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Xiangyu Li,* Shixin Zhang,* Miaomiao Zhang, Ge Li, Bo Yang, Xinyue Lu, Lesheng Teng, Youxin Li, Fengying Sun School of Life Sciences, Jilin University, Changchun, People’s Republic of China*These authors contributed equally to this workCorrespondence: Fengying Sun, School of Life Sciences, Jilin University, 2699 Qianjin Street, Changchun, Jilin, 130012, People’s Republic of China, Tel/Fax +86-431-85155320, Email sunfengying@jlu.edu.cnPurpose: Effective therapy for rheumatoid arthritis (RA) keeps a challenge due to the complex pathogenesis of RA. It is not enough to completely inhibit the process of RA with any single therapy method. The purpose of the research is to compensate for the insufficiency of monotherapy using multiple treatment regimens with different mechanisms.Material and Methods: In this study, we developed a new method to synthesize mesoporous silica nanoparticles hybridized with photosensitizer PCPDTBT (HNs). Branched polyethyleneimine-folic acid (PEI-FA) could be coated on the surface of HNs through electrostatic interactions. It simultaneously blocked the hypoxia-activated prodrug tirapazamine loaded into the mesopores and binded with Mcl-1 siRNA (siMcl-1) that interfered with the expression of the anti-apoptotic protein Mcl-1. Released from the co-delivery nanoparticles (PFHNs/TM) Tirapazamine and siMcl-1 upon exposure to acidic conditions of endosomes/lysosomes in activated macrophages. Under NIR irradiation, photothermal therapy and photodynamic therapy derived from PCPDTBT, hypoxia-activated chemotherapy derived from tirapazamine, and RNAi derived from siMcl-1 were used for the combined treatment for RA by killing activated macrophages. PEI-FA-coated PFHNs/TM exhibited activated macrophage-targeting characteristics, thereby enhancing the in vitro and in vivo NIR-induced combined treatment of RA.Results: The prepared PFHNs/TM have high blood compatibility (far below 5% of hemolysis) and ideal in vitro phototherapy effect while controlling the TPZ release and binding siMcl-1. We prove that PEI-FA-coated PFHNs/TM not only protect the bound siRNA but also are selectively uptaked by activated macrophages through FA receptor-ligand-mediated endocytosis, and effectively silence the target anti-apoptotic protein by siMcl-1 transfection. In vivo, PFHNs/TM have also been revealed to be selectively enriched at the inflammatory site of RA, exhibiting NIR-induced anti-RA efficacy.Conclusion: Overall, these FA-functionalized, pH-responsive PFHNs/TM represent a promising platform for the co-delivery of chemical drugs and nucleic acids for the treatment of RA cooperating with NIR-induced phototherapy.Graphical Abstract: Keywords: rheumatoid arthritis, nano-delivery system, phototherapy, hypoxia-activated chemotherapy, RNA interference

No current RNA-targeted interference tools have been reported to simultaneously up and down-regulate different gene expressions. Here we characterized an RNA-targeted genetic regulatory strategy composed of a flap endonuclease 1 (FEN1) and specific mis-hairpin DNA probes (mis-hpDNA), to realize the orthogonal genetic regulation. By targeting mRNA, the strategy hindered the translation and silenced genes in human cells with efficiencies of ~60%. By targeting miRNA, the strategy prevented the combination of miRNA to its specific mRNA and increased this mRNA expression by about 3-folds. In combination, we simultaneously performed CXCR4 gene knock-down (~50%) and EGFR gene activation (1.5-folds) in human cells. Although the functional property can be further improved, this RNA-targeted orthogonal genetic regulating strategy is complementary to classical tools.

RNA interference (RNAi) is a major cellular mechanism regulating gene expression in which short double-stranded RNA molecules called small interfering RNA (siRNA) mediate sequence-specific mRNA degradation. RNAi technology has recently emerged as a promising therapeutic platform for the effective treatment of various diseases caused by inappropriate gene activity, such as cancer. However, the clinical translation of siRNA therapeutics has been hampered by the major hurdles associated with biological instability and limited delivery efficiency. Based on the various efforts, recent siRNA delivery strategies using cationic lipids and polymers allowed to enhance pharmacokinetics and delivery efficiency, resulting in potent and liver-targeted RNAi therapy. However, non-specific protein adsorption, high liver accumulation, and severe toxicity of cationic nanocarriers still limit the possibility of transfer of siRNA therapeutics from the laboratory to the clinic. One of the promising delivery strategies to overcome the limitations of siRNA therapeutics is carrier-free bioconjugation which is chemically modified and connected with biocompatible molecules such as lipids, peptides, antibodies, aptamers, and polymers. These molecularly engineered siRNA conjugates can be utilized for RNAi delivery to tissues beyond the liver, providing opportunities for clinical translation. This review focused on introducing the recent progress in molecularly engineered siRNA conjugates and their applications toward overcoming the limitations of siRNA for tumor-targeted delivery and therapy.

The molecular mechanisms regulating key fate decisions of hematopoietic stem cells (HSCs) remain incompletely understood. Here, we targeted global shRNA libraries to primary human hematopoietic stem and progenitor cells (HSPCs) to screen for modifiers of self-renewal and differentiation, and identified metastasis-associated 1 (MTA1) as a negative regulator of human HSPC propagation in vitro. Knockdown of MTA1 by independent shRNAs in primary human cord blood (CB) HSPCs led to a cell expansion during culture and a relative accumulation of immature CD34

PIWI proteins and their associated PIWI-interacting RNAs (piRNAs) constitute a small RNA-based adaptive immune system that restricts the deleterious activity of mobile genetic elements to protect genome integrity. Self/nonself discrimination is at the very core of successful defence and relies on complementary base-pairing in RNA-guided immunity. How the millions of piRNA sequences faithfully discriminate between self and nonself and how they adapt to novel genomic invaders remain key outstanding questions in genome biology. This review aims to introduce principles of piRNA silencing in the context of metazoan small RNA pathways. A distinct feature of piRNAs is their origin from single-stranded instead of double-stranded RNA precursors, and piRNAs require a unique set of processing factors. Novel nucleases, helicases and RNA binding proteins have been identified in piRNA biology, and while we are starting to understand some mechanisms of piRNA biogenesis and function, this diverse and prolific class of small RNAs remains full of surprises.

Insect pathogens play significant roles in the biocontrol of medical and agricultural pests. Cui et al. demonstrated that genetically modified (GM) fungi expressing host mosquito miRNAs could enhance the efficacy of the fungus by suppressing the host immune response. This opens avenues for utilisation of cross-kingdom RNAi in biocontrol.

