Comparative analysis of nascent RNA sequencing methods and their applications in studies of cotranscriptional splicing dynamics.

High-throughput detection of nascent RNA is critical for studies of transcription and much more challenging than that of mRNA. Recently, several massively parallel nascent RNA sequencing methods were established in eukaryotic cells. Here, we systematically compared 3 classes of methods on the same pure or crude nuclei preparations: GRO-seq for sequence nuclear run-on RNAs, pNET-seq for sequence RNA polymerase II-associated RNAs, and CB RNA-seq for sequence chromatin-bound (CB) RNAs in Arabidopsis (Arabidopsis thaliana). To improve the resolution of CB RNAs, 3'CB RNA-seq was established to sequence the 3' ends of CB RNAs. In addition, we modified pNET-seq to establish the Chromatin Native Elongation Transcript sequencing (ChrNET) method using chromatin as the starting material for RNA immunoprecipitation. Reproducibility, sensitivity and accuracy in detecting nascent transcripts, experimental procedures, and costs were analyzed, which revealed the strengths and weaknesses of each method. We found that pNET and GRO methods best detected active RNA polymerase II. CB RNA-seq is a simple and cost-effective alternative for nascent RNA studies, due to its high correlation with pNET-seq and GRO-seq. Compared with pNET, ChrNET has higher specificity for nascent RNA capture and lower sequencing cost. 3'CB is sensitive to transcription-coupled splicing. Using these methods, we identified 1,404 unknown transcripts, 4,482 unannotated splicing events, and 60 potential recursive splicing events. This comprehensive comparison of different nascent/chromatin RNA sequencing methods highlights the strengths of each method and serves as a guide for researchers aiming to select a method that best meets their study goals.

The role of histone acetylation in transcriptional regulation and seed development.

Histone acetylation is highly conserved across eukaryotes and has been linked to gene activation since its discovery nearly 60 years ago. Over the past decades, histone acetylation has been evidenced to play crucial roles in plant development and response to various environmental cues. Emerging data indicate that histone acetylation is one of the defining features of "open chromatin," while the role of histone acetylation in transcription remains controversial. In this review, we briefly describe the discovery of histone acetylation, the mechanism of histone acetylation regulating transcription in yeast and mammals, and summarize the research progress of plant histone acetylation. Furthermore, we also emphasize the effect of histone acetylation on seed development and its potential use in plant breeding. A comprehensive knowledge of histone acetylation might provide new and more flexible research perspectives to enhance crop yield and stress resistance.

Degradable biomedical elastomers: paving the future of tissue repair and regenerative medicine.

Degradable biomedical elastomers (DBE), characterized by controlled biodegradability, excellent biocompatibility, tailored elasticity, and favorable network design and processability, have become indispensable in tissue repair. This review critically examines the recent advances of biodegradable elastomers for tissue repair, focusing mainly on degradation mechanisms and evaluation, synthesis and crosslinking methods, microstructure design, processing techniques, and tissue repair applications. The review explores the material composition and cross-linking methods of elastomers used in tissue repair, addressing chemistry-related challenges and structural design considerations. In addition, this review focuses on the processing methods of two- and three-dimensional structures of elastomers, and systematically discusses the contribution of processing methods such as solvent casting, electrostatic spinning, and three-/four-dimensional printing of DBE. Furthermore, we describe recent advances in tissue repair using DBE, and include advances achieved in regenerating different tissues, including nerves, tendons, muscle, cardiac, and bone, highlighting their efficacy and versatility. The review concludes by discussing the current challenges in material selection, biodegradation, bioactivation, and manufacturing in tissue repair, and suggests future research directions. This concise yet comprehensive analysis aims to provide valuable insights and technical guidance for advances in DBE for tissue engineering.

Redox regulation of plant stem cell fate.

