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Highly pathogenic avian influenza (HPAI) viruses cause high mortality rates in wild birds and farmed poultry, and their potential for adaptation to humans remains a major pandemic threat (2). In [...]

Droplet digital polymerase chain reaction (ddPCR) stands out as a highly sensitive diagnostic technique that is gaining traction in infectious disease diagnostics due to its ability to quantitate very low numbers of viral gene copies. By partitioning the sample into thousands of droplets, ddPCR enables precise and absolute quantification without relying on a standard curve. However, current ddPCR systems often exhibit relatively low levels of integration, and the analytical process remains dependent on elaborate workflows for up-front sample preparation. Here, we introduce a fully-integrated system seamlessly combining viral lysis, RNA extraction, emulsification, reverse transcription (RT) ddPCR, and fluorescence readout in a sample-to-answer format. The system comprises a disposable microfluidic cartridge housing buffers and reagents required for the assay, and a centrifugal platform that allows for pneumatic actuation of liquids during rotation, enabling automation of the workflow. Highly monodisperse droplets (∼50 μm in diameter) are produced using centrifugal step emulsification and automatically transferred to an integrated heating module for target amplification. The platform is equipped with a miniature fluorescence imaging system enabling on-chip read-out of droplets after RT-ddPCR. We demonstrate sample-to-answer detection of SARS-CoV-2 N and E genes, along with RNase P endogenous reference, using hydrolysis probes and multiplexed amplification within single droplets for concentrations as low as 0.1 copy per μL. We also tested 14 nasopharyngeal swab specimens from patients and were able to distinguish positive and negative SARS-CoV-2 samples with 100% accuracy, surpassing results obtained by conventional real-time amplification. [ABSTRACT FROM AUTHOR]

The eukaryotic transcriptional Mediator comprises a large core (cMED) and a dissociable CDK8 kinase module (CKM). cMED recruits RNA polymerase II (RNA Pol II) and promotes pre-initiation complex formation in a manner repressed by the CKM through mechanisms presently unknown. Herein, we report cryoelectron microscopy structures of the complete human Mediator and its CKM. The CKM binds to multiple regions on cMED through both MED12 and MED13, including a large intrinsically disordered region (IDR) in the latter. MED12 and MED13 together anchor the CKM to the cMED hook, positioning CDK8 downstream and proximal to the transcription start site. Notably, the MED13 IDR obstructs the recruitment of RNA Pol II/MED26 onto cMED by direct occlusion of their respective binding sites, leading to functional repression of cMED-dependent transcription. Combined with biochemical and functional analyses, these structures provide a conserved mechanistic framework to explain the basis for CKM-mediated repression of cMED function. [Display omitted] • Structures of the complete human Mediator and its CDK8 kinase module (CKM) are presented • CKM binds cMED through MED12 and the intrinsically disordered region (IDR) of MED13 • MED13 IDR blocks RNA Pol II/MED26 from binding to cMED by occupying their binding sites • Human and yeast MED13 share a conserved mechanism for transcriptional repression Chao and Chen et al. present cryo-EM structures of the complete human Mediator and its CDK8 kinase module (CKM). The CKM uses its MED13 IDR to occupy RNA-polymerase-II- and MED26-binding sites, blocking their interactions with Mediator and repressing Mediator-dependent transcription—a mechanism conserved in humans and yeast. [ABSTRACT FROM AUTHOR]

5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression in vivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression. [Display omitted] • 5fC forms chromocenters in the perinucleolar compartment during Xenopus ZGA • 5fC colocalizes with RNA Pol III and accumulates on active tRNA tandem repeats • 5fC promotes Pol III recruitment and tRNA transcription at ZGA • 5fC stimulates transcription of a 5fC-modified tRNA transgene An open question in epigenetics is if 5-formylcytosine (5fC) is merely a byproduct of active demethylation or an instructive mark that regulates gene expression on its own. This work demonstrates 5fC chromocenters in early frog embryos and shows that 5fC acts as an instructive mark to promote RNA Pol III transcription during zygotic genome activation. [ABSTRACT FROM AUTHOR]

AbstractBackgroundMethodsResultsConclusionsThe abnormality of the immune system caused by infection is a contributor to the organ dysfunctions associated with sepsis. The balance between Th17/Treg cells is essential for maintaining immune homeostasis. Bergapten is a natural furocoumarin and has been reported to alleviate the Th17/Treg imbalance. Here, we explored the effects of bergapten on the inflammation and immune state in mouse models of sepsis.The model was established using the cecal ligation and puncture method. Mice were administered 30 mg/kg bergapten. Histological examination, RT-qPCR, enzyme-linked immunosorbent assay, immunoblotting, immunofluorescence, immunohistochemistry, and flow cytometry were used to evaluate the effects of bergapten in vivo.Bergapten ameliorated lung damage, reduced lung wet/dry weight ratio, inhibited myeloperoxidase activity, and reduced inflammatory cell infiltration. Bergapten also restrained sepsis-induced inflammation via inhibition of inflammatory cytokines and NF-κB signaling. These effects were accompanied by the restored Th17/Treg balance induced by bergapten. Bergapten decreased the number of Th17 cells and elevated the number of Tregs, and this effect was mediated by the signal transducer and activator of transcription 5 (STAT5)/Forkhead box P3 (Foxp3) and STAT3/retinoid-related orphan receptor-γt (RORγt) pathways.Bergapten exerted anti-inflammatory effects in acute lung injury by improving the Th17/Treg balance, which suggested a potential of bergapten as an immunomodulatory drug treating sepsis-associated diseases. [ABSTRACT FROM AUTHOR]

Colonizing in the gastrointestinal tract, Escherichia coli confronts diverse acidic challenges and evolves intricate acid resistance strategies for its survival. The lysine-mediated decarboxylation (Cad) system, featuring lysine decarboxylase CadA, lysine/cadaverine antiporter CadB, and transcriptional activator CadC, plays a crucial role in E. coli's adaptation to moderate acidic stress. While the activation of the one-component system CadC and subsequent upregulation of cadBA operon in response to acid and lysine presence have been proposed, the molecular mechanisms governing the transition of CadC from an inactive to an active state remain elusive. Under neutral conditions, CadC is inhibited by forming a complex with lysine-specific permease LysP, stabilized in this inactive state by a disulfide bond. Our study unveils that, in an acidic environment, the disulfide bond in CadC is reduced by the disulfide bond isomerase DsbC, exposing R184 to periplasmic proteases, namely DegQ and DegP. Cleavage at R184 by DegQ and DegP generates an active N-terminal DNA-binding domain of CadC, which binds to the cadBA promoter, resulting in the upregulated transcription of the cadA and cadB genes. Upon activation, CadA decarboxylates lysine, producing cadaverine, subsequently transported extracellularly by CadB. We propose that accumulating cadaverine gradually binds to the CadC pH-sensing domain, preventing cleavage and activation of CadC as a feedback mechanism. The identification of DegP, DegQ, and DsbC completes a comprehensive roadmap for the activation and regulation of the Cad system in response to moderate acidic stress in E. coli. Periplasmic proteases DegP and DegQ along with the disulfide bond isomerase DsbC are implicated in CadC-mediated antiacid response in E. coli. [ABSTRACT FROM AUTHOR]

In insects and mammals, 3D genome topology has been linked to transcriptional states yet whether this link holds for other eukaryotes is unclear. Using both ligation proximity and fluorescence microscopy assays, we show that in Saccharomyces cerevisiae, Heat Shock Response (HSR) genes dispersed across multiple chromosomes and under the control of Heat Shock Factor (Hsf1) rapidly reposition in cells exposed to acute ethanol stress and engage in concerted, Hsf1-dependent intergenic interactions. Accompanying 3D genome reconfiguration is equally rapid formation of Hsf1-containing condensates. However, in contrast to the transience of Hsf1-driven intergenic interactions that peak within 10-20 min and dissipate within 1 hr in the presence of 8.5% (v/v) ethanol, transcriptional condensates are stably maintained for hours. Moreover, under the same conditions, Pol II occupancy of HSR genes, chromatin remodeling, and RNA expression are detectable only later in the response and peak much later (>1 hr). This contrasts with the coordinate response of HSR genes to thermal stress (39°C) where Pol II occupancy, transcription, histone eviction, intergenic interactions, and formation of Hsf1 condensates are all rapid yet transient (peak within 2.5-10 min and dissipate within 1 hr). Therefore, Hsf1 forms condensates, restructures the genome and transcriptionally activates HSR genes in response to both forms of proteotoxic stress but does so with strikingly different kinetics. In cells subjected to ethanol stress, Hsf1 forms condensates and repositions target genes before transcriptionally activating them. [ABSTRACT FROM AUTHOR]

