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Selfish genetic elements contribute to hybrid incompatibility and bias or drive their own transmission1,2. Chromosomal drive typically functions in asymmetric female meiosis, whereas gene drive is normally post-meiotic and typically found in males. Here, using single-molecule and single-pollen genome sequencing, we describe Teosinte Pollen Drive, an instance of gene drive in hybrids between maize (Zea mays ssp. mays) and teosinte mexicana (Z. mays ssp. mexicana) that depends on RNA interference (RNAi). 22-nucleotide small RNAs from a non-coding RNA hairpin in mexicana depend on Dicer-like 2 (Dcl2) and target Teosinte Drive Responder 1 (Tdr1), which encodes a lipase required for pollen viability. Dcl2, Tdr1 and the hairpin are in tight pseudolinkage on chromosome 5, but only when transmitted through the male. Introgression of mexicana into early cultivated maize is thought to have been critical to its geographical dispersal throughout the Americas3, and a tightly linked inversion in mexicana spans a major domestication sweep in modern maize4. A survey of maize traditional varieties and sympatric populations of teosinte mexicana reveals correlated patterns of admixture among unlinked genes required for RNAi on at least four chromosomes that are also subject to gene drive in pollen from synthetic hybrids. Teosinte Pollen Drive probably had a major role in maize domestication and diversification, and offers an explanation for the widespread abundance of self small RNAs in the germ lines of plants and animals.

The clinical use of RNA interference (RNAi) molecular mechanisms has introduced a novel, growing class of RNA therapeutics capable of treating diseases by controlling target gene expression at the posttranscriptional level. With the newly approved nedosiran (Rivfloza), there are now six RNAi-based therapeutics approved by the United States Food and Drug Administration (FDA). Interestingly, five of the six FDA-approved small interfering RNA (siRNA) therapeutics [patisiran (Onpattro), lumasiran (Oxlumo), inclisiran (Leqvio), vutrisiran (Amvuttra), and nedosiran] were revealed to act on the 3'-untranslated regions of target mRNAs, instead of coding sequences, thereby following the common mechanistic action of genome-derived microRNAs (miRNA). Furthermore, three of the FDA-approved siRNA therapeutics [patisiran, givosiran (Givlaari), and nedosiran] induce target mRNA degradation or cleavage via near-complete rather than complete base-pair complementarity. These features along with previous findings confound the currently held characteristics to distinguish siRNAs and miRNAs or biosimilars, of which all converge in the RNAi regulatory pathway action. Herein, we discuss the RNAi mechanism of action and current criteria for distinguishing between miRNAs and siRNAs while summarizing the common and unique chemistry and molecular pharmacology of the six FDA-approved siRNA therapeutics. The term "RNAi" therapeutics, as used previously, provides a coherently unified nomenclature for broader RNAi forms as well as the growing number of therapeutic siRNAs and miRNAs or biosimilars that best aligns with current pharmacological nomenclature by mechanism of action. SIGNIFICANCE STATEMENT: The common and unique chemistry and molecular pharmacology of six FDA-approved siRNA therapeutics are summarized, in which nedosiran is newly approved. We point out rather a surprisingly mechanistic action as miRNAs for five siRNA therapeutics and discuss the differences and similarities between siRNAs and miRNAs that supports using a general and unified term "RNAi" therapeutics to align with current drug nomenclature criteria in pharmacology based on mechanism of action and embraces broader forms and growing number of novel RNAi therapeutics.

Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3-10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (

Genome-derived microRNAs (miRNAs or miRs) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent work has demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely, BioRNAGly and BioRNALeu, respectively, and perform the initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNAGly and BioRNALeu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNAGly/miR-7-5p exhibited greater efficiency to regulate a known target gene expression (EGFR) than BioRNALeu/miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNAGly/miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development.

Global control of infectious diseases depends on the continuous development and deployment of diverse vaccination strategies. Currently available live-attenuated and killed virus vaccines typically take a week or longer to activate specific protection by the adaptive immunity. The mosquito-transmitted Nodamura virus (NoV) is attenuated in mice by mutations that prevent expression of the B2 viral suppressor of RNA interference (VSR) and consequently, drastically enhance in vivo production of the virus-targeting small-interfering RNAs. We reported recently that 2 d after immunization with live-attenuated VSR-disabled NoV (NoVΔB2), neonatal mice become fully protected against lethal NoV challenge and develop no detectable infection. Using Rag1-/- mice that produce no mature B and T lymphocytes as a model, here we examined the hypothesis that adaptive immunity is dispensable for the RNAi-based protective immunity activated by NoVΔB2 immunization. We show that immunization of both neonatal and adult Rag1-/- mice with live but not killed NoVΔB2 induces full protection against NoV challenge at 2 or 14 d postimmunization. Moreover, NoVΔB2-induced protective antiviral immunity is virus-specific and remains effective in adult Rag1-/- mice 42 and 90 d after a single-shot immunization. We conclude that immunization with the live-attenuated VSR-disabled RNA virus vaccine activates rapid and long-lasting protective immunity against lethal challenges by a distinct mechanism independent of the adaptive immunity mediated by B and T cells. Future studies are warranted to determine whether additional animal and human viruses attenuated by VSR inactivation induce similar protective immunity in healthy and adaptive immunity-compromised individuals.

Lowering low-density lipoprotein cholesterol (LDL-C) is a cornerstone of reducing risk for atherosclerotic cardiovascular disease. Despite the approval of nonstatin therapies for LDL-C lowering over the past 2 decades, these medications are underused, and most patients are still not at guideline-recommended LDL-C goals. Barriers include poor adherence, clinical inertia, concern for side effects, cost, and complex prior authorization processes. With atherosclerotic cardiovascular disease-related mortality increasing globally, there remains a need for additional therapeutic options for lowering LDL-C as part of an atherosclerotic cardiovascular disease prevention strategy. Following the identification of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a promising therapeutic target, inclisiran was developed using the natural process of RNA interference for robust, sustained prevention of hepatic PCSK9 synthesis. Twice-yearly maintenance subcutaneous inclisiran (following initial loading doses at Day 1 and Day 90) reduces circulating LDL-C levels by ≈50% versus placebo when added to maximally tolerated statins. Long-term safety and tolerability of inclisiran have been assessed, with studies underway to evaluate the effects of inclisiran on cardiovascular outcomes and to provide additional safety and effectiveness data. In 2021,

Conformationally constrained nucleotides, LNA or α-L-LNA, at the 5' terminus of the antisense strand impeded gene silencing of small interfering RNA (siRNA) by hindering phosphorylation, thereby deterring loading into the RNA-induced silencing complex. Installation of a phosphate mimic, (E)-vinyl phosphonate (VP), improved activity considerably. Gene silencing was more efficient when the antisense strand of the siRNA was modified with 5'-VP-α-L-LNA, which adopts a C3'-exo (south) conformation, than when the antisense strand was modified with 5'-VP-LNA, which adopts a C3'-endo (north) pucker. These data underscore the critical role of conformation of nucleotides in RNA interference. [ABSTRACT FROM AUTHOR]

The role of epigenetics in regulating caste polyphenism in social insects has been debated. Here, we tested the importance of histone de/acetylation processes for the maintenance of queen hallmarks like a high fecundity and a long lifespan. To this end, we performed RNA interference experiments against histone deacetylase 3 (HDAC3) in the termite Cryptotermes secundus. Fat body transcriptomes and chemical communication profiles revealed that silencing of HDAC3 leads to signals indicative of queen hallmarks. This includes fostering of queen signalling, defence against ageing and a reduction of life‐shortening IIS (insulin/insulin‐like growth factor signalling) and endocrine JH (juvenile hormone) signalling via Kr‐h1 (Krüppel‐homologue 1). These observed patterns were similar to those of a protein‐enriched diet, which might imply that histone acetylation conveys nutritional effects. Strikingly, in contrast to solitary insects, reduced endocrine JH signalling had no negative effect on fecundity‐related vitellogenesis in the fat bodies. This suggests an uncoupling of longevity pathways from fecundity in fat bodies, which can help explain queens' extraordinary lifespans combined with high fecundity. [ABSTRACT FROM AUTHOR]