Adaptive antiviral immunity in plants is an RNA-based mechanism in which small RNAs derived from both strands of the viral RNA are guides for an Argonaute (AGO) nuclease. The primed AGO specifically targets and silences the viral RNA. In plants this system has diversified to involve mobile small interfering RNAs (siRNAs), an amplification system involving secondary siRNAs and targeting mechanisms involving DNA methylation. Most, if not all, plant viruses encode multifunctional proteins that are suppressors of RNA silencing that may also influence the innate immune system and fine-tune the virus-host interaction. Animal viruses similarly trigger RNA silencing, although it may be masked in differentiated cells by the interferon system and by the action of the virus-encoded suppressor proteins. There is huge potential for RNA silencing to combat viral disease in crops, farm animals, and people, although there are complications associated with the various strategies for siRNA delivery including transgenesis. Alternative approaches could include using breeding or small molecule treatment to enhance the inherent antiviral capacity of infected cells.

RNA interference is triggered in plants by the exogenous application of double-stranded RNA or small interfering RNA (siRNA) to silence the expression of target genes. This approach can potentially provide insights into metabolic pathways and gene function and afford plant protection against viruses and other plant pathogens. However, the effective delivery of biomolecules such as siRNA into plant cells is difficult because of the unique barrier imposed by the plant cell wall. Here, we demonstrate that 40-nm layered double hydroxide (LDH) nanoparticles are rapidly taken up by intact Nicotiana benthamiana leaf cells and by chloroplasts, following their application via infiltration. We also describe the distribution of infiltrated LDH nanoparticles in leaves and demonstrate their translocation through the apoplast and vasculature system. Furthermore, we show that 40-nm LDH nanoparticles can greatly enhance the internalization of nucleic acids by N. benthamiana leaf cells to facilitate siRNA-mediated downregulation of targeted transgene mRNA by >70% within 1 day of exogenous application. Together, our results show that 40-nm LDH nanoparticle is an effective platform for delivery of siRNA into intact plant leaf cells.

Treatment with RNAi against HIV-1 transcripts efficiently inhibits viral replication but induces selection of escape mutants; therefore, the CCR5 coreceptor was suggested as an additional target. Blocking viral and host transcripts improved the antiviral effect. We have used short hairpin RNA (shRNA) targeting the human CCR5 (shCCR5) or the HIV-1 rev (shRev) transcripts to demonstrate distinctive properties of anti-CCR5 shRNA: shCCR5 induced more sustained protection than shRev; partial reduction in CCR5 expression substantially decreased HIV-1 infection, and shCCR5 performed better than shRev in the mixed shRNA-treated and untreated cultures. These observations indicate that CCR5 inhibitors should be conveniently included in HIV-1 gene silencing treatment schedules when only a certain cell fraction is protected to further reduce endogenous virus in a properly ART-treated HIV-1 infected individual.

Due to the rising population globally, and the demand for food, it is critical to significantly increase crop production by 2050. However, climate change estimates show that droughts and heatwaves will become more prevalent in many parts of the world, posing a severe danger to food output.Selective breeding based on genetic diversity is falling short of meeting the expanding need for food and feed. However, the advent of modern plant genetic engineering, genome editing, and synthetic biology provides precise techniques for producing crops capable of sustaining yield under stress situations.As a result, crop varieties with built-in genetic tolerance to environmental challenges are desperately needed. In the recent years, small RNA (sRNA) data has progressed to become one of the most effective approaches for the improvement of crops. So many sRNAs (18-30nt) have been found with the use of hi-tech bioinformatics and sequencing techniques which are involved in the regulation of sequence specific gene noncoding RNAs (short ncRNAs) i.e., microRNA (miRNA) and small interfering RNA (siRNA). Such research outcomes may advance our understanding of the genetic basis of adaptability of plants to various environmental challenges and the genetic variation of plant's tolerance to a number of abiotic stresses.The review article highlights current trends and advances in sRNAs' critical role in responses of plants to drought, heat, cold, and salinity, and also the potential technology that identifies the abiotic stress-regulated sRNAs, and techniques for analyzing and validating the target genes.

International audience; 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.

The "Wanlihong" Meretrix meretrix (WLH-M) clam is a new variety of this species that has a red shell and stronger Vibrio tolerance than ordinary M. meretrix (ORI-M). To investigate the molecular mechanisms responsible for the WLH-M strain's tolerance to Vibrio, we challenged clams with Vibrio parahaemolyticus and then assessed physiological indexes and conducted transcriptome analysis and RNA interference experiments. The mortality, tissue bacterial load, and hemocyte reactive oxygen species level of ORI-M were significantly higher than those of WLH-M, whereas the content and activity of lysozyme were significantly lower. Gene Ontology functional annotation analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that immune and metabolic pathways were enriched in Vibrio-challenged clams. The expressions of the heat shock protein 70 (Hsp70) and serine protease (SP) genes, which are involved in antibacterial immunity, were significantly upregulated in WLH-M but not in ORI-M, while the expression of the kynurenine 3-monooxygenase gene, a proinflammatory factor, was significantly downregulated in WLH-M. RNA interference experiments confirmed that Hsp70 and SP downregulation could result in increased mortality of WLH-M. Therefore, we speculate that Hsp70 and SP may be involved in the antibacterial immunity of WLH-M in vivo. Our data provided a valuable resource for further studies of the antibacterial mechanism of WLH-M and provided a foundation for the breeding of pathogen-resistant strains.

RNA interference (RNAi) has potential application in pest control, and selection of the specific target gene is one of the key steps in RNAi. As an important effector, the zinc finger protein (ZFP) gene has high similarity among aphid species, and may have potential use in an RNAi-based pest control strategy. This study assessed the control efficiency of an RNAi target, MPZC3H10, a CCCH-type ZFP gene, against green peach aphid.ZC3H10 amino acid sequence similarity is more than 97.71% among the five tested aphid species: Myzus persicae, Aphis citricidus, Acyrthosiphon pisum, Diuraphis noxia and Rhopalosiphum maidis. However, no homologous sequence was found in the transcriptome of their ladybeetle predator, Propylaea japonica. Spatial expression patterns revealed that MPZC3H10 showed high expression in the muscle and fat body of M. persicae. The RNAi bioassay revealed that silencing of MPZC3H10 resulted in high mortality (53.33%) in M. persicae. By contrast, there were no observed negative effects on the growth and development of P. japonica when fed on aphids treated with double-stranded RNA (dsRNA) or injected with a "high dose" of dsRNA.Targeting MPZC3H10 showed promising efficiency for green peach aphid control via artificially designed dsRNA, and was safe for the predatory ladybeetle. © 2022 Society of Chemical Industry.

Aberrant activation of interferon (IFN)-γ signaling plays a key role in several autoimmune skin diseases, including lupus erythematosus, alopecia areata, vitiligo, and lichen planus. Here, we identify fully chemically modified small interfering RNAs (siRNAs) that silence the ligand binding chain of the IFN-γ receptor (IFNGR1), for the modulation of IFN-γ signaling. Conjugating these siRNAs to docosanoic acid (DCA) enables productive delivery to all major skin cell types local to the injection site, with a single dose of injection supporting effective IFNGR1 protein reduction for at least 1 month in mice. In an ex vivo model of IFN-γ signaling, DCA-siRNA efficiently inhibits the induction of IFN-γ-inducible chemokines, CXCL9 and CXCL10, in skin biopsies from the injection site. Our data demonstrate that DCA-siRNAs can be engineered for functional gene silencing in skin and establish a path toward siRNA treatment of autoimmune skin diseases.