Maintaining cellular homeostasis in the face of stress conditions is vital for the overall well‐being of an organism. Reactive oxygen species (ROS) are among the most potent cellular stressors and can disrupt the internal redox balance, giving rise to oxidative stress. Elevated levels of ROS can severely affect biomolecules and have been associated with a range of pathophysiological conditions. In response to oxidative stress, yeast activator protein‐1 (Yap1p) undergoes post‐translation modification that results in its nuclear accumulation. YAP1 has a key role in oxidative detoxification by promoting transcription of numerous antioxidant genes. In this study, we identified previously undescribed functions for NCE102, CDA2, and BCS1 in YAP1 expression in response to oxidative stress induced by hydrogen peroxide (H2O2). Deletion mutant strains for these candidates demonstrated increased sensitivity to H2O2. Our follow‐up investigation linked the activity of these genes to YAP1 expression at the level of translation. Under oxidative stress, global cap‐dependent translation is inhibited, prompting stress‐responsive genes like YAP1 to employ alternative modes of translation. We provide evidence that NCE102, CDA2, and BCS1 contribute to cap‐independent translation of YAP1 under oxidative stress. [ABSTRACT FROM AUTHOR]

Current research reports that lactate affects Treg metabolism, although the precise mechanism has only been partially elucidated. In this study, we presented evidence demonstrating that elevated lactate levels enhanced cell proliferation, suppressive capabilities, and oxidative phosphorylation (OXPHOS) in human Tregs. The expression levels of Monocarboxylate Transporters 1/2/4 (MCT1/2/4) regulate intracellular lactate concentration, thereby influencing the varying responses observed in naive Tregs and memory Tregs. Through mitochondrial isolation, sequencing, and analysis of human Tregs, we determined that α-1,3-Mannosyl-Glycoprotein 2-β-N-Acetylglucosaminyltransferase (MGAT1) served as the pivotal driver initiating downstream N-glycosylation events involving progranulin (GRN) and hypoxia-upregulated 1 (HYOU1), consequently enhancing Treg OXPHOS. The mechanism by which MGAT1 was upregulated in mitochondria depended on elevated intracellular lactate that promoted the activation of XBP1s. This, in turn, supported MGAT1 transcription as well as the interaction of lactate with the translocase of the mitochondrial outer membrane 70 (TOM70) import receptor, facilitating MGAT1 translocation into mitochondria. Pretreatment of Tregs with lactate reduced mortality in a xenogeneic graft-versushost disease (GvHD) model. Together, these findings underscored the active regulatory role of lactate in human Treg metabolism through the upregulation of MGAT1 transcription and its facilitated translocation into the mitochondria. [ABSTRACT FROM AUTHOR]

Gallbladder cancer (GBC) is the most common malignant tumor of the biliary system, with poor response to current treatments. Abnormal alternative splicing has been associated with the development of a variety of tumors. Combining the GEO database and GBC mRNA‐seq analysis, it is found high expression of the splicing factor polypyrimidine region‐ binding protein 3 (PTBP3) in GBC. Multi‐omics analysis revealed that PTBP3 promoted exon skipping of interleukin‐18 (IL‐18), resulting in the expression of ΔIL‐18, an isoform specifically expressed in tumors. That ΔIL‐18 promotes GBC immune escape by down‐regulating FBXO38 transcription levels in CD8+T cells to reduce PD‐1 ubiquitin‐mediated degradation is revealed. Using a HuPBMC mouse model, the role of PTBP3 and ΔIL‐18 in promoting GBC growth is confirmed, and showed that an antisense oligonucleotide that blocked ΔIL‐18 production displayed anti‐tumor activity. Furthermore, that the H3K36me3 promotes exon skipping of IL‐18 by recruiting PTBP3 via MRG15 is demonstrated, thereby coupling the processes of IL‐18 transcription and alternative splicing. Interestingly, it is also found that the H3K36 methyltransferase SETD2 binds to hnRNPL, thereby interfering with PTBP3 binding to IL‐18 pre‐mRNA. Overall, this study provides new insights into how aberrant alternative splicing mechanisms affect immune escape, and provides potential new perspectives for improving GBC immunotherapy. [ABSTRACT FROM AUTHOR]

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer and its prognosis remains poor. Although growing numbers of studies have verified the involvement of circular RNAs (circRNAs) in various cancer types, their specific functions in ICC remain elusive. Herein, a circRNA, circUGP2 is identified by circRNA sequencing, which is downregulated in ICC tissues and correlated with patients' prognosis. Moreover, circUGP2 overexpression suppresses tumor progression in vitro and in vivo. Mechanistically, circUGP2 functions as a transcriptional co‐activator of PURB over the expression of ADGRB1. It can also upregulate ADGRB1 expression by sponging miR‐3191‐5p. As a result, ADGRB1 prevents MDM2‐mediated p53 polyubiquitination and thereby activates p53 signaling to inhibit ICC progression. Based on these findings, circUGP2 plasmid is encapsulated into a lipid nanoparticle (LNP) system, which has successfully targeted tumor site and shows superior anti‐tumor effects. In summary, the present study has identified the role of circUGP2 as a tumor suppressor in ICC through regulating ADGRB1/p53 axis, and the application of LNP provides a promising translational strategy for ICC treatment. [ABSTRACT FROM AUTHOR]

Histones methyltransferase NSD3 targeting H3K36 is frequently disordered and mutant in various cancers, while the function of NSD3 during cancer initiation and progression remains unclear. In this study, it is proved that downregulated level of NSD3 is linked to clinical features and poor survival in lung adenocarcinoma. In vivo, NSD3 inhibited the proliferation, immigration, and invasion ability of lung adenocarcinoma. Meanwhile, NSD3 suppressed glycolysis by inhibiting HK2 translation, transcription, glucose uptake, and lactate production in lung adenocarcinoma. Mechanistically, as an intermediary, NSD3 binds to PPP1CB and p‐STAT3 in protein levels, thus forming a trimer to dephosphorylate the level of p‐STAT3 by PPP1CB, leading to the suppression of HK2 transcription. Interestingly, the phosphorylation function of PPP1CB is related to the concentration of carbon dioxide and pH value in the culture environment. Together, this study revealed the critical non‐epigenetic role of NSD3 in the regulation of STAT3‐dependent glycolysis, providing a piece of compelling evidence for targeting the NSD3/PPP1CB/p‐STAT3 in lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Summary Neonatal and adult megakaryocytes differ in proliferative capacity and ploidy levels, and neonatal and adult platelets differ in function, gene expression, and protein content. The mechanisms underlying these differences are incompletely understood. CDK8 and CDK19 are transcriptional kinases part of the CDK‐mediator complex, which regulates gene transcription in a cell‐specific manner. We discovered that cortistatin A, a potent highly selective inhibitor of CDK8/CDK19, significantly reduced cell expansion and increased ploidy in cord blood‐derived megakaryocytes. These phenotypic changes were associated with gene expression changes that partially overlapped developmentally regulated genes. These findings might have relevance for the management of developmental megakaryocyte disorders. [ABSTRACT FROM AUTHOR]

