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Chitosan shows effective nucleic acid delivery. To understand the influence of chitosan's molecular weight, dose, payload, and hyaluronic acid coating on in vivo toxicity, immune stimulation, biodistribution and efficacy, precisely characterized chitosans were formulated with unmodified or chemically modified siRNA to control for innate immune stimulation. The hemocompatibility, cytokine induction, hematological and serological responses were assessed. Body weight, clinical signs, in vivo biodistribution and functional target knockdown were monitored. Hemolysis was found to be dose- and MW-dependent with the HA coating abrogating hemolysis. Compared to cationic lipid nanoparticles, uncoated and HA-coated chitosan nanoparticles did not induce immune stimulation or hematologic toxicity. Liver and kidney biomarkers remained unchanged with chitosan formulations, while high doses of cationic lipid nanoparticles led to increased transaminase levels and a decrease in body weight. Uncoated and HA-coated nanoparticles accumulated in kidneys with functional knockdown for uncoated chitosan formulations reaching 60%, suggesting potential applications in the treatment of kidney diseases. [ABSTRACT FROM AUTHOR]

Receptor protein kinases (RPKs) critically provide the basic infrastructure to sense, perceive, and conduct the signalling events at the cell surface of organisms. The importance of LRR-RLKs has been well studied in plants, but much less information has been reported in oomycetes. In this work, we have silenced the PcLRR-RK3 and characterised its functional importance in Phytophthora capsici. PcLRR-RK3 was predicted to encode signal peptides, leucine-rich repeats, transmembrane, and kinase domains. PcLRR-RK3-silenced transformants showed impaired colony growth, decreased deformed sporangia, and reduced zoospores count. The mycelium of silenced transformants did not penetrate within the host tissues and showed defects in the pathogenicity of P. capsici. Interestingly, gene silencing also weakens the ability of zoospores germination and penetration into host tissues and fails to produce necrotic lesions. Furthermore, PcLRR-RK3 localisation was found to be the plasma membrane of the cell. Altogether, our results revealed that PcLRR-RK3 was required for the regulation of vegetative growth, zoospores penetration, and establishment into host leaf tissues. [ABSTRACT FROM AUTHOR]

SUMMARY: ERFs (ethylene‐responsive factors) are known to play a key role in orchestrating cold stress signal transduction. However, the regulatory mechanisms and target genes of most ERFs are far from being well deciphered. In this study, we identified a cold‐induced ERF, designated as PtrERF110, from trifoliate orange (Poncirus trifoliata L. Raf., also known as Citrus trifoliata L.), an elite cold‐hardy plant. PtrERF110 is a nuclear protein with transcriptional activation activity. Overexpression of PtrERF110 remarkably enhanced cold tolerance in lemon (Citrus limon) and tobacco (Nicotiana tabacum), whereas VIGS (virus‐induced gene silencing)‐mediated knockdown of PtrERF110 drastically impaired the cold tolerance. RNA sequence analysis revealed that PtrERF110 overexpression resulted in global transcriptional reprogramming of a range of stress‐responsive genes. Three of the genes, including PtrERD6L16 (early responsive dehydration 6‐like transporters), PtrSPS4 (sucrose phosphate synthase 4), and PtrUGT80B1 (UDP‐glucose: sterol glycosyltransferases 80B1), were confirmed as direct targets of PtrERF110. Consistently, PtrERF110‐overexpressing plants exhibited higher levels of sugars and sterols compared to their wild type counterparts, whereas the VIGS plants had an opposite trend. Exogenous supply of sucrose restored the cold tolerance of PtrERF110‐silencing plants. In addition, knockdown of PtrSPS4, PtrERD6L16, and PtrUGT80B1 substantially impaired the cold tolerance of P. trifoliata. Taken together, our findings indicate that PtrERF110 positively modulates cold tolerance by directly regulating sugar and sterol synthesis through transcriptionally activating PtrERD6L16, PtrSPS4, and PtrUGT80B1. The regulatory modules (ERF110‐ERD6L16/SPS4/UGT80B1) unraveled in this study advance our understanding of the molecular mechanisms underlying sugar and sterol accumulation in plants subjected to cold stress. Significance Statement: The transcription factor ERF110 of Poncirus trifoliata L. Raf. functions positively in cold tolerance due to modulation of sugar and sterol biosynthesis by regulating SPS4, ERD6L16, and UGT80B1. [ABSTRACT FROM AUTHOR]

Globodera pallida, an obligate sedentary endoparasite, is a major economic pest that causes substantial potato yield losses. This research aimed to study the effects of gene silencing of three FMRFamide-like peptides (FLPs) genes to reduce G. pallida infestation on potato plants by using kaolinite nanoclay as a carrier to deliver dsRNAs via drenching. A dsRNA dosage of 2.0 mg/ml silenced flp-32c by 89.5%, flp-32p by 94.6%, and flp-2 by 94.3%. J2s incubated for 5 and 10 h showed no phenotypic changes. However, J2s of G. pallida efficiently uptake dsRNA of all targeted genes after 15 h of incubation. On the other hand, J2s that had been kept for 24 h had a rigid and straight appearance. Under fluorescence microscopy, all dsRNA-treated nematodes showed fluorescein isothiocyanate (FITC) signals in the mouth, nervous system, and digestive system. The untreated population of J2s did not show any FITC signals and was mobile as usual. The drenching of potato cultivar Kufri Jyoti with the dsRNA-kaolinite formulations induced deformation and premature death of J2s, compared with untreated J2s that entered J3 or J4 stages. This study validates that the nanocarrier-delivered RNAi system could be employed effectively to manage G. pallida infestations. [ABSTRACT FROM AUTHOR]

The predominant genetic defense mechanism against soybean cyst nematode (SCN) in 95% of the North America market is under threat by virulent SCN populations. Usovsky et al. identified GmSNAP02 as an SCN susceptibility gene through fine-mapping of unique bi-parental populations. Loss-of-function of GmSNAP02 confers enhanced resistance to more virulent SCN. [ABSTRACT FROM AUTHOR]

The selenoenzyme type I iodothyronine deiodinase (DIO1) catalyzes removal of iodine atoms from thyroid hormones. Although DIO1 action is reported to be disturbed in several malignancies, no work has been conducted in high‐grade serous ovarian carcinoma (HGSOC), the most lethal gynecologic cancer. We studied DIO1 expression in HGSOC patients [The Cancer Genome Atlas (TCGA) data and tumor tissues], human cell lines (ES‐2 and Kuramochi), normal Chinese hamster ovarian cells (CHO‐K1), and normal human fallopian tube cells (FT282 and FT109). To study its functional role, DIO1 was overexpressed, inhibited [by propylthiouracil (PTU)], or knocked down (KD), and cell count, proliferation, apoptosis, cell viability, and proteomics analysis were performed. Lower DIO1 levels were observed in HGSOC compared to normal cells and tissues. TCGA analyses confirmed that low DIO1 mRNA expression correlated with worse survival and therapy resistance in patients. Silencing or inhibiting the enzyme led to enhanced ovarian cancer proliferation, while an opposite effect was shown following DIO1 ectopic expression. Proteomics analysis in DIO1‐KD cells revealed global changes in proteins that facilitate tumor metabolism and progression. In conclusion, DIO1 expression and ovarian cancer progression are inversely correlated, highlighting a tumor suppressive role for this enzyme and its potential use as a biomarker in this disease. [ABSTRACT FROM AUTHOR]

Ischemic osteonecrosis, particularly glucocorticoid-induced osteonecrosis of the femoral head (GIONFH), is primarily due to the dysfunction of osteogenesis and angiogenesis. miRNA, as a therapeutic system with immense potential, plays a vital role in the treatment of various diseases. However, due to the unique microenvironmental structure of bone tissue, especially in the case of GIONFH, where there is a deficiency in the vascular system, it is challenging to effectively target and deliver to the ischemic osteonecrosis area. A drug delivery system assisted by genetically engineered cell membranes holds promise in addressing the challenge of targeted miRNA delivery. Herein, we leverage the potential of miR-21 in modulating osteogenesis and angiogenesis to design an innovative biomimetic nanoplatform system. First, we employed metal-organic frameworks (MOFs) as the core structure to load miR-21-m (miR-21-m@MOF). The nanoparticles were further coated with the membrane of bone marrow mesenchymal stem cells overexpressing CXCR4 (CM-miR-21-m@MOF), enhancing their ability to target ischemic bone areas via the CXCR4-SDF1 axis. These biomimetic nanocomposites possess both bone-targeting and ischemia-guiding capabilities, actively targeting GIONFH lesions to release miR-21-m into target cells, thereby silencing PTEN gene and activating the PI3K-AKT signaling pathway to regulate osteogenesis and angiogenesis. This innovative miRNA delivery system provides a promising therapeutic avenue for GIONFH and potentially other related ischemic bone diseases. 1. CXCR4-Engineered Membranes Enhance Targeting for Ischemic Osteonecrosis. 2. miR-21-Based Gene Therapy for Regulating Osteogenesis and Angiogenesis. 3. Expanding the Use of Membrane-Cloaked MOF Nanoparticles. [Display omitted] [ABSTRACT FROM AUTHOR]