Despite the importance of stem cells in plant and animal development, the common mechanisms of stem cell maintenance in both systems have remained elusive. Recently, the importance of hydrogen peroxide (H2O2) signaling in priming stem cell differentiation has been extensively studied in animals. Here, we show that different forms of reactive oxygen species (ROS) have antagonistic roles in plant stem cell regulation, which were established by distinct spatiotemporal patterns of ROS-metabolizing enzymes. The superoxide anion (O2·-) is markedly enriched in stem cells to activate WUSCHEL and maintain stemness, whereas H2O2 is more abundant in the differentiating peripheral zone to promote stem cell differentiation. Moreover, H2O2 negatively regulates O2·- biosynthesis in stem cells, and increasing H2O2 levels or scavenging O2·- leads to the termination of stem cells. Our results provide a mechanistic framework for ROS-mediated control of plant stem cell fate and demonstrate that the balance between O2·- and H2O2 is key to stem cell maintenance and differentiation.

Immediate transcriptional responses of Arabidopsis leaves to heat shock.

Plants have evolved efficient mechanisms for adapting to temperature fluctuations, known as heat stress response and heat stress memory. Although the transcriptional regulatory network of plant heat stress response has been established, little is known about the genome-wide transcriptional changes occurring within the first several minutes after heat shock. Here, we investigated the nascent RNA and mature messenger RNA (mRNA) from plant leaf tissues exposed to 5 min of heat shock treatment using global run-on sequencing and RNA sequencing methods. Only a small group of genes were up- or downregulated at both the nascent RNA and mRNA levels. Primed plants that were already exposed to mild heat stress exhibited a more drastic alteration at multiple transcriptional steps than naïve plants that had not experienced heat stress. Upon heat shock, we also observed the following: (i) engaged RNA polymerase II accumulated downstream of transcription start sites; (ii) 5' pausing release was a rate-limiting step for the induction of some heat shock protein genes; (iii) numerous genes switched transcription modes; (iv) pervasive read-through was induced at terminators; and (v) heat stress memory occurs at multiple steps of the transcription cycle, such as at Pol II recruitment, 5' pausing, elongation, and termination.

FT5a interferes with the Dt1-AP1 feedback loop to control flowering time and shoot determinacy in soybean.

Flowering time and stem growth habit determine inflorescence architecture in soybean, which in turn influences seed yield. Dt1, a homolog of Arabidopsis TERMINAL FLOWER 1 (TFL1), is a major controller of stem growth habit, but its underlying molecular mechanisms remain unclear. Here, we demonstrate that Dt1 affects node number and plant height, as well as flowering time, in soybean under long-day conditions. The bZIP transcription factor FDc1 physically interacts with Dt1, and the FDc1-Dt1 complex directly represses the expression of APETALA1 (AP1). We propose that FT5a inhibits Dt1 activity via a competitive interaction with FDc1 and directly upregulates AP1. Moreover, AP1 represses Dt1 expression by directly binding to the Dt1 promoter, suggesting that AP1 and Dt1 form a suppressive regulatory feedback loop to determine the fate of the shoot apical meristem. These findings provide novel insights into the roles of Dt1 and FT5a in controlling the stem growth habit and flowering time in soybean, which determine the adaptability and grain yield of this important crop.

Spontaneous reactivation of a site-specifically placed transgene independent of copy number or DNA methylation.

As millions of seeds are produced from a breeding line, the long-term stability of transgene expression is vital for commercial-scale production of seeds with transgenic traits. Transgenes can be silenced by epigenetic mechanisms, but reactivation of expression can occur as a result of treatment with chromatin modification inhibitors such as 5-azacytidine, from stress such as heat or UV-B, or in mutants that have acquired a defect in gene silencing. Previously, we targeted a gfp reporter gene into the tobacco (Nicotiana tabacum) genome by site-specific recombination but still found some silenced lines among independent integration events. One such line also had a second random copy and both copies showed DNA hypermethylation. To test whether removing the second copy would reactivate gfp expression, two T1 plants were backcrossed to the wild type. Whereas the silenced status was maintained in the progenies from one backcross, spontaneous partial reactivation of gfp expression was found among progenies from a second backcross. However, this reactivation did not correlate with loss of the second random copy or with a significant change in the pattern or amount of DNA hypermethylation. This finding supports the suggestion that gene reactivation does not necessarily involve loss of DNA homology or methylation.

A Pap1-Oxs1 signaling pathway for disulfide stress in Schizosaccharomyces pombe.