The DNA damage response is a highly conserved protective mechanism that enables cells to cope with various lesions in the genome. Extensive studies across different eukaryotic cells have identified the crucial roles played by components required for response to DNA damage. When compared to the essential signal transducers and repair factors in the DNA damage response circuitry, the negative regulators and underlying mechanisms of this circuitry have been relatively under-examined. In this study, we investigated Gst1, a putative glutathione transferase in the fungal pathogen Candida albicans. We found that under stress caused by the DNA damage agent MMS, GST1 expression was significantly upregulated, and this upregulation was further enhanced by the loss of the checkpoint kinases and DNA repair factors. Somewhat counterintuitively, deletion of GST1 conferred increased resistance to MMS, potentially via enhancing the phosphorylation of Rad53. Furthermore, overexpression of RAD53 or deletion of GST1 resulted in upregulated transcription of DNA damage repair genes, including CAS1, RAD7, and RAD30, while repression of RAD7 transcription in the GST1 deletion reversed the strain's heightened resistance to MMS. Finally, Gst1 physically interacted with Rad53, and their interaction weakened in response to MMS-induced stress. Overall, our findings suggest a negative regulatory role for GST1 in DNA damage response in C. albicans, and position Gst1 within the Rad53-mediated signaling pathway. These findings hold significant implications for understanding the mechanisms underlying the DNA damage response in this fungal pathogen and supply new potential targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus has established the importance of the nsP4 residue cysteine 483 in replication. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the coxsackievirus B3 3D polymerase. We rescued each variant in mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to host-specific replication. In C6/36 cells, we found that while the nsP4 variants had replicase function similar to that of wild-type CHIKV, many variants presented changes in protein accumulation and virion production even when viral nonstructural and structural proteins were produced. Finally, we found that WT CHIKV and nsP4 variant replication and protein production could be enhanced in mammalian cells at 28°C, yet growing virus under these conditions led to changes in virus infectivity. Taken together, these studies highlight that distinct nsP4 subdomains are required for proper RNA transcription and translation, having major effects on virion production. Author summary: Chikungunya virus (CHIKV) is a re-emerging alphavirus transmitted to humans by mosquitoes. Its replication relies on a polymerase that incorporates a significant number of errors in the new genomes, making it a good candidate to develop vaccines or antiviral strategies. However, little is known on alphavirus polymerase function in alternate hosts. To begin to understand how the CHIKV polymerase nsP4 functions, we designed a panel of nsP4 variants taking advantage of the conservation of polymerase structure across positive-strand RNA viruses. We discovered that the palm domain and ring finger subdomain of the polymerase were involved in host-specific RNA transcription and virion production. Taken together, these findings add further evidence to the crucial impact of the core palm domain of CHIKV polymerase not only on the transcription of the RNA itself, but also on the complete viral life cycle. [ABSTRACT FROM AUTHOR]

Excessive alarmins S100A8/A9 escalate the inflammation and even exacerbate immune-driven thrombosis and multi-organ damage. However, the regulatory mechanisms of S100A8/A9 expression in infectious diseases remain unclear. In this study, high-dimensional transcriptomic data analyses revealed a high proportion of CD14+FCN1hi macrophages within the pulmonary niche post-severe SARS-CoV-2 infection. By constructing the S100-coexpression gene list and supervised module scoring, we found that CD14+FCN1hi macrophages presented the highest scores of alarmin S100, and possibly served as the trigger and amplifier of inflammation in severe COVID-19. These CD14+FCN1hi cells lacked the positive regulatory activity of transcription factor PPARγ, and lost their differentiation ability towards mature macrophages. Ex vivo experiments further validated that the epithelial cells with high ORF-3a expression promoted the expression and secretion of S100A8/A9 through ANXA1/SAA1-FPR1 signaling. S100A8/A9 heterodimers, as well as the co-localization of S100A8/A9 with microtubules, were both diminished by the FPR1 inhibitor. Phospho-kinase protein array indicated that STAT3 promoted transcription, and PLC-γ and ERK1/2 pathways were involved in the hetero-dimerization and unconventional secretion of S100A8/A9. Our study highlights the pivotal role of FPR1 signaling in the excessive production of S100A8/A9 and provides a promising target for the prevention and control of severe COVID-19 and post-acute COVID-19 sequelae. CD14+FCN1hi macrophages highly express S100A8/S100A9, which is initiated by FPR1 signaling in severe COVID-19. An FPR1 inhibitor prevents the transcription, hetero-dimerization, and secretion of S100A8/A9 through STAT3/PLC-γ/ERK1/2 pathways. [ABSTRACT FROM AUTHOR]

Recent advances in high-resolution mass spectrometry-based proteomics have improved our understanding of lysine acetylation in proteins, including histones and non-histone proteins. Lysine acetylation, a reversible post-translational modification, is catalyzed by lysine acetyltransferases (KATs) and lysine deacetylases (KDACs). Proteins comprising evolutionarily conserved bromodomains (BRDs) recognize these acetylated lysine residues and consequently activate transcription. Lysine acetylation regulates almost all cellular processes, including transcription, cell cycle progression, and metabolic functions. Studies have reported the aberrant expression, translocation, and mutation of genes encoding lysine acetylation regulators in various cancers, including digestive tract cancers. These dysregulated lysine acetylation regulators contribute to the pathogenesis of digestive system cancers by modulating the expression and activity of cancer-related genes or pathways. Several inhibitors targeting KATs, KDACs, and BRDs are currently in preclinical trials and have demonstrated anti-cancer effects. Digestive tract cancers, including encompass esophageal, gastric, colorectal, liver, and pancreatic cancers, represent a group of heterogeneous malignancies. However, these cancers are typically diagnosed at an advanced stage owing to the lack of early symptoms and are consequently associated with poor 5-year survival rates. Thus, there is an urgent need to identify novel biomarkers for early detection, as well as to accurately predict the clinical outcomes and identify effective therapeutic targets for these malignancies. Although the role of lysine acetylation in digestive tract cancers remains unclear, further analysis could improve our understanding of its role in the pathogenesis of digestive tract cancers. This review aims to summarize the implications and pathogenic mechanisms of lysine acetylation dysregulation in digestive tract cancers, as well as its potential clinical applications. [ABSTRACT FROM AUTHOR]

One of the main efforts of scientists to study drug development is the discovery of novel antiviral agents that could be beneficial in the struggle against viruses that cause diseases in humans. Natural products are complex metabolites that are designed and synthesised by different sources in an attempt to optimise nature. Recently, natural products are still a source of biologically active molecules, facilitating drug discovery. A p21-activating kinase PAK1 is a key regulator of cytoskeletal actin assembly, phenotypic signalling, and transcription process which affects a wide range of cellular processes such as cell motility, invasion, metastasis, cell growth, angiogenesis, and cell cycle progression. Most recently, PAK1 was shown to be involved in the progression of coronavirus-caused pulmonary inflammation (lung fibrosis), but clinical data is not currently available yet. This review highlights the naturally occurring compounds that inhibit the oncogenic, melanogenic, and ageing kinase PAK1. Additionally, the potent anti-inflammatory effects of natural products in an attempt to prevent pulmonary fibrosis in COVID-19 have also been discussed. [ABSTRACT FROM AUTHOR]

The interplay between G4s and R-loops are emerging in regulating DNA repair, replication, and transcription. A comprehensive picture of native co-localized G4s and R-loops in living cells is currently lacking. Here, we describe the development of HepG4-seq and an optimized HBD-seq methods, which robustly capture native G4s and R-loops, respectively, in living cells. We successfully employed these methods to establish comprehensive maps of native co-localized G4s and R-loops in human HEK293 cells and mouse embryonic stem cells (mESCs). We discovered that co-localized G4s and R-loops are dynamically altered in a cell type-dependent manner and are largely localized at active promoters and enhancers of transcriptional active genes. We further demonstrated the helicase Dhx9 as a direct and major regulator that modulates the formation and resolution of co-localized G4s and R-loops. Depletion of Dhx9 impaired the self-renewal and differentiation capacities of mESCs by altering the transcription of co-localized G4s and R-loops -associated genes. Taken together, our work established that the endogenous co-localized G4s and R-loops are prevalently persisted in the regulatory regions of active genes and are involved in the transcriptional regulation of their linked genes, opening the door for exploring broader roles of co-localized G4s and R-loops in development and disease. [ABSTRACT FROM AUTHOR]

In response to DNA double-strand damage, ongoing transcription is inhibited to facilitate accurate DNA repair while transcriptional recovery occurs after DNA repair is complete. However, the mechanisms at play and the identity of the transcripts being regulated in this manner are unclear. In contrast to the situation following UV damage, we found that transcriptional recovery after ionizing radiation (IR) occurs in a manner independent of the HIRA histone chaperone. Sequencing of the nascent transcripts identified a programmed transcriptional response, where certain transcripts and pathways are rapidly downregulated after IR, while other transcripts and pathways are upregulated. Specifically, most of the loss of nascent transcripts occurring after IR is due to inhibition of transcriptional initiation of the highly transcribed histone genes and the rDNA. To identify factors responsible for transcriptional inhibition after IR in an unbiased manner, we performed a whole genome gRNA library CRISPR/Cas9 screen. Many of the top hits on our screen were factors required for protein neddylation. However, at short times after inhibition of neddylation, transcriptional inhibition still occurred after IR, even though neddylation was effectively inhibited. Persistent inhibition of neddylation blocked transcriptional inhibition after IR, and it also leads to cell cycle arrest. Indeed, we uncovered that many inhibitors and conditions that lead to cell cycle arrest in G1 or G2 phase also prevent transcriptional inhibition after IR. As such, it appears that transcriptional inhibition after IR occurs preferentially at highly expressed genes in cycling cells. [ABSTRACT FROM AUTHOR]

