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The emerging class of multi-functional proteins known as moonlighters challenges the one protein, one function mentality by demonstrating crosstalk between biological pathways that were previously thought to be functionally discrete. Here, we present new links between amino acid metabolism and chromatin regulation, two biological pathways that are critical for cellular and organismal homeostasis. We discovered that the threonine biosynthetic pathway is required for the transcriptional silencing of ribosomal DNA (rDNA) in Saccharomyces cerevisiae. The enzymes in the pathway promote rDNA silencing through distinct mechanisms as a subset of silencing phenotypes was rescued with exogenous threonine. In addition, we found that a key pathway enzyme, homoserine dehydrogenase, promotes DNA repair through a mechanism involving the MRX complex, a major player in DNA double strand break repair. These data further the understanding of enzymes with non-canonical roles, here demonstrated within the threonine biosynthetic pathway, and provide insight into their roles as potential anti-fungal pharmaceutical targets.

Transcriptional silencing in Saccharomyces cerevisiae is a persistent and highly stable form of gene repression. It involves DNA silencers and repressor proteins that bind nucleosomes. The silenced state is influenced by numerous factors including the concentration of repressors, nature of activators, architecture of regulatory elements, modifying enzymes and the dynamics of chromatin.Silencers function to increase the residence time of repressor Sir proteins at silenced domains while clustering of silenced domains enables increased concentrations of repressors and helps facilitate long-range interactions. The presence of an accessible NDR at the regulatory regions of silenced genes, the cycling of chromatin configurations at regulatory sites, the mobility of Sir proteins, and the non-uniform distribution of the Sir proteins across the silenced domain, all result in silenced chromatin that only stably silences weak promoters and enhancers via changes in transcription burst duration and frequency.These data collectively suggest that silencing is probabilistic and the robustness of silencing is achieved through sub-optimization of many different nodes of action such that a stable expression state is generated and maintained even though individual constituents are in constant flux.

Transcriptional silencing in Saccharomyces cerevisiae involves the generation of a chromatin state that stably represses transcription. Using multiple reporter assays, a diverse set of upstream activating sequence enhancers and core promoters were investigated for their susceptibility to silencing. We show that heterochromatin stably silences only weak and stress-induced regulatory elements but is unable to stably repress housekeeping gene regulatory elements, and the partial repression of these elements did not result in bistable expression states. Permutation analysis of enhancers and promoters indicates that both elements are targets of repression. Chromatin remodelers help specific regulatory elements to resist repression, most probably by altering nucleosome mobility and changing transcription burst duration. The strong enhancers/promoters can be repressed if silencer-bound Sir1 is increased. Together, our data suggest that the heterochromatic locus has been optimized to stably silence the weak mating-type gene regulatory elements but not strong housekeeping gene regulatory sequences.

XIST noncoding RNA promotes the initiation of X chromosome silencing by recruiting the protein SPEN to one X chromosome in female mammals. The SPEN protein is also called SHARP (SMRT and HDAC-associated repressor protein) and MINT (Msx-2 interacting nuclear target) in humans. SPEN recruits N-CoR2 and HDAC3 to initiate histone deacetylation on the X chromosome, leading to the formation of repressive chromatin marks and silencing gene expression. We dissected the contributions of different RNA and protein regions to the formation of a human XIST-SPEN complex in vitro and identified novel sequence and structure determinants that may contribute to X chromosome silencing initiation. Binding of SPEN to XIST RNA requires RRM 4 of the protein, in contrast to the requirement of RRM 3 and RRM 4 for specific binding to SRA RNA. Measurements of SPEN binding to full-length, dimeric, trimeric, or other truncated versions of the A-repeat region revealed that high-affinity binding of XIST to SPEN in vitro requires a minimum of four A-repeat segments. SPEN binding to XIST A-repeat RNA changes the accessibility of the RNA at specific nucleotide sequences, as indicated by changes in RNA reactivity through chemical structure probing. Based on computational modeling, we found that inter-repeat duplexes formed by multiple A-repeats can present an unpaired adenosine in the context of a double-stranded region of RNA. The presence of this specific combination of sequence and structural motifs correlates with high-affinity SPEN binding in vitro. These data provide new information on the molecular basis of the XIST and SPEN interaction.

Gene therapy has recently become a reality in the treatment of cardiovascular diseases. Strategies to modulate gene expression using antisense oligonucleotides or small interfering RNA are proving to be safe and effective in the clinic. Adeno‐associated viral vector‐based gene delivery and CRISPR‐Cas9‐based genome editing have emerged as efficient strategies for gene delivery and repair in humans. Overall, gene therapy holds the promise not only of expanding current treatment options, but also of intervening in previously untackled causal disease mechanisms with little side effects. This scientific statement provides a comprehensive overview of the various modalities of gene therapy used to treat heart failure and some of its risk factors, and their application in the clinical setting. It discusses specifically the possibilities of gene therapy for hereditary heart diseases and (non)‐genetic heart failure. Furthermore, it addresses safety and clinical trial design issues and challenges for future regulatory strategies. [ABSTRACT FROM AUTHOR]

Summary Cross‐kingdom RNA interference (ckRNAi) is a mechanism of interspecies communication where small RNAs (sRNAs) are transported from one organism to another; these sRNAs silence target genes in trans by loading into host AGO proteins. In this work, we investigated the occurrence of ckRNAi in Arbuscular Mycorrhizal Symbiosis (AMS). We used an in silico prediction analysis to identify a sRNA (Rir2216) from the AM fungus Rhizophagus irregularis and its putative plant gene target, the Medicago truncatula MtWRKY69 transcription factor. Heterologous co‐expression assays in Nicotiana benthamiana, 5′ RACE reactions and AGO1‐immunoprecipitation assays from mycorrhizal roots were used to characterize the Rir2216–MtWRKY69 interaction. We further analyzed MtWRKY69 expression profile and the contribution of constitutive and conditional MtWRKY69 expression to AMS. We show that Rir2216 is loaded into an AGO1 silencing complex from the host plant M. truncatula, leading to cleavage of a host target transcript encoding for the MtWRKY69 transcription factor. MtWRKY69 is specifically downregulated in arbusculated cells in mycorrhizal roots and increased levels of MtWRKY69 expression led to a reduced AM colonization level. Our results indicate that MtWRKY69 silencing, mediated by a fungal sRNA, is relevant for AMS; we thus present the first experimental evidence of fungus to plant ckRNAi in AMS. [ABSTRACT FROM AUTHOR]

Summary Numerous herbivore‐induced plant volatiles (HIPVs) play important roles in plant defense. In tea plants (Camellia sinensis), (Z)‐3‐hexenyl acetate (3‐HAC) has been characterized as associated with resistance to herbivores. To date, how tea plants biosynthesize and regulate 3‐HAC to resist herbivores remain unclear. Based on transcriptomes assembled from Ectropis obliqua‐fed leaves, a cDNA encoding BAHD acyltransferase, namely CsCHAT1, was highly induced in leaves fed with E. obliqua. Enzymatic assays showed that CsCHAT1 converted (Z)‐3‐hexenol into 3‐HAC. Further suppression of CsCHAT1 expression reduced the accumulation of 3‐HAC and lowered the resistance of tea plants to E. obliqua, while 3‐HAC replenishment rescued the reduced resistance of CsCHAT1‐silenced tea plants against E. obliqua. Two transcription factors (TFs), CsNAC30 and CsTCP11, were co‐expressed with CsCHAT1. An integrative approach of biochemistry, DNA–protein interaction, gene silencing, and metabolic profiling revealed that the two TFs positively regulated the expression of CsCHAT1. The suppression of either one decreased the production of 3‐HAC and eliminated the resistance of tea plants to E. obliqua. Notably, the suppression of either one considerably impaired JA‐induced 3‐HAC biosynthesis in tea plant. The proposed pathway can be targeted for innovative agro‐biotechnologies protecting tea plants from damage by E. obliqua. [ABSTRACT FROM AUTHOR]

