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The ability to produce pulcherriminic acid is a characteristic feature of yeast species of the pulcherrima clade recently merged under the taxonomic name Metschnikowia pulcherrima. This iron chelator cyclodipeptide forms pulcherrimin, a maroon-red pigment with ferric ions. Its synthesis and secretion into the environment is under the control of closely linked genes referred to as the PUL cluster. The examination of 18 generations of single-cell clones generated from a stock culture of the collection strain 11-1090 (CBS 10359) in this study revealed that the biosynthesis of pulcherriminic acid is reversibly switched on and off during the propagation of cells in a way similar to the epigenetic silencing and activation of gene expression (bimodal active/silent state) in near-heterochromatic regions of other yeast species. As the strain is heterozygous for PUL2 alleles encoding slightly different amino acid sequences and has a plastic genome structure, the efficiency of pulcherriminic acid synthesis in the switched-on state is presumed to depend on which PUL2 allele is active and on structural changes in the genome. The transitions between the active and silent states of pulcherriminic acid synthesis are associated with transitions between the active and silent states of antimicrobial antagonism. This association confirms the primary role of pulcherriminic acid in the antimicrobial antagonism of M. pulcherrima. [ABSTRACT FROM AUTHOR]

The western flower thrips (Frankliniella occidentalis) is a significant agricultural pest, causing severe global yield losses due to extensive feeding damage and the transmission of plant pathogenic viruses. Despite recent advancements in RNA interference (RNAi) in thrips species, its application has been mostly limited to the adult stage. Given the crucial role of first instar larval thrips in acquiring and transmitting orthotospoviruses, achieving gene silencing in these larvae is critical for studying virus entry and acquisition. While thoracic and abdominal injections have proven effective in adult thrips, the low post‐injection survival rate hinders their use in larval thrips. This study addresses this challenge by presenting a microinjection methodology to deliver dsRNA into the hemolymph of first instar larval thrips through the coxa, the first proximal segment of the foreleg. This method significantly improved larval survival rate by preventing detrimental damage to the internal tissues. Significant knockdown of V‐ATPase‐B, cytochrome P450 (CYP3653A2), and apolipophorin‐II/I (ApoLp‐II/I) transcripts was confirmed after 48 and/or 72 h post injection (hpi), corresponding to the first and second instar larval stages, respectively. Silencing CYP3653A2 or ApoLp‐II/I significantly increased larval mortality. These findings demonstrate proof‐of‐principle of gene silencing and associated silencing phenotype (mortality) for first instar larval thrips and highlight the essential role of CYP3653A2 and ApoLp‐II/I in larval vitality. Our RNAi‐based tool offers an opportunity to investigate the molecular mechanisms of thrips‐orthotospovirus interactions, as the virus must be acquired by young larval thrips for successful transmission to plants, thus presenting potential targets for thrips pest management. [ABSTRACT FROM AUTHOR]

Background: To avoid exaggerated inflammation, innate immune cells adapt to become hypo-responsive or "tolerance" in response to successive exposure to stimuli, which is a part of innate immune memory. Polycomb repressive complex 2 (PRC2) mediates the transcriptional repression by catalyzing histone H3 lysine 27 trimethylation (H3K27me3) but little is known about its role in lipopolysaccharide (LPS)-induced tolerance in macrophages. Result: We examined the unexplored roles of EED, a component of the PRC2, in LPS tolerant macrophages. In Eed KO macrophages, significant reduction in H3K27me3 and increased active histone mark, H3K27ac, was observed. Eed KO macrophages exhibited dampened pro-inflammatory cytokine productions (TNF-α and IL-6) while increasing non-tolerizable genes upon LPS tolerance. Pharmacological inhibition of EED also reduced TNF-α and IL-6 during LPS tolerance. Mechanistically, LPS tolerized Eed KO macrophages failed to increase glycolytic activity. RNA-Seq analyses revealed that the hallmarks of hypoxia, TGF-β, and Wnt/β-catenin signaling were enriched in LPS tolerized Eed KO macrophages. Among the upregulated genes, the promoter of Runx3 was found to be associated with EED. Silencing Runx3 in Eed KO macrophages partially rescued the dampened pro-inflammatory response during LPS tolerance. Enrichment of H3K27me3 was decreased in a subset of genes that are upregulated in Eed KO LPS tolerized macrophages, indicating the direct regulatory roles of PRC2 on such genes. Motif enrichment analysis identified the ETS family transcription factor binding sites in the absence of EED in LPS tolerized macrophages. Conclusion: Our results provided mechanistic insight into how the PRC2 via EED regulates LPS tolerance in macrophages by epigenetically silencing genes that play a crucial role during LPS tolerance such as those of the TGF-β/Runx3 axis. [ABSTRACT FROM AUTHOR]