Bemisia tabaci (Gennadius) whitefly (BtWf) is an invasive pest that has already spread worldwide and caused major crop losses. Numerous strategies have been implemented to control their infestation, including the use of insecticides. However, prolonged insecticide exposures have evolved BtWf to resist these chemicals. Such resistance mechanism is known to be regulated at the molecular level and systems biology omics approaches could shed some light on understanding this regulation wholistically. In this review, we discuss the use of various omics techniques (genomics, transcriptomics, proteomics, and metabolomics) to unravel the mechanism of insecticide resistance in BtWf. We summarize key genes, enzymes, and metabolic regulation that are associated with the resistance mechanism and review their impact on BtWf resistance. Evidently, key enzymes involved in the detoxification system such as cytochrome P450 (CYP), glutathione S-transferases (GST), carboxylesterases (COE), UDP-glucuronosyltransferases (UGT), and ATP binding cassette transporters (ABC) family played key roles in the resistance. These genes/proteins can then serve as the foundation for other targeted techniques, such as gene silencing techniques using RNA interference and CRISPR. In the future, such techniques will be useful to knock down detoxifying genes and crucial neutralizing enzymes involved in the resistance mechanism, which could lead to solutions for coping against BtWf infestation. [ABSTRACT FROM AUTHOR]

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Rhododendron, a globally popular ornamental flower, is nevertheless limited in our understanding of the mechanisms underlying its fragrance formation. Notably, terpenoids are the most prevalent volatile metabolite produced by plants. In this study, gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS) and transcriptomics sequencing were conducted to analyze the synthesis mechanisms of terpenoid fragrance compounds of petals in fragrant R. fortunei Lindl. (YJ) and non-fragrant R. "Nova Zembla" (NW). The results identified that (-)-myrtenol, linalool, pinene, myrtenyl acetate, and terpineol were key floral aroma substances in YJ. Furthermore, an analysis of KEGG enrichment and differentially expressed genes (DEGs) revealed that the bud and decay stages exhibited the highest number of enriched DEGs among different aroma types, indicating these as critical stages for the synthesis of terpenoid floral compounds. In this study, a structural gene, denoted as RfFDPS, was identified as a negative regulatory gene for monoterpene accumulation and a positive regulatory gene for sesquiterpene accumulation in YJ. Utilizing subcellular localization technology, we determined that RfFDPS proteins are located in the cytoplasm. A functional analysis through transient expression and gene silencing of RfFDPS demonstrated its ability to regulate the accumulation of monoterpenes and sesquiterpenes. The overexpression of RfFDPS led to an increase in the expression of structural genes related to terpenoid synthesis, resulting in a decrease in monoterpenes and an increase in sesquiterpenes. Conversely, gene silencing had the opposite effect. In conclusion, RfFDPS plays a pivotal role in the synthesis and release of terpenoid volatile compounds in YJ petals, laying a solid theoretical foundation for the cultivation and enhancement of aromatic R. species. [ABSTRACT FROM AUTHOR]

Frontonasal malformations are caused by a failure in the growth of the frontonasal prominence during development. Although genetic studies have identified genes that are crucial for frontonasal development, it remains largely unknown how these genes are regulated during this process. Here, we show that microRNAs, which are short non-coding RNAs capable of targeting their target mRNAs for degradation or silencing their expression, play a crucial role in the regulation of genes related to frontonasal development in mice. Using the Mouse Genome Informatics (MGI) database, we curated a total of 25 mouse genes related to frontonasal malformations, including frontonasal hypoplasia, frontonasal dysplasia, and hypotelorism. MicroRNAs regulating the expression of these genes were predicted through bioinformatic analysis. We then experimentally evaluated the top three candidate miRNAs (miR-338-5p, miR-653-5p, and miR-374c-5p) for their effect on cell proliferation and target gene regulation in O9-1 cells, a neural crest cell line. Overexpression of these miRNAs significantly inhibited cell proliferation, and the genes related to frontonasal malformations (Alx1, Lrp2, and Sirt1 for miR-338-5p; Alx1, Cdc42, Sirt1, and Zic2 for miR-374c-5p; and Fgfr2, Pgap1, Rdh10, Sirt1, and Zic2 for miR-653-5p) were directly regulated by these miRNAs in a dose-dependent manner. Taken together, our results highlight miR-338-5p, miR-653-5p, and miR-374c-5p as pathogenic miRNAs related to the development of frontonasal malformations. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is a regulatory and protective mechanism that plays a crucial role in the growth, development, and control of plant responses to pathogens and abiotic stresses. In spray-induced gene silencing (SIGS), exogenous double-stranded RNAs (dsRNA) are used to efficiently regulate target genes via plant surface treatment. In this study, we aimed to evaluate the effect of specific exogenous dsRNAs on silencing different regions (promoter, protein-coding and intron) of the target SlTRY tomato gene, encoding an R3-type MYB repressor of anthocyanin biosynthesis. We also assessed the impact of targeting different SlTRY regions on the expression of genes involved in anthocyanin and flavonoid biosynthesis. This study demonstrated the critical importance of selecting the appropriate gene target region for dsRNA action. The highest inhibition of the SlTRY gene expression and activation of anthocyanin biosynthesis was achieved by dsRNA complementary to the protein-coding region of SlTRY gene, compared with dsRNAs targeting the SlTRY promoter or intron regions. Silencing the SlTRY gene increased the content of anthocyanins and boosted levels of other substances in the phenylpropanoid pathway, such as caffeoyl putrescine, chlorogenic acid, ferulic acid glucoside, feruloyl quinic acid, and rutin. This study is the first to examine the effects of four different dsRNAs targeting various regions of the SlTRY gene, an important negative regulator of anthocyanin biosynthesis. [ABSTRACT FROM AUTHOR]

Gastrointestinal cancer is one of the greatest challenges for biomedical research, accounting for one-quarter of diagnoses and one-third of deaths nowadays, due to the existence of drug resistance mechanisms that prevent therapeutic efficacy in advanced stages. Nanotechnology has been shown to be an effective strategy for the evasion of this phenomenon, and gene silencing by siRNA makes it possible to decrease the expression of certain genes involved in chemoresistance and tumor progression. Our review analyzed studies published during the last 5 years that combined siRNA gene inhibition and chemotherapy as treatment of different gastrointestinal tumors. This review was carried out by searching PubMed, SCOPUS and WoS databases, where 49 articles were finally selected. The results showed that simultaneous encapsulation of siRNA targeting different genes involved in cancer and chemotherapy were more effective at the preclinical level compared to the administration of both treatments individually. The cytotoxic effect was generated through increased induction of apoptosis derived from the dysregulation of chemoresistance-related pathways, producing a decrease in tumor volume and an increase in survival of mice in in vivo assays. Therefore, the combination of both therapies in the same nanoformulation appears to be an interesting therapeutic strategy for the treatment of gastrointestinal tumors. [ABSTRACT FROM AUTHOR]

Macrophages represent primary players of the innate immune system. Macrophage activation triggers several signaling pathways and is tightly associated with metabolic changes, which drive different immune subsets. Recent studies unveil the role of various metabolic enzymes in macrophage activation. Here, we show that malic enzyme 1 (ME1) is overexpressed in LPS-induced macrophages. Through chromatin immunoprecipitation, we demonstrate that ME1 transcriptional regulation is under control of NF-κB. Furthermore, ME1 activity is also increased in activated human PBMC-derived macrophages. Notably, ME1 gene silencing decreases nitric oxide as well as reactive oxygen species and prostaglandin E2 inflammatory mediators. Therefore, modulating ME1 provides a potential approach for immunometabolic regulation and in turn macrophage function. [ABSTRACT FROM AUTHOR]