We describe a Pap1-Oxs1 pathway for diamide-induced disulfide stress in Schizosaccharomyces pombe, where the nucleocytoplasmic HMG protein Oxs1 acts cooperatively with Pap1 to regulate transcription. Oxs1 and Pap1 form a complex when cells are exposed to diamide or Cd that causes disulfide stress. When examined for promoters up-regulated by diamide, effective Pap1 binding to these targets requires Oxs1, and vice versa. With some genes, each protein alone enhances transcription, but the presence of both exerts an additive positive effect. In other genes, although transcription is induced by diamide, Oxs1 or Pap1 plays a negative role with full de-repression requiring loss of both proteins. In a third class of genes, Oxs1 positively regulates expression, but in its absence, Pap1 plays a negative role. The Oxs1-Pap1 regulatory interaction appears evolutionarily conserved, as heterologous (human, mouse and Arabidopsis) Oxs1 and Pap1-homologues can bind interchangeably with each other in vitro, and at least in the fission yeast, heterologous Oxs1 and Pap1-homologues can substitute for S. pombe Oxs1 and Pap1 to enhance stress tolerance.

Small RNAs prevent transcription-coupled loss of histone H3 lysine 9 methylation in Arabidopsis thaliana.

In eukaryotes, histone H3 lysine 9 methylation (H3K9me) mediates silencing of invasive sequences to prevent deleterious consequences including the expression of aberrant gene products and mobilization of transposons. In Arabidopsis thaliana, H3K9me maintained by SUVH histone methyltransferases (MTases) is associated with cytosine methylation (5meC) maintained by the CMT3 cytosine MTase. The SUVHs contain a 5meC binding domain and CMT3 contains an H3K9me binding domain, suggesting that the SUVH/CMT3 pathway involves an amplification loop between H3K9me and 5meC. However, at loci subject to read-through transcription, the stability of the H3K9me/5meC loop requires a mechanism to counteract transcription-coupled loss of H3K9me. Here we use the duplicated PAI genes, which stably maintain SUVH-dependent H3K9me and CMT3-dependent 5meC despite read-through transcription, to show that when PAI sRNAs are depleted by dicer ribonuclease mutations, PAI H3K9me and 5meC levels are reduced and remaining PAI 5meC is destabilized upon inbreeding. The dicer mutations confer weaker reductions in PAI 5meC levels but similar or stronger reductions in PAI H3K9me levels compared to a cmt3 mutation. This comparison indicates a connection between sRNAs and maintenance of H3K9me independent of CMT3 function. The dicer mutations reduce PAI H3K9me and 5meC levels through a distinct mechanism from the known role of dicer-dependent sRNAs in guiding the DRM2 cytosine MTase because the PAI genes maintain H3K9me and 5meC at levels similar to wild type in a drm2 mutant. Our results support a new role for sRNAs in plants to prevent transcription-coupled loss of H3K9me.

Hydrogels composite optimized for low resistance and loading-unloading hysteresis for flexible biosensors.

With the advancement of wearable and implantable medical devices, hydrogel flexible bioelectronic devices have attracted significant interest due to exhibiting tissue-like mechanical compliance, biocompatibility, and low electrical resistance. In this study, the development and comprehensive performance evaluation of poly(acrylic acid)/ N,N'-bis(acryloyl) cystamine/ 1-butyl-3-ethenylimidazol-1-ium:bromide (PAA/NB/IL) hydrogels designed for flexible sensor applications are introduced. Engineered through a combination of physical and chemical cross-linking strategies, these hydrogels exhibit strong mechanical properties, high biocompatibility, and effective sensing capabilities. At 95 % strain, the compressive modulus of PAA/NB/IL 100 reach up to 3.66 MPa, with the loading-unloading process showing no significant hysteresis loop, indicating strong mechanical stability and elasticity. An increase in the IL content was observed to enlarge the porosity of the hydrogels, thereby influencing their swelling behavior and sensing functionality. Biocompatibility assessments revealed that the hemolysis rate was below 5 %, ensuring their suitability for biomedical applications. Upon implantation in rats, a minimal acute inflammatory response was observed, comparable to that of the biocompatibility control poly(ethylene glycol) diacrylate (PEGDA). These results suggest that PAA/NB/IL hydrogels hold promise as biomaterials for biosensors, offering a balance of mechanical integrity, physiological compatibility, and sensing sensitivity, thereby facilitating advanced healthcare monitoring solutions.