Background: The tree shrew (Tupaia belangeri) is a promising emerging model organism in biomedical studies, notably due to their evolutionary proximity to primates. To enhance our understanding of how DNA methylation is implicated in regulation of gene expression and the X chromosome inactivation (XCI) in tree shrew brains, here we present their first genome-wide, single-base-resolution methylomes integrated with transcriptomes from prefrontal cortices. Results: Genome-wide relationships between DNA methylation and gene expression are consistent with those in other mammals. Interestingly, we observed a clear and significant global reduction (hypomethylation) of DNA methylation across the entire female X chromosome compared to male X. Female hypomethylation does not directly contribute to the gene silencing of the inactivated X chromosome nor does it significantly drive sex-specific gene expression in tree shrews. However, we identified a putative regulatory region in the 5′ end of the X-inactive-specific transcript (Xist) gene, whose pattern of differential DNA methylation strongly relate to its sex-differential expression in tree shrews. Furthermore, differential methylation of this region is conserved across different species. We also provide evidence suggesting that the observed difference between human and tree shrew X-linked promoter methylation is associated with the difference in genomic CpG contents. Conclusions: Our study offers novel information on genomic DNA methylation of tree shrews as well as insights into the evolution of sex chromosome regulation in mammals. Specifically, we show conserved role of DNA methylation in regulation of Xist expression and propose genomic CpG contents as a factor in driving sex-differential DNA methylation of X-linked promoters. [ABSTRACT FROM AUTHOR]

After 4 years of the COVID-19 pandemic, SARS-CoV-2 continues to circulate with epidemic waves caused by evolving new variants. Although the rapid development of vaccines and approved antiviral drugs has reduced virus transmission and mitigated the symptoms of infection, the continuous emergence of new variants and the lack of simple-use (non-hospitalized, easy timing, local delivery, direct acting, and host-targeting) treatment modalities have limited the effectiveness of COVID-19 vaccines and drugs. Therefore, novel therapeutic approaches against SARS-CoV-2 infection are still urgently needed. As a positive-sense single-stranded RNA virus, SARS-CoV-2 is highly susceptible to RNA interference (RNAi). Accordingly, small interfering (si)RNAs targeting different regions of SARS-CoV-2 genome can effectively block the expression and replication of the virus. However, the rapid emergence of new SARS-CoV-2 variants with different genomic mutations has led to the problem of viral escape from the targets of RNAi strategy, which has increased the potential of off-target effects by siRNA and decreased the efficacy of long-term use of siRNA treatment. In our study, we enzymatically generated a set of Dicer-substrate (D)siRNA swarms containing DsiRNAs targeting single or multiple conserved sequences of SARS-CoV-2 genome by using in vitro transcription, replication and Dicer digestion system. Pre-transfection of these DsiRNA swarms into Vero E6-TMPRSS2 cells inhibited the replication of several SARS-CoV-2 variants, including the recent Omicron subvariants BQ.1.1 and XBB.1.5. This in vitro investigation of novel DsiRNA swarms provides solid evidence for the feasibility of this new RNAi strategy in the prevention and treatment of SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

ABSTRACT Salivary proteins secreted by phytophagous insects play pivotal roles in plant–insect interactions. A salivary protein RpSP27, from the stinkbug Riptortus pedestris, a devastating pest on soybean, was selected for studying due to its ability to induce cell death and activate immune responses in plants. RpSP27 localized to the endoplasmic reticulum and triggered reactive oxygen species burst. Virus‐induced gene silencing assays showed RAR1 plays an essential role in RpSP27‐induced cell death in Nicotiana benthamiana. Expression analyses revealed that RpSP27 is predominantly expressed in R. pedestris salivary glands. RNA interference‐mediated silencing of RpSP27 in R. pedestris significantly reduced insect survival rates and altered feeding behavior by decreasing the formation of salivary sheaths on soybeans and reducing probing and feeding duration. Furthermore, the silencing of RpSP27 in R. pedestris mitigated the staygreen syndrome in soybeans, characterized by delayed senescence and pod abnormalities. This study elucidated the role of RpSP27 in the interaction between R. pedestris and soybean, presenting a potential target for pest management strategies to protect soybean crops from the detrimental effects of R. pedestris feeding. [ABSTRACT FROM AUTHOR]

The long non-coding RNA (lncRNA), HAR1A is emerging as a putative tumour suppressor. In non-neoplastic brain cells, REST suppresses HAR1A expression. In gliomas REST acts as an oncogene and is a potential therapeutic target. It is therefore conceivable that REST promotes glioma progression by down-regulating HAR1A. To test this hypothesis, glioma clinical databases were analysed to study: (I) HAR1A/REST correlation; (II) HAR1A and REST prognostic role; (III) molecular pathways associated with these genes. HAR1A expression and subcellular localization were studied in glioblastoma and paediatric glioma cells. REST function was also studied in these cells, by observing the effects of gene silencing on: (I) HAR1A expression; (II) cancer cell proliferation, apoptosis, migration; (III) expression of neural differentiation genes. The same phenotypes (and cell morphology) were studied in HAR1A overexpressing cells. Our results show that REST and HAR1A are negatively correlated in gliomas. Higher REST expression predicts worse prognosis in low-grade gliomas (the opposite is true for HAR1A). REST-silencing induces HAR1A upregulation. HAR1A is primarily detected in the nucleus. REST-silencing dramatically reduces cell proliferation and induces apoptosis, but HAR1A overexpression has no major effect on investigated cell phenotypes. We also show that REST regulates the expression of neural differentiation genes and that its oncogenic function is primarily HAR1A-independent. [ABSTRACT FROM AUTHOR]