BACKGROUND: Aphids (Hemiptera: Aphididae) are notorious sap‐sucking insects that rampantly threaten agricultural production worldwide. Current management against aphids in the field heavily relies on chemical pesticides, which makes economical and eco‐friendly methods urgently needed. Spray‐induced gene silencing (SIGS) offers a powerful and precise approach to pest management. However, the high costs and instability of double‐stranded RNA (dsRNA) regulators applied for downstream RNA interference (RNAi) still limit this strategy. It remains uncertain if RNAi regulators applied in SIGS could extend to small RNA (sRNA), especially miRNA. RESULTS: We chose two sRNA sequences, miR‐9b and miR‐VgR, whose corresponding targets ABCG4 and VgR are both essential for aphid growth and development. The efficacy of these sequences was initially verified by chemically synthetic single‐stranded RNA (syn‐ssRNA). Through spray treatment, we observed a significantly decreased survival number and increased abnormality rate of green peach aphids fed on the host under laboratory conditions. Based on our previous study, we generated transgenic plants expressing artificial miR‐9b (amiR‐9b) and miR‐VgR (amiR‐VgR). Remarkably, plant‐derived amiRNA exerted potent and long‐lasting inhibitory efficacy with merely one percent concentration of chemical synthetics. Notably, the simultaneous application of amiR‐9b and amiR‐VgR exhibited superior inhibitory efficacy. CONCLUSION: We explored the potential use of sRNA‐based biopesticide through SIGS while investigating the dosage requirements. To optimize this strategy, the utilization of plant‐derived amiRNA was proposed. The results suggested that attributed to stability and durability, deploying amiRNA in pest management is a potential and promising solution for the field application. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Abstract Background Cotton serves as a primary source of natural fibers crucial for the textile industry. However, environmental elements such as drought have posed challenges to cotton cultivation, resulting in adverse impacts on both production and fiber quality. Improving cotton’s resilience to drought could mitigate yield losses and foster the expansion of cotton farming. Rab7 protein, widely present in organisms, controls the degradation and recycling of cargo, and has a potential role in biotic and abiotic tolerance. However, comprehensive exploration of the Rab7 gene family in Gossypium remains scarce. Results Herein, we identified a total of 10, 10, 20, and 20 Rab7 genes through genome-wide analysis in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. Collinearity analysis unveiled the pivotal role of whole genome or segmental duplication events in the expansion of GhRab7s. Study of gene architecture, conserved protein motifs, and domains suggested the conservation of structure and function throughout evolution. Exploration of cis-regulatory elements revealed the responsiveness of GhRab7 genes to abiotic stress, corroborated by transcriptome analysis under diverse environmental stresses. Notably, the greatly induced expression of GhRab7B3-A under drought treatment prompted us to investigate its function through virus-induced gene silencing (VIGS) assays. Silencing GhRab7B3-A led to exacerbated dehydration and wilting compared with the control. Additionally, inhibition of stomatal closure, antioxidant enzyme activities and expression patterns of genes responsive to abiotic stress were observed in GhRab7B3-A silenced plants. Conclusions This study sheds light on Rab7 members in cotton, identifies a gene linked to drought stress, and paves the way for additional investigation of Rab7 genes associated with drought stress tolerance.

The selenoenzyme type I iodothyronine deiodinase (DIO1) catalyzes removal of iodine atoms from thyroid hormones. Although DIO1 action is reported to be disturbed in several malignancies, no work has been conducted in high‐grade serous ovarian carcinoma (HGSOC), the most lethal gynecologic cancer. We studied DIO1 expression in HGSOC patients [The Cancer Genome Atlas (TCGA) data and tumor tissues], human cell lines (ES‐2 and Kuramochi), normal Chinese hamster ovarian cells (CHO‐K1), and normal human fallopian tube cells (FT282 and FT109). To study its functional role, DIO1 was overexpressed, inhibited [by propylthiouracil (PTU)], or knocked down (KD), and cell count, proliferation, apoptosis, cell viability, and proteomics analysis were performed. Lower DIO1 levels were observed in HGSOC compared to normal cells and tissues. TCGA analyses confirmed that low DIO1 mRNA expression correlated with worse survival and therapy resistance in patients. Silencing or inhibiting the enzyme led to enhanced ovarian cancer proliferation, while an opposite effect was shown following DIO1 ectopic expression. Proteomics analysis in DIO1‐KD cells revealed global changes in proteins that facilitate tumor metabolism and progression. In conclusion, DIO1 expression and ovarian cancer progression are inversely correlated, highlighting a tumor suppressive role for this enzyme and its potential use as a biomarker in this disease.

Abstract Background Small RNA (sRNAs)- mediated RNA silencing is emerging as a key player in host-microbe interactions. However, its role in fungus-plant interactions relevant to biocontrol of plant diseases is yet to be explored. This study aimed to investigate Dicer (DCL)-mediated endogenous and cross-kingdom gene expression regulation in the biocontrol fungus Clonostachys rosea and wheat roots during interactions. Results C. rosea Δdcl2 strain exhibited significantly higher root colonization than the WT, whereas no significant differences were observed for Δdcl1 strains. Dual RNA-seq revealed the upregulation of CAZymes, membrane transporters, and effector coding genes in C. rosea, whereas wheat roots responded with the upregulation of stress-related genes and the downregulation of growth-related genes. The expression of many of these genes was downregulated in wheat during the interaction with DCL deletion strains, underscoring the influence of fungal DCL genes on wheat defense response. sRNA sequencing identified 18 wheat miRNAs responsive to C. rosea, and three were predicted to target the C. rosea polyketide synthase gene pks29. Two of these miRNAs (mir_17532_x1 and mir_12061_x13) were observed to enter C. rosea from wheat roots with fluorescence analyses and to downregulate the expression of pks29, showing plausible cross-kingdom RNA silencing of the C. rosea gene by wheat miRNAs. Conclusions We provide insights into the mechanisms underlying the interaction between biocontrol fungi and plant roots. Moreover, the study sheds light on the role of sRNA-mediated gene expression regulation in C. rosea-wheat interactions and provides preliminary evidence of cross-kingdom RNA silencing between plants and biocontrol fungi.

Trimethylation of histone H3 lysine 9 (H3K9me3) is crucial for the regulation of gene repression and heterochromatin formation, cell-fate determination and organismal development1. H3K9me3 also provides an essential mechanism for silencing transposable elements1-4. However, previous studies have shown that canonical H3K9me3 readers (for example, HP1 (refs. 5-9) and MPP8 (refs. 10-12)) have limited roles in silencing endogenous retroviruses (ERVs), one of the main transposable element classes in the mammalian genome13. Here we report that trinucleotide-repeat-containing 18 (TNRC18), a poorly understood chromatin regulator, recognizes H3K9me3 to mediate the silencing of ERV class I (ERV1) elements such as LTR12 (ref. 14). Biochemical, biophysical and structural studies identified the carboxy-terminal bromo-adjacent homology (BAH) domain of TNRC18 (TNRC18(BAH)) as an H3K9me3-specific reader. Moreover, the amino-terminal segment of TNRC18 is a platform for the direct recruitment of co-repressors such as HDAC-Sin3-NCoR complexes, thus enforcing optimal repression of the H3K9me3-demarcated ERVs. Point mutagenesis that disrupts the TNRC18(BAH)-mediated H3K9me3 engagement caused neonatal death in mice and, in multiple mammalian cell models, led to derepressed expression of ERVs, which affected the landscape of cis-regulatory elements and, therefore, gene-expression programmes. Collectively, we describe a new H3K9me3-sensing and regulatory pathway that operates to epigenetically silence evolutionarily young ERVs and exert substantial effects on host genome integrity, transcriptomic regulation, immunity and development.

