Gene Silencing Research Articles

Xenotropic and polytropic retrovirus receptor 1 (XPR1) promotes progression of papillary thyroid carcinoma via the BRAF-ERK1/2-P53 signaling pathway.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Xenotropic and polytropic retrovirus receptor 1 (XPR1), identified as a cellular receptor, plays roles in many pathophysiological processes. However, the underlying function and molecular mechanisms of XPR1 in PTC remain unclear. Therefore, this study aimed to elucidate the role of XPR1 in the process of PTC and the potential mechanisms. RNA-sequencing was performed for gene differential expression analysis in PTC patients' tissues. Immunohistochemical assay, real-time PCR, and western blotting assay were used to determine the expression of XPR1, BRAF, and P53 in PTC tissues. The function of XPR1 on the progression of PTC was explored using in vitro and in vivo experiments. The molecular mechanism of XPR1 was investigated using gene silencing, ELISA, immunofluorescence, western blotting, and real-time PCR assays. We found that XPR1 was markedly upregulated in PTC tissues compared to adjacent noncancerous tissues, suggesting that high expression of XPR1 could be correlated with poor patient disease-free survival in PTC. In addition, the expression of BRAF and P53 in PTC tissues was substantially higher than in adjacent noncancerous tissues. Silencing of XPR1 reduced the proliferation, migration, and invasion capacities of TPC-1 cells in vitro and effectively inhibited the tumorigenecity of PTC in vivo. More importantly, silencing of XPR1 in TPC-1 cells significantly decreased the expression of XPR1, BRAF, and P53 both in vitro and in vivo. Interestingly, we demonstrated that XPR1 may positively activate the BRAF-ERK-P53 signaling pathway, further promoting PTC progression. The findings reveal a crucial role of XPR1 in PTC progression and prognosis via the BRAF-ERK1/2-P53 signaling pathway, providing potential therapeutic targets for treating PTC.

G9a/DNMT1 co-targeting inhibits non-small cell lung cancer growth and reprograms tumor cells to respond to cancer-drugs through SCARA5 and AOX1

The treatment of non-small cell lung cancer (NSCLC) patients has significantly improved with recent therapeutic strategies; however, many patients still do not benefit from them. As a result, new treatment approaches are urgently needed. In this study, we evaluated the antitumor efficacy of co-targeting G9a and DNMT1 enzymes and its potential as a cancer drug sensitizer. We observed co-expression and overexpression of G9a and DNMT1 in NSCLC, which were associated with poor prognosis. Co-targeting G9a/DNMT1 with the drug CM-272 reduced proliferation and induced cell death in a panel of human and murine NSCLC cell lines. Additionally, the transcriptomes of these cells were reprogrammed to become highly responsive to chemotherapy (cisplatin), targeted therapy (trametinib), and epigenetic therapy (vorinostat). In vivo, CM-272 reduced tumor volume in human and murine cell-derived cancer models, and this effect was synergistically enhanced by cisplatin. The expression of SCARA5 and AOX1 was induced by CM-272, and both proteins were found to be essential for the antiproliferative response, as gene silencing decreased cytotoxicity. Furthermore, the expression of SCARA5 and AOX1 was positively correlated with each other and inversely correlated with G9a and DNMT1 expression in NSCLC patients. SCARA5 and AOX1 DNA promoters were hypermethylated in NSCLC, and SCARA5 methylation was identified as an epigenetic biomarker in tumors and liquid biopsies from NSCLC patients. Thus, we demonstrate that co-targeting G9a/DNMT1 is a promising strategy to enhance the efficacy of cancer drugs, and SCARA5 methylation could serve as a non-invasive biomarker to monitor tumor progression.