Background: Bone remodeling is a continuous and balanced process which relies on the dynamic equilibrium between osteoclastic bone resorption and osteoblastic bone formation. During osteoclast differentiation, pro-osteoclastogenic and anti-osteoclastogenic genes are selectively targeted by positive and negative transcription regulators, respectively. VprBP, also known as DCAF1, is a recently identified kinase and plays an important role in driving epigenetic gene silencing and oncogenic transformation. However, nothing is currently known about a possible involvement of VprBP in signaling pathways that regulate other cellular processes. Results: We demonstrate that VprBP stimulates RANKL-induced differentiation of osteoclast precursor cells (OCPs) into mature osteoclasts by suppressing the expression of anti-osteoclastogenic genes through phosphorylation of threonine 120 on histone H2A (H2AT120p). H2AT120p is critical for VprBP function, because abrogating VprBP kinase activity toward H2AT120 transcriptionally reactivates anti-osteoclastogenic genes and significantly attenuates osteoclast differentiation. Consistent with this notion, our in vivo studies established the importance of VprBP-mediated H2AT120p in low bone mass phenotypes and osteoporosis caused by overactive osteoclasts. Conclusions: Our data reveal a previously unrecognized function of VprBP in supporting RANKL-induced osteoclast differentiation and the molecular mechanism underlying its action as a negative regulator of anti-osteoclastogenic genes. [ABSTRACT FROM AUTHOR]

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]

The photosystem II (PSII) Subunit P (PsbP) protein is a component of its oxygen-evolving complex, which can oxidize water to produce oxygen using light energy and is critical to the core components and stability of PSII. Using the whole-genome information, the PsbP genes of 10 plant species were comprehensively identified. The expression patterns of wheat PsbP s under Blumeria graminis f. sp. tritici (Bgt) infection were assessed using qRT-PCR, and the functions of TaPsbP s in wheat powdery mildew resistance were studied using barley stripe mosaic virus-induced gene silencing. In total, 122 PsbP genes were divided into 8 classes with similar gene structures. No tandem repeat events were identified in wheat PsbP , suggesting that the PsbP genes in common wheat were donated by its diploid progenitor species. The expression levels of TaPsbP2A-1 , TaPsbP3A-1 , TaPsbP4A-1 , TaPsbP4A-2 , and TaPsbP7A-2 were induced by Bgt. The silencing of TaPsbP4A-1 increased the resistance of common wheat 'Bainong AK58' to Bgt. This study provides valuable information for functional and evolutionary research on the PsbP gene family. [ABSTRACT FROM AUTHOR]

Growth hormone (GH) action in the brain regulates neuroendocrine axes, energy and glucose homeostasis, and several neurological functions. The lateral hypothalamic area (LHA) contains numerous neurons that respond to a systemic GH injection by expressing the phosphorylated STAT5, a GH receptor (GHR) signaling marker. However, the potential role of GHR signaling in the LHA is unknown. In this study, we demonstrated that ∼70% of orexin- and leptin receptor (LepR)-expressing neurons in the LHA are responsive to GH. Male mice carrying inactivation of the Ghr gene in the LHA were generated via bilateral injections of an adeno-associated virus. In ad libitum-fed mice, GHR ablation in LHA neurons did not significantly change energy and glucose homeostasis. Subsequently, mice were subjected to 5 d of 40% food restriction. Food restriction decreased body weight, energy expenditure, and carbohydrate oxidation. These effects were similarly observed in control and LHAΔGHR mice. While food-deprived control mice progressively increased ambulatory/exploratory activity and food-seeking behavior, LHAΔGHR mice did not show hyperactivity induced by food restriction. GHR ablation in the LHA reduced the percentage of orexin neurons expressing c-Fos during food restriction. Additionally, an acute GH injection increased the expression of c-Fos in LHAORX neurons. Inactivation of Ghr in LepR-expressing cells did not prevent hyperactivity in food-deprived mice, whereas whole-brain Ghr knock-out mice showed reduced ambulatory activity during food restriction. Our findings indicate that GHR signaling in the LHA regulates the activity of orexin neurons and is necessary to increase food-seeking behavior in food-deprived male mice. [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]

The molecular mechanisms of plant responses to phytophagous insect eggs are poorly understood, despite their importance in insect–plant interactions. This study investigates the plant defense mechanisms triggered by the eggs of whitefly Bemisia tabaci, a globally significant agricultural pest. A transcriptome comparison of tobacco plants with and without eggs revealed that whitefly eggs may activate the response of defense-related genes, including those involved in the salicylic acid (SA) signaling pathway. SA levels are induced by eggs, resulting in a reduction in egg hatching, which suggests that SA plays a key role in plant resistance to whitefly eggs. Employing Agrobacterium-mediated transient expression, virus-induced gene silencing assays, DNA–protein interaction studies, and bioassays, we elucidate the regulatory mechanisms involved. Pathogenesis-related proteins NtPR1-L1 and NtPR5-L2, downstream of the SA pathway, also affect whitefly egg hatching. The SA-regulated transcription factor NtWRKY70a directly binds to the NtPR1-L1 promoter, enhancing its expression. Moreover, NtPR1-L1 promotes callose deposition, which may impede the eggs' access to water and nutrients. This study establishes the SA-WRKY70-PR-callose axis as a key mechanism linking plant responses and defenses against whitefly eggs, providing new insights into the molecular interactions between plants and insect eggs. [ABSTRACT FROM AUTHOR]