Heterochromatin is a gene-repressive protein-nucleic acid ultrastructure that is initially nucleated by DNA sequences. However, following nucleation, heterochromatin can then propagate along the chromatin template in a sequence-independent manner in a reaction termed spreading. At the heart of this process are enzymes that deposit chemical information on chromatin, which attracts the factors that execute chromatin compaction and transcriptional or co/post-transcriptional gene silencing. Given that these enzymes deposit guiding chemical information on chromatin they are commonly termed writers. While the processes of nucleation and central actions of writers have been extensively studied and reviewed, less is understood about how the spreading process is regulated. We discuss how the chromatin substrate is prepared for heterochromatic spreading, and how trans-acting factors beyond writer enzymes regulate it. We examine mechanisms by which trans-acting factors in Suv39, PRC2, SETDB1 and SIR writer systems regulate spreading of the respective heterochromatic marks across chromatin. While these systems are in some cases evolutionarily and mechanistically quite distant, common mechanisms emerge which these trans-acting factors exploit to tune the spreading reaction.

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco (Nicotiana tabacum cv. Petit Havana) plastid transformants that express a fragment of the nuclear-encoded Phytoene desaturase (PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escapes into the cytoplasm. We found multiple lines of direct evidence that plastid-encoded PDS transgenes affect nuclear PDS gene silencing: knockdown of the nuclear-encoded PDS mRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment-deficient plants. Furthermore, plastid-expressed dsRNA with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs generally, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open additional fields of study in plastid development, compartmentalization, and small RNA biogenesis.

Spray-induced gene silencing (SIGS) is an emerging tool for crop pest protection. It utilizes exogenously applied double-stranded RNA to specifically reduce pest target gene expression using endogenous RNA interference machinery. In this study, SIGS methods were developed and optimized for powdery mildew fungi, which are widespread obligate biotrophic fungi that infect agricultural crops, using the known azole-fungicide target cytochrome P450 51 (CYP51) in the Golovinomyces orontii-Arabidopsis thaliana pathosystem. Additional screening resulted in the identification of conserved gene targets and processes important to powdery mildew proliferation: apoptosis-antagonizing transcription factor in essential cellular metabolism and stress response; lipid catabolism genes lipase a, lipase 1, and acetyl-CoA oxidase in energy production; and genes involved in manipulation of the plant host via abscisic acid metabolism (9-cis-epoxycarotenoid dioxygenase, xanthoxin dehydrogenase, and a putative abscisic acid G-protein coupled receptor) and secretion of the effector protein, effector candidate 2. Powdery mildew is the dominant disease impacting grapes and extensive powdery mildew resistance to applied fungicides has been reported. We therefore developed SIGS for the Erysiphe necator-Vitis vinifera system and tested six successful targets identified using the G. orontii-A. thaliana system. For all targets tested, a similar reduction in powdery mildew disease was observed between systems. This indicates screening of broadly conserved targets in the G. orontii-A. thaliana pathosystem identifies targets and processes for the successful control of other powdery mildew fungi. The efficacy of SIGS on powdery mildew fungi makes SIGS an exciting prospect for commercial powdery mildew control.

Regulatory non-coding RNAs (ncRNAs) are increasingly recognized as integral to the control of biological processes. This is often through the targeted regulation of mRNA expression, but this is by no means the only mechanism through which regulatory ncRNAs act. The Gene Ontology (GO) has long been used for the systematic annotation of protein-coding and ncRNA gene function, but rapid progress in the understanding of ncRNAs meant that the ontology needed to be revised to accurately reflect current knowledge. Here, a targeted effort to revise GO terms used for the annotation of regulatory ncRNAs is described, focusing on microRNAs (miRNAs), long non-coding RNAs (lncRNAs), small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). This paper provides guidance to biocurators annotating ncRNA-mediated processes using the GO and serves as background for researchers wishing to make use of the GO in their studies of ncRNAs and the biological processes they regulate.

Plant Snakin/GASA and defensin peptides are cysteine-rich molecules with a wide range of biological functions. They are included within the large family of plant antimicrobial peptides (AMPs), characterized by their structural stability, broad spectrum of activity, and diverse mechanisms of action. The Dilatata group of Paspalum includes five allotetraploids that share an equivalent genomic formula IIJJ. From RNA-seq data of seedling tissues, we performed an in silico characterization of the defensin and Snakin/GASA genes in these species and diploids with a II and JJ genome formula and studied the evolutionary consequences of polyploidy on the expression of the two AMPs families. A total of 107 defensins (distributed in eight groups) and 145 Snakin/GASA (grouped in three subfamilies) genes were identified. Deletions, duplications and/or gene silencing seem to have mediated the evolution of these genes in the allotetraploid species. In defensin genes, the IIJJ allopolyploids retained the I subgenome defensin copies in some of the identified groups supporting the closeness of their nuclear genome with the I subgenome species. In both AMPs families, orthologous genes in tetraploids exhibit higher similarity to each other than with diploids. This data supports the theory of a single origin for the allotetraploids. Several copies of both defensin and Snakin/GASA genes were detected in the five polyploids which could have arisen due to duplication events occurring independently during the diploidization processes in the allotetraploid taxa. [ABSTRACT FROM AUTHOR]

Abstract Background Sclerotinia sclerotiorum is a highly destructive phytopathogenic fungus that poses a significant threat to a wide array of crops. The current constraints in genetic manipulation techniques impede a thorough comprehension of its pathogenic mechanisms and the development of effective control strategies. Results Herein, we present a highly efficient genetic transformation system for S. sclerotiorum, leveraging the use of fusiform nanoparticles, which are synthesized with FeCl3 and 2,6-diaminopyrimidine (DAP). These nanoparticles, with an average longitude length of 59.00 nm and a positively charged surface, facilitate the direct delivery of exogenous DNA into the mycelial cells of S. sclerotiorum, as well as successful integration with stable expression. Notably, this system circumvents fungal protoplast preparation and tedious recovery processes, streamlining the transformation process considerably. Furthermore, we successfully employed this system to generate S. sclerotiorum strains with silenced oxaloacetate acetylhydrolase-encoding gene Ss-oah1. Conclusions Our findings demonstrate the feasibility of using nanoparticle-mediated delivery as a rapid and reliable tool for genetic modification in S. sclerotiorum. Given its simplicity and high efficiency, it has the potential to significantly propel genetic research in filamentous fungi, offering new avenues for elucidating the intricacies of pathogenicity and developing innovative disease management strategies. Graphical Abstract

Abstract Bacterial diseases are one of the most common issues that result in crop loss worldwide, and the increasing usage of chemical pesticides has caused the occurrence of resistance in pathogenic bacteria and environmental pollution problems. Nanomaterial mediated gene silencing is starting to display powerful efficiency and environmental friendliness for improving plant disease resistance. However, the internalization of nanomaterials and the physiological mechanisms behind nano-improved plant disease resistance are still rarely understood. We engineered the polyethyleneimine (PEI) functionalized gold nanoparticles (PEI-AuNPs) with fluorescent properties and ROS scavenging activity to act as siRNA delivery platforms. Besides the loading, protection, and delivery of nucleic acid molecules in plant mature leaf cells by PEI-AuNPs, its fluorescent property further enables the traceability of the distribution of the loaded nucleic acid molecules in cells. Additionally, the PEI-AuNPs-based RNAi delivery system successfully mediated the silencing of defense-regulated gene AtWRKY1. Compared to control plants, the silenced plants performed better resistance to Pseudomonas syringae, showing a reduced bacterial number, decreased ROS content, increased antioxidant enzyme activities, and improved chlorophyll fluorescence performance. Our results showed the advantages of AuNP-based RNAi technology in improving plant disease resistance, as well as the potential of plant nanobiotechnology to protect agricultural production.

Introduction: Members of the family Geminiviridae have been reported to infect either a monocot plant or a dicot plant, but not both. This study reports a geminivirus, Wheat Dwarf India Virus (WDIV), first identified in wheat, that is capable of infecting both monocot and dicot plants and acting as a viral vector. Objectives: This study was aimed at developing a broad host range viral vector system for reverse genetics and genome editing. Methods: Here we used a wheat isolate of WDIV and Ageratum yellow leaf curl betasatellite (AYLCB) for infectivity assays and vector development. We performed Agrobacterium-mediated inoculation of WDIV and AYLCB in wheat, oat, barley, corn, soybean, and tobacco. To examine the potential of WDIV to act as a viral vector, we modified the WDIV genome and cloned DNA fragments of the phytoene desaturase (PDS) genes from wheat and tobacco, separately. For gene editing experiments, tobacco lines expressing Cas9 were infiltrated with a WDIV-based vector carrying gRNA targeting the PDS gene. Results: About 80 to 90% of plants inoculated with infectious clones of WDIV alone or WDIV together with AYLCB showed mild symptoms, whereas some plants showed more prominent symptoms. WDIV and AYLCB were detected in the systemically infected leaves of all the plant species. Furthermore, the inoculation of the WDIV vector carrying PDS fragments induced silencing of the PDS gene in both wheat and tobacco plants. We also observed high-efficiency genome editing in the Cas9-expressing tobacco plants that were inoculated with WDIV vector-carrying gRNA. Conclusion: Detection of WDIV in naturally infected wheat, barley, and sugarcane in the field and its ability to systemically infect wheat, oat, barley, corn, soybean, and tobacco under laboratory conditions, provides compelling evidence that WDIV is the first geminivirus identified with the capability of infecting both monocot and dicot plant species. The wide host range of WDIV can be exploited for developing a single vector system for high-throughput genome editing in many plant species.