The fall armyworm (FAW), Spodoptera frugiperda is a cold-sensitive species that overwinters in temperate climates without diapause. Overwintering in insects involves rapid cold hardening (RCH), supported by trehalose (TRE), which serves as an intermediate between glycogen (GLY) and glucose (GLU). However, both GLU and TRE help maintain homeostasis under stress. TRE is hydrolyzed by the enzyme trehalase (Treh) into GLU. This study retrieved Sf-Treh1a, Sf-Treh1b, and Sf-Treh2 from the FAW transcriptome analysis. RNA interference (RNAi) targeting these three Treh genes resulted in significant downregulation of mRNA levels and altered survival rates in RNAi-treated FAW larvae following RCH treatment. High-pressure liquid chromatography (HPLC) quantification of TRE and GLU in treated groups suggests that GLU is an essential component of the hemolymph for survival adaptation to cold conditions in S. frugiperda. This study reveals limited cold adaptability of FAW, as evidenced by lower glucose concentration levels. We found that FAW requires alternative molecules, in conjunction with glucose and trehalose for freeze tolerance and survivability. Our study aims to discover the molecular mechanisms that contribute to freeze tolerance in FAW by exploring the roles of trehalose, glucose, and glycogen. [ABSTRACT FROM AUTHOR]

The gut is not only used by insects as an organ for the digestion of food and absorption of nutrients but also as an important barrier against the invasion and proliferation of pathogenic microorganisms. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), an insect‐specific virus, predominantly colonizes the midgut epithelial cells of the silkworm, thereby jeopardizing its normal growth. However, there is limited knowledge of the cellular immune responses to viral infection and whether the infection is promoted or inhibited by different types of cells in the silkworm midgut. In this study, we used single‐nucleus RNA sequencing to identify representative enteroendocrine cells, enterocytes, and muscle cell types in the silkworm midgut. In addition, by analyzing the transcriptional profiles of various subpopulations in the infected and uninfected groups, we found that BmCPV infection suppresses the response of the antiviral pathways and induces the expression of BmHSP70, which plays a role in promoting BmCPV replication. However, certain immune genes in the midgut of the silkworm, such as BmLebocin3, were induced upon viral infection, and downregulation of BmLEB3 using RNA interference promoted BmCPV replication in the midgut of B. mori. These results suggest that viral immune evasion and active host resistance coexist in BmCPV‐infected silkworms. We reveal the richness of cellular diversity in the midgut of B. mori larvae by single‐nucleus RNA sequencing analysis and provide new insights into the complex interactions between the host and the virus at the single‐cell level. [ABSTRACT FROM AUTHOR]

The roles of plant-specific transcription factor family YABBY may vary among different members. OsYABBY6 is a rice YABBY gene, whose function is not well elucidated so far. In this paper, we show that OsYABBY6 is a nucleus-localized protein with transcriptional activation activity. OsYABBY6 is predominantly expressed in the palea and lemma, as well as in the sheath, culm and node. OsYABBY6 RNA interference (RNAi) plants exhibited altered plant height and larger grain size. Under cold treatment, OsYABBY6 overexpression (OE) plants had up-regulated expression of cold responsive genes, and accumulated less reactive oxygen species but more proline compared to wild type, resulting in improved cold tolerance. On the other hand, RNAi plants showed enhanced drought tolerance compared to the wild type by slower water loss, less reactive oxygen species but more proline and soluble sugar accumulation. In addition, endogenous abscisic acid (ABA) level was reduced in OsYABBY6 RNAi plants, and RNAi and OE plants were more and less sensitive to ABA treatment, respectively. Accordingly, we deduce that OsYABBY6 positively regulates cold response but negatively regulates drought response through different pathways. Our study reveals the crucial roles of OsYABBY6 in plant architecture and grain development, as well as in abiotic stress response, providing new insights into the functions of YABBYs in rice. [ABSTRACT FROM AUTHOR]

ABSTRACT Natural indole alkaloids provide important medicinal resources and defences to environmental stresses. The Uncaria genus is a recorded traditional medicinal woody plant with high alkaloids. Genomic insights into alkaloid variation remain elusive. Here, we have dissected the haploid‐resolved chromosomal T2T genome assembly of Uncaria rhynchophylla with a size of ~634 Mb and contig N50 of 27 Mb using PacBio HiFi long‐reads plus Hi‐C reads and anchored the contigs on 22 pairs of confirmed chromosomes. This genome contains 56% repeat sequences and ~29 000 protein‐encoding genes. U. rhynchophylla diverged from a common ancestor shared with Coffea around 20 million years ago and contains expanded and contracted gene families associated with secondary metabolites and defences/resistance to stresses. We constructed the pathway and mined genes for rhynchophylline alkaloid biosynthesis. Fifty‐three alkaloids in this pathway and eight differentially expressed genes are the keys to alkaloid accumulation. Elevated alkaloid levels are driven by high copy numbers of critical genes STRs and SGRs involved in strictosidine synthesis and hydrolysis as evidenced by phylogenetic, expression and RNA interference analyses. These results advance our genetic understanding and guide further breeding improvements, stress adaptation studies and pharmaceutical development. [ABSTRACT FROM AUTHOR]

Neuropeptide F (NPF) and short neuropeptide F (sNPF) are important neuropeptides and mainly affect feeding behaviour of insects. However, the regulation of insect feeding behaviour by NPF and sNPF appears to differ between species, and it is not clear how NPF and sNPF regulate the food intake of the brown planthopper (Nilaparvata lugens). Therefore, the functions of NPF and sNPF in regulating food intake and affecting the growth and antioxidant levels of N. lugens fed on host rice plants were investigated by knocking down NPF and sNPF respectively and simultaneously knocking down both of them by RNA interference. The results showed that NPF and sNPF were mainly expressed in the head of N. lugens, and N. lugens increased food intake after NPF and sNPF were knocked down, which was reflected in the prolonged duration of N4a and N4b waves in the electrical penetration graph (EPG) experiment after knocking down NPF and sNPF. In addition, knocking down NPF and sNPF led to the increase of body weight and mortality of N. lugens, and also led to the increase of antioxidant level of N. lugens. So it was concluded that NPF and sNPF could regulate food intake, maintain body weight stability and oxidative balance in N. lugens. Our study clarified the molecular mechanism of NPF and sNPF regulating feeding behaviour and affect the growth and antioxidant level of N. lugens. [ABSTRACT FROM AUTHOR]

The brown planthopper (BPH) is the most destructive insect pest in rice. Through a stylet, BPH secretes a plethora of salivary proteins into rice phloem cells as a crucial step of infestation. However, how various salivary proteins function in rice cells to promote insect infestation is poorly understood. Among them, one of the salivary proteins is predicted to be a carbonic anhydrase (Nilaparvata lugens carbonic anhydrase [NlCA]). The survival rate of the NlCA- RNA interference (RNAi) BPH insects was extremely low on rice, indicating a vital role of this salivary protein in BPH infestation. We generated NlCA transgenic rice plants and found that NlCA expressed in rice plants could restore the ability of NlCA- RNAi BPH to survive on rice. Next, we produced rice plants expressing the ratiometric pH sensor pHusion and found that NlCA- RNAi BPH induced rapid intracellular acidification of rice cells during feeding. Further analysis revealed that both NlCA- RNAi BPH feeding and artificial lowering of intracellular pH activated plant defense responses and that NlCA-mediated intracellular pH stabilization is linked to diminished defense responses, including reduced callose deposition at the phloem sieve plates and suppressed defense gene expression. Given the importance of pH homeostasis across the kingdoms of life, discovery of NlCA-mediated intracellular pH modulation uncovered a new dimension in the interaction between plants and piercing/sucking insect pests. The crucial role of NlCA for BPH infestation of rice suggests that NlCA is a promising target for chemical or trans-kingdom RNAi-based inactivation for BPH control strategies in plants. • Rapid intracellular acidification is a novel plant defense response • Brown planthopper (BPH) delivers carbonic anhydrase (NlCA) into rice for survival • NlCA counters intracellular acidification to suppress plant defense • pH-responsive regulators of defense responses are predicted to exist in the rice-BPH interaction The brown planthopper (BPH) is the most devastating insect pest in rice. Jiang et al. discover that BPH secretes a salivary carbonic anhydrase (NICA) to regulate the intracellular pH of the rice cell to facilitate its feeding and survival on rice plants and that NICA counters host intracellular pH acidification to diminished defense responses. [ABSTRACT FROM AUTHOR]