Composite patch with negative Poisson's ratio mimicking cardiac mechanical properties: Design, experiment and simulation.

Developing patches that effectively merge intrinsic deformation characteristics of cardiac with superior tunable mechanical properties remains a crucial biomedical pursuit. Currently used traditional block-shaped or mesh patches, typically incorporating a positive Poisson's ratio, often fall short of matching the deformation characteristics of cardiac tissue satisfactorily, thus often diminishing their repairing capability. By introducing auxeticity into the cardiac patches, this study is trying to present a beneficial approach to address these shortcomings of the traditional patches. The patches, featuring the auxetic effect, offer unparalleled conformity to the cardiac complex mechanical challenges. Initially, scaffolds demonstrating the auxetic effect were designed by merging chiral rotation and concave angle units, followed by integrating scaffolds with a composite hydrogel through thermally triggering, ensuring excellent biocompatibility closely mirroring heart tissue. Tensile tests revealed that auxetic patches possessed superior elasticity and strain capacity exceeding cardiac tissue's physiological activity. Notably, Model III showed an equivalent modulus ratio and Poisson's ratio closely toward cardiac tissue, underscoring its outstanding mechanical potential as cardiac patches. Cyclic tensile loading tests demonstrated that Model III withstood continuous heartbeats, showcasing outstanding cyclic loading and recovery capabilities. Numerical simulations further elucidated the deformation and failure mechanisms of these patches, leading to an exploration of influence on mechanical properties with alternative design parameters, which enabled the customization of mechanical strength and Poisson's ratio. Therefore, this research presents substantial potential for designing cardiac auxetic patches that can emulate the deformation properties of cardiac tissue and possess adjustable mechanical parameters.

Study on the influence of shot peening strengthening before shot peen forming on 2024-T351 aluminum alloy fatigue crack growth rate.

It is sparse and inconclusive that research on the subject whether the fatigue life of the structure will be reduced by shot peening strengthening before shot peen forming (S + F), and this study investigates accordingly. First, the crack growth rate test of the machine-processing plate and shot peening strengthening before shot peen forming plate demonstrate that both plates' final crack growth rate and length are similar. However, the test shows the "fluctuation phenomenon" of crack growth rate and the "intersection phenomenon" in the Paris curve. This study is based on a self-developed simulation plugin for crack growth paths. The results verify that "fluctuation" causes the differential distribution of the overall stress intensity factor in the strengthened (4.5% increase compared to machine-processing) and formed (9.8% decrease compared to machine-processing) crater areas of the shot peening strengthening before shot peen forming plate. Comparing to the full coverage strengthening area, the forming area (only 30% coverage) in the early stage of growth as well as the gain amplitude of the residual stress in the late stage of growth gradually decrease and tend to be the same as that of the machine-processing, as validated by the "intersection phenomenon".

Scutellarin prevents acute alcohol-induced liver injury via inhibiting oxidative stress by regulating the Nrf2/HO-1 pathway and inhibiting inflammation by regulating the AKT, p38 MAPK/NF-κB pathways. / ç¯ç›èŠ±ä¹™ç´ é€šè¿‡è°ƒèŠ‚Nrf2/HO-1通路抑制氧化应激以及通过调节AKT、p38 MAPK/NF-κBé€šè·¯æŠ‘åˆ¶ç‚Žç—‡ååº”é¢„é˜²æ€¥æ€§é ’ç²¾æ€§è‚æŸä¼¤.