Double-stranded RNAs (dsRNAs) longer than 30 bp have not been considered desirable RNA interference (RNAi) triggering structures in mammalian cells as they nonspecifically activate innate immune response. However, in earlier studies, not only dsRNA length but also 5'-triphosphate moiety produced by

An optimal RNAi configuration that could restrict gene expression most efficiently was determined. This approach was also used to target PTGS and yielded higher rates of gene-editing events. Although it was characterized long ago, transgene silencing still strongly impairs transgene overexpression, and thus is a major barrier to plant crop gene-editing. The development of strategies that could prevent transgene silencing is therefore essential to the success of gene editing assays. Transgene silencing occurs via the RNA silencing process, which regulates the expression of essential genes and protects the plant from viral infections. The RNA silencing machinery thereby controls central biological processes such as growth, development, genome integrity, and stress resistance. RNA silencing is typically induced by aberrant RNA, that may lack 5' or 3' processing, or may consist in double-stranded or hairpin RNA, and involves DICER and ARGONAUTE family proteins. In this study, RNAi inducing constructs were designed in eleven different configurations and were evaluated for their capacity to induce silencing in Nicotiana spp. using transient and stable transformation assays. Using reporter genes, it was found that the overexpression of a hairpin consisting of a forward tandem inverted repeat that started with an ATG and that was not followed downstream by a transcription terminator, could downregulate gene expression most potently. Furthermore, using this method, the downregulation of the NtSGS3 gene caused a significant increase in transgene expression both in transient and stable transformation assays. This SGS3 silencing approach was also employed in gene-editing assays and caused higher rates of gene-editing events. Taken together, these findings suggested the optimal genetic configuration to cause RNA silencing and showed that this strategy may be used to restrict PTGS during gene-editing experiments.

Objective To explore the genetic changes in the progression of castration-resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC) and the reason why these cancers resist existing therapies. Methods We employed our CRPC cell line microarray and other CRPC or NEPC datasets to screen the target gene NEIL3. Lentiviral transfection and RNA interference were used to construct overexpression and knockdown cell lines. Cell and animal models of radiotherapy were established by using a medical electron linear accelerator. Flow cytometry was used to detect apoptosis or cell cycle progression. Western blot and qPCR were used to detect changes in the protein and RNA levels. Results TCGA and clinical patient datasets indicated that NEIL3 was downregulated in CRPC and NEPC cell lines, and NEIL3 was correlated with a high Gleason score but a good prognosis. Further functional studies demonstrated that NEIL3 had no effect on the proliferation and migration of PCa cells. However, cell and animal radiotherapy models revealed that NEIL3 could facilitate the radiotherapy sensitivity of PCa cells, while loss of NEIL3 activated radiotherapy resistance. Mechanistically, we found that NEIL3 negatively regulated the expression of ATR, and higher NEIL3 expression repressed the ATR/CHK1 pathway, thus regulating the cell cycle. Conclusions We demonstrated that NEIL3 may serve as a diagnostic or therapeutic target for therapy-resistant patients.

Immune-mediated diseases (IMDs) are chronic conditions that have an immune-mediated etiology. Clinically, these diseases appear to be unrelated, but pathogenic pathways have been shown to connect them. While inflammation is a common occurrence in the body, it may either stimulate a favorable immune response to protect against harmful signals or cause illness by damaging cells and tissues. Nanomedicine has tremendous promise for regulating inflammation and treating IMIDs. Various nanoparticles coated with nanotherapeutics have been recently fabricated for effective targeted delivery to inflammatory tissues. RNA interference (RNAi) offers a tremendous genetic approach, particularly if traditional treatments are ineffective against IMDs. In cells, several signaling pathways can be suppressed by using RNAi, which blocks the expression of particular messenger RNAs. Using this molecular approach, the undesirable effects of anti-inflammatory medications can be reduced. Still, there are many problems with using short-interfering RNAs (siRNAs) to treat IMDs, including poor localization of the siRNAs in target tissues, unstable gene expression, and quick removal from the blood. Nanotherapeutics have been widely used in designing siRNA-based carriers because of the restricted therapy options for IMIDs. In this review, we have discussed recent trends in the fabrication of siRNA nanodelivery systems, including lipid-based siRNA nanocarriers, liposomes, and cationic lipids, stable nucleic acid-lipid particles, polymeric-based siRNA nanocarriers, polyethylenimine (PEI)-based nanosystems, chitosan-based nanoformulations, inorganic material-based siRNA nanocarriers, and hybrid-based delivery systems. We have also introduced novel siRNA-based nanocarriers to control IMIDs, such as pulmonary inflammation, psoriasis, inflammatory bowel disease, ulcerative colitis, rheumatoid arthritis, etc. This study will pave the way for new avenues of research into the diagnosis and treatment of IMDs.

Chitin, a major component of insect cuticles, plays a critical role in insect molting and morphogenesis. Thus, coordination of chitin remodeling during insect development requires tight transcriptional control of the chitin metabolism genes involved in chitin synthesis, assembly and degradation. However, the molecular mechanism underlying transcriptional coordination of chitin metabolism genes during beetle development is not yet completely understood.We cloned the full-length cDNA encoding hormone receptor 3 (TcHR3) from Tribolium castaneum and showed a critical role of TcHR3 in modulating chitin metabolism gene expression during molting. Genome-wide transcriptome analysis of HR3-deficient old larvae using RNA sequencing analysis revealed a positive correlation between TcHR3 and transcription of chitin metabolism genes involved in chitin synthesis and degradation. In addition, HR3 overexpression significantly induced the gene promoter activity of N-acetylglucosaminidase 1 (NAG1) involved in chitin degradation and UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) involved in chitin synthesis. Chromatin immunoprecipitation analysis revealed that HR3 could directly bind to HR3-response element of NAG1 and UAP1 promoters. Finally, HR3-deficient late instar larvae and prepupae exhibited defects in larval-larval and larval-pupal molting, respectively, leading to eventual larval death because developing larvae were trapped inside the old cuticle as a result of abnormal chitin metabolism.TcHR3 is a transcriptional regulator of chitin metabolic genes for molting of T. castaneum. Controlling the molting system by TcHR3 might be a new management strategy for selective control of red flour beetle infestation. © 2022 Society of Chemical Industry.