Plant pathogens and other biological pests represent significant obstacles to crop Protection worldwide. Even though there are many effective conventional methods for controlling plant diseases, new methods that are also effective, environmentally safe, and cost-effective are required. While plant breeding has traditionally been used to manipulate the plant genome to develop resistant cultivars for controlling plant diseases, the emergence of genetic engineering has introduced a completely new approach to render plants resistant to bacteria, nematodes, fungi, and viruses. The RNA interference (RNAi) approach has recently emerged as a potentially useful tool for mitigating the inherent risks associated with the development of conventional transgenics. These risks include the use of specific transgenes, gene control sequences, or marker genes. Utilizing RNAi to silence certain genes is a promising solution to this dilemma as disease-resistant transgenic plants can be generated within a legislative structure. Recent investigations have shown that using target double stranded RNAs via an effective vector system can produce significant silencing effects. Both dsRNA-containing crop sprays and transgenic plants carrying RNAi vectors have proven effective in controlling plant diseases that threaten commercially significant crop species. This article discusses the methods and applications of the most recent RNAi technology for reducing plant diseases to ensure sustainable agricultural yields. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa, an antibiotic‐resistant opportunistic pathogen, poses significant challenges in treating infections, particularly in immunocompromised individuals. This review explores current and future innovative approaches to suppress its growth and virulence. We delve into the bacterium's virulence factors, discussing existing strategies like antibiotics, bacteriophages, probiotics, and small‐molecule inhibitors. Additionally, novel approaches, including RNA interference, CRISPR‐Cas systems, and nanotechnology, show promise in preclinical studies. Despite advancements, challenges persist, prompting the need for a multifaceted approach targeting various aspects of P. aeruginosa pathogenesis for effective infection management. This review provides a comprehensive perspective on the status and future directions of innovative strategies against P. aeruginosa. [ABSTRACT FROM AUTHOR]

SUMMARY: Alfalfa is one of the most widely cultivated forage crops worldwide. However, soil salinization restricts alfalfa growth and development and affects global productivity. The plant cell wall is the first barrier against various stresses. Therefore, elucidating the alterations in cell wall architecture is crucial for stress adaptation. This study aimed to clarify the impact of myo‐inositol oxygenase 2 (MsMIOX2) on cell wall pectin and hemicellulose biosynthesis under saline–alkali stress and identify the upstream transcription factors that govern MsMIOX2. MsMIOX2 activation induced cell wall pectin and hemicellulose accumulation under saline–alkali stress. The effects of MsMIOX2 in saline–alkali tolerance were investigated by characterizing its overexpression and RNA interference lines. MsMIOX2 overexpression positively regulated the antioxidant system and photosynthesis in alfalfa under saline–alkali stress. MsMIOX2 exhibited myo‐inositol oxygenase activity, which increased polysaccharide contents, facilitated pectin and hemicellulose biosynthesis, and extended the cell wall thickness. However, MsMIOX2 RNA interference decreased cell wall thickness and alleviated alfalfa saline–alkali stress tolerance. In addition, MsbZIP53 was identified as an upstream transcriptional MsMIOX2 regulator by yeast one‐hybrid, electrophoretic mobility shift assay, dual‐luciferase, and beta‐glucuronidase assays. MsbZIP53 overexpression increased MsMIOX2 expression, elevated MIOX activity, reinforced the antioxidant system and photosynthesis, and increased saline–alkali stress tolerance in alfalfa. In conclusion, this study presents a novel perspective for elucidating the molecular mechanisms of saline–alkali stress tolerance in alfalfa and emphasizes the potential use of MsMIOX2 in alfalfa breeding. [ABSTRACT FROM AUTHOR]

Objective: This work aims to investigate whether RIP2 silencing in naive CD4+ T cells from lupus-prone mice impacts Th17 cell activity or differentiation in vitro. Methods: Naive CD4+ T cells isolation from MRL/lpr mice's spleens. Three RNA interference target sequences of RIP2 were packaged with lentivirus and transfected into naive CD4+ T cells. The shRIP2 with the highest interference efficiency was selected and transfected into naive CD4+ T cells. Naive CD4+ T cells were cultured under conventional (TGF-β1 and IL-6) and pathogenic (IL-6, IL-23, IL-1β) differentiation environments, respectively. Then, RT-qPCR, Western blot or Flow Cytometry were used for measuring the amounts of RIP2 and IL-17 and the differentiation of Th17 cells in two settings. Results: Under the conventional Th17 (cTh17) cell differentiation environment (TGF-β1 and IL-6), RIP2 deficiency is linked to decreased IL-17A levels (1.00 ± 0.03 vs 0.80 ± 0.03) and attenuated cTh17 cell (2.46 ± 0.08 vs 0.78 ± 0.03) differentiation (all, P < 0.05). Under the pathogenic Th17 (pTh17) cell environment (IL-1β, IL-23, IL-6), RIP2 deficiency is linked to elevated IL-17A levels (1.03 ± 0.05 vs 1.63 ± 0.07) and enhanced pTh17 cell (3.69 ± 0.19 vs 5.49 ± 0.10) differentiation (all, P < 0.05). Conclusion: Our data suggest that RIP2 inhibition induces preferential differentiation of naive CD4+ T cells to pathogenic Th17 cells, while being able to upregulate IL-17A levels in the context of pTh17 cell differentiation. Our study opens up new research areas to reveal the underlying mechanisms and potential therapeutic targets for the prevention and treatment of SLE patients. Key Points • Silencing of RIP2 in naive CD4+T cells from lupus-prone mice promotes pathogenic Th17 (pTh17) cell differentiation and IL-17A production under pTh17 cell (IL-1β, IL-23, and IL-6) conditions. • RIP2 deficiency in naive CD4+ T cells reduces conventional Th17 (cTh17) cell differentiation and IL-17A production under cTh17 cell (TGF-β1 and IL-6) conditions. • RIP2-deficient naive CD4+ T cells preferentially differentiate towards pTh17 cells rather than cTh17 cells in vitro. • Inhibition of RIP2 may be involved in the development of SLE via effects on Th17/IL-17. [ABSTRACT FROM AUTHOR]

Guar is a legume of industrial importance due to the presence of gum (galactomannan) in its seed endosperm. The mannose (M)/galactose (G) ratio in the galactomannan molecule is critical in deciding the physico-chemical properties of guar gum. We report down regulation of galactomannan galactosyltransferase (GMGT) which links G residues to the M backbone in the galactomannan molecule. The RNAi cassette containing the sense-antisense sequence for gmgt was delivered into guar variety RGC-936 using Agrobacterium tumefaciens-mediated gene transfer. Maximum number of putative transformed shoots regenerated from the cotyledonary node explants cultured in vitro on Murashige and Skoog (MS) medium containing 1.5 mg/L indole-3-acetic acid (IAA), 4.0 mg/L butyric acid (BA) and 1.0 mg/L gibberellic acid (GA3). In vitro regenerated shoots were rooted on half-strength MS medium containing 4% sucrose and 2 mg/L indole-3-butyric acid (IBA). The quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) analysis showed reduced levels of GMGT expression in the seed endosperm of 35 days after flowering (DAF) stage pods. The present findings are expected to be beneficial in taking up gene characterization and agronomic traits improvement studies in guar. [ABSTRACT FROM AUTHOR]

