Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α.

Exercise benefits the human body in many ways. Irisin is secreted by muscle, increased with exercise, and conveys physiological benefits, including improved cognition and resistance to neurodegeneration. Irisin acts via αV integrins; however, a mechanistic understanding of how small polypeptides like irisin can signal through integrins is poorly understood. Using mass spectrometry and cryo-EM, we demonstrate that the extracellular heat shock protein 90α (eHsp90α) is secreted by muscle with exercise and activates integrin αVß5. This allows for high-affinity irisin binding and signaling through an Hsp90α/αV/ß5 complex. By including hydrogen/deuterium exchange data, we generate and experimentally validate a 2.98 Å RMSD irisin/αVß5 complex docking model. Irisin binds very tightly to an alternative interface on αVß5 distinct from that used by known ligands. These data elucidate a non-canonical mechanism by which a small polypeptide hormone like irisin can function through an integrin receptor.

A machine learning enhanced EMS mutagenesis probability map for efficient identification of causal mutations in Caenorhabditis elegans.

Chemical mutagenesis-driven forward genetic screens are pivotal in unveiling gene functions, yet identifying causal mutations behind phenotypes remains laborious, hindering their high-throughput application. Here, we reveal a non-uniform mutation rate caused by Ethyl Methane Sulfonate (EMS) mutagenesis in the C. elegans genome, indicating that mutation frequency is influenced by proximate sequence context and chromatin status. Leveraging these factors, we developed a machine learning enhanced pipeline to create a comprehensive EMS mutagenesis probability map for the C. elegans genome. This map operates on the principle that causative mutations are enriched in genetic screens targeting specific phenotypes among random mutations. Applying this map to Whole Genome Sequencing (WGS) data of genetic suppressors that rescue a C. elegans ciliary kinesin mutant, we successfully pinpointed causal mutations without generating recombinant inbred lines. This method can be adapted in other species, offering a scalable approach for identifying causal genes and revitalizing the effectiveness of forward genetic screens.

Molecular mechanism of the metal-independent production of hydroxyl radicals by thiourea dioxide and H2O2.

It is well-known that highly reactive hydroxyl radicals (HO•) can be produced by the classic Fenton system and our recently discovered haloquinone/H2O2 system, but rarely from thiol-derivatives. Here, we found, unexpectedly, that HO• can be generated from H2O2 and thiourea dioxide (TUO2), a widely used and environmentally friendly bleaching agent. A carbon-centered radical and sulfite were detected and identified as the transient intermediates, and urea and sulfate as the final products, with the complementary application of electron spin-trapping, oxygen-18 isotope labeling coupled with HPLC/MS analysis. Density functional theory calculations were conducted to further elucidate the detailed pathways for HO• production. Taken together, we proposed that the molecular mechanism for HO• generation by TUO2/H2O2: TUO2 tautomerizes from sulfinic acid into ketone isomer (TUO2-K) through proton transfer, then a nucleophilic addition of H2O2 on the S atom of TUO2-K, forming a S-hydroperoxide intermediate TUO2-OOH, which dissociates homolytically to produce HO•. Our findings represent the first experimental and computational study on an unprecedented new molecular mechanism of HO• production from simple thiol-derived sulfinic acids, which may have broad chemical, environmental, and biomedical significance for future research on the application of the well-known bleaching agent and its analogs.

Supramolecular coordination platinum metallacycle-based multilevel wound dressing for bacteria sensing and wound healing.

The exploitation of novel wound healing methods with real-time infection sensing and high spatiotemporal precision is highly important for human health. Pt-based metal-organic cycles/cages (MOCs) have been employed as multifunctional antibacterial agents due to their superior Pt-related therapeutic efficiency, various functional subunits and specific geometries. However, how to rationally apply these nanoscale MOCs on the macroscale with controllable therapeutic output is still challenging. Here, a centimeter-scale Pt MOC film was constructed via multistage assembly and subsequently coated on a N,N'-dimethylated dipyridinium thiazolo[5,4-d]thiazole (MPT)-stained silk fabric to form a smart wound dressing for bacterial sensing and wound healing. The MPT on silk fabric could be used to monitor wound infection in real-time through the bacteria-mediated reduction of MPT to its radical form via a color change. The MPT radical also exhibited an excellent photothermal effect under 660 nm light irradiation, which could not only be applied for photothermal therapy but also induce the disassembly of the Pt MOC film suprastructure. The highly ordered Pt MOC film suprastructure exhibited high biosafety, while it also showed improved antibacterial efficiency after thermally induced disassembly. In vitro and in vivo studies revealed that the combination of the Pt MOC film and MPT-stained silk can provide real-time information on wound infection for timely treatment through noninvasive techniques. This study paves the way for bacterial sensing and wound healing with centimeter-scale metal-organic materials.