Male germ cells share a common origin across animal species, therefore they likely retain a conserved genetic program that defines their cellular identity. However, the unique evolutionary dynamics of male germ cells coupled with their widespread leaky transcription pose significant obstacles to the identification of the core spermatogenic program. Through network analysis of the spermatocyte transcriptome of vertebrate and invertebrate species, we describe the conserved evolutionary origin of metazoan male germ cells at the molecular level. We estimate the average functional requirement of a metazoan male germ cell to correspond to the expression of approximately 10,000 protein- coding genes, a third of which defines a genetic scaffold of deeply conserved genes that has been retained throughout evolution. Such scaffold contains a set of 79 functional associations between 104 gene expression regulators that represent a core component of the conserved genetic program of metazoan spermatogenesis. By genetically interfering with the acquisition and maintenance of male germ cell identity, we uncover 161 previously unknown spermatogenesis genes and three new potential genetic causes of human infertility. These findings emphasize the importance of evolutionary history on human reproductive disease and establish a cross- species analytical pipeline that can be repurposed to other cell types and pathologies. [ABSTRACT FROM AUTHOR]

Infectious laryngotracheitis (ILT) remains a significant concern for the poultry industry worldwide due to its impact on animal welfare and its substantial economic consequences. The disease is caused by the alphaherpesvirus, infectious laryngotracheitis virus (ILTV). This study investigated in vitro host-virus interactions of a glycoprotein G (gG) deletion mutant vaccine strain of ILTV (ΔgG ILTV), and its parent wild-type strain (CSW-1 ILTV). Inoculations were performed separately for the two strains of ILTV using both a primary (chicken embryonic kidney, CEK) and a continuous culture (leghorn male hepatoma, LMH) of chicken cells. Transcriptome analysis was performed at 12 hours post infection. Each cell-type displayed distinct effects on host and viral gene transcription, with a greater number of viral and host genes differentially transcribed in CEK cells and LMH cells, respectively. Both cell-types infected with either strain demonstrated enrichment of pathways related to signalling, and gene ontologies (GO) associated with chemotaxis. Infection with either strain upregulated both SOCS proteins and certain proto-oncogenes, which may contribute to prolonged viral persistence by promoting immunosuppression and preventing apoptosis, respectively. Patterns of gene transcription related to cytokines, chemokines, endosomal TLRs, and interferon responses, as well as pathways associated with histone acetylation, transport, and extracellular matrix organization were similar within each cell type, regardless of the viral strain. In CEK cells, GO terms and pathways were downregulated uniquely after CSW-1 ILTV infection, indicating a viral-strain specific effect in this cell-type. Overall, this study highlights that the observed differences in host and ILTV gene transcription in vitro were more strongly influenced by the cell-types used rather than the presence or absence of gG. This underscores the importance of cell-line selection in studying host-virus interactions and interpreting experimental results. [ABSTRACT FROM AUTHOR]

AbstractBotrytis cinerea, an airborne plant pathogen, holds the potential to synthesize sesquiterpenes, which have been used for the industrial production of abscisic acid. Previously, through our genetic technology, we obtained strain ZX2, whose main product 1´,4´-trans-ABA-diol is physiologically active in plants. In this study, 50 L of fed fermentation was carried out with ZX2 strain to study the stability of expression of TUA, TUB, ATC, EF-1, GAPDH, UCE and GTP genes. Four kinds of software (GeNorm, NormFinder, BestKeeper and Delta Ct) were used to analyze the expression stability of candidate genes, and finally the best reference gene was screened by RefFinder. Based on the results, the ACT was the most stable gene. It was used to normalize the expression levels of two genes related to 1´,4´-trans-ABA-diol production (hmgr and bcaba3) when fed-batch fermentation. Guide the selection of appropriate internal reference genes during the fermentation process to accurately quantify the relative transcription levels of target genes in B.cinerea ZX2. [ABSTRACT FROM AUTHOR]

Accurate grading of IDH-mutant gliomas defines patient prognosis and guides the treatment path. Histological grading is challenging, and aside from CDKN2A/B homozygous deletions in IDH-mutant astrocytomas, there are no other objective molecular markers used for grading. RNA-sequencing was conducted on primary IDH-mutant astrocytomas (n = 138) included in the prospective CATNON trial, which was performed to assess the prognostic effect of adjuvant and concurrent temozolomide. We integrated the RNA-sequencing data with matched DNA-methylation and NGS data. We also used multi-omics data from IDH-mutant astrocytomas included in the TCGA dataset and validated results on matched primary and recurrent samples from the GLASS-NL study. Since discrete classes do not adequately capture grading of these tumours, we utilised DNA-methylation profiles to generate a Continuous Grading Coefficient (CGC) based on classification scores from a CNS-tumour classifier. CGC was an independent predictor of survival outperforming current WHO-CNS5 and methylation-based classification. Our RNA-sequencing analysis revealed four distinct transcription clusters that were associated with (i) upregulation of cell cycling genes; (ii) downregulation of glial differentiation genes; (iii) upregulation of embryonic development genes (e.g. HOX, PAX, and TBX) and (iv) upregulation of extracellular matrix genes. The upregulation of embryonic development genes was associated with a specific increase of CpG island methylation near these genes. Higher grade IDH-mutant astrocytomas have DNA-methylation signatures that, on the RNA level, are associated with increased cell cycling, tumour cell de-differentiation and extracellular matrix remodelling. These combined molecular signatures can serve as an objective marker for grading of IDH-mutant astrocytomas. [ABSTRACT FROM AUTHOR]

The mammalian imprinted Dlk1-Dio3 domain contains multiple lncRNAs, mRNAs, the largest miRNA cluster in the genome and four differentially methylated regions (DMRs), and deletion of maternally expressed RNA within this locus results in embryonic lethality, but the mechanism by which this occurs is not clear. Here, we optimized the model of maternally expressed RNAs transcription termination in the domain and found that the cause of embryonic death was apoptosis in the embryo, particularly in the liver. We generated a mouse model of maternally expressed RNAs silencing in the Dlk1-Dio3 domain by inserting a 3 × polyA termination sequence into the Gtl2 locus. By analyzing RNA-seq data of mouse embryos combined with histological analysis, we found that silencing of maternally expressed RNAs in the domain activated apoptosis, causing vascular rupture of the fetal liver, resulting in hemorrhage and injury. Mechanistically, termination of Gtl2 transcription results in the silencing of maternally expressed RNAs and activation of paternally expressed genes in the interval, and it is the gene itself rather than the IG-DMR and Gtl2-DMR that causes the aforementioned phenotypes. In conclusion, these findings illuminate a novel mechanism by which the silencing of maternally expressed RNAs within Dlk1-Dio3 domain leads to hepatic hemorrhage and embryonic death through the activation of apoptosis. [ABSTRACT FROM AUTHOR]

Summary Class 2 Type V‐A CRISPR‐Cas (Cas12a) nucleases are powerful genome editing tools, particularly effective in A/T‐rich genomic regions, complementing the widely used CRISPR‐Cas9 in plants. To enhance the utility of Cas12a, we investigate three Cas12a orthologs—Mb3Cas12a, PrCas12a, and HkCas12a—in plants. Protospacer adjacent motif (PAM) requirements, editing efficiencies, and editing profiles are compared in rice. Among these orthologs, Mb3Cas12a exhibits high editing efficiency at target sites with a simpler, relaxed TTV PAM which is less restrictive than the canonical TTTV PAM of LbCas12a and AsCas12a. To optimize Mb3Cas12a, we develop an efficient single transcription unit (STU) system by refining the linker between Mb3Cas12a and CRISPR RNA (crRNA), nuclear localization signal (NLS), and direct repeat (DR). This optimized system enables precise genome editing in rice, particularly for fine‐tuning target gene expression by editing promoter regions. Further, we introduced Arginine (R) substitutions at Aspartic acid (D) 172, Asparagine (N) 573, and Lysine (K) 579 of Mb3Cas12a, creating two temperature‐tolerant variants: Mb3Cas12a‐R (D172R) and Mb3Cas12a‐RRR (D172R/N573R/K579R). These variants demonstrate significantly improved editing efficiency at lower temperatures (22 °C and 28 °C) in rice cells, with Mb3Cas12a‐RRR showing the best performance. We extend this approach by developing efficient Mb3Cas12a‐RRR STU systems in maize and tomato, achieving biallelic mutants targeting single or multiple genes in T0 lines cultivated at 28 °C and 25 °C, respectively. This study significantly expands Cas12a's targeting capabilities in plant genome editing, providing valuable tools for future research and practical applications. [ABSTRACT FROM AUTHOR]