Objective: Visceral adiposity is associated with increased proinflammatory activity, insulin resistance, diabetes risk, and mortality rate. Numerous individual genes have been associated with obesity, but studies investigating gene regulatory networks in human visceral obesity have been lacking. Methods: We analyzed gene regulatory networks in human visceral adipose tissue (VAT) from 48 and 11 Chinese patients with and without obesity, respectively, using gene coexpression and gene regulatory network construction from RNA‐sequencing data. We also conducted RNA interference‐based functional tests on selected genes for effects on adipocyte differentiation. Results: A scale‐free gene coexpression network was constructed from 360 differentially expressed genes between VAT samples from patients with and without obesity (absolute log fold change > 1, false discovery rate [FDR] < 0.05), with edge probability > 0.8. Gene regulatory network analysis identified candidate transcription factors associated with differentially expressed genes. A total of 15 subnetworks (communities) displayed altered connectivity patterns between obesity and nonobesity networks. Genes in proinflammatory pathways showed increased network connectivity in VAT samples with obesity, whereas the oxidative phosphorylation pathway displayed reduced connectivity (enrichment FDR < 0.05). Functional screening via RNA interference identified genes such as SOX30, SIRPB1, and OSBPL3 as potential network‐derived candidates influencing adipocyte differentiation. Conclusions: This approach highlights the network architecture in human obesity, identifies novel candidate genes, and generates new hypotheses regarding network‐assisted gene regulation in VAT. [ABSTRACT FROM AUTHOR]

BACKGROUND: Vegetative insecticidal proteins (Vips) are widely used in pest management, but Vip tolerance poses a significant threat. DNA methylation plays important roles in regulating the response of biological organisms to environmental stress, and it may also regulate fall armyworm (FAW, Spodoptera frugiperda) Vip3Aa tolerance. RESULTS: In this study, a DNA methylation map was developed for FAW, and its function in regulating FAW Vip3Aa tolerance was explored. The FAW genome‐wide DNA methylation map showed that exons were preferred regions for DNA methylation and housekeeping genes were highly methylated. FAW was screened using Vip3Aa for ten generations, and bioassays indicated that Vip3Aa tolerance increased trans‐generationally. A comparison of DNA methylation maps between Vip3Aa‐tolerant and ‐susceptible strains showed that gene body methylation was positively correlated with gene expression level. FAW exhibits significant variation in DNA methylation among individuals, and Vip3Aa screening induces epigenetic variation based on DNA methylation. Moreover, the study demonstrated that a reduction in methylation density within the gene body of a 3′5′‐cyclic nucleotide phosphodiesterase gene resulted in decreased expression and increased tolerance of FAW to Vip3Aa, which was validated through RNA interference experiments. CONCLUSION: The DNA methylation map and mechanism of Vip3Aa tolerance improve our understanding of DNA methylation and its function in Lepidoptera and provide a new perspective for developing pest management strategies. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Calliptamus italicus L. is a major pest in Xinjiang grassland. The diapause overwintering strategy is one of the important reasons for the large population of this pest. This study investigated the function of the genes associated with the release of diapause (DIB, JHE and CAM) in Calliptamus italicus by RNA interference (RNAi) technology to aid in its biological control. RESULTS: The expression levels of DIB and its downstream‐associated genes (EcR and FTZ‐F1) in the eggs injected with dsDIB for 12 h decreased by 96.6%, 55.8% and 81.8%, respectively. Diapause began to terminate on day 3, and development was almost complete on day 6. However, the head was significantly smaller. The expression levels of JHE and its downstream‐associated genes (JHEH and VgR) at 48 h after dsJHE treatment decreased by 76.5%, 85.6% and 85.9%, respectively. The termination of diapause occured on day 3 of incubation. The development was basically complete on day 6, but the yolk had been incompletely absorbed. The expression of CAM and its downstream‐associated genes (CAMK4 and MYL) at 24 h after dsCAM treatment decreased by 42.4%, 95.3% and 82.7%, respectively. Diapause termination was completed on day 4 for incubation, and development was abnormal on day 6. The absorption of yolk was incomplete. CONCLUSION: DIB, JHE and CAM can delay the diapause termination of Calliptamus italicus eggs to different degrees and can be developed as potential target genes for its biological control. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: The RNA interference (RNAi) efficiency of double‐stranded RNA (dsRNA) delivery to insects by various methods is different and the reduced efficacy of feeding dsRNA is partly due to the presence of DNA/RNA non‐specific endonuclease in the insect gut. However, the mechanism leading to the low RNAi efficiency of Nilaparvata lugens by feeding remains elusive. RESULTS: In this study, we identified a putatively DNA/RNA non‐specific endonuclease gene in the N. lugens genome database that was highly expressed in the first nymphal instar and the midgut. Different expression levels of NldsRNase after feeding and injection suggested that NldsRNase might interfere with oral RNAi in N. lugens. A co‐delivery RNAi strategy further revealed that the presence of NldsRNase reduces RNAi efficiency. In vitro dsRNA degradation experiments also showed that the stability of dsRNA was higher in a gut mixture from nymphs injected with dsNldsRNase. These results support the idea that the low oral RNAi response observed in N. lugens is likely due to the presence of NldsRNase. CONCLUSIONS: Our study provides insight into the differences in RNAi response between the injection and feeding of dsRNA in N. lugens and sheds light on the mechanisms underlying the reduced efficacy of RNAi via feeding. These findings may help to inform the development of more‐effective RNAi‐based strategies controlling N. lugens and other insect pests. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Grapholita molesta is an important and harmful fruit pest worldwide, with widespread feeding hosts. Trypsin, an indispensable hydrolytic digestive protease in the insect gut, is crucial in digestion, growth and development. We analyzed the characteristics of the trypsin‐encoding genes, screened for the optimal dose of RNAi mediated by nanocarriers, and investigated various indices of larval growth and development of G. molesta. RESULTS: Gut content (GC) and RNase A degraded double‐stranded RNA (dsRNA), with a faster degradation rate at higher concentrations. Star polycation (SPc) nanomaterials protected dsGFP from degradation by anion–cation binding and did not migrate through agarose gel. The key conserved motifs of the trypsin‐encoding genes were similar, exhibiting high homology with those in other lepidopteran insects. An interference efficiency of ≈70% was achieved with SPc nanomaterial‐mediated RNA interference with 0.05 μg dsRNA. The efficiency of continuous interference was stable. Trypsin activity, body weight of 8‐day‐old larvae, pupal weight and emergence rate were significantly reduced, and the larval stage was significantly prolonged. CONCLUSION: The investigated trypsin gene is a key target gene in the growth and development of G. molesta. We investigated the efficiency and convenience of feeding SPc nanomaterials in a functional study of insects. Our results provide valuable data for the development of efficient trypsin‐targeting pesticides. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: The transcriptomic profile of cellular senescence is strongly associated with distinct cell types, the specific stressors triggering senescence, and temporal progression through senescence stages. This implies the potential necessity of conducting separate investigations for each cell type and a stressor inducing senescence. To elucidate the molecular mechanism that drives endoplasmic reticulum (ER) stress-induced cellular senescence in MCF-7 breast cancer cells, with a particular emphasis on the ATF6α branch of the unfolded protein response. We conducted transcriptomic analysis on MCF-7 cells by ectopic expression of ATF6α. Methods: Transcriptomic sequencing was conducted on MCF-7 cells at 6 and 9 hours post senescence induction through ATF6α ectopic expression. Comprehensive analyses encompassing enriched functional annotation, canonical pathway analysis, gene network analysis, upstream regulator analysis and gene set enrichment analysis were performed on Differentially Expressed Genes (DEGs) at 6 and 9 hours as well as time-related DEGs. Regulators and their targets identified from the upstream regulator analysis were validated through RNA interference, and their impact on cellular senescence was assessed by senescence-associated β-galactosidase staining. Results: ATF6α ectopic expression resulted in the identification of 12 and 79 DEGs at 6 and 9 hours, respectively, employing criteria of a false discovery rate < 0.05 and a lower fold change (FC) cutoff |log2FC| > 1. Various analyses highlighted the involvement of the UPR and/or ER Stress Pathway. Upstream regulator analysis of 9 hour-DEGs identified six regulators and eleven target genes associated with processes related to cytostasis and 'cell viability and cell death of connective tissue cells.' Validation confirmed the significance of MAP2K1/2, GPAT4, and PDGF-BB among the regulators and DDIT3, PPP1R15A, and IL6 among the targets. Conclusion: Transcriptomic analyses and validation reveal the importance of the MAP2K1/2/GPAT4-DDIT3 pathway in driving cellular senescence following ATF6α ectopic expression in MCF-7 cells. This study contributes to our understanding of the initial molecular events underlying ER stress-induced cellular senescence in breast cancer cells, providing a foundation for exploring cell type- and stressor-specific responses in cellular senescence induction. [ABSTRACT FROM AUTHOR]