MicroRNAs (miRNAs) are small regulatory non-coding RNAs that are involved in a variety of biological processes such as immunity, cell signaling and development by regulating gene expression. The whitefly Bemisia tabaci is a polyphagous vector that transmits many plant viruses causing economic damage to crops worldwide. In this study, we characterized and analyzed the expression of the miRNA core genes Argonaute-1 (Ago1) and Dicer-1 (Dcr1) in B. tabaci and explored the effect of their silencing on the insect's fitness.Our results showed that Ago1 and Dcr1 are differentially expressed in different tissues and developmental stages of B. tabaci. To determine the function of the miRNA pathway in B. tabaci, we silenced Ago1 and Dcr1 using specific double-stranded RNAs to the genes. RNA interference (RNAi) of Ago1 and Dcr1 decreased the expression level of the core genes and reduced the abundance of Let-7 and miR-184 miRNAs. Silencing of the miRNA pathway core gene also negatively affected the biology of B. tabaci by reducing fertility, fecundity and survival of this insect pest.Together, our results showed that silencing the miRNA pathway core genes reduced the miRNA levels followed by reduced fecundity and survival of B. tabaci, which highlighted the importance of the miRNA pathway in this insect. The miRNA core genes are attractive targets for developing an RNAi-based strategy for targeting this notorious insect pest. © 2022 Society of Chemical Industry.

Whitefly and the viruses they transmit pose a serious threat to crops globally. Recently, Jain et al. showed that BioClay-mediated double-stranded RNA (dsRNA) spray provides an eco-friendly approach to controlling whitefly. This 'transgene-free next-generation' insect-specific crop protection strategy may help to reduce the use of chemical pesticides for controlling whitefly.

Despite extensive understanding of antiviral RNAi in plants, whether and how natural variation in components of RNAi contributes to antiviral immunity remains obscure. Liu et al. recently identified novel positive and negative antiviral RNAi regulators, supporting RNAi's principal role in the dynamic virus-host coevolution in natural ecosystems.

Recent studies demonstrated that the progression and metastasis of lung cancer were associated with human antigen R (HuR), a post-transcriptional RNA-binding protein that stabilize and regulate the expression of many tumor-related genes. Although HuR was shown to affect the expressions of epithelial cadherin (E-cadherin), a tumor migration suppressor, in airway epithelial cells, esophageal squamous and colon cancer cells, direct evaluation for the effect and mechanism of HuR on the migration and invasion of lung cancer cells is not documented. In this study, HuR was knocked down via RNA interference and overexpressed using recombinant plasmid in adenocarcinomic human alveolar basal epithelial A549 cells. No apparent inhibition of cell viability was observed. HuR knocked down significantly suppressed A549 migration and invasion in scratch wound healing and transwell assays, with an increase in E-cadherin expression, while the overexpression of HuR notably facilitated A549 migration and invasion, with a decrease in E-cadherin level. In addition, immunoprecipitation study showed that HuR directly interacted with Snail, a repressor of E-cadherin, and upregulated the expression of Snail in A549 cells. These combined results suggested that the effect of HuR on A549 migration and invasion was realized by stabilizing and increasing the expression of Snail, which in-turn interfered with the expression of E-cadherin. The finding of this study revealed direct evidence that HuR affected the migration and invasion of lung cancer cells via regulating E-cadherin and Snail, providing an additional reference and mechanistic clue for further researches and therapeutic strategies in treating lung cancer.

RNA-mediated interference (RNAi) is a conserved mechanism that uses small RNAs (sRNAs) to silence gene expression. In the Caenorhabditis elegans germline, transcripts targeted by sRNAs are used as templates for sRNA amplification to propagate silencing into the next generation. Here we show that RNAi leads to heritable changes in the distribution of nascent and mature transcripts that correlate with two parallel sRNA amplification loops. The first loop, dependent on the nuclear Argonaute HRDE-1, targets nascent transcripts and reduces but does not eliminate productive transcription at the locus. The second loop, dependent on the conserved helicase ZNFX-1, targets mature transcripts and concentrates them in perinuclear condensates. ZNFX-1 interacts with sRNA-targeted transcripts that have acquired poly(UG) tails and is required to sustain pUGylation and robust sRNA amplification in the inheriting generation. By maintaining a pool of transcripts for amplification, ZNFX-1 prevents premature extinction of the RNAi response and extends silencing into the next generation.

Cuticular proteins (CPs) are known to play important roles in insect development and defence responses. The loss of CP genes can lead to changes in insect morphology and sensitivity to the external environment. In this study, we identified the AccCPR2 gene, which belongs to the CPR family (including the RR consensus motif) of CPs, and explored its function in the response of Apis cerana cerana to adverse external stresses. Our results demonstrated that AccCPR2 was highly expressed in the late pupal stage and epidermis, and the expression of AccCPR2 may be induced or inhibited under different stressors. RNA interference experiments showed that knockdown of AccCPR2 reduced the activity of antioxidant enzymes, led to the accumulation of oxidative damage and suppressed the expression of several antioxidant genes. In addition, knockdown of AccCPR2 also reduced the pesticide resistance of A. cerana cerana. The overexpression of AccCPR2 in a prokaryotic system further confirmed its role in resistance to various stresses. In summary, AccCPR2 may play pivotal roles in the normal development and environmental stress response of A. cerana cerana. This study also enriched the theoretical knowledge of the resistance biology of bees.

Mosquito-borne arboviruses, such as dengue virus, West Nile virus, Zika virus and yellow fever virus, impose a tremendous cost on the health of populations around the world. As a result, much effort has gone into the study of the impact of these viruses on human infections. Comparatively less effort, however, has been made to study the way these viruses interact with mosquitoes themselves. As ingested arboviruses infect their midgut and subsequently other tissue, the mosquito mounts a multifaceted innate immune response. RNA interference, the central intracellular antiviral defense mechanism in mosquitoes and other invertebrates can be induced and modulated through outside triggers (small RNAs) and treatments (transgenesis or viral-vector delivery). Accordingly, modulation of this facet of the mosquito’s immune system would thereby suggest a practical strategy for vector control. However, this requires a detailed understanding of mosquitoes’ endogenous small RNAs and their effects on the mosquito and viral proliferation. This paper provides an up-to-date overview of the mosquito’s immune system along with novel data describing miRNA profiles for Aedes aegypti and Culex quinquefasiatus in Grenada, West Indies.

Host-induced gene silencing (HIGS) refers to the silencing of genes in pathogens and pests by expressing homologous double-stranded RNAs (dsRNA) or artificial microRNAs (amiRNAs) in the host plant. The discovery of such trans-kingdom RNA silencing has enabled the development of RNA interference-based approaches for controlling diverse crop pathogens and pests. Although HIGS is a promising strategy, the mechanisms by which these regulatory RNAs translocate from plants to pathogens, and how they induce gene silencing in pathogens, are poorly understood. This lack of understanding has led to large variability in the efficacy of various HIGS treatments. This variability is likely due to multiple factors, such as the ability of the target pathogen or pest to take up and/or process RNA from the host, the specific genes and target sequences selected in the pathogen or pest for silencing, and where, when, and how the dsRNAs or amiRNAs are produced and translocated. In this review, we summarize what is currently known about the molecular mechanisms underlying HIGS, identify key unanswered questions, and explore strategies for improving the efficacy and reproducibility of HIGS treatments in the control of crop diseases.