Eimeria maxima (APU1 and APU2) differ in virulence for chickens, due in part to the greater fecundity of the former. In a previous study, RNA-seq was used to identify a transcripts upregulated in E. maxima APU1 compared to E. maxima APU2. In this study, 2 of these upregulated genes (EMWEY 23530 and EMWEY 48910) were characterized by first confirming upregulation using quantitative RT-PCR. For both EMWEY 23530 and EMWEY 48910, RNA transcription was fairly consistent during sporulation. The extent of differential expression was about 2-fold log2 higher in APU-1 compared to APU-2 (peaking at 18 h for EMWEY 23530 and 0 h for EMWEY 48910). EMWEY 23530 and EMWEY 48910 cDNA were cloned and expressed as polyHis-fusion proteins in Escherichia coli. The observed size of recombinant EMWEY 23530 was 24 kDa; the observed size of recombinant EMWEY 48910 was 35 kDa, which are consistent with the predicted size based on the coding sequences. Immunostaining 2D gel blots of E. maxima APU1 and APU2 oocyst/sporocyst protein with antisera specific for EMWEY 23530 identified a 33.5 kDa protein with a pH 7.4 isoelectric point (Emax p33.5). Similar 2D gel blot analysis with EMWEY 48910 identified a 41 kDa protein with a pH 7.2 isoelectric point (Emax p41). The intensity of Emax p33.5 and Emax p41 was noticeably greater in oocyst/sporocyst proteins from E. maxima APU1 compared to E. maxima APU2. This was corroborated by ELISA wherein equal amounts of total E. maxima APU1 and APU2 protein were probed with serial dilutions of anti-rEmax p33.5 or anti-rEmax p41. Immunofluorescence (IFA) staining of permeabilized unsporulated E. maxima APU1 and APU2 oocysts revealed Emax p33.5 to be localized in one end of oocysts, while Emax p41 appeared on the surface of oocysts. After sporulation, the p33.5 and p41 antigens appeared loosely associated with sporocysts. Taken together, these data confirm excess expression of two proteins in the E. maxima strain characterized by greater fecundity and virulence, and may provide insight into basis for phenotypic differences among different E. maxima. [ABSTRACT FROM AUTHOR]

Background: Existing studies have already revealed the involvement of C–C chemokine receptor type 7 (CCR7) in diverse human cancers, including esophageal cell squamous carcinoma (ESCA). Our current study, aims to explore the relevant mechanisms implicated. Methods: ESCA cell lines were collected for CCR7 expression quantification using western blot. Following the transfection, the viability, migration and invasion of ESCA cells were evaluated via cell counting kit-8 and Transwell assays. The specific molecular mechanisms underlying the effects of CCR7 in ESCA cells were explored via calculating the expressions of proteins related to metastasis and Janus kinase 2/signal transduction and transcription activation 3 (JAK2/STAT3) signaling pathway via western blot. The correlation between CCR7 and metastasis-related proteins was explored via Pearson's correlation test. Results: CCR7 was high-expressed in ESCA cells and CCR7 knockdown repressed the viability, migration and invasion of ESCA cells, concurrent with the increased expression of E-cadherin (E-cad, which was also known as CDH1 and lowly expressed in ESCA cells) and the decreased expressions of vimentin (Vim, which was highly expressed in ESCA cells) and matrix metalloproteinase-9 (MMP-9, which was also highly expressed in ESCA cells). Meanwhile, CCR7 was positively correlated with Vim and MMP-9 yet negatively correlated with E-cad in ESCA cells, which indicated that CCR7 has a role in promoting tumor progression in ESCA cells. Besides, the phosphorylation of STAT3 and JAK2 in ESCA cells was elevated, which was diminished following CCR7 knockdown. Conclusion: This study proves the modulation of CCR7 on ESCA in vitro, which was achieved via JAK2/STAT3 signaling pathway. Our discovery will provide new therapeutic basis and insights for ESCA. [ABSTRACT FROM AUTHOR]

The integrity of the actively transcribed genome against helix‐distorting DNA lesions relies on a multilayered cellular response that enhances Transcription‐Coupled Nucleotide Excision Repair (TC‐NER). When defective, TC‐NER is causatively associated with Cockayne‐Syndrome (CS), a rare severe human progeroid disorder. Although the presence of unresolved transcription‐blocking lesions is considered a driver of the aging process, the molecular features of the transcription‐driven response to genotoxic stress in CS‐B cells remain largely unknown. Here, an in‐depth view of the transcriptional and associated chromatin dynamics that occur in CS‐B cells illuminates the role of CSB therein. By employing high‐throughput genome‐wide approaches, we observed that absence of a functional CSB protein results in a delay in transcription progression, more positioned +1 nucleosomes, and less dynamic chromatin structure, compared to normal cells. We found that early after exposure to UV, CS‐B cells released RNA polymerase II (RNAPII) from promoter‐proximal pause sites into elongation. However, the magnitude of this response and the progression of RNAPII were reduced compared to normal counterparts. Notably, we detected increased post‐UV retainment of unprocessed nascent RNA transcripts and chromatin‐associated elongating RNAPII molecules. Contrary to the prevailing models, we found that transcription initiation is operational in CS‐B fibroblasts early after UV and that chromatin accessibility showed a marginal increase. Our study provides robust evidence for the role of CSB in shaping the transcription and chromatin landscape both in homeostasis and in response to genotoxic insults, which is independent of its known role in TC‐NER, and which may underlie major aspects of the CS phenotype. [ABSTRACT FROM AUTHOR]

Methyl‐CpG binding protein 2 (MeCP2) is an important X‐linked DNA methylation reader and a key heterochromatin organizer. The expression level of MeCP2 is crucial, as indicated by the observation that loss‐of‐function mutations of MECP2 cause Rett syndrome, whereas an extra copy spanning the MECP2 locus results in MECP2 duplication syndrome, both being progressive neurodevelopmental disorders. Our previous study demonstrated that MeCP2 protein expression is rapidly induced by renal ischemia–reperfusion injury (IRI) and protects the kidney from IRI through transcriptionally repressing the interleukin‐6 (IL‐6)/signal transducer and activator of transcription 3 signaling pathway. However, the mechanisms underlying the upregulation of MeCP2 have remained elusive. Here, by using two hypoxia cell models, hypoxia and reoxygenation and cobalt chloride stimulation, we confirmed that the removal of lysine 48‐linked ubiquitination from MeCP2 prevented its proteasome‐dependent degradation under hypoxic conditions. Through unbiased screening based on a deubiquitinating enzymes library, we identified ubiquitin‐specific protease 15 (USP15) as a stabilizer of MeCP2. Further studies revealed that USP15 could attenuate hypoxia‐induced MeCP2 degradation by cleaving lysine 48‐linked ubiquitin chains from MeCP2, primarily targeting its C‐terminal domain. Consistently, USP15 inhibited hypoxia‐induced signal transducer and activator of transcription 3 activation, resulting in reduced transcription of IL‐6 downstream genes. In summary, our study reveals an important role for USP15 in the maintenance of MeCP2 stability and the regulation of IL‐6 signaling. [ABSTRACT FROM AUTHOR]

Background: Batten disease is a group of rare inherited neurodegenerative diseases. Juvenile CLN3 disease is the most prevalent type, and the most common pathogenic variant shared by most patients is the "1-kb" deletion which removes two internal coding exons (7 and 8) in CLN3. Previously, we identified two transcripts in patient fibroblasts homozygous for the 1-kb deletion: the 'major' and 'minor' transcripts. To understand the full variety of disease transcripts and their role in disease pathogenesis, it is necessary to first investigate CLN3 transcription in "healthy" samples without juvenile CLN3 disease. Methods: We leveraged PacBio long-read RNA sequencing datasets from ENCODE to investigate the full range of CLN3 transcripts across various tissues and cell types in human control samples. Then we sought to validate their existence using data from different sources. Results: We found that a readthrough gene affects the quantification and annotation of CLN3. After taking this into account, we detected over 100 novel CLN3 transcripts, with no dominantly expressed CLN3 transcript. The most abundant transcript has median usage of 42.9%. Surprisingly, the known disease-associated 'major' transcripts are detected. Together, they have median usage of 1.5% across 22 samples. Furthermore, we identified 48 CLN3 ORFs, of which 26 are novel. The predominant ORF that encodes the canonical CLN3 protein isoform has median usage of 66.7%, meaning around one-third of CLN3 transcripts encode protein isoforms with different stretches of amino acids. The same ORFs could be found with alternative UTRs. Moreover, we were able to validate the translational potential of certain transcripts using public mass spectrometry data. Conclusion: Overall, these findings provide valuable insights into the complexity of CLN3 transcription, highlighting the importance of studying both canonical and non-canonical CLN3 protein isoforms as well as the regulatory role of UTRs to fully comprehend the regulation and function(s) of CLN3. This knowledge is essential for investigating the impact of the 1-kb deletion and rare pathogenic variants on CLN3 transcription and disease pathogenesis. [ABSTRACT FROM AUTHOR]