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

Methylation of the DNA genome plays an important role in viral gene inactivation. However, the role of DNA methylation in human bocavirus (HBoV) remains unclear. In this study, the HBoV1 genomic DNA was found extensively methylated at the CHG and CHH sites. Inhibiting DNA methylation with 5-aza-2′-deoxycytidine (DAC) altered the methylation status and reduced viral DNA production, while enhanced the RNA splicing at D1 and D3 sites and the polyadenylation at the proximal polyadenylation site, (pA)p. Knockdown of DNA methyltransferase 1 (DNMT1) had the same effect on viral DNA synthesis and RNA processing as the DAC treatment, indicating that DNMT1 is the major host methyltransferase involved in viral DNA methylation. In addition, the nonstructural protein NS1 promoted DNMT1 degradation through the ubiquitin-proteasome pathway to regulate viral replication and RNA processing. Collectively, the results suggest that DNA methylation and DNMT1 facilitate HBoV replication and are essential for appropriate NS1 localization in the nucleus. DNMT1 degradation through NS1 promotes the virus RNA processing, leading to viral protein expression. Author summary: DNA methylation is an important epigenetic mark involved in both host biological processes and virus replication cycle. The mechanism underlying the precisely organized viral DNA replication and RNA processing in parvovirus infection is still largely unknown. This study illustrates that these processes are regulated by DNMT1 mediated epigenetic mechanisms. DNA methylation and DNMT1 facilitate HBoV DNA replication but repress its RNA processing. NS1, besides the initiation of DNA replication, promotes the viral RNA processing through DNMT1 degradation by the proteasome pathway. Our finding also indicates that the DNMT1 mediated DNA methylation system is a promising target for preventing HBoV infection. [ABSTRACT FROM AUTHOR]

Background: Echinocandin B (ECB) is a key precursor of the antifungal drug anidulafungin and its biosynthesis occurs via ani gene cluster in Aspergillus nidulans NRRL8112. Strain improvement for industrial ECB production has mainly relied on mutation breeding due to the lack of genetic tools. Results: Here, a CRISPR-base-editing tool was developed in A. nidulans NRRL8112 for simultaneous inactivation of the nkuA gene and two marker genes, pryoA and riboB, which enabled efficient genetic manipulation. Then, in-vivo plasmid assembly was harnessed for ani gene expression screening, identifying the rate-limiting enzyme AniA and a pathway-specific transcription factor AniJ. Stepwise titer enhancement was achieved by overexpressing aniA and/or aniJ, and ECB production reached 1.5 g/L during 5-L fed-batch fermentation, an increase of ~ 30-fold compared with the parent strain. Conclusion: This study, for the first time, revealed the regulatory mechanism of ECB biosynthesis and harnessed genetic engineering for the development of an efficient ECB-producing strain. [ABSTRACT FROM AUTHOR]

Background: Cellular metabolism regulates the pluripotency of embryonic stem cells (ESCs). Yet, how metabolism regulates the transition among different pluripotent states remains elusive. It has been shown that protein lactylation, which uses lactate, a metabolic product of glycolysis, as a substrate, plays a critical role in various biological events. Here we focused on that glycolysis regulates the conversion between ESCs and 2-cell-like cells (2CLCs) through protein lactylation. Methods: RNA-seq revealed the activation of 2-cell (2C) genes by suppression of Ldh. Stable isotope labeling by amino acids in cell culture (SILAC) coupled with lactylated peptide enrichment and quantitative mass spectrometric analysis was carried out to investigate the mechanism how protein lactylation regulates the pluripotent-to-2C transition. And we focused on Hdac1. Lactylation of Hdac1 required for silencing 2C genes was proved by quantitative reverse-transcription PCR (qRT-PCR), immunofluorescence (IF), Western blot and chimeric embryos. Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) and in vitro deacetylation assay confirmed lactylation of Hdac1 promoting its binding at 2C genes and enhancing its deacetylase activity, thereby facilitating the removal of H3K27ac and the silencing of 2C genes. Results: We found that inhibition or depletion of Ldha, the enzyme converting pyruvate to lactate, leads to the activation of 2C genes, as well as reduced global lactylation in ESCs. To investigate the mechanism how protein lactylation regulates the pluripotent-to-2C transition, quantitative lactylome analysis was performed, and 1716 lactylated proteins were identified. We then focused on Hdac1, a histone deacetylase involved in the silencing of 2C genes. Lactylation of Hdac1 promotes its binding at 2C genes and enhances its deacetylase activity, thus facilitating the removal of H3K27ac and the silencing of 2C genes. Conclusions: In summary, our study reveals a mechanistic link between cellular metabolism and pluripotency regulation through protein lactylation. Our research is the first time to reveal that quantitative lactylome analysis in mouse ESCs. We found that lactylated Hdac1 promotes its binding at 2C genes and enhances its deacetylase activity, thus facilitating the removal of H3K27ac and the silencing of 2C genes. [ABSTRACT FROM AUTHOR]

ABSTRACT Cytoplasmic male sterile (CMS) lines play a crucial role in utilization of heterosis in crop plants. However, the mechanism underlying the manipulation of male sterility in cotton by long non‐coding RNA (lncRNA) and brassinosteroids (BRs) remains elusive. Here, using an integrative approach combining lncRNA transcriptomic profiles with virus‐induced gene silencing experiments, we identify a flower bud‐specific lncRNA in the maintainer line 2074B, lncRNA67, negatively modulating with male sterility in upland cotton (Gossypium hirsutum). lncRNA67 positively regulates cytochrome P274B (GhCYP724B), which acted as an eTM (endogenous target mimic) for miR3367. The suppression of GhCYP724B induced symptoms of BR deficiency and male semi‐sterility in upland cotton as well as in tobacco, which resulted from a reduction in the endogenous BR contents. GhCYP724B regulates BRs synthesis by interacting with GhDIM and GhCYP90B, two BRs biosynthesis proteins. Additionally, GhCYP724B suppressed a unique chimeric open reading frame (Aorf27) in 2074A mitochondrial genome. Ectopic expression of Aorf27 in yeast inhibited cellular growth, and over expression of Aorf27 in tobacco showed male sterility. Overall, the results proved that the miR3367–lncRNA67–GhCYP724B module positively regulates male sterility by modulating BRs biosynthesis. The findings uncovered the function of lncRNA67–GhCYP724B in male sterility, providing a new mechanism for understanding male sterility in upland cotton. [ABSTRACT FROM AUTHOR]

Background: Lung adenocarcinoma (LUAD) accounts for the highest proportion of lung cancers; however, specific biomarkers are lacking for diagnosis, treatment, and prognostic assessment. Cell division cycle-associated 8 (CDCA8) is a cell cycle regulator with elevated expression in various cancers. However, the association between CDCA8 expression and LUAD prognosis remains unclear. Methods: The association between CDCA8 and LUAD prognosis was evaluated based on the The Cancer Genome Atlas (TCGA) dataset, and CDCA8 related functions were determined using gene enrichment and gene ontology analyses. We also analyzed the association between CDCA8 expression and immune cell infiltration. Immunohistochemistry was used to determine the differential expression of CDCA8 in tumors and controls. Finally, we evaluated the differences in the sensitivity of different levels of CDCA8 to different anticancer drugs in LUAD. Results: CDCA8 expression was significantly higher in primary LUAD tumors than in normal tissues (P < 0.001). Moreover, Kaplan–Meier survival analysis demonstrated that high CDCA8 expression predicted poor survival in patients with LUAD (P = 0.006). The receiver operating characteristic (ROC) curves indicated that CDCA8 was an effective guide for the diagnosis of LUAD. Functional annotation indicated that CDCA8 might be involved in functions such as p53 stabilization, nucleotide metabolism, RNA-mediated gene silencing, and the G2/M phase checkpoint. Immune infiltration results suggested that CDCA8 was positively correlated with Th2 cells and Tgd and negatively correlated with Eosinophils and Mast cells (P < 0.01). In addition, elevated expression of CDCA8 may increase the sensitivity of patients to certain anticancer drugs. Conclusions: CDCA8 upregulation is significantly associated with poor survival and immune infiltration in patients with LUAD. Our study suggests that CDCA8 can be used as a biomarker for LUAD prognosis and a reference for personalized medication. [ABSTRACT FROM AUTHOR]