The WD40 domain-containing protein Ehd5 positively regulates flowering in rice (Oryza sativa).

Heading date (flowering time), which greatly influences regional and seasonal adaptability in rice (Oryza sativa), is regulated by many genes in different photoperiod pathways. Here, we characterized a heading date gene, Early heading date 5 (Ehd5), using a modified bulked segregant analysis method. The ehd5 mutant showed late flowering under both short-day and long-day conditions, as well as reduced yield, compared to the wild type. Ehd5, which encodes a WD40 domain-containing protein, is induced by light and follows a circadian rhythm expression pattern. Transcriptome analysis revealed that Ehd5 acts upstream of the flowering genes Early heading date 1 (Ehd1), RICE FLOWERING LOCUS T 1 (RFT1), and Heading date 3a (Hd3a). Functional analysis showed that Ehd5 directly interacts with Rice outermost cell-specific gene 4 (Roc4) and Grain number, plant height, and heading date 8 (Ghd8), which might affect the formation of Ghd7-Ghd8 complexes, resulting in increased expression of Ehd1, Hd3a, and RFT1. In a nutshell, these results demonstrate that Ehd5 functions as a positive regulator of rice flowering and provide insight into the molecular mechanisms underlying heading date.

An open-source drug discovery platform enables ultra-large virtual screens.

On average, an approved drug currently costs US$2-3 billion and takes more than 10 years to develop1. In part, this is due to expensive and time-consuming wet-laboratory experiments, poor initial hit compounds and the high attrition rates in the (pre-)clinical phases. Structure-based virtual screening has the potential to mitigate these problems. With structure-based virtual screening, the quality of the hits improves with the number of compounds screened2. However, despite the fact that large databases of compounds exist, the ability to carry out large-scale structure-based virtual screening on computer clusters in an accessible, efficient and flexible manner has remained difficult. Here we describe VirtualFlow, a highly automated and versatile open-source platform with perfect scaling behaviour that is able to prepare and efficiently screen ultra-large libraries of compounds. VirtualFlow is able to use a variety of the most powerful docking programs. Using VirtualFlow, we prepared one of the largest and freely available ready-to-dock ligand libraries, with more than 1.4 billion commercially available molecules. To demonstrate the power of VirtualFlow, we screened more than 1 billion compounds and identified a set of structurally diverse molecules that bind to KEAP1 with submicromolar affinity. One of the lead inhibitors (iKeap1) engages KEAP1 with nanomolar affinity (dissociation constant (Kd) = 114 nM) and disrupts the interaction between KEAP1 and the transcription factor NRF2. This illustrates the potential of VirtualFlow to access vast regions of the chemical space and identify molecules that bind with high affinity to target proteins.

Epigenetic marks or not? The discovery of novel DNA modifications in eukaryotes.

DNA modifications add another layer of complexity to the eukaryotic genome to regulate gene expression, playing critical roles as epigenetic marks. In eukaryotes, the study of DNA epigenetic modifications has been confined to 5mC and its derivatives for decades. However, rapid developing approaches have witnessed the expansion of DNA modification reservoirs during the past several years, including the identification of 6mA, 5gmC, 4mC, and 4acC in diverse organisms. However, whether these DNA modifications function as epigenetic marks requires careful consideration. In this review, we try to present a panorama of all the DNA epigenetic modifications in eukaryotes, emphasizing recent breakthroughs in the identification of novel DNA modifications. The characterization of their roles in transcriptional regulation as potential epigenetic marks is summarized. More importantly, the pathways for generating or eliminating these DNA modifications, as well as the proteins involved are comprehensively dissected. Furthermore, we briefly discuss the potential challenges and perspectives, which should be taken into account while investigating novel DNA modifications.