In insects, tyrosine hydroxylase (TH) plays essential roles in cuticle tanning and cuticle pigmentation. Plagiodera versicolora (Coleoptera: Chrysomelidae) is a leaf‐eating forest pest in salicaceous trees worldwide. However, the function of PverTH in P. versicolora is still unknown. In this study, we obtained a PverTH gene from transcriptome analysis. The expression analysis of PverTH showed that the highest expression was found in epidermis of larvae. In this study, we used RNA interference (RNAi) technology to knockdown the PverTH gene. The results showed that ingestion of dsTH led to cuticle coloration became lighter in larvae, pupae and adults. Knockdown of PverTH gene inhibited larval growth, and consequently caused higher mortality. In addition, RNAi of TH disrupted the cuticle tanning, caused lower pupation rate, lower eclosion rate and higher deformity rate. This study indicates that PverTH is vital for the cuticular pigments and cuticle tanning. Moreover, this research suggested that the development of PverTH gene as a potential target gene to control P. versicolora. [ABSTRACT FROM AUTHOR]

Background: Repetitive transcranial magnetic stimulation (rTMS) has been used to treat various neurological disorders. However, the molecular mechanism underlying the therapeutic effect of rTMS on Parkinson's disease (PD) has not been fully elucidated. Neuroinflammation like regulatory T-cells (Tregs) appears to be a key modulator of disease progression in PD. If rTMS affects the peripheral Tregs in PD remains unknown. Methods: Here, we conducted a prospective clinical study (Chinese ClinicalTrials. gov: ChiCTR 2100051140) involving 54 PD patients who received 10-day rTMS (10 Hz) stimulation on the primary motor cortex (M1) region or sham treatment. Clinical and function assessment as well as flow cytology study were undertaken in 54 PD patients who were consecutively recruited from the department of neurology at Zhujiang hospital between September 2021 and January 2022. Subsequently, we implemented flow cytometry analysis to examine the Tregs population in spleen of MPTP-induced PD mice that received rTMS or sham treatment, along with quantitative proteomic approach reveal novel molecular targets for Parkinson's disease, and finally, the RNA interference method verifies the role of these new molecular targets in the treatment of PD. Results: We demonstrated that a 10-day rTMS treatment on the M1 motor cortex significantly improved motor dysfunction in PD patients. The beneficial effects persisted for up to 40 days, and were associated with an increase in peripheral Tregs. There was a positive correlation between Tregs and motor improvements in PD cases. Similarly, a 10-day rTMS treatment on the brains of MPTP-induced PD mice significantly ameliorated motor symptoms. rTMS reversed the downregulation of circulating Tregs and tyrosine hydroxylase neurons in these mice. It also increased anti-inflammatory mediators, deactivated microglia, and decreased inflammatory cytokines. These effects were blocked by administration of a Treg inhibitor anti-CD25 antibody in MPTP-induced PD mice. Quantitative proteomic analysis identified TLR4, TH, Slc6a3 and especially Syt6 as the hub node proteins related to Tregs and rTMS therapy. Lastly, we validated the role of Treg and rTMS-related protein syt6 in MPTP mice using the virus interference method. Conclusions: Our clinical and experimental studies suggest that rTMS improves motor function by modulating the function of Tregs and suppressing toxic neuroinflammation. Hub node proteins (especially Syt6) may be potential therapeutic targets. Trial registration: Chinese ClinicalTrials, ChiCTR2100051140. Registered 15 December 2021, https://www.chictr.org.cn/bin/project/edit?pid=133691 rTMS is a safe and non-invasive method for Parkinson's disease. In this study, we showed the proportion of CD4+CD25+CD127- regulatory T-cells (Tregs) in the peripheral blood was significantly increased after rTMS treatment. Similar effects of rTMS treatment were verified in MPTP-induced PD mice. Proteomic analysis and RNA interference analyses identified TLR4, TH, Slc6a3 and especially Syt6 as hub node proteins that can be modulated by rTMS therapy in PD. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) has emerged as an eco‐friendly alternative to classic pesticides for pest control. This review highlights the importance of identifying the best target genes for RNAi‐mediated pest control. We argue that the knowledge‐based approach to predicting effective targets is limited by our current gaps of knowledge, making unbiased screening a superior method for discovering the best target processes and genes. We emphasize the recent evidence that suggests targeting conserved basic cellular processes, such as protein degradation and translation, is more effective than targeting the classic pesticide target processes. We support these claims by comparing the efficacy of previously reported RNAi target genes and classic insecticide targets with data from our genome‐wide RNAi screen in the red flour beetle, Tribolium castaneum. Finally, we provide practical advice for identifying excellent target genes in other pests, where large‐scale RNAi screenings are typically challenging. [ABSTRACT FROM AUTHOR]

Introduction: Cells generate mechanical forces mainly through myosin motor activity on the actin cytoskeleton. In C. elegans, actomyosin stress fibers drive contractility of the smooth muscle-like cells of the spermatheca, a distensible, tube-shaped tissue in the hermaphrodite reproductive system and the site of oocyte fertilization. Stretching of the spermathecal cells by oocyte entry triggers activation of the small GTPase Rho. In this study, we asked how forces are distributed in vivo, and explored how spermathecal tissue responds to alterations in myosin activity. Methods: In animals expressing GFP labeled actin or apical membrane complexes, we severed these structures using femtosecond laser ablation and quantified retractions. RNA interference was used to deplete key contractility regulators. Results: We show that the basal actomyosin fibers are under tension in the occupied spermatheca. Reducing actomyosin contractility by depletion of the phospholipase C-e/PLC-1 or non-muscle myosin II/NMY-1, leads to distended spermathecae occupied by one or more embryos, but does not alter tension on the basal actomyosin fibers. However, activating myosin through depletion of the Rho GAP SPV-1 increases tension on the actomyosin fibers, consistent with earlier studies showing Rho drives spermathecal contractility. On the inner surface of the spermathecal tube, tension on the apical junctions is decreased by depletion of PLC-1 and NMY-1. Surprisingly, when basal contractility is increased through SPV-1 depletion, the tension on apical junctions also decreases, with the most significant effect on the junctions aligned in perpendicular to the axis of the spermatheca. Discussion: Our results suggest that much of the tension on the basal actin fibers in the occupied spermatheca is due to the presence of the embryo. Additionally, increased tension on the outer basal surface may compress the apical side, leading to lower tensions apically. The three dimensional shape of the spermatheca plays a role in force distribution and contractility during ovulation. [ABSTRACT FROM AUTHOR]

The red palm weevil, Rhynchophorus ferrugineus (Oliver, 1970) (Coleoptera: Dryophthoridae) is the most devastating insect-pest of palm trees worldwide. Synthetic insecticides are the most preferred tool for the management of RPW. Alternatively, RNA interference (RNAi) mediated silencing of crucial genes provides reasonable control of insect pests. Recently, we have targeted four important genes; ecdysone receptor (EcR), serine carboxypeptidase (SCP), actin and chitin-binding peritrophin (CBP) in the 3rd and 5th instar larvae RPW. The results from 20 days trial showed that the survival rate of 3rd instar larvae fed on SCP and actin dsRNAs exhibited the lowest survival (12–68%). While, in the 5th instar larvae, the lowest survival rate (24%) was recorded for SCP after 20 days of incubation. Similarly, the weight of the 3rd and 5th instar larvae treated with SCP and actin was significantly reduced to 2.30–2.36 g and 4.64–4.78 g after 6 days of dsRNA exposure. The larval duration was also decreased significantly in the larvae treated with all the dsRNA treatments. The qRT-PCR results confirmed a significant suppression of the targeted genes as 90–97% and 85–93% in the 3rd and 5th instar larvae, respectively. The results suggest that the SCP and the actin genes can be promising targets to mediate RNAi-based control of RPW. [ABSTRACT FROM AUTHOR]