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by hypermethylation of expanded CGG repeats (>200) in the FMR1 gene leading to gene silencing and loss of Fragile X Messenger Ribonucleoprotein (FMRP) expression. FMRP plays important roles in neuronal function, and loss of FMRP in mouse and human FXS cell models leads to aberrant synaptic signaling and hyperexcitability. Multiple drug candidates have advanced into clinical trials for FXS, but no efficacious treatment has been identified to date, possibly as a consequence of poor translation from pre-clinical animal models to human. Here, we use a high resolution all-optical electrophysiology platform applied to multiple FXS patient-derived and CRISPR/Cas9-generated isogenic neuronal cell lines to develop a multi-parametric FXS disease phenotype. This neurophysiological phenotype was optimized and validated into a high throughput assay based on the amount of FMRP re-expression and the number of healthy neurons in a mosaic network necessary for functional rescue. The resulting highly sensitive and multiparameter functional assay can now be applied as a discovery platform to explore new therapeutic approaches for the treatment of FXS. Deep functional characterization of Fragile X syndrome patient and isogenic neurons using all-optical electrophysiology and machine learning identifies a validated, FMR1-dependent cellular phenotype compatible with high throughput drug screening. [ABSTRACT FROM AUTHOR]

ABSTRACT Drought is an important factor that affects plant anthocyanin biosynthesis. However, the underlying molecular mechanisms remain elusive. Ethylene response factors (ERFs) are pivotal regulators in plant growth and environmental responses, particularly in anthocyanin biosynthesis. This study investigated the leaf colour transition from green to red in Malus spectabilis under drought conditions. This transition was primarily attributed to the accumulation of anthocyanins, specifically cyanidin‐3,5‐diglucoside and cyanidin‐3‐O‐galactoside. Our findings elucidate the pivotal role of MsERF17 in drought‐induced anthocyanin biosynthesis. Biochemical and molecular analyses showed that MsERF17 positively regulates anthocyanin synthesis by binding to promoters of MsbHLH3 and MsF3′ H, thereby activating their expression. Moreover, transient overexpression and virus‐induced gene silencing of MsERF17 in fruit peel and leaves, respectively, regulated anthocyanin synthesis. The stable transformation of calli further corroborated the positive regulatory function of MsERF17 in anthocyanin biosynthesis. Our results provide novel insights into the mechanism by which MsERF17, induced by ethylene, promotes anthocyanin accumulation through the positive regulation of MsbHLH3 and MsF3'H expression under drought conditions in M. spectabilis leaves. [ABSTRACT FROM AUTHOR]

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. Here, we present the cryoelectron microscopy (cryo-EM) structure of an HP1α dimer bound to an H2A.Z-nucleosome, revealing two distinct HP1α-nucleosome interfaces. The primary HP1α binding site is located at the N terminus of histone H3, specifically at the trimethylated lysine 9 (K9me3) region, while a secondary binding site is situated near histone H2B, close to nucleosome superhelical location 4 (SHL4). Our biochemical data further demonstrates that HP1α binding influences the dynamics of DNA on the nucleosome. It promotes DNA unwrapping near the nucleosome entry and exit sites while concurrently restricting DNA accessibility in the vicinity of SHL4. Our study offers a model for HP1α-mediated heterochromatin maintenance and gene silencing. It also sheds light on the H3K9me-independent role of HP1 in responding to DNA damage. [Display omitted] • The cryo-EM structure of a human HP1α dimer bound to an H2A.Z nucleosome was determined • H3K9me-independent binding of HP1α is mediated by chromodomain-H2B interactions • HP1α promotes DNA unwrapping near the nucleosome entry/exit site The HP1 protein plays a crucial role in maintaining and spreading heterochromatin. Sokolova et al. employed an integrative structural biology approach to determine the structure of human HP1 α in complex with an H2A.Z nucleosome. [ABSTRACT FROM AUTHOR]

Regulating secondary metabolite (SM) in Myxococcus xanthus bears the potential to influence the formation of important natural products with various biological activities. The authors of this study have previously found that the detectable levels of two proteins (4‐hydroxyphenylpyruvate dioxygenase [HppD] and a Hsp90‐like protein  [HtpG]) are affected by ROK inactivation. As evidence, the current study was designed to elucidate the possible role of these two proteins in regulating the SMs’ biosynthesis in this bacterium. To begin with, inactivation of the corresponding genes was carried out, and two mutant strains (M. xanthus hppD− and htpG−) were constructed. Subsequently, high‐performance liquid chromatography coupled with mass spectrometry analysis for the metabolic extracts of the mutants revealed a significant reduction in the production of several SMs, like DKxanthene, myxalamide A, and myxochromide A, in comparison to the wild type. Furthermore, electrophoretic mobility shift assays using purified ROK protein suggested a direct binding on the genes’ promoter region encoding the two proteins under study. It is therefore possible to conclude that hppD and htpG genes are implicated in the bacterium SMs’ biosynthetic regulatory cascade, which seems to be directly regulated by the ROK protein. The present study provides additional evidence to a previous investigation showing the pleiotropic regulatory role of ROK on the production of SMs in M. xanthus. [ABSTRACT FROM AUTHOR]

With the advent of gene editing technologies like CRISPR/Cas9, it has become possible to edit genomic regions of interest for research and therapeutic purposes. These technologies have also been adapted to alter gene expression without changing their DNA sequence, allowing epigenetic edits. While genetic editors make edits by cutting the genome at specified regions, epigenetic editors leverage the same targeting mechanism but act based on the epigenetic modifier fused to them, such as a methyltransferase. Here, we discuss two recently employed epigenetic editors (epi‐editors) that silenced target genes involved in disease to mitigate their effects. Neumann et al. reported the construction of an epigenetic editor called CHARM that could methylate and silence the prion gene in mouse brains and subsequently switch itself off. Additionally, Capelluti et al. developed an epi‐editor called EvoETR that knocked down Pcsk9 in the murine liver to reduce LDL levels. We aim to highlight the design principles underlying the design of these epi‐editors to inform future editor designs. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is one of the primary contributors to cancer-related fatalities, with up to 80% of advanced CRC cases exhibiting mutations in the p53 gene. Unfortunately, the development of new compounds targeting mutant p53 is quite limited. The anticancer effects of Dehydroepiandrosterone (DHEA) on various cancers have been reported. However, the suppressive effect of DHEA on CRC cells harboring wild-type or mutant p53 gene remains controversial. This study emphasized revealing the suppressive mechanism and the effect of DHEA on CRC cell tumorigenesis in the presence of wild-type or mutant p53 gene. We demonstrate that DHEA causes CRC cell death and cell cycle arrest in a dose and time-dependent manner. Notably, DHEA exhibits similar inhibitory effects on CRC cells regardless of the p53 gene status. Further study reveals that DHEA induces endoplasmic reticulum (ER) stress and triggers PERK/eIF2/ATF4/CHOP UPR signaling pathway to activate autophagy followed by apoptosis, which was confirmed by suppression of 4-phenylbutyric acid (an ER stress inhibitor) or knockdown either ATF4 or CHOP. DHEA-induced apoptosis was attenuated by silencing ATG5 gene in either p53+/+ or p53−/− CRC cells, indicating autophagy regulation of apoptosis. Furthermore, DHEA treatment accompanied by bafilomycin A1 (a blocker of autophagosome degradation) leads to the accumulation of ATF4, CHOP, DR5, and p21 levels in CRC cells, implying that the degradative autophagy machinery regulates these four molecules. Consistently, DHEA demonstrates its inhibitory effect by suppressing CRC tumor formation in vivo. Altogether, we provide compelling evidence that DHEA is a potential therapeutic candidate for CRC patient treatment regardless of the p53 status through ER stress-PERK-autophagy-apoptosis axis. [ABSTRACT FROM AUTHOR]