Using metabolomics to explore the effects of epigenetic-modification strategies on the metabolites of Acanthus ilicifolius L. endophytic fungi against ovarian cancer

Ovarian cancer is a serious threat to women's health and safety. So far, people have discovered more than 130 small molecule compounds of natural origin for anti-tumor, of which approximately 50% are of microbial origin. The Acanthus ilicifolius L. species is primarily distributed in the Guangdong, Hainan, and Guangxi regions of China and grows in tidally accessible coastal areas. Recent studies have revealed that Acanthus ilicifolius L. extracts are endowed with a range of pharmacological properties, including anti-inflammatory, hepatoprotective, antioxidant, and antitumor activities. Endophytic fungi are commonly found in the healthy tissue and organs of medicinal plants. These fungi and the plants they inhabit form mutually beneficial symbiotic relationships. Endophytic fungi produce a series of secondary metabolites, with active substances having shown great economic value and applications prospects in drug research and development as well as for the biological control of plant diseases. Secondary metabolites production by endophytic fungi is regulated by specific gene clusters, and several techniques have been used to stimulate the secondary metabolic processes of fungi, including epigenetic-modification and OSMAC (one strain many compounds) strategies, co-culturing, and gene modification. Among these, epigenetic modification has been shown to be effective; this strategy involves the addition of small-molecule epigenetic modifiers to the culture medium, thereby activating silenced biosynthetic gene clusters without altering the DNA sequences of the fungi. This approach facilitates the expression of silenced genes in endophytic fungi, thereby increasing the number and diversity of secondary metabolites. Furthermore, it assists in overcoming the inhibition of microbial secondary-metabolite synthesis under laboratory conditions, and enhances silenced-gene expressions. The advent of novel analytical techniques and bioinformatics has provided a comprehensive, multifaceted, and holistic understanding of fungal metabolism through the development of metabolomics as a research platform. However, few studies have combined anti-ovarian cancer-activity screening with metabolomic approaches in the search for activity-differentiating metabolites from endophytic fungi under the intervention of epigenetic modifiers. Herein, we investigated the impact of epigenetic modifiers on the secondary metabolites of the endophytic Diaporthe goulteri fungus from Acanthus ilicifolius L. to determine their potential anti-ovarian cancer activities. Crude extracts were obtained by controlling three variables: the number of fermentation days, the type of epigenetic modifier, and its concentration, with activities screened using the CCK-8 (cell counting kit-8) method. Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was subsequently employed for non-targeted metabolomic analysis. A multivariate statistical analysis model was constructed using principal component analysis and orthogonal partial least squares-discriminant analysis, which combines model and variable importance projection, with qualitative screening performed and significant changes (variable importance in the projection (VIP)≥1; P<0. 05) determined. Fifteen differential metabolites were identified in the fungal and epigenetic modification group, primarily comprising polyketides, amino acids, derivatives, alkaloids, and organic acids, including prenderol, glycine, valine, 2-ethylcaproic acid, rubratoxin B, finasteride, 6-silaspiro[5.5]undecane, 1-(2-nitrophenoxy)octane, heptadecene, 1-pentadecene, 11-ketoetiocholanolone, 3-(1-ethyl-1,3,3-trimethyl-2,3-dihydro-1H-inden-5-yl)butanal, N2-benzoylarginine, tabutrex, (3aR,6S,6aS)-6-(4-hydroxy-2-methoxy-2-butanyl)-4,4-dimethylhexahydro-1(2H)-pentalenone, and 8-aminoquinoline. The expressions of prenderol, 1-(2-nitrophenoxy)octane, 3-(1-ethyl-1,3,3-trimethyl-2,3-dihydro-1H-inden-5-yl)butanal, N2-benzoylarginine, and 8-aminoquinoline were downregulated, whereas the expressions of the remaining 10 substances were upregulated. Polyketides were the main components that exhibited higher expressions. This study showed that latent active differential metabolites can be searched by combining anti-ovarian cancer-activity screening with metabolomics analysis, thereby providing a reference for the further development of Acanthus ilicifolius L. resources and the subsequent targeted isolation of active compounds.

Systematical characterization of Rab7 gene family in Gossypium and potential functions of GhRab7B3-A gene in drought tolerance

BackgroundCotton 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.ResultsHerein, 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.ConclusionsThis 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.