Bacillus licheniformis 24 (BL24) is an efficient, non-pathogenic producer of 2,3-butanediol (2,3-BD). However, during inulin fermentation, the strain produces large amounts of exopolysaccharides (EPS), which interfere with the process' performance. The present study aims to investigate the effect that inactivation of the sacB gene, encoding levansucrase in BL24, has on 2,3-BD production efficiency. Knockout of the sacB gene was accomplished via insertional inactivation. The sacB-knockout variant formed 0.57 g/L EPS from sucrose and 0.7–0.8 g/L EPS from glucose and fructose, a 15- and 2.5-fold reduction relative to the wild type, respectively. Likewise, during batch fermentation with soluble inulin Frutafit® CLR, the mutant BLΔsacB produced significantly less EPS than the wild type, allowing the maintenance of pH at values favoring 2,3-BD synthesis. At pH 6.50, BLΔsacB reached a record titer of 128.7 g/L 2,3-BD, with productivity of 1.65 g/L/h, and a yield of 85.8% of the theoretical maximum. The obtained concentration of 2,3-BD is two-fold higher compared to that of the wild type. Subsequent RT-qPCR assays confirmed a successful sacB knockout. Three of the genes involved in inulin hydrolysis (sacA, sacC, and fruA) maintained their expression levels compared to the wild type, while that of levB increased. Although total EPS accumulation could not be completely eliminated via sacB gene knockout alone, the overall reduction in EPS content has enabled the highest yield of 2,3-BD from inulin to date, a promising result for the industrial production from inulin-rich substrates. [ABSTRACT FROM AUTHOR]

Targeting epigenetics is a new strategy to treat cancer and develop novel epigenetic drugs with anti-tumor activity. DNA methyltransferases transfer the methyl group from S-adenosyl-L-methionine (SAM) to the cytosine residue in a CpG island, leading to the transcription silencing of the gene. Hypermethylation can frequently be observed in several tumor types. Hence, the inhibition of DNMT1 has become a novel approach to cure cancer. In this study, virtual screening and molecular docking were performed for more than 11,000 ligands from the ZINC15 database to discover new hypomethylation agents. Four candidate compounds were further tested for their effects on DNMT1 in silico and in vitro. Compounds 2 and 4 showed the best DNMT1 inhibitory activity, but only compound 4 was able to inhibit the growth of several cancer cell lines. The hypomethylation of the luciferase gene by compound 4 was verified by a CMV- luciferase assay using KG-1 cells. Additionally, compound 4 suppressed cell migration in a dose- and time-dependent manner in the wound healing assay. Moreover, cell cycle analyses demonstrated that compound 4 arrested CCRF-CEM cells and MDA-MB-468 cells in the G0/G1 phase. Also, compound 4 significantly induced early and late apoptosis in a dose-dependent manner. In conclusion, we introduce compound 4 as a novel DNMT1 inhibitor with anticancer activity. [ABSTRACT FROM AUTHOR]

Strategies in genetic and pharmacological modulation of innate immunity to enhance oncolytic virotherapy (OV) efficacy are being explored. We have recently characterized the ability for vanadium-based compounds, a class of pan-phosphatase (PP) inhibitors, to potentiate OVs. We next sought to identify PPs that could be targeted to enhance OVs, akin to vanadium. By conducting a high-throughput screen of a library of silencing RNA (siRNA) targeting human PPs, we uncovered several PPs that robustly enhanced infectivity and oncolysis of the oncolytic vesicular stomatitis virus (VSV∆51). Knockdown of our top validated hit, lysosomal acid phosphatase 2 (ACP2), increased VSV∆51 viral titers by over 20-fold. In silico analysis by RNA sequencing revealed ACP2 to regulate antiviral type I interferon (IFN-1) signaling pathways, similar to vanadium. To further exploit this mechanism for therapeutic gain, we encoded a short-hairpin RNA (shRNA) against ACP2 into oncolytic vesicular stomatitis virus (VSV∆51) under a miR-30 promoter. This bioengineered OV demonstrated expression of the miR-30 promoter, knockdown of ACP2, repression and ultimately, showed markedly enhanced viral VSV∆51 particle production compared to its non-targeting control counterpart. Altogether, this study identifies IFN-1 regulating PP targets, namely ACP2, that may prove instrumental in increasing the therapeutic efficacy of OVs. [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]