Triple‐negative breast cancer (TNBC) remains a significant challenge in terms of treatment, with limited efficacy of chemotherapy due to side effects and acquired drug resistance. In this study, a threose nucleic acid (TNA)‐mediated antisense approach is employed to target therapeutic Akt genes for TNBC therapy. Specifically, two new TNA strands (anti‐Akt2 and anti‐Akt3) are designed and synthesized that specifically target Akt2 and Akt3 mRNAs. These TNAs exhibit exceptional enzymatic resistance, high specificity, enhance binding affinity with their target RNA molecules, and improve cellular uptake efficiency compared to natural nucleic acids. In both 2D and 3D TNBC cell models, the TNAs effectively inhibit the expression of their target mRNA and protein, surpassing the effects of scrambled TNAs. Moreover, when administered to TNBC‐bearing animals in combination with lipid nanoparticles, the targeted anti‐Akt TNAs lead to reduced tumor sizes and decreased target protein expression compared to control groups. Silencing the corresponding Akt genes also promotes apoptotic responses in TNBC and suppresses tumor cell proliferation in vivo. This study introduces a novel approach to TNBC therapy utilizing TNA polymers as antisense materials. Compared to conventional miRNA‐ and siRNA‐based treatments, the TNA system holds promise as a cost‐effective and scalable platform for TNBC treatment, owing to its remarkable enzymatic resistance, inexpensive synthetic reagents, and simple production procedures. It is anticipated that this TNA‐based polymeric system, which targets anti‐apoptotic proteins involved in breast tumor development and progression, can represent a significant advancement in the clinical development of effective antisense materials for TNBC, a cancer type that lacks effective targeted therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Genes involved in iron deficiency responses have been well characterized in Arabidopsis thaliana, but their roles in crop species have not been well explored. Reliance on model species may fail to identify novel iron stress mechanisms present within crop species, likely selected by hundreds of years of selection. Fiskeby III (PI 438471) is a soybean line from Sweden that demonstrates high levels of resilience to numerous stresses. Earlier Fiskeby III studies have identified a suite of genes responding to iron deficiency stress in Fiskeby III that are also associated with Arabidopsis iron deficiency responses. We were interested in determining how canonical iron genes function in Fiskeby III under normal and iron stress conditions. To investigate this, we used virus-induced gene silencing to knock down gene expression of three iron deficiency response genes (FER-like iron deficiency induced transcription factor (FIT), elongated hypocotyl 5 (HY5) and popeye (PYE)) in Fiskeby III. Analyses of RNAseq data generated from silenced plants in iron-sufficient and -deficient conditions found silencing FIT and HY5 altered general stress responses but did not impact iron deficiency tolerance, confirming Fiskeby III utilizes novel mechanisms to tolerate iron deficiency stress. [ABSTRACT FROM AUTHOR]

The MYB transcription factor family plays a crucial regulatory role in plant growth, development, biological progress, and stress responses. Here, we identified a R2R3-MYB transcription factor gene, SlMYB78-like, from tomato and characterized its function by gene silencing via RNA interference (RNAi). The results exhibited that the silencing of SlMYB78-like reduced the sensitivity of tomato seedlings to exogenous ABA. In addition, when exposed to drought and salt stresses, the RNAi lines grown in soil showed decreased tolerance, with lower ABA accumulation, relative water content, and chlorophyll content while displaying higher relative conductivity and malondialdehyde (MDA) content than the wild type. Moreover, the expression of genes related to chlorophyll biosynthesis, photosynthesis, and ABA biosynthesis/response were down-regulated in SlMYB78-like-silenced lines. Notably, the transcript level of SlCYP707-A2, which encodes a protein involved in ABA degradation, was up-regulated significantly after stresses. The transient expression assay Dual-luciferase (Dual-LUC) and a yeast one-hybrid (Y1H) assay demonstrated that SlMYB78-like bound to the promoter of SlCYP707-A2. Additionally, the physical interaction between SlMYB78-like and SlDREB3, which functioned in ABA signaling transduction, was identified through yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Collectively, our study illustrates that SlMYB78-like participates in the abiotic stress response via the ABA pathway. [ABSTRACT FROM AUTHOR]

Overexposure to ultraviolet (UV) radiation accelerates skin aging, resulting in wrinkle formation, reduced skin elasticity, and hyperpigmentation. UV irradiation induces increased matrix metalloproteinases (MMPs) that degrade collagen in the extracellular matrix. Skin aging is also accompanied by epigenetic alterations such as promoter methylation by DNA methyltransferases, leading to the activation or suppression of gene expression. Although carnitine acetyltransferase (CRAT) is implicated in aging, the effect of UV on the expression of CRAT and regulatory mechanisms of UV-induced MMP-1 expression remain unknown. We investigated changes in CRAT expression upon UV irradiation and its effect on MMP-1 expression. Primary human dermal fibroblasts were UV irradiated with either control or 5-AZA-dC. CRAT knockdown or overexpression was performed to investigate its effect on MMP-1 expression. The mRNA level was analyzed by quantitative real-time PCR, and protein level by western blotting. The expression of CRAT was decreased in UV-irradiated human skin in vivo and in human dermal fibroblasts in vitro. CRAT was downregulated upon UV irradiation by hypermethylation, and treatment with 5-Aza-2′-deoxycytidine, a DNA methyltransferase inhibitor, reversed UV-induced downregulation of CRAT. CRAT knockdown activated the JNK, ERK, and p38 MAPK signaling pathways, which increased MMP-1 expression. Stable overexpression of CRAT alleviated UV-induced MMP-1 induction. CRAT downregulation caused by promoter hypermethylation may play an important role in UV-induced skin aging via upregulation of MMP-1 expression. • CRAT is significantly downregulated by promoter hypermethylation. • CRAT affects skin photoaging by inducing MMP-1 via the ERK, JNK, and p38 pathways. • Epigenetic regulation of CRAT may enable therapeutic approaches for anti-photoaging. [ABSTRACT FROM AUTHOR]