Secretory Proteins Are Involved in the Parasitism of Melon by Phelipanche aegyptiaca During the Attachment Stage

Parasitic plants represent a significant challenge in global agriculture, with Broomrape (Orobanche/Phelipanche spp.) being a notable example of a holoparasitic species that targets the roots of host plants. This study employed comparative transcriptomics to investigate the mechanisms underlying the parasitism of P. aegyptiaca on melon, focusing on both resistant and susceptible interactions. The findings indicate that the critical phase of P. aegyptiaca parasitism occurs during the post-attachment stage. It is suggested that peptidases may play a role in the development of invasive cells, while cell wall-degrading enzymes (CWDEs) are likely involved in cell wall modification and degradation, and transferases, elicitors, and effectors may play a role in immune regulation. In this study, 25 tobacco rattle virus (TRV) recombinant vectors were successfully constructed and functionally validated using a host-induced gene silencing assay to explore the functions of candidate-secreted effector proteins. The results revealed that silencing Cluster-107894.0, Cluster-11592.0, and Cluster-12482.0 significantly decreased the parasitism rate of P. aegyptiaca on Nicotiana benthamiana. Notably, Cluster-107849.0 encodes a cellulase with hydrolase activity, Cluster-11592.0 encodes a periodic-dependent kinase inhibitor with phosphoprotein activity, and Cluster-12482.0 encodes a glucan 1,3-β-glucosidase with hydrolase activity. These findings potentially offer a novel theoretical framework and justification for understanding host–parasite plant interactions, and suggest new avenues for developing crop varieties resistant to parasitic infestation.

Ectopic enhancer-enhancer interactions as causal forces driving RNA-directed DNA methylation in gene regulatory regions.

Cis-regulatory elements (CREs) are integral to the spatiotemporal and quantitative expression dynamics of target genes, thus directly influencing phenotypic variation and evolution. However, many of these CREs become highly susceptible to transcriptional silencing when in a transgenic state, particularly when organised as tandem repeats. We investigated the mechanism of this phenomenon and found that three of the six selected flower-specific CREs were prone to transcriptional silencing when in a transgenic context. We determined that this silencing was caused by the ectopic expression of non-coding RNAs (ncRNAs), which were processed into 24-nt small interfering RNAs (siRNAs) that drove RNA-directed DNA methylation (RdDM). Detailed analyses revealed that aberrant ncRNA transcription within the AGAMOUS enhancer (AGe) in a transgenic context was significantly enhanced by an adjacent CaMV35S enhancer (35Se). This particular enhancer is known to mis-activate the regulatory activities of various CREs, including the AGe. Furthermore, an insertion of 35Se approximately 3.5 kb upstream of the AGe in its genomic locus also resulted in the ectopic induction of ncRNA/siRNA production and de novo methylation specifically in the AGe, but not other regions, as well as the production of mutant flowers. This confirmed that interactions between the 35Se and AGe can induce RdDM activity in both genomic and transgenic states. These findings highlight a novel epigenetic role for CRE-CRE interactions in plants, shedding light on the underlying forces driving hypermethylation in transgenes, duplicate genes/enhancers, and repetitive transposons, in which interactions between CREs are inevitable.

Clr4SUV39H1 ubiquitination and non-coding RNA mediate transcriptional silencing of heterochromatin via Swi6 phase separation

Transcriptional silencing by RNAi paradoxically relies on transcription, but how the transition from transcription to silencing is achieved has remained unclear. The Cryptic Loci Regulator complex (CLRC) in Schizosaccharomyces pombe is a cullin-ring E3 ligase required for silencing that is recruited by RNAi. We found that the E2 ubiquitin conjugating enzyme Ubc4 interacts with CLRC and mono-ubiquitinates the histone H3K9 methyltransferase Clr4SUV39H1, promoting the transition from co-transcriptional gene silencing (H3K9me2) to transcriptional gene silencing (H3K9me3). Ubiquitination of Clr4 occurs in an intrinsically disordered region (Clr4IDR), which undergoes liquid droplet formation in vitro, along with Swi6HP1 the effector of transcriptional gene silencing. Our data suggests that phase separation is exquisitely sensitive to non-coding RNA (ncRNA) which promotes self-association of Clr4, chromatin association, and di-, but not tri- methylation instead. Ubc4-CLRC also targets the transcriptional co-activator Bdf2BRD4, down-regulating centromeric transcription and small RNA (sRNA) production. The deubiquitinase Ubp3 counteracts both activities.