SUMMARY Pustule formation is pivotal for the development of the Xanthomonas citri subsp. citri (Xcc)‐induced citrus canker disease (CCD). Although our previous study demonstrated that the exogenous application of abscisic acid (ABA) facilitated pustule formation induced by Xcc, the precise mechanism remains elusive. The 9‐cis‐epoxycarotenoid dioxygenase (NCED) is a crucial enzyme in ABA biosynthesis. This study explored the role of citrus CsNCED1‐1 in CCD resistance through overexpression and RNA interference of CsNCED1‐1 in Wanjincheng orange (Citrus sinensis). Our findings indicated that CsNCED1‐1 negatively modulated CCD resistance by fostering ABA accumulation, concomitant with an increase in jasmonic acid (JA) and a decrease in salicylic acid (SA). Plants overexpressing CsNCED1‐1 displayed shortened leaves with smaller and denser stomata along with irregular and increased palisade cells. CsLOB1 is a known susceptibility gene for CCD, and CsbZIP40 positively influences resistance to this disease. We further confirmed that CsLOB1 promoted and CsbZIP40 suppressed the transcription of CsNCED1‐1 by directly binding to the CsNCED1‐1 promoter. Notably, CsbZIP40 and CsLOB1 showed a competitive relationship in the regulation of CsNCED1‐1 expression, with CsbZIP40 exhibiting greater competitiveness. Overall, our findings highlight that CsNCED1‐1 promotes susceptibility to citrus canker by disrupting JA‐ and SA‐mediated defense mechanisms and triggering the proliferation and remodeling of palisade cells, thereby facilitating pathogen colonization and pustule formation. This study offers novel insights into the regulatory mechanisms underlying citrus canker resistance and the role of CsNCED1‐1 in citrus. [ABSTRACT FROM AUTHOR]

The growing global food demand, coupled with the limitations of traditional pest control methods, has driven the search for innovative and sustainable solutions in agricultural pest management. In this review, we highlight polymeric nanocarriers for their potential to deliver double-stranded RNA (dsRNA) and control pests through the gene-silencing mechanism of RNA interference (RNAi). Polymer-dsRNA systems have shown promise in protecting dsRNA, facilitating cellular uptake, and ensuring precise release. Despite these advances, challenges such as scalability, cost-efficiency, regulatory approval, and public acceptance persist, necessitating further research to overcome these obstacles and fully unlock the potential of RNAi in sustainable agriculture. Application of RNAi-based technology for crop pest control is hampered by the lacking of efficient system for the delivery of dsRNA molecules to pests or plants. Here, the authors review polymer systems for dsRNA delivery and provide perspectives on their application in sustainable agriculture production. [ABSTRACT FROM AUTHOR]

Background: Epigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage. Methods: KDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay. Results: RSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation. Conclusions: Overall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy. [ABSTRACT FROM AUTHOR]

As a powerful eukaryotic expression vector, the baculovirus expression vector system (BEVS) is widely applied to the production of heterogeneous proteins for research and pharmaceutical purposes, while optimization of BEVS remains a work in progress for membrane or secreted protein expression. In this study, the impact of the signal peptide (SP) derived from Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 protein on protein expression, secretion, and the endoplasmic reticulum-associated degradation (ERAD) pathway were investigated in BmN cells and BEVS. Transient expression studies in BmN cells revealed that SP alters the localization and expression levels of recombinant proteins, reducing intracellular accumulation while enhancing secretion efficiency. Quantitative analysis demonstrated that SP-mediated secretion was markedly higher compared to controls, albeit with lower total expression levels. Further exploration into SP-mediated ERAD pathway activation showed increased expression of BiP and other ERAD-associated genes (PDI, UFD1, S1P, and ASK1), correlating with higher SP-driven protein expression levels. RNA interference (RNAi) experiments elucidated that knockdown of ERAD-associated genes enhances both the secretion efficiency of SP-guided proteins and the infectivity of BmNPV. Particularly, interference with BiP demonstrated the most pronounced effect on protein secretion enhancement. Viral infection experiments further supported these findings, showing upregulated ERAD-associated genes during BmNPV infection, indicating their role in viral protein processing and infectivity. In conclusion, this study elucidates the complex interplay between SP-mediated protein secretion, ERAD pathway activation, and viral infectivity in BmNPV-infected cells. These insights suggest strategies for optimizing recombinant protein production and viral protein processing in baculovirus expression systems, with potential implications for biotechnological and biomedical applications. Further research could refine our understanding and manipulation of protein secretion pathways in insect cell-based expression systems. [ABSTRACT FROM AUTHOR]

Skin mast cells (MCs) mediate acute allergic reactions in the cutaneous environment and contribute to chronic dermatoses, including urticaria, and atopic or contact dermatitis. The cAMP response element binding protein (CREB), an evolutionarily well conserved transcription factor (TF) with over 4,000 binding sites in the genome, was recently found to form a feedforward loop with KIT, maintaining MC survival. The most selective MC function is degranulation with its acute release of prestored mediators. Herein, we asked whether CREB contributes to the expression and function of the degranulation-competent receptors FcεRI and MRGPRX2. Interference with CREB by pharmacological inhibition (CREBi, 666-15) or RNA interference only slightly affected the expression of these receptors, while KIT was strongly attenuated. Interestingly, MRGPRX2 surface expression moderately increased following CREB-knockdown, whereas MRGPRX2-dependent exocytosis simultaneously decreased. FcεRI expression and function were regulated consistently, although the effect was stronger at the functional level. Preformed MC mediators (tryptase, histamine, β-hexosaminidase) remained comparable following CREB attenuation, suggesting that granule synthesis did not rely on CREB function. Collectively, in contrast to KIT, FcεRI and MRGPRX2 moderately depend on unperturbed CREB function. Nevertheless, CREB is required to maintain MC releasability irrespective of stimulus, insinuating that CREB may operate by safeguarding the degranulation machinery. To our knowledge, CREB is the first factor identified to regulate MRGPRX2 expression and function in opposite direction. Overall, the ancient TF is an indispensable component of skin MCs, orchestrating not only survival and proliferation but also their secretory competence. [ABSTRACT FROM AUTHOR]

Background: Saline-alkaline water aquaculture has become a key way to mitigate the reduction of freshwater aquaculture space and meet the increasing global demand for aquatic products. To enhance the comprehensive utilization capability of saline-alkaline water, it is necessary to understand the regulatory mechanisms of aquatic animals coping with saline-alkaline water. In this study, our objective was to elucidate the function of proline metabolism in the alkaline adaptation of Nile tilapia (Oreochromis niloticus). Results: Expose Nile tilapia to alkaline water of different alkalinity for 2 weeks to observe changes in its growth performance and proline metabolism. Meanwhile, to further clarify the role of proline metabolism, RNA interference experiments were conducted to disrupt the normal operation of proline metabolic axis by knocking down pycr (pyrroline-5-carboxylate reductases), the final rate-limiting enzyme in proline synthesis. The results showed that both the synthesis and degradation of proline were enhanced under carbonate alkalinity stress, and the environmental alkalinity impaired the growth performance of tilapia, and the higher the alkalinity, the greater the impairment. Moreover, environmental alkalinity caused oxidative stress in tilapia, enhanced ion transport, ammonia metabolism, and altered the intensity and form of energy metabolism in tilapia. When the expression level of the pycr gene decreased, the proline metabolism could not operate normally, and the ion transport, antioxidant defense system, and energy metabolism were severely damaged, ultimately leading to liver damage and a decreased survival rate of tilapia under alkalinity stress. Conclusions: The results indicated that proline metabolism plays an important role in the alkaline adaptation of Nile tilapia and is a key regulatory process in various biochemical and physiological processes. [ABSTRACT FROM AUTHOR]

Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54's potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation. [ABSTRACT FROM AUTHOR]