Alcoholic liver disease (ALD) is the most frequent liver disease worldwide, resulting in severe harm to personal health and posing a serious burden to public health. Based on the reported antioxidant and anti-inflammatory capacities of scutellarin (SCU), this study investigated its protective role in male BALB/c mice with acute alcoholic liver injury after oral administration (10, 25, and 50 mg/kg). The results indicated that SCU could lessen serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and improve the histopathological changes in acute alcoholic liver; it reduced alcohol-induced malondialdehyde (MDA) content and increased glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) activity. Furthermore, SCU decreased tumor necrosis factor-|α (TNF-|α), interleukin-6 (IL-6), and IL-|1ß messenger RNA (mRNA) expression levels, weakened inducible nitric oxide synthase (iNOS) activity, and inhibited nucleotide-binding oligomerization domain (NOD)|-like receptor protein 3 (NLRP3) inflammasome activation. Mechanistically, SCU suppressed cytochrome P450 family 2 subfamily E member 1 (CYP2E1) upregulation triggered by alcohol, increased the expression of oxidative stress-related nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) pathways, and suppressed the inflammation-related degradation of inhibitor of nuclear factor-|κB (NF-|κB)|-|α (IκBα) as well as activation of NF|-|κB by mediating the protein kinase B (AKT) and p38 mitogen-activated protein kinase (MAPK) pathways. These findings demonstrate that SCU protects against acute alcoholic liver injury via inhibiting oxidative stress by regulating the Nrf2/HO-1 pathway and suppressing inflammation by regulating the AKT, p38 MAPK/NF-κB pathways.

DDM1-mediated R-loop resolution and H2A.Z exclusion facilitates heterochromatin formation in Arabidopsis.

Programmed constitutive heterochromatin silencing is essential for eukaryotic genome regulation, yet the initial step of this process is ambiguous. A large proportion of R-loops (RNA:DNA hybrids) had been unexpectedly identified within Arabidopsis pericentromeric heterochromatin with unknown functions. Through a genome-wide R-loop profiling screen, we find that DDM1 (decrease in DNA methylation 1) is the primary restrictor of pericentromeric R-loops via its RNA:DNA helicase activity. Low levels of pericentromeric R-loops resolved by DDM1 cotranscriptionally can facilitate constitutive heterochromatin silencing. Furthermore, we demonstrate that DDM1 physically excludes histone H2A variant H2A.Z and promotes H2A.W deposition for faithful heterochromatin initiation soon after R-loop clearance. The dual functions of DDM1 in R-loop resolution and H2A.Z eviction are essential for sperm nuclei structure maintenance in mature pollen. Our work unravels the cotranscriptional R-loop resolution coupled with accurate H2A variants deposition is the primary step of constitutive heterochromatin silencing in Arabidopsis, which might be conserved across eukaryotes.

Printable and Flexible Humidity Sensor Based on Graphene -Oxide-Supported MoTe2 Nanosheets for Multifunctional Applications.

This study focuses on a novel humidity sensor composed of graphene-oxide (GO)-supported MoTe2 nanosheets. Conductive Ag electrodes were formed on PET substrates by inkjet printing. A thin film of GO-MoTe2 was deposited on the Ag electrode used for adsorbing humidity. The experiment's results demonstrate that MoTe2 are attached to GO nanosheets uniformly and tightly. The capacitive output of the sensors with various ratios of GO/MoTe2 has been tested for different levels of humidity (11.3-97.3%RH) at room temperature (25 °C). As a consequence, the obtained hybrid film exhibits superior sensitivity (94.12 pF/%RH). The structural integrity and interaction of different components were discussed to afford the prominent humidity sensitivity performance. Under the bending condition, the output curve of the sensor has no obvious fluctuation. This work provides a low-cost way to build flexible humidity sensors with high-performance in environmental monitoring and healthcare.

OsTOC1 plays dual roles in the regulation of plant circadian clock by functioning as a direct transcription activator or repressor.

Plant clock function relies on precise timing of gene expression through complex regulatory networks consisting of activators and repressors at the core of oscillators. Although TIMING OF CAB EXPRESSION 1 (TOC1) has been recognized as a repressor involved in shaping oscillations and regulating clock-driven processes, its potential to directly activate gene expression remains unclear. In this study, we find that OsTOC1 primarily acts as a transcriptional repressor for core clock components, including OsLHY and OsGI. Here, we show that OsTOC1 possesses the ability to directly activate the expression of circadian target genes. Through binding to the promoters of OsTGAL3a/b, transient activation of OsTOC1 induces the expression of OsTGAL3a/b, indicating its role as an activator contributing to pathogen resistance. Moreover, TOC1 participates in regulating multiple yield-related traits in rice. These findings suggest that TOC1's function as a transcriptional repressor is not inherent, providing flexibility to circadian regulations, particularly in outputs.