Systematic Analysis of Cinnamyl Alcohol Dehydrogenase Family in Cassava and Validation of MeCAD13 and MeCAD28 in Lignin Synthesis and Postharvest Physiological Deterioration

Cassava (Manihot esculenta Crantz) is used as a biomass energy material and an effective supplement for food and feed. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step of lignin biosynthesis and is responsible for various stresses. However, systematic investigations of the CAD gene family in cassava have been poorly understood. In this study, a genome-wide survey and bioinformatics analysis of CAD gene family was performed, transcriptomics, qRT-PCR, gene silencing and stress of yeast cell were used for excavate and validate the candidate MeCADs gene. 36 MeCADs genes unevenly distributed across 12 chromosomes were identified. Through phylogenetic analyses alongside their Arabidopsis counterparts, these MeCADs were divided into four groups, each containing a similar structure and conserved motifs. Interestingly, transcriptome data analysis revealed that 32 MeCAD genes were involved in the postharvest physiological deterioration (PPD) process, whereas 27 MeCAD genes showed significant changes. Additionally, the relative quantitative analysis of 6 MeCAD genes demonstrated that they were sensitive to PPD, suggesting that they may be involved in the regulation of PPD. Silencing MeCAD13 and MeCAD28 further showed that lignin content significantly decreased in the leaves. The wound-stress tolerance of transgenic yeast cells was enhanced after transformation with MeCAD13 and MeCAD28. MeCAD13 and MeCAD28 may play positive roles in lignin biosynthesis and PPD response, respectively. These results provided a systematic functional analysis of MeCADs in cassava and paved a new way to genetically modify lignin biosynthesis and PPD tolerance.

Hippocampal SENP3 mediates chronic stress-induced depression-like behaviors by impairing the CREB-BDNF signaling.

Impaired signaling between cyclic adenosine monophosphate response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in the hippocampus is generally considered to be the cause of depression. The mechanisms underlying the impairment of CREB-BDNF signaling under stress conditions are largely unclear. Small ubiquitin-like modifier (SUMO) specific peptidase 3 (SENP3) is a molecule that can regulate SUMOylation of target proteins related to synaptic plasticity. Its dynamic changes have been reported to be associated with neuronal damage in various models of central nervous disorders such as cerebral ischemia and traumatic brain injury. However, its role in depression is completely unknown. This problem was addressed in the present study. Our results showed that chronic unpredictable stress (CUS) triggered a specific increase in SENP3 expression in the hippocampus of non-stressed mice. Overexpression of SENP3 in the hippocampus of non-stressed mice elicited depression-like behaviors in the tail suspension test, forced swimming test, and sucrose preference test, accompanied by impairment of the CREB-BDNF signaling cascade in the hippocampus. Conversely, genetic silencing of SENP3 in the hippocampus suppressed the development of depression-like behaviors. Furthermore, infusion of SENP3-shRNA into the hippocampus failed to suppress CUS-induced depression-like behaviors when mice received genetic silencing CREB or BDNF in the hippocampus or inhibition of the BDNF receptor by K252a. Taken together, these results suggest that abnormally elevated SENP3 in the hippocampus leads to the development of depression-like behavior by impairing the CREB-BDNF signaling cascade. SENP3 in the hippocampus could be a promising target for the development of new antidepressants.

Recognition of a salivary effector by the TNL protein RCSP promotes effector-triggered immunity and systemic resistance in Nicotiana benthamiana.

Insects secret chemosensory proteins (CSPs) into plant cells as potential effector proteins during feeding. The molecular mechanisms underlying how CSPs activate plant immunity remain largely unknown. We show that CSPs from six distinct insect orders induce dwarfism when overexpressed in Nicotiana benthamiana. Agrobacterium-mediated transient expression of Nilaparvata lugens CSP11 (NlCSP11) triggered cell death and plant dwarfism, both of which were dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), neuregulin 1 (NRG1) and SENESCENCE-ASSOCIATED GENE 101 (SAG101), indicating the activation of effector-triggered immunity (ETI) in N. benthamiana. Overexpression of NlCSP11 led to stronger systemic resistance against Pseudomonas syringae DC3000 lacking effector HopQ1-1 and tobacco mosaic virus, and induced higher accumulation of salicylic acid (SA) in uninfiltrated leaves compared to another effector XopQ that is recognized by a Toll-interleukin-1 receptor (TIR) domain nucleotide-binding leucine-rich repeat receptor (TNL) called ROQ1 in N. benthamiana. Consistently, NlCSP11-induced dwarfism and systemic resistance, but not cell death, were abolished in N. benthamiana transgenic line expressing the SA-degrading enzyme NahG. Through large-scale virus-induced gene silencing screening, we identified a TNL protein that mediates the recognition of CSPs (RCSP), including aphid effector MP10 that triggers resistance against aphids in N. benthamiana. Co-immunoprecipitation, bimolecular fluorescence complementation and AlphaFold2 prediction unveiled an interaction between NlCSP11 and RCSP. Interestingly, RCSP does not contain the conserved catalytic glutamic acid in the TIR domain, which is required for TNL function. Our findings point to enhanced ETI and systemic resistance by a TNL protein via hyperactivation of the SA pathway. Moreover, RCSP is the first TNL identified to recognize an insect effector.