During the wound healing process, the activation of signal transducer and activator of transcription 3 (STAT3) is considered crucial for the migration and proliferation of epithelial cells, as well as for establishing the inflammatory environment. However, an excessive STAT3 activation aggravates scar formation. Here we show that 450 nm blue light and 630 nm red light can differentially regulate the phosphorylation of STAT3 (p-STAT3) and its downstream cytokines in keratinocytes. Further mechanistic studies reveal that red light promotes wound healing by activating the PI3 kinase p110 beta (PI3Kβ)/STAT3 signaling axis, while blue light inhibits p-STAT3 at the wound site by modulating cytochrome c-P450 (CYT-P450) activity and reactive oxygen species (ROS) generation. In a mouse scar model, skin wound healing can be significantly accelerated with red light followed by blue light to reduce scar formation. In summary, our study presents a potential strategy for regulating epithelial cell p-STAT3 through visible light to address skin scarring issues and elucidates the underlying mechanisms. In mouse keratinocytes, upregulation of PI3Kβ by red light promotes p-STAT3 and accelerates skin wound healing, whereas ROS induced by blue light inhibits p-STAT3 and alleviates scar formation. [ABSTRACT FROM AUTHOR]

Indoxyl sulfate (IS), a uremic toxin, is a harmful factor that damages kidneys. Chronic heat stress in laying hens causes renal injury; however, whether IS accumulation is involved in this injury remains unknown. We selected 20 Boris brown laying hens (27 weeks old) and randomly assigned them to two groups (n = 10), one group was exposed to chronic heat stress (32 °C for 4 weeks), whereas the other was maintained at 24 °C. Chronic heat exposure significantly increased plasma and renal IS concentrations (P < 0.05). Exposure to heat also increased renal expression of the aryl hydrocarbon receptor (AhR) and its target genes (CYP1A4 and CYP1B1). Furthermore, chronic heat exposure tended to increase the 2-thiobarbituric acid reactive substances content (P = 0.08) and significantly decreased the antioxidant capacity in the kidney, while increasing the transcription levels of nuclear factor κB and fibrosis-related genes (COLA1A1, αSMA, TGF-β, Smad3, and VCAM-1) and the area of renal fibrosis. Our results indicate that chronic heat exposure induces systemic and renal IS accumulation in laying hens. This accumulated IS may activate the AhR pathway and chronically disrupt the oxidative stress status and antioxidant activity, thus promoting renal fibrosis and dysfunction in laying hens. [ABSTRACT FROM AUTHOR]

Background: Hyperactive RNA Polymerase I (Pol I) transcription is canonical in cancer, associated with malignant proliferation, poor prognosis, epithelial-mesenchymal transition, and chemotherapy resistance. Despite its significance, the molecular mechanisms underlying Pol I hyperactivity remain unclear. This study aims to elucidate the role of long noncoding RNAs (lncRNAs) in regulating Pol I transcription in lung adenocarcinoma (LUAD). Methods: Bioinformatics analyses were applied to identify lncRNAs interacting with Pol I transcriptional machinery. Fluorescence in situ hybridization was employed to examine the nucleolar localization of candidate lncRNA in LUAD cells. RNA immunoprecipitation assay validated the interaction between candidate lncRNA and Pol I components. Chromatin isolation by RNA purification and Chromatin Immunoprecipitation (ChIP) were utilized to confirm the interactions of candidate lncRNA with Pol I transcriptional machinery and the rDNA core promoter. Functional analyses, including lncRNA knock-in and knockdown, inhibition of Pol I transcription, quantitative PCR, cell proliferation, clonogenicity, apoptosis, cell cycle, wound-healing, and invasion assays, were performed to determine the effect of candidate lncRNA on Pol I transcription and associated malignant phenotypes in LUAD cells. ChIP assays and luminometry were used to investigate the transcriptional regulation of the candidate lncRNA. Results: We demonstrate that oncogenic LINC01116 scaffolds essential Pol I transcription factors TAF1A and TAF1D, to the ribosomal DNA promoter, and upregulate Pol I transcription. Crucially, LINC01116-driven Pol I transcription activation is essential for its oncogenic activities. Inhibition of Pol I transcription abrogated LINC01116-induced oncogenic phenotypes, including increased proliferation, cell cycle progression, clonogenicity, reduced apoptosis, increased migration and invasion, and drug sensitivity. Conversely, LINC01116 knockdown reversed these effects. Additionally, we show that LINC01116 upregulation in LUAD is driven by the oncogene c-Myc, a known Pol I transcription activator, indicating a functional regulatory feedback loop within the c-Myc-LINC01116-Pol I transcription axis. Conclusion: Collectively, our findings reveal, for the first time, that LINC01116 enhances Pol I transcription by scaffolding essential transcription factors to the ribosomal DNA promoter, thereby driving oncogenic activities in LUAD. We propose the c-Myc-LINC01116-Pol I axis as a critical oncogenic pathway and a potential therapeutic target for modulating Pol I transcription in LUAD. [ABSTRACT FROM AUTHOR]

SUMMARY Plant trichomes are an excellent model for studying cell differentiation and development, providing crucial defenses against biotic and abiotic stresses. There is a well‐established inverse relationship between trichome density and aphid prevalence, indicating that higher trichome density leads to reduced aphid infestations. Here we present the cloning and characterization of a dominant quantitative trait locus, HIC (hirsute cotton), which significantly enhances cotton trichome density. This enhancement leads to markedly improved resistance against cotton aphids. The HIC encodes an HD‐ZIP IV transcriptional activator, crucial for trichome initiation. Overexpression of HIC leads to a substantial increase in trichome density, while knockdown of HIC results in a marked decrease in density, confirming its role in trichome regulation. We identified a variant in the HIC promoter (−810 bp A to C) that increases transcription of HIC and trichome density in hirsute cotton compared with Gossypium hirsutum cultivars with fewer or no trichomes. Interestingly, although the −810 variant in the HIC promoter is the same in G. barbadense and hirsute cotton, the presence of a copia‐like retrotransposon insertion in the coding region of HIC in G. barbadense causes premature transcription termination. Further analysis revealed that HIC positively regulates trichome density by directly targeting the EXPANSIN A2 gene, which is involved in cell wall development. Taken together, our results underscore the pivotal function of HIC as a primary regulator during the initial phases of trichome formation, and its prospective utility in enhancing aphid resistance in superior cotton cultivars via selective breeding. [ABSTRACT FROM AUTHOR]

Aging is characterized by the accumulation of proteins that display amyloid-like behavior. However, the molecular mechanisms by which these proteins arise remain unclear. Here, we demonstrate that amyloid-like proteins are produced in a variety of human cell types, including stem cells, brain organoids and fully differentiated neurons by mistakes that occur in messenger RNA molecules. Some of these mistakes generate mutant proteins already known to cause disease, while others generate proteins that have not been observed before. Moreover, we show that these mistakes increase when cells are exposed to DNA damage, a major hallmark of human aging. When taken together, these experiments suggest a mechanistic link between the normal aging process and age-related diseases. Amyloid-like proteins are central to age-related diseases, such as Alzheimer's and Parkinson's. Here, the authors show that transcription errors can produce mutant proteins with enhanced amyloid- and prion-like properties in human cells. [ABSTRACT FROM AUTHOR]

SGEF (also known as ARHGEF26), a RhoG specific GEF, can form a ternary complex with the Scribble polarity complex proteins Scribble and Dlg1, which regulates the formation and maintenance of adherens junctions and barrier function of epithelial cells. Notably, silencing SGEF results in a dramatic downregulation of both E-cadherin and ZO-1 (also known as TJP1) protein levels. However, the molecular mechanisms involved in the regulation of this pathway are not known. Here, we describe a novel signaling pathway governed by the Scribble–SGEF–Dlg1 complex. Our results show that the three members of the ternary complex are required to maintain the stability of the apical junctions, ZO-1 protein levels and tight junction (TJ) permeability. In contrast, only SGEF is necessary to regulate E-cadherin levels. The absence of SGEF destabilizes the E-cadherin–catenin complex at the membrane, triggering a positive feedback loop that exacerbates the phenotype through the repression of E-cadherin transcription in a process that involves the internalization of E-cadherin by endocytosis, β-catenin signaling and the transcriptional repressor Slug (also known as SNAI2). [ABSTRACT FROM AUTHOR]