A four-step biosynthetic pathway involving C-3 oxidation-reduction reactions from cycloastragenol to astragaloside IV in Astragalus membranaceus.

Astragaloside IV is a significant chemical component derived from the medicinal plant Astragalus membranaceus. Despite the characterization of several glycosyltransferases from A. membranaceus, the complete biosynthetic pathway of astragaloside IV has not been fully elucidated. In this study, we propose a biosynthetic pathway for astragaloside IV that involves a sequence of oxidation-reduction reactions. The biosynthesis pathway from cycloastragenol to astragaloside IV encompasses four key steps: C-3 oxidation, 6-O-glucosylation, C-3 reduction, and 3-O-xylosylation. We identified a hydroxysteroid dehydrogenase AmHSD1 from A. membranaceus. AmHSD1 catalyzes the C-3 oxidation of cycloastragenol, yielding cycloastragenol-3-one, and the C-3 reduction of cycloastragenol-3-one-6-O-glucoside, resulting in cycloastragenol-6-O-glucoside. Additionally, the glycosyltransferases AmGT8 and AmGT1, previously reported by our groups, were identified as catalyzing the 6-O-glucosylation and 3-O-xylosylation steps, respectively. Astragaloside IV was successfully synthesized in transient expression in Nicotiana benthamiana using the combination of AmHSD1, AmGT8 and AmGT1. These results support the proposed four-step biosynthetic pathway and suggest that AmHSD1 probably plays a crucial role in the biosynthesis of astragaloside IV within A. membranaceus.

The Chromosome-level Genome Provides Insights into the Evolution and Adaptation of Extreme Aggression.

Extremely aggressive behavior, as the special pattern, is rare in most species and characteristic as contestants severely injured or killed ending the combat. Current studies of extreme aggression are mainly from the perspectives of behavioral ecology and evolution, while lacked the aspects of molecular evolutionary biology. Here, a high-quality chromosome-level genome of the parasitoid Anastatus disparis was provided, in which the males exhibit extreme mate-competition aggression. The integrated multiomics analysis highlighted that neurotransmitter dopamine overexpression, energy metabolism (especially from lipid), and antibacterial activity are likely major aspects of evolutionary formation and adaptation for extreme aggression in A. disparis. Conclusively, our study provided new perspectives for molecular evolutionary studies of extreme aggression as well as a valuable genomic resource in Hymenoptera.

PIF1 helicase promotes break-induced replication in mammalian cells.

Break-induced replication (BIR) is a specialized homologous-recombination pathway for DNA double-strand break (DSB) repair, which often induces genome instability. In this study, we establish EGFP-based recombination reporters to systematically study BIR in mammalian cells and demonstrate an important role of human PIF1 helicase in promoting BIR. We show that at endonuclease cleavage sites, PIF1-dependent BIR is used for homology-initiated recombination requiring long track DNA synthesis, but not short track gene conversion (STGC). We also show that structure formation-prone AT-rich DNA sequences derived from common fragile sites (CFS-ATs) induce BIR upon replication stress and oncogenic stress, and PCNA-dependent loading of PIF1 onto collapsed/broken forks is critical for BIR activation. At broken replication forks, even STGC-mediated repair of double-ended DSBs depends on POLD3 and PIF1, revealing an unexpected mechanism of BIR activation upon replication stress that differs from the conventional BIR activation model requiring DSB end sensing at endonuclease-generated breaks. Furthermore, loss of PIF1 is synthetically lethal with loss of FANCM, which is involved in protecting CFS-ATs. The breast cancer-associated PIF1 mutant L319P is defective in BIR, suggesting a direct link of BIR to oncogenic processes.

Advancing single-cell RNA-seq data analysis through the fusion of multi-layer perceptron and graph neural network.