Approximately 25% of newly diagnosed AML patients display an internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene. Although both multi-targeted and FLT3 specific tyrosine kinase inhibitors (TKIs) are being utilized for clinical therapy, drug resistance, short remission periods, and high relapse rates are challenges that still need to be tackled. RNA interference (RNAi), mediated by short interfering RNA (siRNA), presents a mechanistically distinct therapeutic platform with the potential of personalization due to its gene sequence-driven mechanism of action. This study explored the use of a non-viral approach for delivery of FLT3 siRNA (siFLT3) in FLT3-ITD positive AML cell lines and primary cells as well as the feasibility of combining this treatment with drugs currently used in the clinic. Treatment of AML cell lines with FLT3 siRNA nanocomplexes resulted in prominent reduction in cell proliferation rates and induction of apoptosis. Quantitative analysis of relative mRNA transcript levels revealed downregulation of the FLT3 gene, which was accompanied by a similar decline in FLT3 protein levels. Moreover, an impact on leukemic stem cells was observed in a small pool of primary AML samples through significantly reduced colony numbers. An absence of a molecular response post-treatment with lipopolymer/siFLT3 complexes in peripheral blood mononuclear cells, obtained from healthy individuals, denoted a passive selectivity of the complexes towards malignant cells. The effect of combining lipopolymer/siFLT3 complexes with daunorubucin and FLT3 targeting TKI gilteritinib led to a significant augmentation of anti-leukemic activity. These findings demonstrate the promising potential of RNAi implemented with lipopolymer complexes for AML molecular therapy. The study prospectively supports the addition of RNAi therapy to current treatment modalities available to target the heterogeneity prevalent in AML. We show that a clinically validated target, the FLT3 gene, can be eradicated in leukemia cells using non-viral RNAi. We validated these lipopolymers as effective vehicles to deliver nucleic acids to leukemic cells. The potency of the lipopolymers was superior to that of the 'gold-standard' delivery agent, lipid nanoparticles (LNPs), which are not effective in leukemia cells at clinically relevant doses. Mechanistic studies were undertaken to probe structure-function relationships for effective biomaterial formulations. Cellular and molecular responses to siRNA treatment have been characterized in cell models, including leukemia patient-derived cells. The use of the siRNA therapy with clinically used chemotherapy was demonstrated. [Display omitted] [ABSTRACT FROM AUTHOR]

The γ-aminobutyric acid (GABA) is a widely distributed neurotransmitter in living organisms, known for its inhibitory role in animals. GABA exerts calming effects on the mind, lowers blood pressure in animals, and enhances stress resistance during the growth and development of plants. Enhancing GABA content in plants has become a focal point of current research. In plants, GABA is synthesized through two metabolic pathways, the GABA shunt and the polyamine degradation pathway, with the GABA shunt being the primary route. Extensive studies have investigated the regulatory mechanisms governing GABA synthesis. At the genetic level, GABA production and degradation can be modulated by gene overexpression, signaling molecule-induced expression, transcription factor regulation, and RNA interference. Additionally, at the level of transporter proteins, increased activity of GABA transporters and proline transporters enhances the transport of glutamate and GABA. The activity of glutamate decarboxylase, a key enzyme in GABA synthesis, along with various external factors, also influences GABA synthesis. This paper summarizes the biological functions, metabolic pathways, and regulatory mechanisms of GABA, providing a theoretical foundation for further research on GABA in plants. [ABSTRACT FROM AUTHOR]

Pleurotus tuber-regium (Fr.) Sing. can evade oxygen by forming sclerotia under oxidative stress, consequently averting the development of hyperoxidative state, during which the expression level of catalase gene (PtCat) is significantly up-regulated. To investigate the relationship between the catalase gene and sclerotia formation, over-expression and interference strains of the PtCat gene were obtained by Agrobacterium tumefaciens-mediated transformation for phenotypic analysis. In the absence of hydrogen peroxide (H2O2) stress, a minor difference was observed in the mycelial growth rate and the activity of antioxidant enzymes between the over-expression and interference strains. However, when exposed to 1–2 mM H2O2, the colony diameter of the over-expression strain was approximately 2–3× that of the interference strain after 8 days of culturing. The catalase activity of the over-expression strain increased by 1000 U/g under 2 mM H2O2 stress, while the interference strain increased by only 250 U/g. After one month of cultivation, the interference strain formed an oval sclerotium measuring 3.5 cm on the long axis and 2 cm on the short axis, while the over-expression strain did not form sclerotia. Therefore, it is concluded that catalase activity regulates the formation of sclerotia in P. tuber-regium. [ABSTRACT FROM AUTHOR]

Pleurotus ostreatus is a crucial commercial mushroom widely cultivated for diverse uses. Scientists have worked on breeding disease-resistant and high-yielding varieties to secure food supply. Studies on the molecular genetic mechanism of growth and development can provide valuable information to facilitate crop breeding programs by genetic engineering. Aegerolysins are pore-forming proteins widely distributed in both prokaryotes and eukaryotes, which are reported to have haemolytic activity and be involved in the early stages of fructification. The present study aimed to explore biological function of a differential expressed aegerolysin gene PriA in P. ostreatus. The expression level of PriA gene was higher in primordium and fruiting body than that in mycelium. The PriA expression in overexpression (OE) and RNAi interference (RNAi) strains was detected by qRT-PCR. The RNAi strains grew at slightly slower rates and advanced producing yellow pigments than the wild type, while OE strains showed no prominent phenotypic characteristics. Furthermore, Pseudomonas tolaasii infection assays showed that the PriA OE strains could enhance mycelia and caps resistance to P. tolaasii. These data demonstrate PriA from P. ostreatus play an essential role in mycelial development and increase antagonism against P. tolaasii. Our study provides some reference information on interactions between edible fungi and pathogenic bacteria and offers a new resistance-conferring gene for breeding. [ABSTRACT FROM AUTHOR]

Spiders are an abundant group of natural enemies preying on insect pests in agroecosystem. But their potential in biological control has not been fully realized due to difficult mass production. One hindrance is the intense intraspecific aggression in spiders. Neurotransmitters such as serotonin play important roles in modulating aggression. Here, we investigated the regulatory function of serotonin (5‐hydroxytryptamine [5‐HT]) signaling in the intraspecific aggression in a wandering spider Pardosa pseudoannulata (Araneae, Lycosidae). The aggression was quantified with 5 escalated aggression behaviors as approach, chasing, lunging, boxing, and biting. Virgin (VG) females exhibited higher aggression levels but less 5‐HT content than post‐reproductive (PR) females. Systemic increase of 5‐HT via 5‐HT injection decreased aggression, while decrease of 5‐HT via RNA interference (RNAi) of the tryptophan hydroxylase gene, increased aggression. The involvement of the four 5‐HT receptors were determined via individual or combined RNAi. Co‐RNAi of the three 5‐HT1 genes increased overall aggression with decreased incidents of approach, chasing, lunging, and increased biting. RNAi of 5‐HT1B decreased approach and increased biting, whereas RNAi of 5‐HT1A or 5‐HT1C did not affect aggression. RNAi of 5‐HT7 decreased approach only. Therefore, different 5‐HT receptor types contribute to different aspects of the inhibitory effects of 5‐HT on aggression and provide several pharmacological targets for manipulating spider aggression. 5‐HT injection did not affect spiders’ predation on their insect prey, the brown planthopper Nilaparvata lugens. The findings reveal 1 neuronal mechanism regulating intraspecific aggression in spiders and provide an insight in developing aggression suppression strategies for spider mass rearing. [ABSTRACT FROM AUTHOR]

The Chinese cordyceps, a unique parasitic complex of Thitarodes/Hepialus ghost moths and Ophiocordyceps sinensis fungus in the Tibetan Plateau, is a highly valuable biological resource for medicine and health foods in Asian countries. Efficient system for artificial cultivation of Chinese cordyceps relies on understanding the gene functions involved in the induction of growing blastospores into hyphae in the larval hemolymph of insect host, during O. sinensis infection. Transcriptome analysis and ribonucleic acid interference (RNA interference) method were employed to identify the key differentially expressed genes and to demonstrate their functions in Thitarodes xiaojinensis. Key larval genes critical for O. sinensis blastospore development or filamentation were identified. Nine of the 20 top upregulated genes encoded cuticles proteins, indicating that these proteins highly activated when the larval hemolymph was full of blastospores. Small interfering RNA (siRNA) knockdown of five larval genes such as Flightin, larval cuticle protein LCP-30, 26-hydroxylase (CYP18A1), cuticle protein 18.6, isoform B, and probable chitinase 3 significantly stimulated the dimorphic transition from blastospores to prehyphae in O. sinensis in the larval hemolymph after 120 h after injection. The expressions of these genes determined by quantitative real-time PCR were suppressed in various levels from 38.64% to 91.54%, compared to the controls. These results demonstrated that injection of the siRNAs of key upregulated genes into the larval hemolymph containing high load of blastospores caused the gene silence in T. xiaojinensis larvae and induced the fungal transition from blastospores to prehyphae, providing novel knowledge on the regulation of O. sinensis fungal dimorphism by Thitarodes host and cues for further study of Thitarodes biology and commercial cultivation of Chinese cordyceps. [ABSTRACT FROM AUTHOR]