Since its inception, antisense oligonucleotides (ASOs) have been extensively used in functional genomics. Recent advancements in chemical modification and delivery technology extend its applications to the targeted management of crop pests. ASO was conceptualized in the early 1970s and first applied to inhibit retrovirus replication in chickens. The principles of ASOs rely on target complementarity by hybridizing with the mRNA and inhibiting the function. However, the application of ASO received little attention because of its high instability in the biological environment. Initial ASOs, featuring phosphorothioate linkages, laid the groundwork for subsequent advancements, by addressing challenges such as nuclease degradation and cellular uptake limitations. The second generation introduced 2'-modifications to enhance stability and specificity while allowing reversible gene regulation. Third-generation ASOs incorporated innovative chemical modifications like locked nucleic acids (LNAs), peptide nucleic acids (PNAs), phosphoramidate morpholino oligomers (PMOs), 2′-deoxy-2′-fluoro-d-arabinonucleic acid (FANA), cyclohexene nucleic acids (CeNAs), and tricyclo-DNA (tcDNA), offering enhanced efficacy and stability. ASO technology continues to evolve, promising precision medicine and customized therapies for genetic diseases. In agriculture, ASOs selectively target essential genes in pests or pathogens, which would be a novel option for providing precise crop protection and reducing reliance on conventional pesticides. Recent studies demonstrated successful ASO application in thrips, psyllids, Gypsy moths, scale insects, citrus greening, and Zebra-chip diseases. Additionally, ASOs hold the potential for improving crop yield and quality by modulating gene expression related to plant growth, development, and stress responses. They can enhance abiotic stress tolerance, regulate genes associated with specific traits, and offer alternative strategies for managing diseases, such as targeting S-genes in tomatoes for resistance against bacterial spots. These applications antecede the oligo-based pesticides that will revolutionize future agriculture by reducing pesticide uses and harmful impacts on the ecosystem. Further advancements in environmental stability, field efficacy, delivery formulation, and environmental safety would mold this technology into a novel tool for sustainable crop protection. This review highlights the fundamentals of ASO, recent advancements in design chemistry, delivery systems, validation and optimization process, and its potential in customized pest management. This would serve as a ready reckoner for future ASO research and application. [ABSTRACT FROM AUTHOR]

SUMMARY: Cold stress restricts the metabolic and physiological activities of plants, thereby affecting their growth and development. Although broad‐complex, tramtrack, and bric‐à‐brac (BTB) proteins are essential for diverse biological processes and stress responses, the mechanisms underlying BTB‐mediated cold responses remain not fully understood. Here, we characterize the function of the cold‐induced SlBTB19 protein in tomato (Solanum lycopersicum). Overexpression of SlBTB19 resulted in increased plant sensitivity to cold stress, whereas SlBTB19 knockout mutants exhibited a cold‐tolerance phenotype. Further analyses, including protein–protein interaction studies and cell‐free degradation assays, revealed that SlBTB19 interacts with and destabilizes the transcription factor SlWRKY2. Using virus‐induced gene silencing (VIGS) to silence SlWRKY2 in both wild‐type and slbtb19 mutants, we provided genetic evidence that SlWRKY2 acts downstream of SlBTB19 in regulating cold tolerance. Importantly, we demonstrated that SlWRKY2 positively regulates cold tolerance in a CRT/DRE binding factor (CBF)‐dependent manner. Under cold stress, SlWRKY2 binds to the W‐box in the CBF1 and CBF3 promoters, directly activating their expression. In summary, our findings identify a SlBTB19‐SlWRKY2 module that negatively regulates the CBF‐dependent cold tolerance pathway in tomato. Significance Statement: Our findings not only contribute to the broader understanding of the molecular basis of plant responses to cold stress but also highlight the importance of post‐transcriptional mechanisms in regulating these responses. In summary, we proposed an updated model: A cold‐induced BTB family protein SlBTB19 negatively regulates cold tolerance. SlBTB19 interacts with and facilitates the proteolysis of the SlWRKY2 protein. SlWRKY2 is a positive regulator of cold tolerance, which activates cold‐responsive genes SlCBFs. [ABSTRACT FROM AUTHOR]

SUMMARY: "Jiaobai" is a symbiont of Zizania latifolia and Ustilago esculenta, producing fleshy galls as a popular vegetable in South and East Asia. Current "Jiaobai" cultivars exhibit abundant variation in their gall formation date; however, the underlying mechanism is not clear. In this study, a strict short‐day (SD) "Jiaobai" line "YD‐3" was used. Plants were treated with two day‐length regimes [14 h/10 h (day/night) (control) and 8 h/16 h (day/night) (SD)] from 100 to 130 days after planting. The gall swelling rate of the two treatments and another early SD treatment (from 60 to 90 days after planting), together with the contingent flowering plants in the experiment population, revealed that SD can improve both gall enlargement and flowering of "Jiaobai" plants. Comparison of RNA sequencing data among control, SD swelling, and SD flowering treatments of leaves and meristems indicated that SD promotion of "Jiaobai" swelling is conducted by the CONSTANS (CO)‐FLOWERING LOCUS T (FT) pathway, similar but not identical to the SD‐induced flowering pathway in Z latifolia and rice. "Virus‐induced gene silencing", "Yeast one‐hybrid assay" and "Dual‐luciferase assay" showed that a FT gene, ZlGsd1, is critical in SD promotion of gall formation and is positively regulated by a CO gene, ZlCOL1. Our study elucidated how photoperiod affects the formation of a unique organ produced by plant‐fungus symbiosis. The difference in SD response between "Jiaobai" and rice, as well as their potential applications in breeding of "Jiaobai" and rice, were also discussed. [ABSTRACT FROM AUTHOR]

The article discusses the virulence mechanisms of Xanthomonas oryzae pv. oryzae, a bacterium causing bacterial leaf blight in rice. It focuses on the type III secretion system (T3SS) and the regulation of hrp genes, essential for bacterial virulence. The study identifies key regulators, such as HrpG and HrpX, and explores the role of xylose in inducing hrp gene expression. Additionally, novel regulators like GamR and XylR are discovered, shedding light on the complex regulatory network controlling bacterial growth and virulence. [Extracted from the article]

[Display omitted] • Acid ceramidase (ASAH1) is an enzyme in sphingolipid metabolism that converts pro-survival ceramide into sphingosine and its role in colorectal cancer (CRC) is not known. • ASAH1 is over expressed in human murine CRC and silencing ASAH1 induces immunological cell death (ICD) in CRC cell lines. • ASAH1 silencing also modulate the glutathione system and induces mitochondrial stress. • Combination of ASAH1 inhibitor along with check point therapy (anti PD-1) sensitize the tumor cells to immunotherapy. Acid ceramidase (hereafter referred as ASAH1) is an enzyme in sphingolipid metabolism that converts pro-survival ceramide into sphingosine. ASAH1 has been shown to be overexpressed in certain cancers. However, the role of ASAH1 in colorectal cancer still remain elusive. The present study is aimed to understand how ASAH1 regulates colorectal cancer (CRC) progression and resistance to checkpoint inhibitor therapy. Both pharmacological and genetic silencing of ASAH1 was used in the study. In vitro experiments were done on human and mouse CRC cell lines. The in vivo studies were conducted in NOD-SCID and BALB/c mice models. The combination of ASAH1 inhibitor and checkpoint inhibitor was tested using a syngeneic tumor model of CRC. Transcriptomic and metabolomic analyses were done to understand the effect of ASAH1 silencing. ASAH1 is overexpressed in human CRC cases, and silencing the expression resulted in the induction of immunological cell death (ICD) and mitochondrial stress. The ASAH1 inhibitor (LCL-521), either as monotherapy or in combination with an anti-PD-1 antibody, resulted in reduction of tumors and, through induction of type I and II interferon response, activation of M1 macrophages and T cells, leading to enhanced infiltration of cytotoxic T cells. Our findings supported that the combination of LCL-521 and ICIs, which enhances the antitumor responses, and ASAH1 can be a druggable target in CRC. [ABSTRACT FROM AUTHOR]