The advancement of single-cell sequencing technology has smoothed the ability to do biological studies at the cellular level. Nevertheless, single-cell RNA sequencing (scRNA-seq) data presents several obstacles due to the considerable heterogeneity, sparsity and complexity. Although many machine-learning models have been devised to tackle these difficulties, there is still a need to enhance their efficiency and accuracy. Current deep learning methods often fail to fully exploit the intrinsic interconnections within cells, resulting in unsatisfactory results. Given these obstacles, we propose a unique approach for analyzing scRNA-seq data called scMPN. This methodology integrates multi-layer perceptron and graph neural network, including attention network, to execute gene imputation and cell clustering tasks. In order to evaluate the gene imputation performance of scMPN, several metrics like cosine similarity, median L1 distance and root mean square error are used. These metrics are utilized to compare the efficacy of scMPN with other existing approaches. This research utilizes criteria such as adjusted mutual information, normalized mutual information and integrity score to assess the efficacy of cell clustering across different approaches. The superiority of scMPN over current single-cell data processing techniques in cell clustering and gene imputation investigations is shown by the experimental findings obtained from four datasets with gold-standard cell labels. This observation demonstrates the efficacy of our suggested methodology in using deep learning methodologies to enhance the interpretation of scRNA-seq data.

Natural polymorphisms in ZMET2 encoding a DNA methyltransferase modulate the number of husk layers in maize.

DNA methylation affects agronomic traits and the environmental adaptability of crops, but the natural polymorphisms in DNA methylation-related genes and their contributions to phenotypic variation in maize (Zea mays) remain elusive. Here, we show that a polymorphic 10-bp insertion/deletion variant in the 3'UTR of Zea methyltransferase2 (ZMET2) alters its transcript level and accounts for variation in the number of maize husk layers. ZMET2 encodes a chromomethylase and is required for maintaining genome-wide DNA methylation in the CHG sequence context. Disruption of ZMET2 increased the number of husk layers and resulted in thousands of differentially methylated regions, a proportion of which were also distinguishable in natural ZMET2 alleles. Population genetic analyses indicated that ZMET2 was a target of selection and might play a role in the spread of maize from tropical to temperate regions. Our results provide important insights into the natural variation of ZMET2 that confers both global and locus-specific effects on DNA methylation, which contribute to phenotypic diversity in maize.

Rab2A regulates the progression of nonalcoholic fatty liver disease downstream of AMPK-TBC1D1 axis by stabilizing PPARγ.

Nonalcoholic fatty liver disease (NAFLD) affects approximately a quarter of the population worldwide, and persistent overnutrition is one of the major causes. However, the underlying molecular basis has not been fully elucidated, and no specific drug has been approved for this disease. Here, we identify a regulatory mechanism that reveals a novel function of Rab2A in the progression of NAFLD based on energy status and PPARγ. The mechanistic analysis shows that nutrition repletion suppresses the phosphorylation of AMPK-TBC1D1 signaling, augments the level of GTP-bound Rab2A, and then increases the protein stability of PPARγ, which ultimately promotes the hepatic accumulation of lipids in vitro and in vivo. Furthermore, we found that blocking the AMPK-TBC1D1 pathway in TBC1D1S231A-knock-in (KI) mice led to a markedly increased GTP-bound Rab2A and subsequent fatty liver in aged mice. Our studies also showed that inhibition of Rab2A expression alleviated hepatic lipid deposition in western diet-induced obesity (DIO) mice by reducing the protein level of PPARγ and the expression of PPARγ target genes. Our findings not only reveal a new molecular mechanism regulating the progression of NAFLD during persistent overnutrition but also have potential implications for drug discovery to combat this disease.

Cellular and molecular characteristics of stromal Lkb1 deficiency-induced gastrointestinal polyposis based on single-cell RNA sequencing.

Liver kinase B1 (Lkb1), encoded by serine/threonine kinase (Stk11), is a serine/threonine kinase and tumor suppressor that is strongly implicated in Peutz-Jeghers syndrome (PJS). Numerous studies have shown that mesenchymal-specific Lkb1 is sufficient for the development of PJS-like polyps in mice. However, the cellular origin and components of these Lkb1-associated polyps and underlying mechanisms remain elusive. In this study, we generated tamoxifen-inducible Lkb1flox/flox;Myh11-Cre/ERT2 and Lkb1flox/flox;PDGFRα-Cre/ERT2 mice, performed single-cell RNA sequencing (scRNA-seq) and imaging-based lineage tracing, and aimed to investigate the cellular complexity of gastrointestinal polyps associated with PJS. We found that Lkb1flox/+;Myh11-Cre/ERT2 mice developed gastrointestinal polyps starting at 9 months after tamoxifen treatment. scRNA-seq revealed aberrant stem cell-like characteristics of epithelial cells from polyp tissues of Lkb1flox/+;Myh11-Cre/ERT2 mice. The Lkb1-associated polyps were further characterized by a branching smooth muscle core, abundant extracellular matrix deposition, and high immune cell infiltration. In addition, the Spp1-Cd44 or Spp1-Itga8/Itgb1 axes were identified as important interactions among epithelial, mesenchymal, and immune compartments in Lkb1-associated polyps. These characteristics of gastrointestinal polyps were also demonstrated in another mouse model, tamoxifen-inducible Lkb1flox/flox;PDGFRα-Cre/ERT2 mice, which developed obvious gastrointestinal polyps as early as 2-3 months after tamoxifen treatment. Our findings further confirm the critical role of mesenchymal Lkb1/Stk11 in gastrointestinal polyposis and provide novel insight into the cellular complexity of Lkb1-associated polyp biology. © 2024 The Pathological Society of Great Britain and Ireland.