Double Braking Effects of Nanomedicine on Mitochondrial Permeability Transition Pore for Treating Idiopathic Pulmonary Fibrosis.

Mitochondrial permeability transition pore (mPTP) opening is a key hallmark of injured type II alveolar epithelial cells (AECIIs) in idiopathic pulmonary fibrosis (IPF). Inhibiting mPTP opening in AECIIs is considered a potential IPF treatment. Herein, a "double braking" strategy on mPTP by cyclosporin A (CsA) derived ionizable lipid with 3D structure (3D-lipid) binding cyclophilin D (CypD) and siRNA downregulating mitochondrial calcium uniporter (MCU) expression is proposed for treating IPF. 3D-lipid and MCU targeting siRNA (siMCU) are co-assembled to form stable 3D-LNP/siMCU nanoparticles (NPs), along with helper lipids. In vitro results demonstrated that these NPs effectively inhibit mPTP opening by 3D-lipid binding with CypD and siRNA downregulating MCU expression, thereby decreasing damage-associated molecular patterns (DAMPs) release and suppressing epithelial-to-mesenchymal transition (EMT) process in bleomycin-induced A549 cells. In vivo results revealed that 3D-LNP/siMCU NPs effectively ameliorated collagen deposition, pro-fibrotic factors secretion, and fibroblast activation in bleomycin-induced pulmonary fibrosis (PF) mouse models. Moreover, compared to the commercial MC3-based formulation, optimized Opt-MC3/siRNA NPs with incorporating 3D-lipid as the fifth component, showed superior therapeutic efficacy against PF due to their enhanced stability and higher gene silencing efficiency. Overall, the nanomedicine containing 3D-lipid and siMCU will be a promising and potential approach for IPF treatment.

Peroxisomal Localization of Benzyl Alcohol O-Benzoyltransferase HSR201 Is Mediated by a Non-canonical Peroxisomal Targeting Signal and Required for Salicylic Acid Biosynthesis.

The phytohormone salicylic acid (SA) regulates plant responses to various types of environmental stress, particularly pathogen infections. We previously revealed that the benzyl alcohol O-benzoyltransferase HSR201 was required for pathogen signal-induced SA synthesis, and its overexpression together with NtCNL, encoding a cinnamate-coenzyme A ligase, was sufficient for the production of significant amounts of SA in tobacco. We herein examined the subcellular localization of HSR201 and found that it fused to a yellow fluorescent protein localized in peroxisomes. Most peroxisomal matrix proteins possess peroxisomal targeting signal type-1 (PTS1) located at the extreme C terminus or PTS2 located at the N terminus; however, a bioinformatics analysis failed to identify similar signals for HSR201. Deletion and mutation analyses of HSR201 identified one essential (extreme C-terminal Leu46°) and three important (Ile455, Ile456 and Ala459) amino acid residues for its peroxisomal localization. The virus-induced gene silencing (VIGS) of PEX5, a PTS1 receptor, but not PEX7, a PTS2 receptor, compromised the peroxisomal targeting of HSR201 in Nicotiana benthamiana. When overexpressed with NtCNL, HSR201 mutants with reduced or non-peroxisomal targeting induced lower SA levels than the wild type; however, these mutations did not affect the protein stability or activity of HSR201. VIGS of the HSR201 homolog compromised pathogen signal-induced SA accumulation in N. benthamiana, which was complemented by the HSR201 wild type, but not the mutant with non-peroxisomal targeting. These results suggest that the peroxisomal localization of HSR201 is mediated by a non-canonical PTS1 and required for SA biosynthesis.