Some fungi have demonstrated the ability to adapt rapidly to changing environments by exhibiting morphological plasticity, a trait influenced by species and environmental factors. Here, an anamorphic yeast strain IOJ-3 exhibited unique sectorization characteristics, naturally producing diverse filamentous sectors when cultivated on potato dextrose agar (PDA) medium or natural culture medium for durations exceeding 13 days. The strain IOJ-3 and its filamentous sectors were identified as Dothiora sorbi. The morphology of the sectors was consistent and heritable. The life cycle of strain IOJ-3 was investigated through microscopic observation, emphasizing the development of conidiogenous cells as a crucial stage, from which filamentous sectors originate. Some physiological characteristics of IOJ-3 and filamentous sectors are compared, and strain IOJ-3 has a higher antibiotic tolerance than two filamentous sectors, IOJ-3a expands faster on the culture medium, and IOJ-3b can penetrate cellophane. A transcriptomic analysis was conducted to investigate the differentially expressed genes between the yeast form IOJ-3 and its two filamentous sectors, revealing a total of 594 genes that exhibited consistent differential expression relative to IOJ-3, including 44 silencing genes in IOJ-3 that were activated. Gene Ontology analysis indicated that these differentially expressed genes were primarily associated with the cellular component category. Furthermore, adding 5-Azacytidine accelerated filamentous sectorization and increased the proportion of filamentous cells of strain IOJ-3 in PD liquid media, suggesting that the filamentous sectorization observed in strain IOJ-3 is linked to processes of DNA demethylation. In conclusion, this study sheds light on the biological characteristics of D. sorbi regarding morphological transitions and provides substantial direction for exploring genes related to fungal filamentous development. [ABSTRACT FROM AUTHOR]

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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. [ABSTRACT FROM AUTHOR]

Caffeoyl coenzyme A-O-methyltransferase (CCoAOMT) has a critical function in the lignin biosynthesis pathway. However, its functions in cotton are not clear. In this research, we observed 50 CCoAOMT genes from four cotton species, including two diploids (Gossypium arboretum, 9, and Gossypium raimondii, 8) and two tetraploids (Gossypium hirsutum, 16, and Gossypium barbadense, 17), performed bioinformatic analysis, and focused on the involvement and functions of GhCCoAOMT7 in lignin synthesis of Gossypium hirsutum. CCoAOMT proteins were divided into four subgroups based on the phylogenetic tree analysis. Motif analysis revealed that all CCoAOMT proteins possess conserved Methyltransf_3 domains, and conserved structural features were identified based on the genes' exon-intron organization. A synteny analysis suggested that segmental duplications were the primary cause in the expansion of the CCoAOMT genes family. Transcriptomic data analysis of GhCCoAOMTs revealed that GhCCoAOMT2, GhCCoAOMT7, and GhCCoAOMT14 were highly expressed in stems. Subcellular localization experiments of GhCCoAOMT2, GhCCoAOMT7, and GhCCoAOMT14 showed that GhCCoAOMT2, GhCCoAOMT7, and GhCCoAOMT14 were localized in the nucleus and plasma membrane. However, there are no cis-regulatory elements related to lignin synthesis in the GhCCoAOMT7 gene promoter. GhCCoAOMT7 expression was inhibited by virus-induced gene silencing technology to obtain gene silencing lines, the suppression of GhCCoAOMT7 expression resulted in a 56% reduction in the lignin content in cotton stems, and the phloroglucinol staining area corresponding to the xylem was significantly decreased, indicating that GhCCoAOMT7 positively regulates lignin synthesis. Our results provided fundamental information regarding CCoAOMTs and highlighted their potential functions in cotton lignin biosynthesis and lignification. [ABSTRACT FROM AUTHOR]

Lily bulbils, advantageous axillary organs used for asexual reproduction, have an underexplored developmental mechanism. Gibberellins are known to participate in bulbil development, but the regulatory mechanisms remain unclear. In this study, exogenous gibberellin (GA3) significantly increased the bulbil length, width, and weight by raising the endogenous gibberellin levels and elongating the scale cells. Transcriptomic analysis identified LlGA20ox2, a key gibberellin biosynthesis gene, which was upregulated during bulbil development and significantly responsive to GA3 treatment. Given the similarities in bulbil and bulblet development, we determined the roles of LlGA20ox2 using a bulblet system. Silencing LlGA20ox2 in bulblets inhibited development by reducing the cell length, while overexpression increased the bulblet length and width. In the gibberellin signaling pathway, we identified two key genes, LlGID1C and LlCIGR2. Silencing these genes resulted in phenotypes similar to LlGA20ox2, inhibiting bulblet development. Further transcriptomic analysis revealed that gibberellin-responsive genes were enriched in the glucuronate pathway, pentose phosphate pathway and galactose metabolism pathways. Most of these differentially expressed genes responded to gibberellin and were highly expressed in later stages of bulbil development, suggesting their involvement in gibberellin-regulated bulbil growth. In conclusion, we preliminarily explored the mechanisms of gibberellin regulation in bulbil development, offering significant commercial potential for new lily reproductive organs. [ABSTRACT FROM AUTHOR]

Kenaf (Hibiscus cannabinus L.) is an important fiber crop, which can be applied for the restoration of saline-alkali land. The objective of our study was to investigate the impacts of exogenous glutathione (GSH) on physiological and biochemical properties, ion balance, and DNA methylation of kenaf under salt stress. We used Hoagland nutrient solution containing 200 mM NaCl to simulate salt stress, and found the growth of kenaf seedlings was substantially hindered. 100 μM GSH pretreatment effectively increased the plant height, stem diameter, main root length, and fresh weight under salt stress, as well as reduced the uptake of Na+ and Cl− and promoted the uptake of K+. Besides, exogenous GSH pretreatment protected kenaf plants from salt-induced adversities by reducing the ROS-induced oxidative damage, enhancing the contents of chlorophyll, proline, and soluble sugar. Salinity reduced the total DNA methylation level in kenaf genome, triggering higher mRNA expressions of HcGLP3, HcDOF1.4, HcULP3, HcVHA, HcPP2C39, and HcSRF6. However, GSH addition enhanced the total DNA methylation level. We further utilized virus-induced genes silencing technique to confirm that HcGLP3 played a positive role in the response of kenaf to salinity. Taken together, exogenous GSH could enhance salt tolerance in kenaf by mediating modulation of oxidative stress response and DNA methylation. [ABSTRACT FROM AUTHOR]

Bulbils, originating from axillary meristem, are known to have a significant impact on the propagation of Lilium lancifolium. Transcription factor ASYMMETRIC LEAVES 1 has been shown to be involved in the regulation of bulbil formation based on the transcriptome data of L. lancifolium. The present investigation involved the cloning of the LlAS1 gene from L. lancifolium by RT-PCR and further be characterized. The open reading frame of LlAS1 comprised 1035 bp, which encoded 344 amino acids. The LlAS1 protein contained two conserved SANT domains in series at the N-terminus. Phylogenetic analysis revealed that LlAS1 belongs to the monocot group and was closely related to the AS1 of Musa acuminata subsp. malaccensis. Expression analysis showed that LlAS1 was strongly expressed in bulbil, especially in primary bulbils. It was highly expressed during the process of bulbil primordium establishment and bulbil formation. Transient overexpression and virus-induced gene silencing (VIGS) of LlAS1 in leaf axils significantly promoted and inhibited bulbil formation of L. lancifolium, respectively. The findings of the study indicated that LlAS1 was positively correlated with bulbil formation of L. lancifolium, laying a foundation for further understanding the regulation of LlAS1 gene for bulbil formation and application in molecular genetic improvement of lilies. [ABSTRACT FROM AUTHOR]

Objectives: The role of matrix metalloproteinases (MMPs) in Skin Cutaneous Melanoma (SKCM) development and progression is unclear so far. This comprehensive study delved into the intricate role of MMPs in SKCM development and progression. Methods: RT-qPCR, bisulfite sequencing, and WES analyzed MMP gene expression, promoter methylation, and mutations in SKCM cell lines. TCGA datasets validated findings. DrugBank and molecular docking identified potential regulatory drugs, and cell line experiments confirmed the role of key MMP genes in tumorigenesis. Results: Our findings unveiled significant up-regulation of MMP9, MMP12, MMP14, and MMP16, coupled with hypomethylation of their promoters in SKCM cell lines, implicating their involvement in disease progression. Mutational analysis highlighted a low frequency of mutations in these genes, indicating less involvement of mutations in the expression regulatory mechanisms. Prognostic assessments showcased a significant correlation between elevated expression of these genes and poor overall survival (OS) in SKCM patients. Additionally, functional experiments involving gene silencing revealed a potential impact on cellular proliferation, further emphasizing the significance of MMP9, MMP12, MMP14, and MMP16 in SKCM pathobiology. Conclusion: This study identifies Estradiol and Calcitriol as potential drugs for modulating MMP expression in SKCM, highlighting MMP9, MMP12, MMP14, and MMP16 as key diagnostic and prognostic biomarkers. [ABSTRACT FROM AUTHOR]

