surface methodology (RSM), formulations were refined for encapsulation efficiency, particle size, polydispersity index, and zeta potential. Both types of CNs demonstrated high encapsulation efficiency (>95 %), small sizes (<300 nm), and stable zeta potential (>20 mV). Shrimp-derived CNs provided superior RNase protection and controlled release, while squid-derived CNs showed a burst release. Incorporated into feed, both types of CNs remained stable for over a month. Shrimp-derived CNs offered greater dsRNA protection (>70 %) and improved efficacy against white spot syndrome virus (WSSV), significantly reducing mortality. These results position shrimp-derived CNs as promising dsRNA carriers for combating WSSV in shrimp aquaculture., Competing Interests: Declaration of competing interest Nuttawee Niamsiri reports financial support was provided by Mahidol University (Fundamental Fund: fiscal year 2023 by National Science Research and Innovation Fund (NSRF)). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Seminal fluid proteins (SFPs) in insect males are critical for reproduction, being transported into female ovary. In Nilaparvata lugens, a significant rice pest, seven spermary-specific carboxylesterases (CarEs) were found abundantly in SFPs, with over-expression in males of an imidacloprid-resistant (RES) strain compared to a susceptible (SUS) strain. This study aimed to evaluate roles of spermary-specific CarEs in N. lugens reproduction, especially in the reproduction disadvantage in RES. Sublethal doses of three insecticides significantly impacted N. lugens reproduction, concomitant with up-regulation of these CarE genes in males. When RNAi against these CarE genes in SUS males did not affect reproduction, RNAi suppression of insecticide-induced up-regulation worsened the reproductive decline caused by insecticides, indicating a reproduction benefit from the transient up-regulation induced by insecticides. However, constitutive overexpression of these CarEs in RES males did not enhance reproduction and instead imposed a fitness cost. RNAi against part CarE genes in RES males increased reproduction. Collectively, the transient up-regulation and constitutive overexpression of these spermary-expressed CarE genes had the opposite effects on N. lugens reproduction. We need to find a balance between these opposite effects in insect pest control and insecticide resistance management, especially for insecticides targeting reproduction., Competing Interests: Declaration of competing interest The authors declare no conflict of interest regarding the content of this paper. This manuscript is original and has not been submitted for publication elsewhere. All authors have consented to the submission of this manuscript for publication., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Gastrulation involves multiple, physically-coupled tissue rearrangements. During Drosophila gastrulation, posterior midgut (PMG) invagination promotes both germband extension and hindgut invagination, but whether the normal epithelial rearrangement of PMG invagination is required for morphogenesis of the connected tissues has been unclear. In steppke mutants, epithelial organization of the PMG primordium is strongly disrupted. Despite this disruption, germband extension and hindgut invagination are remarkably effective, and involve myosin network inductions known to promote their wild-type remodelling. Known tissue-autonomous signaling could explain the planar-polarized, junctional myosin networks of the germband, but pushing forces from PMG invagination have been implicated in inducing apical myosin networks of the hindgut primordium. To confirm that the wave of hindgut primordium myosin accumulations is due to mechanical effects, rather than diffusive signalling, we analyzed α-catenin RNAi embryos, in which all of the epithelial tissues initially form but then lose cell-cell adhesion, and observed strongly diminished hindgut primordium myosin accumulations. Thus, alternate mechanical changes in steppke mutants seem to circumvent the lack of normal PMG invagination to induce hindgut myosin networks and invagination. Overall, both germband extension and hindgut invagination are robust to experimental disruption of the PMG invagination, and, although the processes occur with some abnormalities in steppke mutants, there is remarkable redundancy in the multi-tissue system. Such redundancy could allow complex morphogenetic processes to change over evolutionary time., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The KRAS/ERK pathway is crucial in cancer progression and chemotherapy resistance, yet its upstream regulatory mechanism remains elusive. We identified MSI2 as a new promoter of chemotherapy resistance in cancers. MSI2 directly binds to a specific class of mature miRNAs by recognizing the 'UAG' motif and interacts with the essential effector AGO2, highlighting MSI2 as a novel regulatory factor within the miRNA pathway. Specifically, MSI2 recruits UAG-miRNA miR-30a-3p to facilitate its loading onto AGO2, efficiently inhibiting the expression of CGRRF1. Further analysis reveals that CGRRF1 functions as a new ubiquitin E3 ligase for KRAS, mediating the ubiquitination and proteasome degradation of KRAS. Consequently, a novel regulatory axis involving MSI2-AGO2/miR-30a-3p-CGRRF1 positively regulates the KRAS/ERK pathway. Remarkably, platinum-based chemotherapy drugs significantly enhance the levels of phosphorylated ERK1/2 (p-ERK1/2) in cancer cells, and the EGFR inhibitor Gefitinib also increases p-ERK1/2 levels in Gefitinib-resistant cancer cells. Combining small-molecule inhibitors targeting MSI2, such as Ro 08-2750, efficiently alleviated chemoresistance in tumor cells exposed to Platinum and Gefitinib. These findings suggest that MSI2 could be a novel therapeutic target for developing strategies to counteract cancer resistance to treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)