Protamine-Mediated Tangles Produce Extreme Deoxyribonucleic Acid Compaction.

In sperm cells, protamine replaces histones to compact DNA 10-20 times more than in somatic cells. To characterize the extreme compaction, we employed confocal microscopy and optical tweezers to determine the conformations and stability of protamine-bound λ-DNA. Confocal images show increasing compaction of λ-DNA at increasing protamine concentration. In the presence of protamine, single λ-DNA molecules form tangles that withstand forces strong enough (∼55 pN) for strand separation and shorten the contour length by up to 40% even at high forces, as well as bends and loops that rupture at 10-40 pN forces. Strand separation nucleates tangles, implicating protamine interactions with DNA bases. Molecular dynamics simulations show that Arg sidechains of protamine each form hydrogen bonds with multiple bases, frequently in the form of a wedge between the two strands of DNA. Protamine may participate in both local and higher-order chromatin organization, leading to extreme compaction and global transcription silencing.

RNA interference protocols for gene silencing in the spittlebug Philaenus spumarius, vector of Xylella fastidiosa

RNA interference (RNAi) is double stranded RNA (dsRNA)-based gene silencing mechanism. Exogenous dsRNAs application to crops has raised as a powerful tool to control agricultural pests. In particular, several sap-feeder are important plant pathogens vectors, such as Philaenus spumarius, known as main vector of Xylella fastidiosa (Xf), causal agent of olive quick decline syndrome (OQDS) in southern Italy. Here, dsATP synthase beta (dsATP), dsLaccase (dsLacc) and dsGreen Fluorescent Protein (dsGFP) as control, were provided to spittlebug adults by microinjection or to nymphs fed on dsRNA-treated plant shoots. Treated insects were collected at different time points to monitor silencing efficiency over time, describing significant reduction of transcript levels from 8 to 24 days post treatment. Downregulation of target genes ranged from 2- to 16-fold compared to the corresponding dsGFP controls, where highest silencing effects were generally noticed for ATP synthase beta. Sequencing of libraries obtained from total smallRNA (sRNA) showed the generation of dsRNA-derived sRNAs by RNAi pathway, with majority of reads mapping exclusively on the correspondent dsRNA. Also, we characterized components of a functional RNAi machinery in P. spumarius. Further research is needed to clarify such mechanism, screen effective target lethal genes to reduce vector population and improve delivery strategies.

The Role of Exogenous microRNAs on Human Health: The Plant–Human Trans-Kingdom Hypothesis

MicroRNAs (miRNAs) are small, endogenous, single-stranded RNAs that act on gene silencing at the post-transcriptional level by binding to a target messenger RNA (mRNA), leading to its degradation or inhibiting translation into functional proteins. The key role of miRNAs in development, proliferation, differentiation andapoptosis has been deeply investigated, revealing that deregulation in their expression is critical in various diseases, such as metabolic disorders and cancer. Since these small molecules initially evolved as a mechanism of protection against viruses and transposable elements, the fascinating hypothesis that they can move between organisms both of the same or different species has been postulated. Trans-kingdom is the term used to define the migration that occurs between species. This mechanism has been well analyzed between plants and their pests, in order to boost defense and increase pathogenicity, respectively. Intriguingly, in the last decades, the plant–human trans-kingdom migration via food intake hypothesis arose. In particular, various studies highlighted the ability of exogenous miRNAs, abundant in the mainly consumed plant-derived food, to enter the human body affecting gene expression. Notably, plant miRNAs can resist the strict conditions of the gastrointestinal tract through a methylation step that occurs during miRNA maturation, conferring high stability to these small molecules. Recent studies observed the anti-tumoral, immune modulator and anti-inflammatory abilities of trans-kingdom interaction between plant and human. Here, we depict the existing knowledge and discuss the fascinating plant–human trans-kingdom interaction, highlighting first the eventual role of plant miRNAs from foods on our somatic gene identity card and then the potential impact of using plant miRNAs as novel therapeutic avenues.

Quantitative Characterization of Gene Regulatory Circuits Associated With Fungal Secondary Metabolism to Discover Novel Natural Products.