Limonium bicolor , known horticulturally as sea lavender, is a typical recretohalophyte with salt glands in its leaf epidermis that secrete excess Na+ out of the plant. Although many genes have been proposed to contribute to salt gland initiation and development, a detailed analysis of alternative splicing, alternative polyadenylation patterns, and long non-coding RNAs (lncRNAs) has been lacking. Here, we applied single-molecule long-read mRNA isoform sequencing (Iso-seq) to explore the complexity of the L. bicolor transcriptome in leaves during salt gland initiation (stage A) and salt gland differentiation (stage B) based on the reference genome. We identified alternative splicing events and the use of alternative poly(A) sites unique to stage A or stage B, leading to the hypothesis that they might contribute to the differentiation of salt glands. Based on the Iso-seq data and RNA in situ hybridization of candidate genes, we selected the lncRNA Btranscript_153392 for gene editing and virus-induced gene silencing to dissect its function. In the absence of this transcript, we observed fewer salt glands on the leaf epidermis, leading to diminished salt secretion and salt tolerance. Our data provide transcriptome resources for unraveling the mechanisms behind salt gland development and furthering crop transformation efforts towards enhanced survivability in saline soils. [ABSTRACT FROM AUTHOR]

Antibacterial drug resistance poses a significant challenge to modern healthcare systems, threatening our ability to effectively treat bacterial infections. This review aims to provide a comprehensive overview of the types and mechanisms of antibacterial drug resistance. To achieve this aim, a thorough literature search was conducted to identify key studies and reviews on antibacterial resistance mechanisms, strategies and next-generation antimicrobials to contain antimicrobial resistance. In this review, types of resistance and major mechanisms of antibacterial resistance with examples including target site modifications, decreased influx, increased efflux pumps, and enzymatic inactivation of antibacterials has been discussed. Moreover, biofilm formation, and horizontal gene transfer methods has also been included. Furthermore, measures (interventions) taken to control antimicrobial resistance and next-generation antimicrobials have been discussed in detail. Overall, this review provides valuable insights into the diverse mechanisms employed by bacteria to resist the effects of antibacterial drugs, with the aim of informing future research and guiding antimicrobial stewardship efforts. [ABSTRACT FROM AUTHOR]

Nonsense‐mediated decay (NMD) and autophagy play pivotal roles in restricting virus infection in plants. However, the interconnection between these two pathways in viral infections has not been explored. Here, it is shown that overexpression of NbSMG7 and NbUPF3 attenuates cucumber green mottle mosaic virus (CGMMV) infection by recognizing the viral internal termination codon and vice versa. NbSMG7 is subjected to autophagic degradation, which is executed by its interaction with one of the autophagy‐related proteins, NbATG8i. Mutation of the ATG8 interacting motif (AIM) in NbSMG7 (SMG7mAIM1) abolishes the interaction and comprises its autophagic degradation. Silencing of NbSMG7 and NbATG8i, or NbUPF3 and NbATG8i, compared to silencing each gene individually, leads to more virus accumulations, but overexpression of NbSMG7 and NbATG8i fails to achieve more potent virus inhibition. When CGMMV is co‐inoculated with NbSMG7mAIM1 or with NbUPF3, compared to co‐inoculating with NbSMG7 in NbATG8i transgene plants, the inoculated plants exhibit milder viral phenotypes. These findings reveal that NMD‐mediated virus inhibition is impaired by the autophagic degradation of SMG7 in a negative feedback loop, and a novel regulatory interplay between NMD and autophagy is uncovered, providing insights that are valuable in optimizing strategies to harness NMD and autophagy for combating viral infections. [ABSTRACT FROM AUTHOR]

This article explores the molecular characteristics and growth patterns of meningiomas in patients with NF2-related schwannomatosis (NF2-SWN). The study involved 11 patients with NF2-SWN and a total of 23 resected meningiomas. The researchers used various techniques, including whole-exome sequencing, RNA sequencing, DNA methylation analysis, and immunohistochemistry, to assess the clinical and molecular characteristics of the tumors. The findings suggest that epigenetic mechanisms, particularly methylation clustering and immune activity, may play a role in the growth patterns of meningiomas in NF2-SWN patients. The study also highlights the presence of macrophage infiltration and immune response in these tumors. [Extracted from the article]

Background: Sclerotinia sclerotiorum is a highly destructive phytopathogenic fungus that poses a significant threat to a wide array of crops. The current constraints in genetic manipulation techniques impede a thorough comprehension of its pathogenic mechanisms and the development of effective control strategies. Results: Herein, we present a highly efficient genetic transformation system for S. sclerotiorum, leveraging the use of fusiform nanoparticles, which are synthesized with FeCl3 and 2,6-diaminopyrimidine (DAP). These nanoparticles, with an average longitude length of 59.00 nm and a positively charged surface, facilitate the direct delivery of exogenous DNA into the mycelial cells of S. sclerotiorum, as well as successful integration with stable expression. Notably, this system circumvents fungal protoplast preparation and tedious recovery processes, streamlining the transformation process considerably. Furthermore, we successfully employed this system to generate S. sclerotiorum strains with silenced oxaloacetate acetylhydrolase-encoding gene Ss-oah1. Conclusions: Our findings demonstrate the feasibility of using nanoparticle-mediated delivery as a rapid and reliable tool for genetic modification in S. sclerotiorum. Given its simplicity and high efficiency, it has the potential to significantly propel genetic research in filamentous fungi, offering new avenues for elucidating the intricacies of pathogenicity and developing innovative disease management strategies. [ABSTRACT FROM AUTHOR]

Royal Jelly (RJ) is a natural substance produced by honeybees, serving not only as nutrition for bee brood and queens but also as a functional food due to its health-promoting properties. Despite its well-known broad-spectrum antibacterial activity, the precise molecular mechanism underlying its antibacterial action has remained elusive. In this study, we investigated the impact of RJ on the bacteria model MG1655 at its half-maximal inhibitory concentration, employing LC–MS/MS to analyze proteomic changes. The differentially expressed proteins were found to primarily contribute to the suppression of gene expression processes, specifically transcription and translation, disrupting nutrition and energy metabolism, and inducing oxidative stress. Notably, RJ treatment led to a marked inhibition of superoxide dismutase and catalase activities, resulting in heightened oxidative damage and lipid peroxidation. Furthermore, through a protein–protein interaction network analysis using the STRING database, we identified CRP and IHF as crucial host regulators responsive to RJ. These regulators were found to play a pivotal role in suppressing essential hub genes associated with energy production and antioxidant capabilities. Our findings significantly contribute to the understanding of RJ's antibacterial mechanism, highlighting its potential as a natural alternative to conventional antibiotics. The identification of CRP and IHF as central players highlights the intricate regulatory networks involved in RJ's action, offering new targets for developing innovative antimicrobial strategies. [ABSTRACT FROM AUTHOR]