Insect cytochrome P450 monooxygenases (P450s) play a crucial role in phytochemical metabolism and tolerance. Three P450 genes (TcCYP4Q3, TcCYP4Q5, and TcCYP4Q7) are associated with the response of eugenol in Tribolium castaneum. However, the responding mechanisms of these P450 genes to eugenol remain unknown.Here, spatiotemporal expression profiling revealed that TcCYP4Q3 and TcCYP4Q5 were most highly expressed in late adult, while TcCYP4Q7 was predominantly expressed in late larva; and all of these three P450 genes were mainly expressed in the fat body of larvae. Furthermore, the expressions of these three P450 genes were significantly up-regulated after exposure to eugenol, and depletion of them enhanced the susceptibility of beetles to eugenol. Interestingly, RNA interference (RNAi) against the CncC gene, a transcription factor of CncC signaling pathway associated with regulation of insect P450s in response to phytochemicals, reduced the transcripts of these three P450 genes following exposure to eugenol. Investigation of CncC signaling pathway showed that this pathway could be activated by eugenol.Altogether, the results indicate that these three P450 genes are regulated by CncC signaling pathway to participate in the susceptibility of Tribolium castaneum to phytochemicals. These findings will aid implications for the development of novel therapeutics to control pest. © 2022 Society of Chemical Industry.

Sclerotinia sclerotiorum is generally considered one of the most economically damaging pathogens in oilseed rape (Brassica napus). Breeding for Sclerotinia resistance is challenging, as no immune germplasm available in B. napus. It is desirable to develop new breeding strategies. In the present study, host-induced gene silencing (HIGS), developed based on RNA interference (RNAi), was applied to protect B. napus from S. sclerotiorum infection. Three pathogenicity genes, the endo-polygalacturonase gene (SsPG1), cellobiohydrolase gene (SsCBH), and oxaloacetate acetylhydrolase gene (SsOAH1), were chosen as HIGS targets. Co-incubation of synthesized double-stranded RNAs (dsRNAs) with S. sclerotiorum in liquid medium significantly reduced the transcript levels of the target genes. Application to plant surfaces of dsRNA targeting the three genes conferred effective protection against S. sclerotiorum. Stable transgenic B. napus plants expressing small interfering RNAs with sequence identity to SsPG1, SsCBH, and SsOAH1 were generated. HIGS transgenic B. napus prevented the expression of S. sclerotiorum target genes, slowed pathogenicity-factor accumulation, impeded fungal growth, and suppressed appressorium formation, thereby conferring resistance to S. sclerotiorum. Simultaneous silencing of SsPG1, SsCBH, and SsOAH1 by stable expression of a chimeric hairpin RNAi construct in B. napus led to enhanced protection phenotypes (with disease lesion size reduced by 36.8%–43.7%). We conclude that HIGS of pathogenic-factor genes of S. sclerotiorum is a promising strategy for controlling Sclerotinia rot in oilseed rape.

The insulin-like peptide (ILP) family is well known for regulating reproduction in invertebrates, while its role in mollusks remains largely unknown. In this study, we first isolated and characterized the ILP gene in the cuttlefish Sepiella japonica. The full-length SjILP cDNA obtained was 926 bp and encoded a precursor protein of 161 amino acids. The precursor protein consisted of a signal peptide, a B chain, a C-peptide, and an A chain. It possessed the typical features of ILP proteins, including two cleavage sites (KR) and eight conserved cysteines. To define the function of SjILP, the expression of SjILP in different tissues and ovarian development stages were analyzed using qRT-PCR. SjILP was mainly expressed in the ovary, and its gene expression correlated with ovarian development. Furthermore, silencing SjILP using RNA interference (RNAi) dramatically decreased the expression levels of four ovarian-development-related genes (vitellogenin1, vitellogenin2, cathepsin L1-like, and follistatin). These data suggest the critical role of SjILP in the regulation of ovarian development in S. japonica.

Phosphoenolpyruvate carboxylase (PEPC) is a carboxylating enzyme with important roles in plant metabolism. Most studies in C-4 plants have focused on photosynthetic PEPC, but less is known about non-photosynthetic PEPC isozymes, especially with respect to their physiological functions. In this work, we analyzed the precise roles of the sorghum (Sorghum bicolor) PPC3 isozyme by the use of knock-down lines with the SbPPC3 gene silenced (Ppc3 lines). Ppc3 plants showed reduced stomatal conductance and plant size, a delay in flowering time, and reduced seed production. In addition, silenced plants accumulated stress indicators such as Asn, citrate, malate, and sucrose in roots and showed higher citrate synthase activity, even in control conditions. Salinity further affected stomatal conductance and yield and had a deeper impact on central metabolism in silenced plants compared to wild type, more notably in roots, with Ppc3 plants showing higher nitrate reductase and NADH-glutamate synthase activity in roots and the accumulation of molecules with a higher N/C ratio. Taken together, our results show that although SbPPC3 is predominantly a root protein, its absence causes deep changes in plant physiology and metabolism in roots and leaves, negatively affecting maximal stomatal opening, growth, productivity, and stress responses in sorghum plants. The consequences of SbPPC3 silencing suggest that this protein, and maybe orthologs in other plants, could be an important target to improve plant growth, productivity, and resistance to salt stress and other stresses where non-photosynthetic PEPCs may be implicated. This research was supported by the Junta de Andalucía (P12-FQM-489 and PAI group BIO298), the Basque Government (IT932-16), and the Ministerio de Economía, Industria y Competitividad (AGL2012-35708 and AGL2016-75413-P).

Comparing the developmental mechanisms of segmentation among insects with different modes of embryogenesis provides insights on how the function of segmentation genes evolved. Functional analysis of eve by genetic mutants shows that the Drosophila pair-rule gene, even-skipped (eve), contributes to initial segmental patterning. However, eve orthologs tends to have diverse functions in other insects. To compare the evolutionary functional divergence of this gene, we evaluated eve function in a phylogenetically basal insect, the cricket Gryllus bimaculatus. To investigate the phenotypic effects of eve gene knock-out, we generated CRISPR/Cas9 system-mediated mutant strains of the cricket. CRISPR/Cas9 mutagenesis of multiple independent sites in the eve coding region revealed that eve null mutant embryos were defective in forming the gnathal, thoracic, and abdominal segments, consequently shortening the anterior-posterior axis. In contrast, the structures of the anterior and posterior ends (e.g., antenna, labrum, and cercus) formed normally. Hox gene expression in the gnathal, thoracic, and abdominal segments was detected in the mutant embryos. Overall, this study showed that Gryllus eve plays an important role in embryonic elongation and the formation of segmental boundaries in the gnathal to abdominal region of crickets. In the light of studies on other species, the eve function shown in Gryllus might be ancestral in insects.