Microbial genetic circuits are vital for regulating gene expression and synthesizing bioactive compounds. However, assessing their strength and timing, especially in multicellular fungi, remains challenging. Here, an advanced microfluidic platform is combined with a mathematical model enabling precise characterization of fungal gene regulatory circuits (GRCs) at the single-cell level. Utilizing this platform, the expression intensity and timing of 30 transcription factor-promoter combinations derived from two representative fungal GRCs, using the model fungus Aspergillus nidulans are determined. As a proof of concept, the selected GRC combination is utilized to successfully refactor the biosynthetic pathways of bioactive molecules, precisely control their production, and activate the expression of the silenced biosynthetic gene clusters (BGCs). This study provides insights into microbial gene regulation and highlights the potential of platform in fungal synthetic biology applications and the discovery of novel natural products.

Activation, regulation, and effects of the mitochondrial unfolded protein response in endothelial cells

Abstract Introduction Endoplasmic reticulum stress and the resulting unfolded protein response (UPRER) play significant roles in endothelial dysfunction and atherosclerosis. However, the understanding of the mitochondrial unfolded protein response (UPRMito) in endothelial dysfunction is limited. This study aims to characterize the activation, regulation, and effects of the UPRMito to provide a comprehensive understanding of endothelial UPRMito. Methods and Results RNA sequencing and quantitative proteomic analysis were employed to investigate the impact of stressors on mitochondrial (MitoBlock-6, CDDO) and endoplasmic (Tunicamycin) protein homeostasis on the transcriptome and proteome of Human Coronary Artery Endothelial cells (HCAEC, Fig.1). The analysis identified 12,745 unique transcripts and 7,891 unique proteins. Mitocarta annotation highlighted the regulatory effect induced by these stressors, particularly on mitochondrial proteins and transcripts (Fig. 2). Geneset enrichment analyses revealed upregulation of pathways regulated by activating transcription factors 4/5 (ATF4/5) and the integrated stress response marker CHOP. Analysis of protein expression changes unveiled that 21 proteins were upregulated by both CDDO and MitoBlock-6, including essential mitochondrial chaperones (HSP10 and HSP60). In contrast, the UPRER stressor Tunicamycin did not induce canonical UPRMito activation. Additionally, 142 proteins were commonly downregulated, affecting crucial biological processes such as mitochondrial translation, mitochondrial gene expression, and ribosome biogenesis. Importantly, NAD+ADP-ribosyltransferase activity was highly downregulated, involving PARP proteins with roles in DNA damage repair. On a functional level, mitochondrial stressors led to dose-dependent alterations in HCAEC viability, apoptosis, and reactive oxygen species formation (ROS) measured by resazurin-based assays, caspase 3/7 activity, and MitoSox staining, respectively. Low doses and short incubations with mitochondrial stressors promoted endothelial cell health through cytoprotective UPRMito pathways, while prolonged incubation led to unresolvable mitochondrial stress (exemplarily shown in Fig. 3). SiRNA-mediated gene silencing of the UPRMito regulators ATF4 and ATF5 promoted mitochondrial ROS formation in cells treated with mitochondrial stressors. These effects were particularly more pronounced after ATF5-Knockdown, suggesting that ATF5 is the main regulator of the UPRMito in HCAEC (Fig. 4). Moreover, high-dose UPRMito stressors led to an impairment in mitochondrial membrane integrity with cytosolic release of pro-inflammatory mtDNA. Conclusion UPRMito and UPRER lead to a different stress response. Depending on the underlying conditions, both stress responses can exert both cytoprotective and cytotoxic effects. Further studies are required to analyze the in vivo relevance and possible therapeutic exploitation of the UPRMito in endothelial dysfunction and atherosclerosis.Figures

Targeting the histone methyltransferase SETD7 rescues diabetes-induced impairment of angiogenic response by transcriptional repression of semaphorin 3G