The health risks associated with microplastics have attracted widespread attention. Polystyrene microplastics (PS‐MPs) can induce damage to cardiac tissue, while pyroptosis‐mediated injury to the vascular endothelial plays a vital role in the pathogenesis of cardiovascular diseases. The study intended to explore the role and mechanism of NLR family pyrin domain containing 3 (NLRP3) mediated pyroptosis in PS‐MPs causing the injury of vascular endothelial cells. In vivo, Wistar rats were exposed to 0.5, 5, and 50 mg/kg/d 0.5 μm PS‐MPs. In vitro, the human vascular endothelial cells (HUVECs) were used for mechanistic studies. siRNA was used for silencing the NILRP3 gene. H&E staining and flow cytometry were performed to examine the vascular injury and cell membrane damage. The oxidative stress was detected by flow cytometry, immunofluorescence, and corresponding kits. ELISA were used to measure the levels of inflammatory factors. Real‐time PCR and western blot were used to measure the expression of pyroptosis signaling pathway. In rats, PS‐MPs could cause vascular damage, oxidative stress, and inflammatory response, and activated the pyroptosis signaling pathway. HUVECs exposure to PS‐MPs, the vitality decreased in a dose‐dependent manner, ROS and MDA were significantly increased while SOD was decreased. PS‐MPs induced the onset of pyroptosis signaling pathway in HUVECs. Cell membrane damage and the levels of IL‐Iβ and IL‐18 in HUVECs significantly increased, those are symbols for the development of pyroptosis. Inhibition of NLRP3‐mediated pyroptosis effectively protected HUVECs from PS‐MPs‐induced damage. Pyroptosis played a vital role in controlling the vascular endothelial injury caused by PS‐MPs. [ABSTRACT FROM AUTHOR]

The rice leaf folder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), is a major migratory pest in rice agriculture. This pest is characterised by its larvae's ability to fold rice leaves using silk, a behaviour that culminates in the formation of a silken cocoon during the pupal stage. The fibroin light chain (CmFib‐L) gene is crucial for silk production, yet its specific function in C. medinalis has reminded elusive. This study presents a comprehensive analysis of the CmFib‐L gene, revealing its complete open reading frame (ORF) and expression patterns. Notably, the gene is highly expressed in the fifth‐instar larvae and the silk gland, which are critical stages for silk production. Our experiments demonstrate that silencing the CmFib‐L gene leads to a reduction in pupal weight, an extension of the pupal stage and a disorganised silk cocoon. Furthermore, the larval behaviour of leaf folding and spinning is significantly impaired when the expression of CmFib‐L is downregulated. These findings not only show the importance of fibroin light chain in silk production but also reveal a new target gene to regulate and control the behaviour and development of C. medinalis. [ABSTRACT FROM AUTHOR]

ERP29 gene encodes a chaperone protein critical for protein folding and secretion. Previous study linked ERP29 inhibition to an elevated risk of pharynx squamous cell carcinoma (PSCC) and reduced patients' survival. However, ERP29 role in PSCC progression remains unknown. Here, we investigated ERP29 impact on PSCC progression in cisplatin (CDDP)-sensitive (FaDu and LAU-2063), CDDP-treated (FaDu-CDDP), and CDDP-resistant (FaDu-R) cells. ERP29 silencing decreased necrosis and increased migration in CDDP-sensitive, treated, and resistant cells; and reduced E-cadherin and increased vimentin immunoexpression in CDDP-sensitive 3D-spheroids. During CDDP treatment, ERP29 silencing enhanced proliferation. In CDDP-sensitive cells, ERP29 silencing upregulated genes associated with WNT, MAPK, and PI3K/AKT signaling pathways while downregulating CASP9 expression. During CDDP treatment, ERP29 silencing downregulated MDM2 and CASP9 expression. In CDDP-resistant cells, ERP29 silencing upregulated SOS1, MAPK1, AKT1, ITGAV, and CCNE1, while downregulating KRAS, JUN, MDM2, and CASP9 expression. In addition, inhibition of microRNA miR-4421 increased ERP29 expression and decreased MAPK1, AKT1, and JUN expression in CDDP-sensitive cells, as well as SOS1, MAPK1, AKT1, and ITGAV in CDDP-resistant cells. Lower ERP29 and higher miR-4421 expressions were predictive of poor survival, suggesting a potential therapeutic use for miR-4421 inhibitors. Upon validation, these findings may contribute to targeted therapies for PSCC based on ensuring ERP29 expression. [ABSTRACT FROM AUTHOR]

ABSTRACT 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. [ABSTRACT FROM AUTHOR]

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. Here the authors investigate how the E3 ubiquitin ligase Cryptic Loci Regulator Complex mediates the transition from RNAi-dependent co-transcriptional gene silencing to transcriptional gene silencing suggesting the involvement of phase separation in the transition during heterochromatin formation. [ABSTRACT FROM AUTHOR]

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

ABSTRACT The abscisic acid (ABA) signaling pathway is essential for plant response to abiotic stresses and can be modulated positively or negatively by MAPKKK proteins. This study focuses on the functional characterization of CaMEKK17, a MAPKKK previously recognized for its rapid induction under drought stress. Functional analyses demonstrated that CaMEKK17 is an active serine/threonine kinase with a conserved catalytic domain that is crucial for its kinase activity. CaMEKK17 silencing in pepper plants resulted in reduced drought tolerance, characterized by increased transpirational water loss and impaired ABA‐mediated stomatal closure. Conversely, CaMEKK17 overexpression in Arabidopsis increased kinase activity, enhancing ABA sensitivity and drought tolerance. Further investigation revealed that CaMEKK17 interacts with pepper group A type 2C protein phosphatases (PP2Cs), particularly CaAITP1 and CaAIPP1, which inhibit its kinase activity. Protein–protein interactions mediated inhibition by CaAITP1, whereas CaAIPP1 relied on its phosphatase activity. Double gene silencing of CaMEKK17 and CaAITP1 demonstrated that CaMEKK17 functions downstream of CaAITP1 in ABA‐mediated drought tolerance. Taken together, our findings suggest that CaMEKK17 positively modulates drought tolerance in pepper plants but may be inhibited by PP2Cs. [ABSTRACT FROM AUTHOR]

In plants, recognition between resistance gene (R) and virus induces a local hypersensitive response (HR), which is accompanied by systemic acquired resistance (SAR). The dominant resistance gene N in tobacco confers resistance to tobacco mosaic virus (TMV) at both locally inoculated tissues and systemically infected tissues. However, the mechanisms underlying HR- and SAR-mediated viral inhibition are not fully revealed. In this study, we find that Nicotiana glutinosa RNA-binding protein (NgRBP) is an RNA silencing suppressor which enhances TMV-triggered HR. Stronger HR could result in stronger local and systemic RNA silencing as well as SAR. Enhanced RNA silencing in the systemically infected leaves induced by the NgRBP gene is compromised by transient expression of NahG. These results indicate that RNA silencing is activated by HR and plays a crucial role in local and systemic virus resistance. Our results reveal a crosstalk between N gene-mediated virus resistance and RNA silencing which would deepen our understanding of the established HR and SAR models. [ABSTRACT FROM AUTHOR]