Efficient and Safe In Vivo Treatment of Primary Hyperoxaluria Type 1 via LNP-CRISPR/Cas9-mediated glycolate oxidase disruption.

Primary hyperoxaluria type 1 (PH1) is a severe genetic metabolic disorder caused by mutations in the AGXT gene, leading to defects in enzymes crucial for glyoxylate metabolism. PH1 is characterized by severe, potentially life-threatening manifestations due to excessive oxalate accumulation, which leads to calcium oxalate crystal deposits in the kidneys and, ultimately, renal failure and systemic oxalosis. Existing substrate reduction therapies, such as inhibition of liver-specific glycolate oxidase (GO) encoded by HAO1 using siRNA or CRISPR/Cas9 delivered by adeno-associated virus (AAV), either require repeated dosing or have raised safety concerns. To address these limitations, our study employed lipid nanoparticles (LNPs) for CRISPR/Cas9 delivery to rapidly generate a PH1 mouse model and validate the therapeutic efficacy of LNP-CRISPR/Cas9 targeting the Hao1 gene. The LNP-CRISPR/Cas9 system exhibited efficient editing of the Hao1 gene, significantly reducing GO expression and lowering urinary oxalate levels in treated PH1 mice. Notably, these effects persisted for 12 months with no significant off-target effects, liver-induced toxicity, or substantial immune responses, highlighting the approach's safety and specificity. Furthermore, the developed humanized mouse model validated the efficacy of our therapeutic strategy. These findings support LNP-CRISPR/Cas9 targeting HAO1 as a promising and safer alternative for PH1 treatment with a single administration.

Sleeping Beauty mRNA-LNP enables stable rAAV transgene expression in mouse and NHP hepatocytes and improves vector potency.

Recombinant adeno-associated virus (rAAV) vector gene delivery systems have demonstrated great promise in clinical trials but continue to face durability and dose-related challenges. Unlike rAAV gene therapy, integrating gene addition approaches can provide curative expression in mitotically active cells and pediatric populations. We explored a novel in vivo delivery approach based on an engineered transposase, Sleeping Beauty (SB100X), delivered as an mRNA within a lipid nanoparticle (LNP), in combination with an rAAV-delivered transposable transgene. This combinatorial approach achieved correction of ornithine transcarbamylase deficiency in the neonatal Spfash mouse model following a single delivery to dividing hepatocytes in the newborn liver. Correction remained stable into adulthood, while a conventional rAAV approach resulted in a return to the disease state. In non-human primates, integration by transposition, mediated by this technology, improved gene expression 10-fold over conventional rAAV-mediated gene transfer while requiring 5-fold less vector. Additionally, integration site analysis confirmed a random profile while specifically targeting TA dinucleotides across the genome. Together, these findings demonstrate that transposable elements can improve rAAV-delivered therapies by lowering the vector dose requirement and associated toxicity while expanding target cell types.

The novel GATA1-interacting protein HES6 is an essential transcriptional cofactor for human erythropoiesis.