The plant hormone ethylene is indispensable to the ripening of climacteric fruits. Although extensive studies have been conducted on ethylene signaling, the ethylene response factor (ERF)-mediated transcriptional regulation of ethylene biosynthesis in pear fruits remains to be fully elucidated. We here constructed, sequenced, and analyzed transcriptome libraries in ethephon-treated and 1-MCP-treated Cuiguan pear fruits. In total, 721 fruit ripening-associated differentially expressed genes were identified. Among them, two key genes exhibited positive correlations: the 1-aminocyclopropane-1-carboxylic acid synthase (ACS)-encoding gene PbrACS3 and the ERF-encoding gene named PbrERF114. PbrERF114 overexpression increased ethylene production as well as the PbrACS3 expression level. Conversely, virus-induced gene silencing downregulated PbrERF114, thereby decreasing ethylene production and reducing PbrACS3 expression levels. Notably, PbrERF114 could directly interact with PbrACS3 and PbrERF24 promoters, thus inducing their expression. However, it did not result in an enhancement in luciferase activity, which is regulated by the PbrACS1b or PbrACO1 promoter. PbrERF24 could directly bind to PbrACO1 as well as PbrACS3 to promote ethylene synthesis. In conclusion, PbrERF114 can directly and indirectly mediate ethylene biosynthesis by transcriptionally regulating PbrACS3 and PbrERF24, respectively, thereby triggering a signaling cascade that induces the expression of both PbrACS3 and PbrACO1. [ABSTRACT FROM AUTHOR]

SUMMARY: Long non‐coding RNAs (lncRNAs) play an important role in various biological processes in plants. However, there have been few reports on the evolutionary signatures of lncRNAs in closely related cotton species. The lncRNA transcription patterns in two tetraploid cotton species and their putative diploid ancestors were compared in this paper. By performing deep RNA sequencing, we identified 280 429 lncRNAs from 21 tissues in four cotton species. lncRNA transcription evolves more rapidly than mRNAs, and exhibits more severe turnover phenomenon in diploid species compared to that in tetraploid species. Evolutionarily conserved lncRNAs exhibit higher expression levels, and lower tissue specificity compared with species‐specific lncRNAs. Remarkably, tissue expression of homologous lncRNAs in Gossypium hirsutum and G. barbadense exhibited similar patterns, suggesting that these lncRNAs may be functionally conserved and selectively maintained during domestication. An orthologous lncRNA, lncR4682, was identified and validated in fibers of G. hirsutum and G. barbadense with the highest conservatism and expression abundance. Through virus‐induced gene silencing in upland cotton, we found that lncR4682 and its target genes GHPAS2 and GHKCS19 positively regulated fiber elongation. In summary, the present study provides a systematic analysis of lncRNAs in four closely related cotton species, extending the understanding of transcriptional conservation of lncRNAs across cotton species. In addition, LncR4682‐PAS2‐KCS19 contributes to cotton fiber elongation by participating in the biosynthesis of very long‐chain fatty acids. Significance Statement: This study explores the evolutionary convergency and divergency of lncRNAs in four cotton species during the polyploidy and identifies the role of lncR4682‐PAS2‐KCS19 module in fiber elongation. [ABSTRACT FROM AUTHOR]

The use of combinatorial siRNAs shows great promise for drug discovery, but the identification of safe and effective siRNA combinations remains challenging. Here, we develop a massively multiplexed technology for systematic screening of siRNA-based cocktail therapeutics. We employ composite micro-carriers that are responsive to near infrared light and magnetic field to achieve photoporation-facilitated siRNA transfection to individual cells. Thus, randomized gene silencing by different siRNA formulations can be performed with high-throughput single-cell-based analyses. For screening anti-cancer siRNA cocktails, we test more than 1300 siRNA combinations for knocking down multiple genes related to tumor growth, discovering effective 3-siRNA formulations with an emphasis on the critical role of inhibiting Cyclin D1 and survivin, along with their complementary targets for synergic efficacy. This approach enables orders of magnitude reduction in time and cost associated with largescale siRNA screening, and resolves key insights to siRNA pharmacology that are not permissive to existing methods. Combined use of multiple siRNAs promises a new path for drug development. Here, the authors demonstrate a multiplexed combinatorial screening of thousands of siRNA formulations through highthroughput single-cell gene silencing, discovering the most effective anti-cancer siRNA cocktail therapeutics. [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]

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]

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]

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]

A protein-expressing citrus tristeza virus-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus-induced gene silencing system intended for use with studies of California citrus. Virus-induced gene silencing constructs engineered with a truncated Citrus macrophylla PHYTOENE DESATURASE (CmPDS) gene sequence in the sense or antisense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two nonsynonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3, were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 weeks postinoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8- to 12-month postinoculation window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 months postinoculation, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Obligate biotrophic powdery mildew fungi infect a wide range of economically important plants. These fungi often deliver effector proteins into the host tissues to suppress plant immunity and sustain infection. The phytohormone salicylic acid (SA) is one of the most important signals that activate plant immunity against pathogens. However, how powdery mildew effectors interact with host SA signalling is poorly understood. Isochorismatase (ISC) effectors from two other filamentous pathogens have been found to inhibit host SA biosynthesis by hydrolysing isochorismate, the main SA precursor in the plant cytosol. Here, we identified an ISC effector, named EqIsc1, from the rubber tree powdery mildew fungus Erysiphe quercicola. In ISC enzyme assays, EqIsc1 displayed ISC activity by transferring isochorismate to 2,3‐dihydro‐2,3‐dihydroxybenzoate in vitro and in transgenic Nicotiana benthamiana plants. In EqIsc1‐expressing transgenic Arabidopsis thaliana, SA biosynthesis and SA‐mediated immune response were significantly inhibited. In addition, we developed an electroporation‐mediated transformation method for the genetic manipulation of E. quercicola. Inoculation of rubber tree leaves with EqIsc1‐silenced E. quercicola strain induced SA‐mediated immunity. We also detected the translocation of EqIsc1 into the plant cytosol during the interaction between E. quercicola and its host. Taken together, our results suggest that a powdery mildew effector functions as an ISC enzyme to hydrolyse isochorismate in the host cytosol, altering the SA biosynthesis and immune response. [ABSTRACT FROM AUTHOR]