Stigmaeopsis nanjingensis (Ma and Yuan) (Acari: Tetranychidae) is an important pest of bamboo—feeding behavior and silk production by the female adult mites is seriously harmful to bamboo leaves. Due to its small size, silking and cocooning, its management is difficult. This study discusses a fast and easy method for management of the pest by disturbing the spinning behavior. Stigmaeopsis nanjingensis is host specific and feeds only on bamboo leaves. Leaf margins of bamboo are highly hydrophobic, which makes dsRNA difficult to immerse. Hence, it is a challenge to apply the commonly used feeding method to inhibit gene expression in mites. In this study, we deliver dsRNA to interfere with the expression of fibroin by body wall permeation with a nanocarrier-based delivery system. The dsRNA/nanocarrier formulation droplets could enter the body cavity within 2 min after falling on the mite. The fibroin silencing efficiency was 75.4%, and the results of electron microscopy showed that dsRNA/nanocarrier damage the morphological structure of the silk thread. This study demonstrated the effectiveness of a nanocarrier-based percutaneous dsRNA delivery system in S. nanjingensis and its effect on the fibroin gene that influences the spinning behavior of S. nanjingensis. These findings may provide a new delivery system for RNAi-based control of spider mites that utilize protective webbing in the field.

Introduction Investigation into the action mechanisms of plant secondary metabolites against pests is a vital strategy for the development of novel promising biopesticides. Scoparone (isolated from Artemisia capillaris), a renewable plant-derived bioresource, displays potent acaricidal activities against mites, but its targets of action remain unclear. Objectives This study aimed to systematically explore the potential molecular targets of scoparone against Tetranychus cinnabarinus and provide insights to guide the future application of scoparone as an agent for the management of agricultural mite pests worldwide. Methods The mechanism and potential targets of scoparone against mites were investigated using RNA-seq analysis; RNA interference (RNAi) assays; bioassays; and [Ca2+]i, pull-down and electrophysiological recording assays. Results RNA-seq analysis identified Ca2+ signalling pathway genes, specifically 5 calmodulin (CaM1–5) genes and 1 each of L-, T-, N-type voltage-gated Ca2+ channel (VGCC) genes, as candidate target genes for scoparone against mites. Furthermore, RNAi and electrophysiological data showed that the CaM1- and L-VGCC-mediated Ca2+ signalling pathways were activated by scoparone. Interestingly, by promoting the interaction between CaM1 and the IQ motif (a consensus CaM-binding domain of L-VGCC), CaM1 markedly enhanced the activating effect of scoparone on L-VGCC. Pull-down assays further demonstrated that CaM interacted with the IQ motif, triggering L-VGCC opening. Importantly, mutation of the IQ motif significantly weakened CaM1 binding and eliminated the CaM1-mediated enhancement of scoparone-induced L-VGCC activation, indicating that the effect of scoparone was dependent on the CaM1–IQ interaction. Conclusion This study demonstrates, for the first time, that the acaricidal compound scoparone targets the interface between CaM1 and L-VGCC and activates the CaM-binding site, located in the IQ motif at the L-VGCC C-terminus. This work may contribute to the development of target-specific green acaricidal compounds based on L-VGCC.

Grapevine leafroll-associated virus 2 (GLRaV-2) is a prevalent virus associated with grapevine leafroll disease, but the molecular mechanism underlying GLRaV-2 infection is largely unclear. Here, we report that 24-kDa protein (p24), an RNA-silencing suppressor (RSS) encoded by GLRaV-2, promotes GLRaV-2 accumulation via interaction with the B3 DNA-binding domain of grapevine (Vitis vinifera) RELATED TO ABSCISIC ACID INSENSITIVE3/VIVIPAROUS1 (VvRAV1), a transcription factor belonging to the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) superfamily. Salicylic acid-inducible VvRAV1 positively regulates the grapevine pathogenesis-related protein 1 (VvPR1) gene by directly binding its promoter, indicating that VvRAV1 may function in the regulation of host basal defense responses. p24 hijacks VvRAV1 to the cytoplasm and employs the protein to sequester 21-nt double-stranded siRNA together, thereby enhancing its own RSS activity. Moreover, p24 enters the nucleus via interaction with VvRAV1 and weakens the latter’s binding affinity to the VvPR1 promoter, leading to decreased expression of VvPR1. Our results provide a mechanism by which a viral RSS interferes with both the antiviral RNA silencing and the AP2/ERF-mediated defense responses via the targeting of one specific host factor.

RNA interference (RNAi) selectively targets genes and silences their expression in vivo, causing developmental defects, mortality and altered behavior. Consequently, RNAi has emerged as a promising research area for insect pest management. However, it is not yet a viable alternative over conventional pesticides despite several theoretical advantages in safety and specificity. As a first step toward a more standardized approach, a machine learning algorithm was used to identify factors that predict trial efficacy. Current research on RNAi for pest management is highly variable and relatively unstandardized. The applied random forest model was able to reliably predict mortality ranges based on bioassay parameters with 72.6% accuracy. Response time and target gene were the most important variables in the model, followed by applied dose, double-stranded RNA (dsRNA) construct size and target species, further supported by generalized linear mixed effect modeling. Our results identified informative trends, supporting the idea that basic principles of toxicology apply to RNAi bioassays and provide initial guidelines standardizing future research similar to studies of traditional insecticides. We advocate for training that integrates genetic, organismal, and toxicological approaches to accelerate the development of RNAi as an effective tool for pest management. © 2022 The Authors. Pest Management Science published by John WileySons Ltd on behalf of Society of Chemical Industry.

MicroRNAs (miRNAs) are a class of small, mature, noncoding RNA that lead to posttranscriptional gene silencing to regulate gene expression. miRNAs are instrumental in biological processes such as cell development, cell differentiation, cell proliferation, and cell apoptosis. The miRNA-mediated gene silencing is an important part of the regulation of gene expression in many kinds of diseases. miR-155, one of the best-characterized miRNAs, has been found to be closely related to physiological and pathological processes. What is more, miR-155 can be used as a potential therapeutic target for inflammatory diseases. We analyze the articles about miR-155 for nearly five years, review the advanced study on the function of miR-155 in different inflammatory cells like T cells, B cells, DCs, and macrophages, and then summarize the biological functions of miR-155 in different inflammatory cells. The widespread involvement of miR-155 in human diseases has led to a novel therapeutic approach between Chinese and Western medicine.

Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt small interfering RNAs (siRNAs), heterochromatic siRNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs), respectively. The former small RNAs are linked to silencing of transposable elements and heterochromatic repeats, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient ‘eudicot-type’ DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 5′ phosphates and 3′ overhangs, consistent with the structural properties of their in vivo double-stranded RNA substrates. Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5, which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution.

Corneal neovascularization (CNV) is a common disease that affects the vision ability of more than 1 million people annually. Small interfering RNA (siRNA) delivery nanoparticle platforms are a promising therapeutic modality for CNV treatment. However, the efficient delivery of siRNA into cells and the effective release of siRNA from delivery vehicles in a particular cell type challenge effective RNAi clinical application for CNV suppression. This study reports the design of a novel reactive oxygen species (ROS)-responsive lipid nanoparticle for siRNA delivery into corneal lesions for enhanced RNAi as a potential CNV treatment. We demonstrated that lipid nanoparticles could efficiently deliver siRNA into human umbilical vein endothelial cells and release siRNA for enhanced gene silencing by using the upregulated ROS of CNV to promote lipid nanoparticle degradation. Moreover, the subconjunctival injection of siRNA nanocomplexes into corneal lesions effectively knocked down vascular endothelial growth factor expression and suppressed CNV formation in an alkali burn model. Thus, we believe that the strategy of using ROS-responsive lipid nanoparticles for enhanced RNAi in CNV could be further extended to a promising clinical therapeutic approach to attenuate CNV formation.