Normal erythropoiesis requires the precise regulation of gene expression patterns, and transcription cofactors play a vital role in this process. Deregulation of cofactors has emerged as a key mechanism contributing to erythroid disorders. Through gene expression profiling, we found HES6 as an abundant cofactor expressed at gene level during human erythropoiesis. HES6 physically interacted with GATA1 and influenced the interaction of GATA1 with FOG1. Knockdown of HES6 impaired human erythropoiesis by decreasing GATA1 expression. Chromatin immunoprecipitation and RNA sequencing revealed a rich set of HES6- and GATA1-co-regulated genes involved in erythroid-related pathways. We also discovered a positive feedback loop composed of HES6, GATA1 and STAT1 in the regulation of erythropoiesis. Notably, erythropoietin (EPO) stimulation led to up-regulation of these loop components. Increased expression levels of loop components were observed in CD34+ cells of polycythemia vera patients. Interference by either HES6 knockdown or inhibition of STAT1 activity suppressed proliferation of erythroid cells with the JAK2V617F mutation. We further explored the impact of HES6 on polycythemia vera phenotypes in mice. The identification of the HES6-GATA1 regulatory loop and its regulation by EPO provides novel insights into human erythropoiesis regulated by EPO/EPOR and a potential therapeutic target for the management of polycythemia vera.

An antagonistic pleiotropic gene regulates the reproduction and longevity tradeoff.

The antagonistic pleiotropy theory of aging proposes that genes enhancing fitness in early life limit the lifespan, but the molecular evidence remains underexplored. By profiling translatome changes in Caenorhabditis elegans during starvation recovery, we find that an open reading frame (ORF) trl-1 "hidden" within an annotated pseudogene significantly translates upon refeeding. trl-1 mutant animals increase brood sizes but shorten the lifespan and specifically impair germline deficiency­induced longevity. The loss of trl-1 abnormally up-regulates the translation of vitellogenin that produces copious yolk to provision eggs, whereas vitellogenin overexpression is known to reduce the lifespan. We show that the TRL-1 protein undergoes liquid­liquid phase separation (LLPS), through which TRL-1 granules recruit vitellogenin messenger RNA and inhibit its translation. These results indicate that trl-1 functions as an antagonistic pleiotropic gene to regulate the reproduction­longevity tradeoff by optimizing nutrient production for the next generation.

Chaotropic Effect-Induced Sol-Gel Transition and Radical Stabilization for Bacterially Sensitive Near-Infrared Photothermal Therapy.

Deep-seated bacterial infections (DBIs) are stubborn and deeply penetrate tissues. Eliminating deep-seated bacteria and promoting tissue regeneration remain great challenges. Here, a novel radical-containing hydrogel (SFT-B Gel) cross-linked by a chaotropic effect was designed for the sensing of DBIs and near-infrared photothermal therapy (NIR-II PTT). A silk fibroin solution stained with 4,4',4″-(1,3,5-triazine-2,4,6-triyl)tris(1-methylpyridin-1-ium) (TPT3+) was employed as the backbone, which could be cross-linked by a closo-dodecaborate cluster (B12H122-) through a chaotropic effect to form the SFT-B Gel. More interestingly, the SFT-B Gel exhibited the ability to sense DBIs, which could generate a TPT2+• radical with obvious color changes in the presence of bacteria. The radical-containing SFT-B Gel (SFT-B★ Gel) possessed strong NIR-II absorption and a remarkable photothermal effect, thus demonstrating excellent NIR-II PTT antibacterial activity for the treatment of DBIs. This work provides a new approach for the construction of intelligent hydrogels with unique properties using a chaotropic effect.

Defect Engineering of Ultrasmall TiO2 Nanoparticles Enables Highly Efficient Photocatalysts for Solar H2 Production from Woody Biomass.

The conversion of woody biomass to H2 through photocatalysis provides a sustainable strategy to generate renewable hydrogen fuel but was limited by the slow decomposition rate of woody biomass. Here, we fabricate ultrasmall TiO2 nanoparticles with tunable concentration of oxygen vacancy defects (VO-TiO2) as highly efficient photocatalysts for photocatalytic conversion of woody biomass to H2. Owing to the positive role of oxygen vacancy in reducing energy barrier for the generation of •OH which was the critical species to oxidize woody biomass, the obtained VO-TiO2 achieves rapid photocatalytic conversion of α-cellulose and poplar wood chip to H2 in the presence of Pt nanoclusters as the cocatalyst. As expected, the highest H2 generation rate in α-cellulose and poplar wood chip system respectively achieve 1146 and 59 µmol h-1 g-1, and an apparent quantum yield of 4.89% at 380 nm was obtained in α-cellulose aqueous solution.