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive wheat diseases. The plant hormone abscisic acid (ABA) plays a key regulatory role in plant response to stress. ABA‐, stress‐, ripening‐induced proteins (ASR) have been shown to be abundantly induced in response to biotic and abiotic stresses to protect plants from damage. However, the function of wheat ASR2‐like protein (TaASR2L) in plants under biotic stress remains unclear. In this study, transient silencing of TaASR2L using a virus‐induced gene silencing system substantially reduced wheat resistance to Pst. TaASR2L interaction with serine/arginine‐rich splicing factor SR30‐like (TaSR30) was validated mainly in the nucleus. Knockdown of TaSR30 expression substantially reduced wheat resistance to Pst. Overexpression of TaASR2L and TaSR30 demonstrated that they can promote the expression of ABA‐ and resistance‐related genes to enhance wheat resistance to Pst. In addition, the expression levels of TaSR30 and TaASR2L were substantially increased by exogenous ABA, and the resistance of wheat to Pst was increased, and the expression of PR genes was induced. Therefore, these results suggest that TaASR2L interacts with TaSR30 by promoting PR genes expression and enhancing wheat resistance to Pst. [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]

[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]

Amphenicol antibiotics, such as chloramphenicol (CHL), thiamphenicol (TAP), and florfenicol (Ff), are high-risk emerging pollutants. Their extensive usage in aquaculture, livestock, and poultry farming has led to an increase in bacterial antibiotic resistance and facilitated the spread of resistance genes. Yet, limited research has been conducted on the co-resistance of CHL, TAP, and Ff. Herein, a novel amidase AphA was identified from a pure cultured strain that can concurrently mediate the hydrolytic inactivation of CHL, TAP, and Ff, yielding products p-nitrophenylserinol, thiamphenicol amine (TAP-amine), and florfenicol amine (Ff-amine), respectively. The antibacterial activity of these antibiotic hydrolysates exhibited a significant reduction or complete loss in comparison to the parent compounds. Notably, AphA shared less than 26% amino acid sequence identity with previously reported enzymes and exhibited high conservation within the sphingomonad species. Through enzymatic kinetic analysis, the AphA exhibited markedly superior affinity and catalytic activity toward Ff in comparison to CHL and TAP. Site-directed mutagenesis analysis revealed the indispensability of catalytic triad residues, particularly serine 153 and histidine 277, in forming crucial hydrogen bonds essential for AphA’s hydrolytic activity. Comparative genomic analysis showed that aphA genes in some species are closely adjacent to various transposable elements, indicating that there is a high potential risk of horizontal gene transfer (HGT). This study established a hydrolysis resistance mechanism of amphenicol antibiotics in sphingomonads, which offers theoretical guidance and a novel marker gene for assessing the prevalent risk of amphenicol antibiotics in the environment. IMPORTANCE Amphenicol antibiotics are pervasive emerging contaminants that present a substantial threat to ecological systems. Few studies have elucidated resistance genes or mechanisms that can act on CHL, TAP, and Ff simultaneously. The results of this study fill this knowledge gap and identify a novel amidase AphA from the bacterium Sphingobium yanoikuyae B1, which mediates three typical amphenicol antibiotic inactivation, and the molecular mechanism is elucidated. The diverse types of transposable elements were identified in the flanking regions of the aphA gene, indicating the risk of horizontal transfer of this antibiotic resistance genes (ARG). These findings offer new insights into the bacterial resistance to amphenicol antibiotics. The gene reported herein can be utilized as a novel genetic diagnostic marker for monitoring the environmental fate of amphenicol antibiotics, thereby enriching risk assessment efforts within the context of antibiotic resistance. [ABSTRACT FROM AUTHOR]

Development of genome-editing tools in diverse microbial species is an important step both in understanding the roles of those microbes in different environments, and in engineering microbes for a variety of applications. Freshwater-specific clades of Actinobacteria are ubiquitous and abundant in surface freshwaters worldwide. Here, we show that Rhodoluna lacicola and Aurantimicrobium photophilum, which represent widespread clades of freshwater Actinobacteria, are naturally transformable. We also show that gene inactivation via double homologous recombination and replacement of the target gene with antibiotic selection markers can be used in both strains, making them convenient and broadly accessible model organisms for freshwater systems. We further show that in both strains, the predicted phytoene synthase is the only phytoene synthase, and its inactivation prevents the synthesis of all pigments. The tools developed here enable targeted modification of the genomes of some of the most abundant microbes in freshwater communities. These genome-editing tools will enable hypothesis testing about the genetics and (eco)physiology of freshwater Actinobacteria and broaden the available model systems for engineering freshwater microbial communities. IMPORTANCE To advance bioproduction or bioremediation in large, unsupervised environmental systems such as ponds, wastewater lagoons, or groundwater systems, it will be necessary to develop diverse genetically amenable microbial model organisms. Although we already genetically modify a few key species, tools for engineering more microbial taxa, with different natural phenotypes, will enable us to genetically engineer multispecies consortia or even complex communities. Developing genetic tools for modifying freshwater bacteria is particularly important, as wastewater, production ponds or raceways, and contaminated surface water are all freshwater systems where microbial communities are already deployed to do work, and the outputs could potentially be enhanced by genetic modifications. Here, we demonstrate that common tools for genome editing can be used to inactivate specific genes in two representatives of a very widespread, environmentally relevant group of Actinobacteria. These Actinobacteria are found in almost all tested surface freshwater environments, where they co-occur with primary producers, and genome-editing tools in these species are thus a step on the way to engineering microbial consortia in freshwater environments. [ABSTRACT FROM AUTHOR]