Worldwide, pine forests have been threatened by a devastating pine wood disease caused by Bursaphelenchus xylophilus, with Monochamus saltuarius being a newly recorded vector of the disease in Northeast China. The olfactory system plays important roles in both feeding and oviposition during the adult stage of M. saltuarius, and olfactory gene function research is essential for gaining an understanding of the olfactory mechanisms of this pest. However, there is limited information available regarding olfactory gene functions in this pest. In the present study, we selected 7 olfactory genes, including 2 chemosensory proteins, 2 odorant‐binding proteins, the odorant co‐receptor and 2 odorant receptors, which were relatively highly expressed during the adult stage. We silenced these genes by RNA interference (RNAi), and real‐time quantitative PCR was used to detect their expression levels after double‐stranded RNA (dsRNA) injection. The results indicate that these genes were significantly downregulated at 2 d post‐dsRNA injection, and this was sustained until 5 d post‐injection. Electroantennography tests indicated that the knockdown of MsalOBP14 and MsalOrco impaired the olfactory response of M. saltuarius to 11 host volatiles and 1 sex pheromone compound. Y‐tube experiments further confirmed that downregulated MsalOBP14 and MsalOrco expression led to olfactory dysfunction in M. saltuarius, which significantly lost selectivity. The results indicate that MsalOBP14 and MsalOrco play critical roles in sex communication and host volatile detection in M. saltuarius, and possibly represent 2 effective targets for controlling this forest pest through olfactory disruption. [ABSTRACT FROM AUTHOR]

Background: Determining the impact of somatic mutations requires understanding the functional relationship of genes acquiring mutations; however, it is largely unknown how mutations in functionally related genes influence each other. Methods: We employed non-synonymous-to-synonymous or dNdS ratios to evaluate the evolutionary dependency (ED) of gene pairs, assuming a mutation in one gene of a gene pair can affect the evolutionary fitness of mutations in its partner genes as mutation context. We employed PanCancer- and tumor type-specific mutational profiles to infer the ED of gene pairs and evaluated their biological relevance with respect to gene dependency and drug sensitivity. Results: We propose that dNdS ratios of gene pairs and their derived cdNS (context-dependent dNdS) scores as measure of ED distinguishing gene pairs either as synergistic (SYN) or antagonistic (ANT). Mutation contexts can induce substantial changes in the evolutionary fitness of mutations in the paired genes, e.g., IDH1 and IDH2 mutation contexts lead to substantial increase and decrease of dNdS ratios of ATRX indels and IDH1 missense mutations corresponding to SYN and ANT relationship with positive and negative cdNS scores, respectively. The impact of gene silencing or knock-outs on cell viability (genetic dependencies) often depends on ED, suggesting that ED can guide the selection of candidates for synthetic lethality such as TCF7L2-KRAS mutations. Using cell line-based drug sensitivity data, the effects of targeted agents on cell lines are often associated with mutations of genes exhibiting ED with the target genes, informing drug sensitizing or resistant mutations for targeted inhibitors, e.g., PRSS1 and CTCF mutations as resistant mutations to EGFR and BRAF inhibitors for lung adenocarcinomas and melanomas, respectively. Conclusions: We propose that the ED of gene pairs evaluated by dNdS ratios can advance our understanding of the functional relationship of genes with potential biological and clinical implications. [ABSTRACT FROM AUTHOR]

AbstractBackgroundObjectivesMethodsResultsConclusionInterferons (IFNs) represent a first-line defense against viruses and other pathogens. It has been shown that an impaired and uncontrolled release of these glycoproteins can result in tissue damage and explain severe progression of coronavirus disease 2019 (COVID-19). However, their potential role in Long-COVID syndrome (LC) remains debateable.The objective of the present study is to shed further light on the possible role of IFNs (and related genes) gene expression patterns in the progression of COVID-19 and LC patients.We carried out a multi-cohort study by analyzing the IFN gene expression patterns (using different IFN gene signatures) in five cohorts of acute COVID-19 (n = 541 samples) and LC patients (n = 188), and compared them to patterns observed in three autoimmune diseases (systemic lupus erythematous [n = 242], systemic sclerosis [n = 91], and Sjögren’s syndrome [n = 282]).The data show that, while the interferon signatures are strongly upregulated in severe COVID-19 patients and autoimmune diseases, it decays with the time from symptoms onset and in LC patients. Differential pathway analysis of IFN-related terms indicates an over activation in autoimmune diseases (IFN-I/II) and severe COVID-19 (IFN-I/II/III), while these pathways are mostly inactivated or downregulated in LC (IFN-I/III). By analyzing six proteomic LC datasets, we did not find evidence of a role of IFNs in this condition.Our findings suggest a potential role of cytokine exhaustion mediated by IFN gene expression inactivation as a possible driver of LC. [ABSTRACT FROM AUTHOR]

SUMMARY: FLAGELLIN SENSING 2 (FLS2) encodes a pattern recognition receptor that perceives bacterial flagellin. While putative FLS2 orthologs are broadly conserved in plants, their functional characterization remains limited. Here, we report the identification of orthologs in cucumber (Cucumis sativus) and melon (C. melo), named CsFLS2 and CmFLS2, respectively. Homology searching identified CsFLS2, and virus‐induced gene silencing (VIGS) demonstrated that CsFLS2 is required for flg22‐triggered ROS generation. Interestingly, genome re‐sequencing of melon cv. Lennon and subsequent genomic PCR revealed that Lennon has two CmFLS2 haplotypes, haplotype I encoding full‐length CmFLS2 and haplotype II encoding a truncated form. We show that VIGS‐mediated knockdown of CmFLS2 haplotype I resulted in a significant reduction in both flg22‐triggered ROS generation and immunity to a bacterial pathogen in melon cv. Lennon. Remarkably, genomic PCR of CmFLS2 revealed that 68% of tested commercial melon cultivars possess only CmFLS2 haplotype II: these cultivars thus lack functional CmFLS2. To explore evolutionary aspects of CmFLS2 haplotype II occurrence, we genotyped the CmFLS2 locus in 142 melon accessions by genomic PCR and analyzed 437 released sequences. The results suggest that CmFLS2 haplotype II is derived from C. melo subsp. melo. Furthermore, we suggest that the proportion of CmFLS2 haplotype II increased among the improved melo group compared with the primitive melo group. Collectively, these findings suggest that the deleted FLS2 locus generated in the primitive melo subspecies expanded after domestication, resulting in the spread of commercial melon cultivars defective in flagellin recognition, which is critical for bacterial immunity. [ABSTRACT FROM AUTHOR]

Cyanobacteria are important primary producers, contributing to 25% of the global carbon fixation through photosynthesis. They serve as model organisms to study the photosynthesis, and are important cell factories for synthetic biology. To enable efficient genetic dissection and metabolic engineering in cyanobacteria, effective and accurate genetic manipulation tools are required. However, genetic manipulation in cyanobacteria by the conventional homologous recombination-based method and the recently developed CRISPR-Cas gene editing system require complicated cloning steps, especially during multi-site editing and single base mutation. This restricts the extensive research on cyanobacteria and reduces its application potential. In this study, a highly efficient and convenient cytosine base editing system was developed which allows rapid and precise C → T point mutation and gene inactivation in the genomes of Synechocystis and Anabaena. This base editing system also enables efficient multiplex editing and can be easily cured after editing by sucrose counter-selection. This work will expand the knowledge base regarding the engineering of cyanobacteria. The findings of this study will encourage the biotechnological applications of cyanobacteria. An efficient cytosine base editor was developed in cyanobacteria, which allows rapid and precise C → T point mutation and gene inactivation. This genome editing tool will accelerate the metabolic engineering and fundamental researches in cyanobacteria. [ABSTRACT FROM AUTHOR]

Metabolic changes involving the tricarboxylic acid (TCA) cycle have been linked to different non-metabolic cell processes. Among them, apart from cancer and immunity, emerges the DNA damage response (DDR) and specifically DNA damage repair. The oncometabolites succinate, fumarate and 2-hydroxyglutarate (2HG) increase reactive oxygen species levels and create pseudohypoxia conditions that induce DNA damage and/or inhibit DNA repair. Additionally, by influencing DDR modulation, they establish direct relationships with DNA repair on at least four different pathways. The AlkB pathway deals with the removal of N-alkylation DNA and RNA damage that is inhibited by fumarate and 2HG. The MGMT pathway acts in the removal of O-alkylation DNA damage, and it is inhibited by the silencing of the MGMT gene promoter by 2HG and succinate. The other two pathways deal with the repair of double-strand breaks (DSBs) but with opposite effects: the FH pathway, which uses fumarate to help with the repair of this damage, and the chromatin remodeling pathway, in which oncometabolites inhibit its repair by impairing the homologous recombination repair (HRR) system. Since oncometabolites inhibit DNA repair, their removal from tumor cells will not always generate a positive response in cancer therapy. In fact, their presence contributes to longer survival and/or sensitization against tumor therapy in some cancer patients. [ABSTRACT FROM AUTHOR]