The study is aimed at investigating the effect of the FLOT2 gene on invasion and metastasis of colorectal cancer (CRC) cells and the corresponding molecular mechanism by preparing polylysine-silicon nanoparticles. Specifically, polylysine was used to modify the silica nanoparticles prepared by the emulsification method to obtain polylysine-silicon nanoparticles. The characterization of polylysine-silicon nanoparticles was completed by nanoparticle size analyzer, laser particle size potentiometer, and transmission microscope. The influence of polylysine-silicon nanoparticles on the survival rate of CRC cell line HT-29 was detected using the method of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). The FLOT2-siRNA expression vector was constructed and transfected with HT-29. The HT-29 transfected with empty plasmid was used as the negative control (NC). Western Blot (WB) and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect expression levels of FLOT2 gene and epithelial-mesenchymal transition- (EMT-) related genes. Transwell invasion assay, Transwell migration assay, and CCK8 assay were used to detect the cell invasion, migration, and proliferation. The results showed that the average particle size of polylysine-silicon nanoparticles was 30 nm, the potential was 19.65 mV, the particle size was 65.8 nm, and the dispersion coefficient was 0.103. At the same concentration, the toxicity of silicon nanoparticles to HT-29 was significantly lower than that of liposome reagent, and the transfection efficiency was 60%, higher than that of liposome reagent (40%). The mRNA level and protein expression of the FLOT2 gene in the FLOT2-siRNA group were significantly lower than those in the NC group ( P < 0.01 ). The optical density (OD) value of the NC group and the blank control (CK) group were significantly higher than that of FLOT2-siRNA cells ( P < 0.01 ). The OD value of FLOT2-siRNA cells was lower than that of NC cells at 48 h, 72 h, and 96 h ( P < 0.01 ). The mRNA levels and protein expressions of MMP2 and vimentin in the FLOT2-siRNA group were significantly lower than those in the NC group and CK group ( P < 0.01 ). The mRNA level and protein expression of the E-cadherin gene in the FLOT2-siRNA group were significantly higher than those in the NC group and CK group ( P < 0.01 ). In conclusion, an RNA interference plasmid with high transfection efficiency and low cytotoxicity was established based on nanotechnology. siRNA-mediated FLOT2 protein inhibits the invasion, migration, and proliferation of CRC cells by regulating the expression changes of EMT-related genes, which provides a scientific basis for clinical treatment of CRC.

In the context of relatively sufficient research that annotated WNT1 inducible signaling pathway protein 1 (WISP1) as a promoting factor in tumor progression of breast cancer, and identified the effects of ultrasound microbubble technology on enhancing the transfection efficiency and achieving better gene interference, this study managed to investigate the effects of ultrasound microbubble-mediated siWISP1 transfection on proliferation and metastasis of breast cancer cells. To achieve our research objectives, the expression of WISP1 in breast cancer tissues was retrieved from GEPIA website, and the viability of breast cancer cells (SK-BR-3 and MCF7) was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for ultrasound intensity screening. After the transfection of siWISP1 by ultrasound microbubble or lipofectamine 6000, the content of WISP1 secreted by cells was detected through Enzyme-linked immunosorbent assay (ELISA), and WISP1 expression in cells was determined by quantitative reverse transcription polymerase-chain reaction (qRT-PCR). Besides, the cell invasion, migration, and proliferation were evaluated by wound healing, transwell, and EdU assays, respectively. In accordance with experimental results, WISP1 was highly expressed in breast cancer tissues, and the 1 W/cm

Nucleic acid-based therapeutics that control gene expression have been steadily progressing towards achieving their full clinical potential throughout the last few decades. Rapid progress has been achieved in RNAi-based therapy by optimizing high specificity and gene silencing efficiency using chemically modified siRNAs. Since 2018, four siRNA drugs - patisiran, givosiran, lumasiran, and inclisiran, were approved by the US FDA, providing a testament to the promise of RNAi therapeutics. Despite these promising results, safe and efficient siRNA delivery at the target site remains a major obstacle for efficient siRNA-based therapeutics. In this review, we have outlined the synergistic effects of emerging dual ribose modifications, including 2',4'- and 2',5'-modifications, 5'

Immature dendritic cells (imDCs) are activated and mature to initiate an adaptive immune response, resulting in allograft rejection and transplantation failure. Myeloid differentiation factor 88 (Myd88) is a key factor in the Toll-like receptor (TLR) signaling pathway. Here, we investigated the effect of Myd88 silencing on DC function and immune response. CD34 + cells were isolated from the bone marrow of rhesus monkeys by the immunomagnetic bead method and then infected with an adenovirus expressing Myd88-specific short hairpin RNA (sh-Myd88). sh-NC (nontargeting negative control)- or sh-Myd88-infected DCs were treated with lipopolysaccharide (LPS) for another 48 h to induce DCS maturation. The maturation of DCs was identified by immunofluorescence staining for MHCII, CD80, and CD86. DC apoptosis was examined using Annexin V/PI staining. DC-related cytokine levels (IFN-γ and IL-12) were assessed by ELISA. A mixed lymphocyte reaction (MLR) was performed to test the effect of Myd88-silenced DCs on T lymphocytes in vitro. The results showed that compared with control or sh-NC-infected DCs, Myd88-silenced DCs had lower MHCII, CD80, CD86, and DC-related cytokine (IFN-γ and IL-12) levels. Myd88 did not affect the apoptosis of DCs. MLR demonstrated that Myd88 silencing could effectively block LPS-activated T cell proliferation in vitro. These data were consistent with the characteristics of tolerogenic DCs. In conclusion, our data indicated that Myd88 silencing could inhibit the maturation of imDCs and alleviate immune rejection, which provides a reference for immune tolerance in clinical liver transplantation.

Small interfering RNAs (siRNAs) produced from template double-stranded RNAs (dsRNAs) can activate the immune system in transgenic plants by detecting virus transcripts to degrade. In the present study, an RNA interference (RNAi) gene silencing mechanism was used for the development of transgenic potato plants resistant to potato virus Y (PVY), the most harmful viral disease. Three RNAi gene constructs were designed based on the coat protein (CP) and the untranslated region parts of the PVY genome, being highly conserved among all strains of the PVY viruses. Transgenic potato plants were generated using Agrobacterium containing pCAMRNAiCP, pCAMRNAiUR, and pCAMRNAiCP-UR constructs. The transgene insertions were confirmed by molecular analysis containing polymerase chain reaction (PCR) and southern blotting. The resistance of transgenic plants to PVY virus was determined using bioassay and evaluating the amount of viral RNA in plants by RT-PCR, dot blotting of PVY coating protein, and enzyme-linked immunosorbent assay (ELISA). Bioassay analysis revealed that more than 67% of transgenic potato plants were resistant to PVY compared with the non-transgenic plants, which showed viral disease symptoms. No phenotypic abnormalities were observed in transgenic plants. Out of six lines in southern blot analysis, four lines had one copy of the transgene and two lines had two copies of the target genes. No correlation was detected between the copy number of the genes and the resistance level of the plant to PVY. Transgenic lines obtained from all three constructs indicated more or less similar levels of resistance against viral infection; however, CP-UR lines exhibited relatively high resistance followed by CP and UR expressing lines, respectively. Meanwhile, some lines showed a delay in symptoms 35 days after infection which were classified as susceptible.