Transposable element-initiated enhancer-like elements generate the subgenome-biased spike specificity of polyploid wheat.

Transposable elements (TEs) comprise ~85% of the common wheat genome, which are highly diverse among subgenomes, possibly contribute to polyploid plasticity, but the causality is only assumed. Here, by integrating data from gene expression cap analysis and epigenome profiling via hidden Markov model in common wheat, we detect a large proportion of enhancer-like elements (ELEs) derived from TEs producing nascent noncoding transcripts, namely ELE-RNAs, which are well indicative of the regulatory activity of ELEs. Quantifying ELE-RNA transcriptome across typical developmental stages reveals that TE-initiated ELE-RNAs are mainly from RLG_famc7.3 specifically expanded in subgenome A. Acquisition of spike-specific transcription factor binding likely confers spike-specific expression of RLG_famc7.3-initiated ELE-RNAs. Knockdown of RLG_famc7.3-initiated ELE-RNAs resulted in global downregulation of spike-specific genes and abnormal spike development. These findings link TE expansion to regulatory specificity and polyploid developmental plasticity, highlighting the functional impact of TE-driven regulatory innovation on polyploid evolution.

LHP1-mediated epigenetic buffering of subgenome diversity and defense responses confers genome plasticity and adaptability in allopolyploid wheat.

Polyploidization is a major driver of genome diversification and environmental adaptation. However, the merger of different genomes may result in genomic conflicts, raising a major question regarding how genetic diversity is interpreted and regulated to enable environmental plasticity. By analyzing the genome-wide binding of 191 trans-factors in allopolyploid wheat, we identified like heterochromatin protein 1 (LHP1) as a master regulator of subgenome-diversified genes. Transcriptomic and epigenomic analyses of LHP1 mutants reveal its role in buffering the expression of subgenome-diversified defense genes by controlling H3K27me3 homeostasis. Stripe rust infection releases latent subgenomic variations by eliminating H3K27me3-related repression. The simultaneous inactivation of LHP1 homoeologs by CRISPR-Cas9 confers robust stripe rust resistance in wheat seedlings. The conditional repression of subgenome-diversified defenses ensures developmental plasticity to external changes, while also promoting neutral-to-non-neutral selection transitions and adaptive evolution. These findings establish an LHP1-mediated buffering system at the intersection of genotypes, environments, and phenotypes in polyploid wheat. Manipulating the epigenetic buffering capacity offers a tool to harness cryptic subgenomic variations for crop improvement.

Nanocomposite conductive tough hydrogel based on metal coordination reinforced covalent Pluronic F-127 micelle network for human motion sensing.

The design of conductive hydrogels integrating anti-fatigue, high sensitivity, strong mechanical property and good sterilization performance remains a challenge. We innovatively introduced metal coordination in covalently crosslinked Pluronic F-127 micelle network and synthesized nanocomposite conductive tough hydrogel through the combination of covalent crosslinking, metal coordination and silver nanowire reinforcement. Compared with pure diacylated PF127 hydrogel (PF127), the tensile strength of PF-AA-AM-Al3+/Ag0.25 hydrogel reaching 1.4 MPa was about 10 times than that of PF127. The toughness of PF-AA-AM-Al3+/Ag0.25 reaches 1.88 MJ/m3. Compared with PF-AA-AM-Al3+, the introduction of silver nanowires increased the fatigue life of PF-AA-AM-Al3+/Ag0.25 by 200% (31837 cycles), 170% (12804 cycles) and 1022% (511 cycles) under 100%, 120% and 150% ultimate tensile strains, respectively. Besides, the PF-AA-AM-Al3+/Ag0.25 showed strain sensitivity to small deformation (Gauge factor = 2.42) in wearable tests on hands and knees. In addition, the PF-AA-AM-Al3+/Ag0.25 had good cytocompatibility and antibacterial performance that bacteria killing ratio of 98% to S. aureus and 99% to E. coli. Finally, a viscoelastic numerical constitutive model was established based on finite element method to study the damage failure history of the material. Comparative analysis showed that local stress concentration was the main factor leading to the failure of hydrogel.