In recent years, RNA interference (RNAi) has emerged as a potential tool to manage the pest population in agricultural fields and storage systems, however so far it has not come up in a big way. RNAi is a basic molecular biology mechanism occurring in all eukaryotic cells wherein a dsRNA (specific to a particular target messenger RNA) is used to inhibit/silence/knockdown gene expression. This dsRNA undergoes nucleolytic cleavage and several modifications to bind the complementary mRNA and induce endo-nucleolytic cleavage or translation repression. This basic molecular biological phenomenon can be exploited to silence the key genes (virulence and disease progression, toxin production, pest-specific key genes) in pests thus challenging their fecundity and survival. This mechanism is suitable for managing most insects (lepidopteran, dipteran, coleopteran, hemipteran, etc.) and nematodes. Application of RNAi differs based on the mode of delivery system of dsRNA to the pest. The efficiency and efficacy of RNAi depend upon targeted genes, type of dsRNA produced, length of dsRNA, feeding methods, stability of dsRNA in insect guts, and easy uptake by gut epithelium, etc. However, this technology is not utilised up to its full potential and therefore the present review is aimed to assess the possibility of RNAi in agricultural pest control and the way forward. [ABSTRACT FROM AUTHOR]

Argonaute (AGO), a component of RNA-induced silencing complexes (RISCs), is a representative RNA-binding protein (RBP) known to bind with mature microRNAs (miRNAs) and is directly involved in post-transcriptional gene silencing. However, despite the biological significance of miRNAs, the roles of other miRNA-binding proteins (miRBPs) remain unclear in the regulation of miRNA loading, dissociation from RISCs and extracellular release. In this study, we performed protein arrays to profile miRBPs and identify 118 RBPsthat directly bind to miRNAs. Among those proteins, the RBP quaking (QKI) inhibits extracellular release of the mature microRNA let-7b by controlling the loading of let-7b into extracellular vesicles via additional miRBPs such as AUF1 (also known as hnRNPD) and hnRNPK. The enhanced extracellular release of let-7b after QKI depletion activates Toll-like receptor 7 (TLR7) and promotes the production of proinflammatory cytokines in recipient cells, leading to brain inflammation in the mouse cortex. Thus, this study reveals the contribution of QKI to the inhibition of brain inflammation via regulation of extracellular let-7b release. [ABSTRACT FROM AUTHOR]

Simple Summary: The brown planthopper is a major pest threatening rice crops in Asia, making it essential to identify target genes for RNAi-based pest control strategies. The Cleavage Stimulation Factor (CstF) complex, which plays a key role in mRNA 3′ end processing, is composed of several critical genes, and mutations in these genes can cause severe developmental defects, indicating their potential as targets. In this study, we identified homologs of the human CstF complex genes (NlCstF50, NlCstF64, and NlCstF77) in Nilaparvata lugens and investigated their functions using RNAi techniques. These genes are expressed throughout all developmental stages and various tissues, with particularly high levels in eggs and testes. Targeting these genes with RNAi resulted in significant mortality, decreased egg production, and lower hatch rates. These results suggest that NlCstF50, NlCstF64, and NlCstF77 could be effective targets for RNAi-based pest control strategies. The Cleavage Stimulation Factor (CstF) complex, consisting of three subunits, is essential for the 3′ end processing of precursor messenger RNA (pre-mRNA). In mammals, this complex includes CstF50, CstF64, and CstF77, named according to their molecular weights, and these proteins are conserved across many organisms. However, the functional roles of the three CstF genes (NlCstF50, NlCstF64, and NlCstF77) in Nilaparvata lugens, a major rice pest, have not been fully explored. This study identified and characterized the sequences of these genes, with proteins encoded by NlCstF50, NlCstF64, and NlCstF77 consisting of 439, 419, and 732 amino acids, respectively. These proteins are conserved among various insect species. Spatio-temporal expression analysis revealed that these genes are expressed at all developmental stages and in various tissues, with peak levels in eggs and testes. RNA interference (RNAi) targeting one or all three NlCstF genes resulted in a reduction in gene expression by 68% to 90% at 72 h post-injection, indicating that multi-gene dsRNA can achieve similar silencing outcomes as single-gene dsRNA. Knocking down one or all three NlCstF genes caused significant lethal phenotypes and molting disruptions. Mortality rates increased from 62.5% (dsNlCstF50) to 95.4% (dsNlCstF(50+64+77)). Additionally, silencing these genes reduced the number of eggs laid per female and hatch rates. These results highlight the critical role of NlCstF genes in the development and reproduction of N. lugens, suggesting their potential as targets for RNAi-based pest control strategies. [ABSTRACT FROM AUTHOR]