After a meal, excess nutrients are stored within adipose tissue as triglycerides in structures called lipid droplets. Previous genome-wide RNAi screens have identified that mRNA splicing factor genes are required for normal lipid droplet formation in Drosophila cells. We have previously shown that mRNA splicing factors called serine/arginine-rich (SR) proteins are important for triglyceride storage in the Drosophila fat body. SR proteins shuttle in and out of the nucleus with the help of proteins called Transportins (Tnpo-SR); however, whether this transport is important for SR protein-mediated regulation of lipid storage is unknown. The purpose of this study is to characterize the role of Tnpo-SR proteins in regulating lipid storage in the Drosophila fat body. Decreasing Tnpo-SR in the adult fat body resulted in an increase in triglyceride storage and consistent with this phenotype, Tnpo-SR-RNAi flies also have increased starvation resistance. In addition, the lipid accumulation in Tnpo-SR-RNAi flies is the result of increased triglyceride stored in each fat body cell and not due to increased food consumption. Interestingly, the splicing of CPT1, an enzyme important for the β-oxidation of fatty acids, is altered in Tnpo-SR-RNAi fat bodies. The isoform that produces the less catalytically active form of CPT1 accumulates in fat bodies where Tnpo-SR levels are decreased, suggesting a decrease in lipid breakdown, potentially causing the excess triglyceride storage observed in these flies. Together, these data suggest that the transport of splicing proteins in and out of the nucleus is important for proper triglyceride storage in the Drosophila fat body.

The development of long-lived immune memory cells against pathogens is critical for the success of vaccines to establish protection against future infections. However, the mechanisms governing the long-term survival of immune memory cells remain to be elucidated. In this article, we show that the maintenance mitochondrial homeostasis by autophagy is critical for restricting metabolic functions to protect IgG memory B cell survival. Knockout of mitochondrial autophagy genes, Nix and Bnip3, leads to mitochondrial accumulation and increases in oxidative phosphorylation and fatty acid synthesis, resulting in the loss of IgG+ memory B cells in mice. Inhibiting fatty acid synthesis or silencing necroptosis gene Ripk3 rescued Nix−/−Bnip3−/− IgG memory B cells, indicating that mitochondrial autophagy is important for limiting metabolic functions to prevent cell death. Our results suggest a critical role for mitochondrial autophagy in the maintenance of immunological memory by protecting the metabolic quiescence and longevity of memory B cells.

RNA interference is a mechanism of suppressing gene expression in plants, animals and fungi. This regulation mechanism involves three main enzymes, Dicers (Dcr), Argonautes (Ago) and RNA Dependent RNA Polymerases (Rdrp) allowing to produce smallRNAs. RNA interference and smallRNAs have a role in the plant-microorganisms interaction, either in a pathogenic or in a symbiotic relationships. Alternaria brassicicola is a pathogenic fungus of the Brassicaceae plants. During plant infection, it is able to transmit itself vertically and horizontally, giving advantages for new infection and dissemination. To investigate RNA interference and the presence of smallRNAs in A. brassicicola, an in silico analysis was achieved. Two DCR, 4 AGO and 3 RDRP genes were identified comforting the presence of smallRNAs in A. brassicicola. SmallRNA sequencing from wild-type strain and DCR deleted mutants allowed the identifcation of 17 miRNAs in A. brassicicola. The synthesis of these miRNAs is only weakly influenced by the inactivation of DCR genes suggesting the possible existence of an alternative Dicer-independent miRNA synthesis pathway. Target's prediction of A. brassicicola miRNAs identified genes in the fungus and in the plant model Arabidopsis thaliana. Some miRNAs were predicted to target A. thaliana genes involved in the methylation of histone and in the disease resistance.

Harmonia axyridis presents remarkable appendage regeneration capacity and can therefore be considered as an emerging regeneration research model. Amino acid sequences of the Janus kinase Hopscotch (Hahop) and the transcription factor STAT (HaStat), the main components of the JAK/STAT signaling pathway, conserved with their homologs in other models. The expression levels of these two genes were continuously up-regulated during the appendage regeneration process. To identify the functions of JAK/STAT signaling, we performed RNAi experiments of Hahop and HaStat in H. axyridis, and found regeneration defects following in Hahop

Primary hyperoxaluria type 1 (PH1) is a rare, progressive, genetic disease with limited treatment options. We report the efficacy and safety of lumasiran, an RNA interference therapeutic, in infants and young children with PH1.This single-arm, open-label, phase 3 study evaluated lumasiran in patients aged6 years with PH1 and an estimated glomerular filtration rate45 mL/min/1.73 mAll patients (N = 18) completed the 6-month primary analysis period. Median age at consent was 50.1 months. Least-squares mean percent reduction in spot UOx:Cr was 72.0%. At month 6, 50% of patients (9/18) achieved spot UOx:Cr ≤1.5× upper limit of normal. Least-squares mean percent reduction in plasma oxalate was 31.7%. The most common treatment-related adverse events were transient, mild, injection-site reactions.Lumasiran showed rapid, sustained reduction in spot UOx:Cr and plasma oxalate and acceptable safety in patients aged6 years with PH1, establishing RNA interference therapies as safe, effective treatment options for infants and young children.

Dark green islands (DGIs) are the outcome of post-transcriptional gene silencing (PTGS) in antiviral immunity, but their characteristics related to PTGS remain largely unknown. In this study, the cucumber mosaic virus (CMV) was inoculated on Nicotiana tabacum plants to explore the PTGS features of DGIs. Our results showed that higher expressions of PTGS-associated genes, especially NtAGO1, present in DGIs. To investigate the role of NtAGO1 in the generation and the antiviral effect of DGIs, NtAGO1 was then over-expressed or knocked out in N. tabacum plants through agrobacterium-mediated genetic transformation. The results showed that more DGIs with larger areas appeared on NtAGO1 over-expressed plants, accompanied by less virus accumulation, less reactive oxygen species production, and seldom membrane damage, whereas fewer DGIs appeared on NtAGO1 knockout plants with more damage on infected plants. In addition, the NtAGO1-participated antiviral process could promote the transduction of the salicylic acid-mediated defense pathway. Taken together, our results indicate that DGIs are maintained by a stronger PTGS mechanism, and NtAGO1 positively regulates the generation and viral resistance of DGIs in N. tabacum.