CCHC-type zinc finger proteins (CCHC-ZFPs), ubiquitous across plant species, are integral to their growth, development, hormonal regulation, and stress adaptation. Roses (Rosa sp.), as one of the most significant and extensively cultivated ornamentals, account for more than 30% of the global cut-flower market. Despite its significance, the CCHC gene family in roses (Rosa sp.) remains unexplored. This investigation identified and categorized 41 CCHC gene members located on seven chromosomes of rose into 14 subfamilies through motif distribution and phylogenetic analyses involving ten additional plant species, including Ginkgo biloba, Ostreococcus lucimarinus, Arabidopsis thaliana, and others. This study revealed that dispersed duplication likely plays a crucial role in the diversification of the CCHC genes, with the Ka/Ks ratio suggesting a history of strong purifying selection. Promoter analysis highlighted a rich presence of cis-acting regulatory elements linked to both abiotic and biotic stress responses. Differential expression analysis under drought conditions grouped the 41 CCHC gene members into five distinct clusters, with those in group 4 exhibiting pronounced regulation in roots and leaves under severe drought. Furthermore, virus-induced gene silencing (VIGS) of the RcCCHC25 member from group 4 compromised drought resilience in rose foliage. This comprehensive analysis lays the groundwork for further investigations into the functional dynamics of the CCHC gene family in rose physiology and stress responses. [ABSTRACT FROM AUTHOR]

The identification of specialized metabolites isolated from microorganisms is urgently needed to determine their roles in treating cancer and controlling multidrug-resistant pathogens. Naphthoquinones act as anticancer agents in various types of cancers, but some toxicity indicators have been limited in their appropriate application. In this context, new isofuranonaphthoquinones (ifnq) that are less toxic to humans could be promising lead compounds for developing anticancer drugs. The aim of this study is to identify and characterize novel furanonaphthoquinones (fnqs) from Nocardia sp. CS682 and to evaluate their potential therapeutic applications. Analysis of the genome of Nocardia sp. CS682 revealed the presence of a furanonaphthoquinone (fnq) gene cluster, which displays a similar genetic organization and high nucleotide sequence identity to the ifnq gene cluster from Streptomyces sp. RI-77, a producer of the naphthoquinones JBIR-76 and JBIR-77. In this study, the overexpression of the Streptomyces antibiotic regulatory protein (SARP) in Nocardia sp. CS682DR (nargenicin gene-deleted mutant) explicitly produced new fnqs, namely, NOC-IBR1 and NOC-IBR2. Subsequently, the role of the SARP regulator was confirmed by gene inactivation using CRISPR-Cas9 and complementation studies. Furthermore, antioxidant, antimicrobial, and cytotoxicity assays were performed for the isolated compounds, and it was found that NOC-IBR2 exhibited superior activities to NOC-IBR1. In addition, a flexible methyltransferase substrate, ThnM3, was found to be involved in terminal methylation of NOC-IBR1, which was confirmed by in vitro enzyme assays. Thus, this study supports the importance of genome mining and genome editing approaches for exploring new specialized metabolites in a rare actinomycete called Nocardia. [ABSTRACT FROM AUTHOR]

Myelin-forming oligodendrocytes in the vertebrate nervous system co-express the transcription factor Sox10 and its paralog Sox8. While Sox10 plays crucial roles throughout all stages of oligodendrocyte development, including terminal differentiation, the loss of Sox8 results in only mild and transient perturbations. Here, we aimed to elucidate the roles and interrelationships of these transcription factors in fully differentiated oligodendrocytes and myelin maintenance in adults. For that purpose, we conducted targeted deletions of Sox10, Sox8, or both in the brains of two-month-old mice. Three weeks post-deletion, none of the resulting mouse mutants exhibited significant alterations in oligodendrocyte numbers, myelin sheath counts, myelin ultrastructure, or myelin protein levels in the corpus callosum, despite efficient gene inactivation. However, differences were observed in the myelin gene expression in mice with Sox10 or combined Sox8/Sox10 deletion. RNA-sequencing analysis on dissected corpus callosum confirmed substantial alterations in the oligodendrocyte expression profile in mice with combined deletion and more subtle changes in mice with Sox10 deletion alone. Notably, Sox8 deletion did not affect any aspects of the expression profile related to the differentiated state of oligodendrocytes or myelin integrity. These findings extend our understanding of the roles of Sox8 and Sox10 in oligodendrocytes into adulthood and have important implications for the functional relationship between the paralogs and the underlying molecular mechanisms. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) is a severe disease with substantial economic consequences for the swine industry. The DEAD-box helicase 3 (DDX3X) is an RNA helicase that plays a crucial role in regulating RNA metabolism, immunological response, and even RNA virus infection. However, it is unclear whether it contributes to PRRSV infection. Recent studies have found that the expression of DDX3X considerably increases in Marc-145 cells when infected with live PRRSV strains Ch-1R and SD16; however, it was observed that inactivated viruses did not lead to any changes. By using the RK-33 inhibitor or DDX3X-specific siRNAs to reduce DDX3X expression, there was a significant decrease in the production of PRRSV progenies. In contrast, the overexpression of DDX3X in host cells substantially increased the proliferation of PRRSV. A combination of transcriptomics and metabolomics investigations revealed that in PRRSV-infected cells, DDX3X gene silencing severely affected biological processes such as ferroptosis, the FoxO signalling pathway, and glutathione metabolism. The subsequent transmission electron microscopy (TEM) imaging displayed the typical ferroptosis features in PRRSV-infected cells, such as mitochondrial shrinkage, reduction or disappearance of mitochondrial cristae, and cytoplasmic membrane rupture. Conversely, the mitochondrial morphology was unchanged in DDX3X-inhibited cells. Furthermore, silencing of the DDX3X gene changed the expression of ferroptosis-related genes and inhibited the virus proliferation, while the drug-induced ferroptosis inversely promoted PRRSV replication. In summary, these results present an updated perspective of how PRRSV infection uses DDX3X for self-replication, potentially leading to ferroptosis via various mechanisms that promote PRRSV replication. [ABSTRACT FROM AUTHOR]

The application of double-stranded RNAs (dsRNAs) to plant surfaces has emerged as a promising tool for manipulating gene expression in plants and pathogens, offering new opportunities for crop improvement. While research has shown the capability of exogenous dsRNAs to silence genes, the full spectrum of their impact, particularly on the intricate network of microRNAs (miRNAs), remains largely unexplored. Here, we show that the exogenous application of chalcone synthase (CHS)-encoding dsRNA to the rosette leaves of Arabidopsis thaliana induced extensive alterations in the miRNA profile, while non-specific bacterial neomycin phosphotransferase II (NPTII) dsRNA had a minimal effect. Two days after treatment, we detected 60 differentially expressed miRNAs among the 428 miRNAs found in the A. thaliana genome. A total of 59 miRNAs were significantly changed after AtCHS-dsRNA treatment compared with water and NPTII-dsRNA, and 1 miRNA was significantly changed after AtCHS-dsRNA and NPTII-dsRNA compared with the water control. A comprehensive functional enrichment analysis revealed 17 major GO categories enriched among the genes potentially targeted by the up- and downregulated miRNAs. These categories included processes such as aromatic compound biosynthesis (a pathway directly related to CHS activity), heterocycle biosynthesis, RNA metabolism and biosynthesis, DNA transcription, and plant development. Several predicted targets of upregulated and downregulated miRNAs, including APETALA2, SCL27, SOD1, GRF1, AGO2, PHB, and PHV, were verified by qRT-PCR. The analysis showed a negative correlation between the expression of miRNAs and the expression of their predicted targets. Thus, exogenous plant gene-specific dsRNAs induce substantial changes in the plant miRNA composition, ultimately affecting the expression of a wide range of genes. These findings have profound implications for our understanding of the effects of exogenously induced RNA interference, which can have broader effects beyond targeted mRNA degradation, affecting the expression of other genes through miRNA regulation. [ABSTRACT FROM AUTHOR]