Disease resistance is often associated with compromised plant growth and yield due to defense‐growth tradeoffs. However, key components and mechanisms underlying the defense‐growth tradeoffs are rarely explored in maize. In this study, we find that ZmSKI3, a putative subunit of the SUPERKILLER (SKI) complex that mediates the 3′‐5′ degradation of RNA, regulates both plant development and disease resistance in maize. The Zmski3 mutants showed retarded plant growth and constitutively activated defense responses, while the ZmSKI3 overexpression lines are more susceptible to Curvularia lunata and Bipolaris maydis. Consistently, the expression of defense‐related genes was generally up‐regulated, while expressions of growth‐related genes were mostly down‐regulated in leaves of the Zmski3‐1 mutant compared to that of wild type. In addition, 223 differentially expressed genes that are up‐regulated in Zmski3‐1 mutant but down‐regulated in the ZmSKI3 overexpression line are identified as potential target genes of ZmSKI3. Moreover, small interfering RNAs targeting the transcripts of the defense‐ and growth‐related genes are differentially accumulated, likely to combat the increase of defense‐related transcripts but decrease of growth‐related transcripts in Zmski3‐1 mutant. Taken together, our study indicates that plant growth and immunity could be regulated by both ZmSKI3‐mediated RNA decay and post‐transcriptional gene silencing in maize. [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]

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]

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]

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]

The Chinese gentian, Gentiana sino-ornata produces brilliant blue flowers. To investigate the biological function and transcriptional regulation mechanism of the anthocyanin 5-O-acyltransferase gene (Gs5AT) in the corolla, it is beneficial to analyse the mechanism of blue flower colour presentation. In this investigation, we obtained the CDS and promoter sequences of the gene Gs5AT. Yeast one-hybrid experiments were used to identify the transcription factor GsbHLH7 that activates the gene Gs5AT. According to quantitive reverse transcription polymerase chain reaction analysis, the expression of the gene Gs5AT was significantly and positively correlated with the gene GsbHLH7. The colour phenotype of the flowers was significantly altered by the virus-induced gene silencing transduction of Gs5AT and GsbHLH7 , with GsbHLH7 silencing producing more pronounced changes in the corolla colour than Gs5AT. The expression of GsF3′5′H , GsDFR , GsANS , Gs3GT , and Gs5GT all fell to varying degrees after GsbHLH7 silencing, indicating that GsbHLH7 may regulate transcription of these genes as well as Gs5AT. The results of this study indicate that Gs5AT was positively regulated by the GsbHLH7 , and thus affects the colour presentation of the blue corolla. The Chinese gentian, Gentiana sino-ornata , is a commercially important flower that produces brilliant blue blossoms. We identified the responsible gene, Gs5AT , and discovered that another gene, GsbHLH7 , can regulate the expression of the first gene. This study provides a further basis for molecular breeding of blue flowers. [ABSTRACT FROM AUTHOR]

To analyze the genes involved in orchid floral development, a homeodomain-leucine zipper II gene PaHAT14 , which is specifically and highly expressed in perianth during early flower development, was identified from Phalaenopsis. Transgenic Arabidopsis plants expressing 35S:: PaHAT14 and 35S:: PaHAT14  + SRDX (fused with the repressor motif SRDX) exhibited similar altered phenotypes, including small leaves, early flowering and bending petals with increased cuticle production. This suggests that PaHAT14 acts as a repressor. In contrast, transgenic Arabidopsis plants expressing 35S:: PaHAT14  + VP16 (fused with the activation domain VP16) exhibited curled leaves, late flowering and folded petals with decreased cuticle production within hardly opened flowers. Additionally, the expression of the ERF gene DEWAX2 , which negatively regulates cuticular wax biosynthesis, was downregulated in 35S:: PaHAT14 and 35S:: PaHAT14  + SRDX transgenic Arabidopsis , while it was upregulated in 35S:: PaHAT14  + VP16 transgenic Arabidopsis. Furthermore, transient overexpression of PaHAT14 in Phalaenopsis petal/sepal increased cuticle deposition due to the downregulation of PaERF105 , a Phalaenopsis DEWAX2 ortholog. On the other hand, transient overexpression of PaERF105 decreased cuticle deposition, whereas cuticle deposition increased and the rate of epidermal water loss was reduced in PaERF105 virus-induced gene silencing Phalaenopsis flowers. Moreover, ectopic expression of PaERF105 not only produced phenotypes similar to those in 35S:: PaHAT14  + VP16 Arabidopsis but also compensated for the altered phenotypes observed in 35S:: PaHAT14 and 35S:: PaHAT14  + SRDX Arabidopsis. These results suggest that PaHAT14 promotes cuticle deposition by negatively regulating downstream gene PaERF105 in orchid flowers. [ABSTRACT FROM AUTHOR]