Liquid–liquid phase separation (LLPS) is responsible for the emergence of intracellular membrane‐less organelles and the development of coacervate protocells. Benefitting from the advantages of simplicity, precision, programmability, and noninvasiveness, light has become an effective tool to regulate the assembly dynamics of LLPS, and mediate various biochemical processes associated with LLPS. In this review, recent advances in optically controlling membrane‐less organelles within living organisms are summarized, thereby modulating a series of biological processes including irreversible protein aggregation pathologies, transcription activation, metabolic flux, genomic rearrangements, and enzymatic reactions. Among these, the intracellular systems (i.e., optoDroplet, Corelet, PixELL, CasDrop, and other optogenetic systems) that enable the photo‐mediated control over biomolecular condensation are highlighted. The design of photoactive complex coacervate protocells in laboratory settings by utilizing photochromic molecules such as azobenzene and diarylethene is further discussed. This review is expected to provide in‐depth insights into phase separation‐associated biochemical processes, bio‐metabolism, and diseases. [ABSTRACT FROM AUTHOR]

The remarkable efficacy of COVID-19 vaccines has established mRNA as a highly promising biomedical technology. However, the adequate application of mRNA therapeutics necessitates additional measures to mitigate the inherent immunogenicity, which is predominantly caused by dsRNA. As a byproduct of the in vitro transcription of mRNA, dsRNA was reported to be originated through several distinct mechanisms, including the extension of 3′ loop-back hairpins, the extension of hybridized abortive transcripts, and promoter-independent transcription. The intricate mechanisms involved pose a dilemma as the reduction in dsRNA results in a concomitant decrease in other critical quality attributes of mRNA. Here, we demonstrate that the promoter binding motifs of T7 RNA polymerase directly impact the production of promoter-independent transcription-based dsRNA. Specifically, the G753A mutation significantly reduces the formation of dsRNA byproducts, which can further combine with modified nucleotides to enhance the effectiveness of dsRNA mitigation and with previously reported high-integrity mutation K389A to minimize side effects. Accordingly, the present study reports a cost-effective approach to synthesize high-purity, less immunostimulatory mRNA by using an engineered T7 RNA polymerase mutant. [ABSTRACT FROM AUTHOR]

Nucleotide metabolism is the ultimate and most critical link in the self-replication process of tumors, including gastric cancer (GC). However, in clinical treatment, classic antitumor drugs such as 5-fluorouracil (5-FU) are mostly metabolic analogs of purines or pyrimidines, which lack specificity for tumor cells and therefore have significant side effects. It is unclear whether there are other drugs that can target nucleotide metabolism, except for nucleic acid analogs. Here, we found that a natural compound, dehydroabietylamine (DHAA), significantly reduced the viability and proliferation of GC cells and organoids. DHAA disrupts the purine and pyrimidine metabolism of GC cells, causing DNA damage and further inducing apoptosis. DHAA treatment decreased transcription and protein levels of key enzymes involved in the nucleotide metabolism pathway, with significant reductions in the expression of pyrimidine metabolism key enzymes CAD, DHODH, and purine metabolism key enzymes PAICS. We also found that DHAA directly binds to and reduces the expression of Forkhead box K2 (FOXK2), a common transcription factor for these metabolic enzymes. Ultimately, DHAA was shown to delay tumorigenesis in K19-Wnt1/C2mE transgenic mice model and reduce levels of CAD, DHODH, and PAICS in vivo. We demonstrate that DHAA exerts an anticancer effect on GC by targeting transcription factor FOXK2, reducing transcription of key genes for nucleotide metabolism and impairing nucleotide biosynthesis, thus DHAA is a promising candidate for GC therapy. [ABSTRACT FROM AUTHOR]

DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C•G-to-T•A conversions in mitochondrial DNA (mtDNA). DdCBEs work in pairs, with each arm composed of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA are predicted to remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5′-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this requirement, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5′-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs containing TALE proteins engineered to recognize all 5′ bases. These modified DdCBEs are herein referred to as αDdCBEs. Notably, 5′-T-noncompliant canonical DdCBEs efficiently edited mtDNA at diverse loci. However, they were frequently outperformed by αDdCBEs, which exhibited significant improvements in activity and specificity, regardless of the most 5′ bases of their TALE binding sites. Furthermore, we showed that αDdCBEs are compatible with the enhanced DddAtox variants DddA6 and DddA11, and we validated TALE shifting with αDdCBEs as an effective approach to optimize base editing outcomes. Overall, αDdCBEs enable efficient, specific, and unconstrained mitochondrial base editing. [ABSTRACT FROM AUTHOR]

Insects produce silk to form cocoons, nests, and webs, which are important for their survival and reproduction. However, little is known about the molecular mechanism of silk protein synthesis at the translation level. The solute carrier family 7 (SLC7) genes are involved in activating the target of rapamycin complex 1 (TORC1) signaling pathway and protein translation process, but the physiological roles of SLC7 genes in silk‐producing insects have not been reported. Here, we found that amino acid signaling regulates silk protein synthesis and larval development via the L‐type amino acid transporter 1 (LAT1; also known as SLC7A5) in Bombyx mori. A total of 12 SLC7 homologs were identified in the silkworm genome, among which BmSLC7A5 was found to be a silk gland‐enriched gene and may be involved in leucine transport. Bioinformatics analysis indicated that SLC7A5 displays high homology and a close phylogenetic relationship in silk‐producing insects. Subsequently, we found that leucine treatment significantly increased silk protein synthesis by improving the transcription and protein levels of silk genes. Furthermore, systemic and silk gland‐specific knockout of BmSLC7A5 led to decreased silk protein synthesis by inhibiting TORC1 signaling, and somatic mutation also resulted in arrested development from the 5th instar to the early pupal stage. Altogether, our study reveals that BmSLC7A5 is involved in regulating silk protein synthesis and larval development by affecting the TORC1 signaling pathway, which provides a new strategy and target for improving silk yield. [ABSTRACT FROM AUTHOR]

Increased synthesis and deposition of collagen (COL) in the extracellular matrix (ECM) of equine endometrium contributes to endometrosis. Toll‐like receptors (TLRs) are transmembrane receptors involved in the innate immune response, recognized for their role in antigen recognition and previously associated with equine endometritis. The TLRs not only recognize pathogen‐associated molecular patterns but also regulate inflammations, fibrosis and cancer. The aim of this study was to explore the relationship between TLR expression at different stages of Kenney and Doig's (K‐D) grading and COL1 expression during the follicular phase of the oestrous cycle. Forty samples of endometrial tissues were collected post‐mortem from mares on the follicular phase of the oestrous cycle (10 samples of each K‐D category). Relative mRNA transcription of TLR‐2, TLR‐4 and COL1A2 genes was assessed using qPCR, and COL1 protein expression by Western blot analysis. The COL1A2 transcription increased in category IIB when compared to categories I, IIA and III endometria (p <.01). The relative protein abundance of COL1 showed no significant differences between endometrial categories (p >.05). As for the TLRs mRNA transcription, TLR‐2/‐4 transcripts increased in IIA when compared to the other K‐D endometria categories (p <.05). Our findings suggest that TLRs may be involved in the initiation of the endometrial inflammatory response. Additional studies are needed to explore TLRs' potential role as diagnostic markers for monitoring inflammation progression and fibrosis development, as well as their involvement in the mechanisms underlying fibrotic pathways. [ABSTRACT FROM AUTHOR]