Ralstonia pseudosolanacearum, a notorious phytopathogen, is responsible for causing bacterial wilt, leading to significant economic losses globally in many crops within the Solanaceae family. Despite various cultural and chemical control strategies, managing bacterial wilt remains a substantial challenge. This study demonstrates, for the first time, the effective use of plant-induced bacterial gene silencing against R. pseudosolanacearum, facilitated by Tobacco rattle virusmediated gene silencing, to control bacterial wilt symptoms in Nicotiana benthamiana. The methodology described in this study could be utilized to identify novel phytobacterial virulence factors through both forward and reverse genetic approaches. To validate plant-induced gene silencing, small RNA fractions extracted from plant exudates were employed to silence bacterial gene expression, as indicated by the reduction in the expression of GFP and virulence genes in R. pseudosolanacearum. Furthermore, treatment of human and plant pathogenic Gram-negative and Gram-positive bacteria with plantgenerated small RNAs resulted in the silencing of target genes within 48 hours. Taken together, the results suggest that this technology could be applied under field conditions, offering precise, gene-based control of target bacterial pathogens while preserving the indigenous microbiota. [ABSTRACT FROM AUTHOR]

Summary RNA‐dependent RNA Polymerases (RdRPs) synthesize double‐stranded RNA (dsRNA) from a single‐stranded RNA (ssRNA) template. In plants, dsRNAs produced by RdRPs can be further processed into small interfering RNA (siRNAs) with different lengths, ranging from 21 to 24 nucleotides (nt). These siRNAs play a pivotal role in various biological processes, including antiviral responses, transposable elements silencing, DNA methylation, and the regulation of plant reproduction and development. Recent research has reported significant progress in uncovering the molecular mechanisms of plant RNA‐DEPENDENT RNA POLYMERASE 2 (RDR2), a representative RdRP involved in the RNA‐directed DNA methylation (RdDM) pathway. These discoveries provide a molecular basis underlying the principles of RdRP function and offer insights into potential advancements in crop breeding and antiviral defense strategies. [ABSTRACT FROM AUTHOR]

Virus-Induced Gene Silencing (VIGS) is a versatile tool in plant science, yet its application to non-model species like sunflower demands extensive optimization due to transformation challenges. In this study, we aimed to elucidate the factors that significantly affect the efficiency of Agrobacterium-VIGS in sunflowers. After testing a number of approaches, we concluded that the seed vacuum technique followed by 6 h of co-cultivation produced the most efficient VIGS results. Genotype-dependency analysis revealed varying infection percentages (62–91%) and silencing symptom spreading in different sunflower genotypes. Additionally, we explored the mobility of tobacco rattle virus (TRV) and phenotypic silencing manifestation (photo-bleaching) across different tissues and regions of VIGS-infected sunflower plants. We showed the presence of TRV is not necessarily limited to tissues with observable silencing events. Finally, time-lapse observation demonstrated a more active spreading of the photo-bleached spots in young tissues compared to mature ones. This study not only offers a robust VIGS protocol for sunflowers but also provides valuable insights into genotype-dependent responses and the dynamic nature of silencing events, shedding light on TRV mobility across different plant tissues. [ABSTRACT FROM AUTHOR]

Background and Aims: Poor prognosis in lung cancer is associated with tumor-associated macrophages (TAMs) that exhibit M2clike behaviours that support multiple pathways in the tumour microenvironment. The interplay between epigenetic modifications and the ubiquitin-proteasome pathway involves three key mechanisms: regulation of epigenetic enzymes by ubiquitin, interaction between ubiquitin and epigenetic modifiers, and epigenetic silencing of critical genes involved in cellular processes. Therefore, we investigated the effects of ixazomib, a proteasome inhibitor, on gene expression changes in epigenetic chromatin modification enzymes in a co-culture of M2c macrophages and A549 lung cancer cells. Methods: The IC50 concentration of ixazomib was determined to be 2.19 μM using a real-time cell analyser. THP-1 monocytes were polarised into M0 macrophages with 100 ng/mL phorbol 12-myristate 13-acetate (PMA), rested, and then exposed to 1 mM hydrocortisone to become M2c macrophages. A549 cells were seeded in the lower chamber of a co-culture plate. M2c macrophages were then co-cultured with A549 cells for 24 h with or without 2.19 μM ixazomib. After being isolated, mRNA was converted to cDNA and analysed using a gene panel with RT-PCR. Results: The findings showed that 56 genes had exceptionally high expression levels (up to 1848-fold. Ixazomib downregulated these overexpressed genes. Conclusion: Ixazomib effectively modulates the expression of genes involved in epigenetic chromatin modification in the lung cancer microenvironment, indicating its utility in lung cancer therapy. Further studies are needed to explore the combined use of epigenetic drugs and proteasome inhibitors. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a progressive pulmonary disease that leads to interstitial inflammation, lung damage, and eventually life-threatening complications. Among various pathologic factors, Smad4 is a pivotal molecule involved in the progression and exacerbation of IPF. It mediates nuclear transfer of Smad2/Smad3 complexes and initiates the transcription of fibrosis-promoting genes. Thus, the inhibition of Smad4 expression in pulmonary fibroblasts by small interfering RNAs (siRNAs) might be a promising therapeutic strategy for IPF. Herein, we engineered exosome membranes (EM) by cationic lipid (i.e., DOTAP) to load siRNAs against Smad4 (DOTAP/siSmad4@EM), and investigated their specific delivery to pulmonary fibroblasts for treating IPF in a mouse model via pulmonary administration. As reference nanoscaffolds, undecorated DOTAP/siSmad4 complexes (lipoplexes, consisting of cationic lipid DOTAP and siRNAs) and siSmad4-loaded lipid nanoparticles (DOTAP/siSmad4@lipo, consisting of lipoplexes fused with DPPC-Chol liposomes) were also prepared. The results showed that DOTAP/siSmad4@EM exhibited a higher cellular uptake and gene silencing efficacies in mouse pulmonary fibroblasts (viz., MLg2908) as compared to the two reference nanoscaffolds. Furthermore, the outcomes of the in vivo experiments illustrated that DOTAP/siSmad4@EM could significantly down-regulate the Smad4 expression with augmented anti-fibrosis efficiency. Additionally, the DOTAP/siSmad4@EM conferred excellent biocompatibility with low cytokine levels in bronchoalveolar lavage fluid and proinflammatory responses in the pulmonary area. Taken together, the outcomes of our investigation imply that specific inhibition of Smad4 expression in pulmonary fibroblasts by pulmonary administrated DOTAP/siSmad4@EM is a promising therapeutic strategy for IPF, which could safely and effectively deliver siRNA drugs to the targeted site of action. [ABSTRACT FROM AUTHOR]

The standout characteristic of the orchid perianth is the transformation of the upper median petal into a distinctively formed lip, which gives orchid flowers their typically zygomorphic symmetry and makes them the most popular ornamental plants worldwide. To study orchid flower development, two WUSCHEL -related homeobox (WOX) genes, PaWOX3 and PaWOX3B , were identified in Phalaenopsis. PaWOX3 and PaWOX3B mRNAs accumulate abundantly during early reproductive development and perianths of young buds, significantly decreasing in mature flowers and absent in vegetative leaves and roots. PaWOX3 and PaWOX3B virus-induced gene silencing (VIGS) knockdown in Phalaenopsis significantly reduces floral bud numbers, suggesting that PaWOX3/PaWOX3B may be involved in flower initiation. Transgenic Arabidopsis ectopically expressing repressor forms of PaWOX3/PaWOX3B and their Oncidium ortholog, OnPRS , exhibit lateral organ development defects, implicating these genes likely have function in regulating growth and differentiation for lateral organs. Neither PaWOX3, PaWOX3B single nor PaWOX3 / PaWOX3B double VIGS Phalaenopsis altered the flower morphology. Interestingly, double silencing of PaWOX3 or PaWOX3B with OAGL6-2 , which controlled the identity/formation of lips, altered the symmetry of 'BigLip' produced in OAGL6-2 VIGS. This result indicated that the levels of PaWOX3 / PaWOX3B are still sufficient to maintain the symmetry for the OAGL6-2 VIGS 'BigLip'. However, the symmetry of the OAGL6-2 VIGS 'BigLip' cannot be maintained once the expression of PaWOX3 or PaWOX3B is further reduced. Thus, in addition to controlling lip identity, this study further found that OAGL6-2 could cooperate with functionally redundant PaWOX3 / PaWOX3B in maintaining the symmetric axis of lip. [ABSTRACT FROM AUTHOR]