SUMMARY Citral, a naturally occurring acyclic monoterpene aldehyde, is present in the essential oils of various plants, but only a few produce it in abundance. Despite its importance as a key aroma molecule, knowledge regarding the in‐planta biosynthesis of citral and its metabolic origin remains limited. Here, we have elucidated the functions of an alcohol dehydrogenase (CfADH1) and an aldoketo‐reductase (CfAKR2b) in citral biosynthesis in lemongrass (Cymbopogon flexuosus), one of the most cultivated aromatic crops for its citral‐rich essential oil. Expression of both CfADH1 and CfAKR2b showed correlation with citral accumulation in different developmental stages. Recombinant CfADH1 and CfAKR2b, despite their sequence unrelatedness, catalyzed citral formation from geraniol with NADP cofactor. Virus‐induced gene silencing in lemongrass and transient expression in lemon balm (Melissa officinalis) demonstrated the in‐planta involvement of CfADH1 and CfAKR2b in citral biosynthesis. While CfADH1 exhibited a dual cytosolic/plastidial localization, CfAKR2b was localized to the cytosol. This was supported by higher citral‐forming activity in the cytosolic fraction than in the chloroplast fraction of lemongrass leaf extract. Moreover, feeding lemongrass seedlings with inhibitors specific to the cytosolic mevalonate pathway and the plastidial methylerythritol phosphate pathway, combined with volatile profiling, supported the involvement of both pathways in citral formation. Taken together, our results indicate that high citral production has evolved in lemongrass through the recruitment of phylogenetically distant enzymes localized in both the cytosol and plastids. [ABSTRACT FROM AUTHOR]

RESEARCH HIGHLIGHTSAvian leukosis virus subgroup J (ALV-J) is an alpharetrovirus that infects chickens, causing immunosuppression and a decrease in production performance, leading to substantial economic losses in the poultry industry. ALV-J is also well-known for its oncogenic properties, inducing tumours such as myelomas and haemangiomas in infected chickens. TRIM45 has been identified as a potential tumour suppressor; however, the relationship between TRIM45 expression and ALV-J infection remains to be elucidated. This study aimed to dissect the molecular characteristics of the chicken TRIM45 gene and its modulation during ALV-J infection, as well as its influence on viral replication. We found that the chicken TRIM45 RING domain is significantly different from that of humans and other mammals. TRIM45 is expressed in all chicken tissues, with the highest levels in the heart. Subcellular localization studies indicated a cytoplasmic distribution of TRIM45, forming aggregates within cells. Our findings demonstrate that ALV-J infection significantly upregulates TRIM45 expression in DF-1 cells. To assess the functional role of TRIM45 in ALV-J replication, we employed both gene silencing and overexpression strategies. Strikingly, the overexpression of TRIM45, including a mutant lacking the RING domain, was found to markedly suppress ALV-J replication. In contrast, TRIM45 knockdown via siRNA resulted in an enhanced viral replication, highlighting the importance of TRIM45 limiting ALV-J replication. Mechanistically, overexpression of TRIM45 induces apoptosis in infected cells, independent of its RING domain function. In conclusion, our study demonstrates that chicken TRIM45 acts as a negative regulator of ALV-J replication in vitro by promoting apoptosis in infected cells.Chicken TRIM45 RING domain and protein localization significantly differ from humans.TRIM45 negatively regulates ALV-J replication in vitro.TRIM45 inhibits ALV-J replication by inducing apoptosis in infected cells.Chicken TRIM45 RING domain and protein localization significantly differ from humans.TRIM45 negatively regulates ALV-J replication in vitro.TRIM45 inhibits ALV-J replication by inducing apoptosis in infected cells. [ABSTRACT FROM AUTHOR]

Background: The excavation and utilization of endophytic fungi from medicinal plants is highly important for the development of new drugs. The endophytic fungus Aspergillus tamarii TPD11, which was isolated and obtained by the authors in the previous stage, can produce a variety of polyphyllins with important potential applications in hemostasis, inflammation and antitumor activities; however, the genomic information of TPD11 is still unknown. Results: In this study, we sequenced and assembled the whole genome of the endophytic fungus A. tamarii TPD11, resolved the genome evolutionary relationships of 24 Aspergillus strains, and phylogenetic analysis of the genomes of 16 strains revealed the evolutionary differences between Aspergillus and Penicillium and the mechanisms of genome expansion and contraction. CAZy annotation analysis revealed that TPD11 obtains nutrients mainly by ingesting starch from the host plant. TPD11 has a biosynthesis-related gene cluster for the synthesis of squalestatin S1, and the silencing of this biosynthesis-related gene cluster might increase the content of polyphyllin. Annotation of 11 UDP-glycosyltransferase genes helps to further reveal the biosynthetic pathway of polyphyllin. In addition, secondary metabolism gene cluster and CAZy analyses confirmed the potential probiotic, insecticidal and antimicrobial activities of TPD11 on host plants. Conclusions: This study reveals the intrinsic mechanism by which endophytic fungi increase the content of polyphyllin, which provides a basis for the synthetic synthesis of the natural product polyphyllin. [ABSTRACT FROM AUTHOR]

RNA virus-based episomal vector (REVec), engineered from Borna disease virus, is an innovative gene delivery tool that enables sustained gene expression in transduced cells. However, the difficulty in controlling gene expression and eliminating vectors has limited the practical use of REVec. In this study, we overcome these shortcomings by inserting artificial aptazymes into the untranslated regions of foreign genes carried in vectors or downstream of the viral phosphoprotein gene, which is essential for vector replication. Non-transmissive REVec carrying GuaM8HDV or the P1-F5 aptazyme showed immediate suppression of gene expression in a guanine or theophylline concentration-dependent manner. Continuous compound administration also markedly reduced the percentage of vector-transduced cells and eventually led to the complete elimination of the vectors from the transduced cells. This new REVec is a safe gene delivery technology that allows fine-tuning of gene expression and could be a useful platform for gene therapy and gene-cell therapy, potentially contributing to the cure of many genetic disorders. Key points: • We developed a bornavirus vector capable of silencing transgene expression by insertion of aptazyme • Transgene expression was markedly suppressed in a compound concentration-dependent manner • Artificial aptazyme systems allowed complete elimination of the vector from transduced cells [ABSTRACT FROM AUTHOR]

Deposition of monosodium urate and calcium pyrophosphate (MSU and CPP) micro-crystals is responsible for painful and recurrent inflammation flares in gout and chondrocalcinosis. In these pathologies, the inflammatory reactions are due to the activation of macrophages responsible for releasing various cytokines including IL-1β. The maturation of IL-1β is mediated by the multiprotein NLRP3 inflammasome. Here, we find that activation of the NLRP3 inflammasome by crystals and concomitant production of IL-1β depend on cell volume regulation via activation of the osmo-sensitive LRRC8 anion channels. Both pharmacological inhibition and genetic silencing of LRRC8 abolish NLRP3 inflammasome activation by crystals in vitro and in mouse models of crystal-induced inflammation. Activation of LRRC8 upon MSU/CPP crystal exposure induces ATP release, P2Y receptor activation and intracellular calcium increase necessary for NLRP3 inflammasome activation and IL-1β maturation. We identify a function of the LRRC8 osmo-sensitive anion channels with pathophysiological relevance in the context of joint crystal-induced inflammation. Formation of urate and calcium pyrophosphate micro-crystals is responsible for painful inflammatory flares in gout and chondrocalcinosis. Here the authors show that the osmo-sensitive LRRC8 anion channel is involved with macrophage inflammasome activation by crystals involving cell volume regulation and ATP release leading to P2Y receptor activation. [ABSTRACT FROM AUTHOR]