Genetic biocontrol technologies present promising and eco-friendly strategies for the management of pest and insect-transmitted diseases. Although considerable advancements achieve in gene drive applications targeting mosquitoes, endeavors to combat agricultural pests have been somewhat restricted. Here, we identify that the testis-specific serine/threonine kinases (TSSKs) family is uniquely expressed in the testes of Cydia pomonella, a prominent global invasive species. We further generated male moths with disrupted the expression of TSSKs and those with TSSKs disrupted using RNA interference and CRISPR/Cas9 genetic editing techniques, resulting in significant disruptions in spermiogenesis, decreased sperm motility, and hindered development of eggs. Further explorations into the underlying post-transcriptional regulatory mechanisms reveales the involvement of lnc117962 as a competing endogenous RNA (ceRNA) for miR-3960, thereby regulating TSSKs. Notably, orchard trials demonstrates that the release of male strains can effectively suppress population growth. Our findings indicate that targeting TSSKs could serve as a feasible avenue for managing C. pomonella populations, offering significant insights and potential strategies for controlling invasive pests through genetic sterile insect technique (gSIT) technology. Functional analysis reveals that targeting testisspecific serine/threonine protein kinase (TSSKs) is a feasible avenue for controlling invasive pest Cydia pomonella through genetic sterile insect technique (gSIT) technology. [ABSTRACT FROM AUTHOR]

Juvenile hormones (JHs) play a crucial role in regulating development and reproduction in insects. Most insects predominantly synthesize JH III, which typically involves esterification followed by epoxidation, lepidopteran insects use a pathway of epoxidation followed by esterification. Although hemipteran insects have JH III and JH skipped bisepoxide III (JH SB3), the synthesis pathway and key epoxidases remain unclear. This study was conducted on Aphis craccivora, and demonstrated that corpora allata, microsomes, Ac‐CYP15C1, and Ac‐JHAMT catalyze JH III production in vitro, establishing the pathway of epoxidation followed by esterification. These findings were further confirmed through RNA interference and molecular docking. The presence of JH III and JH SB3 in A. craccivora was identified, and their synthesis pathway was elucidated as follows: Ac‐CYP15C1 oxidizes farnesic acid to JH A, followed by methylation to JH III by Ac‐JHAMT, possibly providing an epoxidation site on the second carbon for JH SB3. This alteration may significantly contribute to the differentiation and functional diversification of JH types in insects. [ABSTRACT FROM AUTHOR]

Breast cancer (BC) is one of the frequent tumors that seriously endanger the physical and mental well-being in women with strong heterogeneity, and its pathogenesis involves multiple risk factors. Depending on the type of BC, hormonal therapy, targeted therapy, and immunotherapy are the current systemic treatment options along with conventional chemotherapy. Despite significant progress in understanding BC pathogenesis and therapeutic options, there is still a need to identify new therapeutic targets and develop more effective treatments. According to recent sequencing and profiling studies, non-coding (nc) RNAs genes are deregulated in human cancers via deletion, amplification, abnormal epigenetic, or transcriptional regulation, and similarly, the expression of many ncRNAs is altered in breast cancer cell lines and tissues. The ability of single ncRNAs to regulate the expression of multiple downstream gene targets and related pathways provides a theoretical basis for studying them for cancer therapeutic drug development and targeted delivery. Therefore, it is far-reaching to explore the role of ncRNAs in tumor development and their potential as therapeutic targets. Here, our review outlines the potential of two major ncRNAs, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) as diagnostic and prognostic biomarkers as well as targets for new therapeutic strategies in breast cancer. [ABSTRACT FROM AUTHOR]

Nanocarrier-based delivery of small interfering RNA (siRNA) has been recognized as a promising approach in cancer treatment. To produce siRNA, short nucleotide sequences are generated exogenously. siRNA inhibits target gene expression in a sequence-specific manner and initiates RNA interference (RNAi) in cells. SiRNA is a recently popularized nucleic acid-based medication that shows unique promise in the treatment of cancer. Before clinical siRNA delivery devices are created, there are still a lot of challenges to be solved. This review covers the potential targets for siRNA drug design, elucidates the characteristics and advantages of siRNA drugs and provides a summary of the available clinical siRNA therapies for cancer treatment. Therapeutically complex siRNA chemical alterations and delivery systems are described, and bio-responsive materials for siRNA release have been classified. This study will support continued advancements in clinical applications of siRNA by acting as a resource for disseminating information for more accurate and effective targeted delivery systems. [ABSTRACT FROM AUTHOR]

Motivation RNA interference (RNAi) has become a widely used experimental approach for post-transcriptional regulation and is increasingly showing its potential as future targeted drugs. However, the prediction of highly efficient siRNAs (small interfering RNAs) is still hindered by dataset biases, the inadequacy of prediction methods, and the presence of off-target effects. To overcome these limitations, we propose an accurate and robust prediction method, OligoFormer, for siRNA design. Results OligoFormer comprises three different modules including thermodynamic calculation, RNA-FM module, and Oligo encoder. Oligo encoder is the core module based on the transformer encoder. Taking siRNA and mRNA sequences as input, OligoFormer can obtain thermodynamic parameters, RNA-FM embedding, and Oligo embedding through these three modules, respectively. We carefully benchmarked OligoFormer against six comparable methods on siRNA efficacy datasets. OligoFormer outperforms all the other methods, with an average improvement of 9% in AUC, 6.6% in PRC, 9.8% in F1 score, and 5.1% in PCC compared to the best method among them in our inter-dataset validation. We also provide a comprehensive pipeline with prediction of siRNA efficacy and off-target effects using PITA score and TargetScan score. The ablation study shows RNA-FM module and thermodynamic parameters improved the performance and accelerated convergence of OligoFormer. The saliency maps by gradient backpropagation and base preference maps show certain base preferences in initial and terminal region of siRNAs. Availability and implementation The source code of OligoFormer is freely available on GitHub at: https://github.com/lulab/OligoFormer. Docker image of OligoFormer is freely available on the docker hub at https://hub.docker.com/r/yilanbai/oligoformer. [ABSTRACT FROM AUTHOR]

Persistent plant viruses are widespread in natural ecosystems. However, little is known about why persistent infection with these viruses may cause little or no harm to their host. Here, we discovered a new polerovirus that persistently infected wild rice plants by deep sequencing and assembly of virus‐derived small‐interfering RNAs (siRNAs). The new virus was named Rice tiller inhibition virus 2 (RTIV2) based on the symptoms developed in cultivated rice varieties following Agrobacterium‐mediated inoculation with an infectious RTIV2 clone. We showed that RTIV2 infection induced antiviral RNA interference (RNAi) in both the wild and cultivated rice plants as well as Nicotiana benthamiana. It is known that virulent virus infection in plants depends on effective suppression of antiviral RNAi by viral suppressors of RNAi (VSRs). Notably, the P0 protein of RTIV2 exhibited weak VSR activity and carries alanine substitutions of two amino acids broadly conserved among diverse poleroviruses. Mixed infection with umbraviruses enhanced RTIV2 accumulation and/or enabled its mechanical transmission in N. benthamiana. Moreover, replacing the alanine at either one or both positions of RTIV2 P0 enhanced the VSR activity in a co‐infiltration assay, and RTIV2 mutants carrying the corresponding substitutions replicated to significantly higher levels in both rice and N. benthamiana plants. Together, our findings show that as a persistent plant virus, RTIV2 carries specific mutations in its VSR gene to weaken viral suppression of antiviral RNAi. Our work reveals a new strategy for persistent viruses to maintain long‐term infection by weak suppression of the host defence response. [ABSTRACT FROM AUTHOR]