Breeding abiotic stress-tolerant varieties of Rosa chinensis is a paramount goal in horticulture. WRKY transcription factors, pivotal in plant responses to diverse stressors, offer potential targets for enhancing stress resilience in R. chinensis. Using bioinformatics and genomic data, we identified RcWRKY transcription factor genes, characterised their chromosomal distribution, phylogenetic relationships, structural attributes, collinearity, and expression patterns in response to saline stress. Leveraging bidirectional database searches, we pinpointed 66 RcWRKY genes, categorised into three groups. All except RcWRKY60 encoded DNA Binding Domain and Zinc Finger Motif regions of the WRKY domain. Expansion of the RcWRKY gene family was propelled by 19 segmental, and 2 tandem, duplications. We unveiled 41 and 15 RcWRKY genes corresponding to 50 AtWRKY and 17 OsWRKY orthologs respectively, indicating postdivergence expansion. Expression analyses under alkaline stress pinpointed significant alterations in 54 RcWRKY genes. Integration of functional roles from their Arabidopsis orthologs and cis -acting elements within their promoters, along with quantitative reverse transcription PCR validation, underscored the importance of RcWRKY27 and 29 in R. chinensis ' alkaline stress response. These findings offer insights into the biological roles of RcWRKY transcription factors, as well as the regulatory dynamics governing R. chinensis ' growth, development, and stress resilience. The China rose (Rosa chinensis) is known as the 'queen of flowers', and is commercially cultivated globally. To enable breeders to develop varieties tolerant to alkaline soils, we investigated the genes involved in their stress response. This research will also contribute to the broader understanding of plant stress responses and offer potential avenues for enhancing stress tolerance in crop species. [ABSTRACT FROM AUTHOR]

Purpose: Dysfunctional lymphangiogenesis is pivotal for various pathological processes including tumor lymph node metastasis which is a crucial cause of therapeutic failure for ESCC. In this study, we aim to elucidate the molecular mechanisms and clinical relevance of Zinc-finger protein ZNF468 in lymphangiogenesis and lymphatic metastasis in ESCC. Methods: Immunohistochemistry, Western blot, Kaplan-Meier plotter analysis and Gene Set Enrichment Analysis were preformed to detect the association of ZNF468 with lymphangiogenesis and poor prognosis in ESCC patients. Foot-pads lymph node metastasis model, tube formation assay, 3D-culture assay and invasion assay were preformed to verify the effect of ZNF468 on lymphangiogenesis and lymph node metastasis. CUT&Tag analysis, immunoprecipitation and mass spectrometry analysis and ChIP-PCR assay were preformed to study the molecular mechanisms of ZNF468 in lymphangiogenesis. Results: We found that ectopic expression of ZNF468 was correlated with higher microlymphatic vessel density in ESCC tissues, leading to poorer prognosis of ESCC patients. ZNF468 enhanced the capacity of lymphangiogenesis and promoted lymphatic metastasis in ESCC both in vitro and in vivo. However, silencing ZNF468 reversed these phenotypes in ESCC. Mechanically, we demonstrated that ZNF468 recruits the histone modification factors (PRMT1/HAT1) to increase the levels of H4R2me2a and H3K9ac, which then leads to the recruitment of the transcription initiation complex on the VEGF-C promoter, ultimately promoting the upregulation of VEGF-C transcription. Strikingly, the promoting effect of lymphatic metastasis induced by ZNF468 in ESCC was abrogated by targeting PRMT1 using Arginine methyltransferase inhibitor-1 or silencing VEGF-C. Furthermore, we found that the activation of PI3K/AKT and ERK1/2 signaling is required for ZNF468-medicated lymphatic metastasis in ESCC. Importantly, the clinical relevance between ZNF468 and VEGF-C were confirmed not only in ESCC samples and but also in multiple cancer types. Conclusion: Our results identified a precise mechanism underlying ZNF468-induced epigenetic upregulation of VEGF-C in facilitating lymphangiogenesis and lymph node metastasis of ESCC, which might provide a novel prognostic biomarker and potential therapeutic for ESCC patients. [ABSTRACT FROM AUTHOR]

Searching for biomarkers of senescence remains necessary and challenging. Reliable and detectable biomarkers can indicate the senescence condition of individuals, the need for intervention in a population, and the effectiveness of that intervention in controlling or delaying senescence progression and senescence‐associated diseases. Therefore, it is of great importance to fulfill the unmet requisites of senescence biomarkers especially when faced with the growing global senescence nowadays. Here, we established that DNA G‐quadruplex (G4) in mitochondrial genome was a reliable hallmark for mesenchymal senescence. Via developing a versatile and efficient mitochondrial G4 (mtG4) probe we revealed that in multiple types of senescence, including chronologically healthy senescence, progeria, and replicative senescence, mtG4 hallmarked aged mesenchymal stem cells. Furthermore, we revealed the underlying mechanisms by which accumulated mtG4, specifically within respiratory chain complex (RCC) I and IV loci, repressed mitochondrial genome transcription, finally impairing mitochondrial respiration and causing mitochondrial dysfunction. Our findings endowed researchers with the visible senescence biomarker based on mitochondrial genome and furthermore revealed the role of mtG4 in inhibiting RCC genes transcription to induce senescence‐associated mitochondrial dysfunction. These findings depicted the crucial roles of mtG4 in predicting and controlling mesenchymal senescence. [ABSTRACT FROM AUTHOR]

Amyloid plaques, a major pathological hallmark of Alzheimer's disease (AD), are caused by an imbalance between the amyloidogenic and non‐amyloidogenic pathways of amyloid precursor protein (APP). BACE1 cleavage of APP is the rate‐limiting step for amyloid‐β production and plaque formation in AD. Although the alteration of BACE1 expression in AD has been investigated, the underlying mechanisms remain unknown. In this study, we determined MEIS2 was notably elevated in AD models and AD patients. Alterations in the expression of MEIS2 can modulate the levels of BACE1. MEIS2 downregulation improved the learning and memory retention of AD mice and decreased the number of amyloid plaques. MEIS2 binds to the BACE1 promoter, positively regulates BACE1 expression, and accelerates APP amyloid degradation in vitro. Therefore, our findings suggest that MEIS2 might be a critical transcription factor in AD, since it regulates BACE1 expression and accelerates BACE1‐mediated APP amyloidogenic cleavage. MEIS2 is a promising early intervention target for AD treatment. [ABSTRACT FROM AUTHOR]

In 2015, the largest recorded harmful algal bloom (HAB) occurred in the Northeast Pacific, causing nearly 100 million dollars in damages to fisheries and killing many protected marine mammals. Dominated by the toxic diatom Pseudo-nitzschia australis, this bloom produced high levels of the neurotoxin domoic acid (DA). Through molecular and transcriptional characterization of 52 near-weekly phytoplankton net-tow samples collected at a bloom hotspot in Monterey Bay, California, we identified active transcription of known DA biosynthesis (dab) genes from the three identified toxigenic species, including P. australis as the primary origin of toxicity. Elevated expression of silicon transporters (sit1) during the bloom supports the previously hypothesized role of dissolved silica (Si) exhaustion in contributing to bloom physiology and toxicity. We find that coexpression of the dabA and sit1 genes serves as a robust predictor of DA one week in advance, potentially enabling the forecasting of DA-producing HABs. We additionally present evidence that low levels of iron could have colimited the diatom population along with low Si. Iron limitation represents an overlooked driver of both toxin production and ecological success of the low-iron-adapted Pseudo-nitzschia genus during the 2015 bloom, and increasing pervasiveness of iron limitation may fuel the escalating magnitude and frequency of toxic Pseudo-nitzschia blooms globally. Our results advance understanding of bloom physiology underlying toxin production, bloom prediction, and the impact of global change on toxic blooms. [ABSTRACT FROM AUTHOR]

ABSTRACT Light is a vital environmental signal that regulates the expression of plastid genes. Plastids are crucial organelles that respond to light, but the effects of light on plastid RNA processing following transcription remain unclear. In this study, we systematically examined the influence of light exposure on plastid RNA processing, focusing on RNA splicing and RNA editing. We demonstrated that light promotes the splicing of transcripts from the plastid genes rps12, ndhA, atpF, petB, and rpl2. Additionally, light increased the editing rate of the accD transcript at nucleotide 794 (accD‐794) and the ndhF transcript at nucleotide 290 (ndhF‐290), while decreasing the editing rate of the clpP transcript at nucleotide 559 (clpP‐559). We have identified key regulators of signaling pathways, such as CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), ELONGATED HYPOCOTYL 5 (HY5), and PHYTOCHROME‐INTERACTING FACTORs (PIFs), as important players in the regulation of plastid RNA splicing and editing. Notably, COP1 was required for GENOMES UNCOUPLED1 (GUN1)‐dependent repression of clpP‐559 editing in the light. We showed that HY5 and PIF1 bind to the promoters of nuclear genes encoding plastid‐localized RNA processing factors in a light‐dependent manner. This study provides insight into the mechanisms underlying light‐mediated post‐transcriptional regulation of plastid gene expression. [ABSTRACT FROM AUTHOR]