Abstract Background Peripheral artery disease (PAD) is highly prevalent in patients with diabetes (DM) and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in DM patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in this setting. Mono-methylation of histone 3 at lysine 4 (H3K4me1) - a specific epigenetic signature induced by the histone methyltransferase SETD7 - favours an open chromatin thus enabling gene transcription. Purpose To investigate whether SETD7-dependent epigenetic changes modulate angiogenic response in diabetes. Methodology Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro assays, namely cell migration and tube formation were employed to study angiogenic properties in HAECs. SETD7 and H3K4me1 levels were investigated by Western blot and Chromatin immunoprecipitation (ChIP). Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor (R)-PFI-2. Mice with streptozotocin-induced diabetes were orally treated with (R)-PFI-2 or vehicle and underwent hindlimb ischemia by femoral artery ligation for 14 days. Blood flow recovery was analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Our experimental findings were also translated in gastrocnemius muscle samples from patients with and without diabetes. Results RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with increased H3K4me1 levels as well as with impaired endothelial cell migration and tube formation. Both SETD7 gene silencing and pharmacological inhibition by (R)PFI-2 rescued hyperglycemia-induced impairment of HAECs migration and tube formation, while SETD7 overexpression blunted the angiogenic response. RNA-seq and ChIP assays showed that SETD7-dependent H3K4me1 regulates the transcription of the angiogenesis inhibitor semaphorin-3G (SEMA-3G). Moreover, SEMA-3G overexpression blunted migration and tube formation in SETD7-depleted HAECs. In diabetic mice with hindlimb ischemia, treatment with (R)-PFI-2 improved limb vascularization and perfusion as compared to vehicle. Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients as compared to controls. Conclusion Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD.

CaWRKY20 Negatively Regulates Plant Resistance to Colletotrichum scovillei in Pepper.

Chili anthracnose, a fungal disease caused by Colletotrichum scovillei, is among the most devastating diseases affecting pepper (Capsicum annuum L.). Although WRKY transcription factors play important roles in plant immunity, it is unknown how WRKY gene family members contribute to pepper plant resistance to C. scovillei. Here, CaWRKY20 was found to negatively regulate pepper resistance to C. scovillei, which was demonstrated by virus-induced gene silencing and transient overexpression in pepper. Moreover, overexpression of CaWRKY20 enhanced susceptibility to C. scovillei in tomato. Additionally, our findings demonstrated that CaWRKY20 can indirectly regulate the expression of salicylic acid (SA)-related defense genes (CaPR1, CaPR10 and CaSAR8.2) as well as reactive oxygen species (ROS)-scavenging enzyme genes (CaCAT, CaPOD and CaSOD) in response to C. scovillei. In addition, CaWRKY20 was found to interact with CaMIEL1 in the nucleus to regulate the defense response to C. scovillei in pepper. Furthermore, CaWRKY20 directly bound to the W-box in the promoter of SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (CaSARD1) and suppressed its expression, resulting in reduced resistance to C. scovillei. These results will clarify the mechanism by which WRKY transcription factors are involved in pepper disease resistance and can thus facilitate molecular breeding for anthracnose-resistant varieties.

Regenerative Outcomes of Combining siCOL1A2 Hydrogel with Acupuncture in a Rat Model of Chronic Intervertebral Disc Degeneration

Intervertebral disc degeneration (IVDD) is a significant cause of chronic pain and disability, necessitating innovative therapeutic strategies. This study investigates the combined effect of a novel siCOL1A2-encapsulated hydrogel and acupuncture on IVDD in a rat model. We developed a hydrogel system, siCOL1A2-encapsulated G5-PBA hydrogel (siCOL1A2@G5-PBA@Gel), designed for sustained siRNA delivery to the degenerated discs and assessed its therapeutic efficacy alongside acupuncture treatment. Key inflammatory genes were identified through RNA-seq analysis, with COL1A2 highlighted as a crucial regulator of inflammatory responses in IVDD. Our in vivo experiments involved treating rats with hydrogel alone, acupuncture alone, and combining both. The treatments were evaluated through behavioral pain assessments, imaging techniques (X-ray and MRI), and histological analyses. Results indicated that the combination therapy significantly alleviated pain, reduced inflammation, and promoted disc regeneration more effectively than individual treatments. The hydrogel proved biocompatible and facilitated targeted gene silencing, while acupuncture enhanced therapeutic outcomes by improving local blood circulation and modulating inflammatory responses. These findings suggest that integrating siCOL1A2 hydrogel with acupuncture offers a promising approach to treating IVDD.