Evolutionary Dynamics and Molecular Mechanisms of HORMA Domain Protein Signaling.

Controlled assembly and disassembly of multi-protein complexes is central to cellular signaling. Proteins of the widespread and functionally diverse HORMA family nucleate assembly of signaling complexes by binding short peptide motifs through a distinctive safety-belt mechanism. HORMA proteins are now understood as key signaling proteins across kingdoms, serving as infection sensors in a bacterial immune system and playing central roles in eukaryotic cell cycle, genome stability, sexual reproduction, and cellular homeostasis pathways. Here, we describe how HORMA proteins' unique ability to adopt multiple conformational states underlies their functions in these diverse contexts. We also outline how a dedicated AAA+ ATPase regulator, Pch2/TRIP13, manipulates HORMA proteins' conformational states to activate or inactivate signaling in different cellular contexts. The emergence of Pch2/TRIP13 as a lynchpin for HORMA protein action in multiple genome-maintenance pathways accounts for its frequent misregulation in human cancers and highlights TRIP13 as a novel therapeutic target.

The SMC-family Wadjet complex protects bacteria from plasmid transformation by recognition and cleavage of closed-circular DNA.

Self versus non-self discrimination is a key element of innate and adaptive immunity across life. In bacteria, CRISPR-Cas and restriction-modification systems recognize non-self nucleic acids through their sequence and their methylation state, respectively. Here, we show that the Wadjet defense system recognizes DNA topology to protect its host against plasmid transformation. By combining cryoelectron microscopy with cross-linking mass spectrometry, we show that Wadjet forms a complex similar to the bacterial condensin complex MukBEF, with a novel nuclease subunit similar to a type II DNA topoisomerase. Wadjet specifically cleaves closed-circular DNA in a reaction requiring ATP hydrolysis by the structural maintenance of chromosome (SMC) ATPase subunit JetC, suggesting that the complex could use DNA loop extrusion to sense its substrate's topology, then specifically activate the nuclease subunit JetD to cleave plasmid DNA. Overall, our data reveal how bacteria have co-opted a DNA maintenance machine to specifically recognize and destroy foreign DNAs through topology sensing.

An E1-E2 fusion protein primes antiviral immune signalling in bacteria.

In all organisms, innate immune pathways sense infection and rapidly activate potent immune responses while avoiding inappropriate activation (autoimmunity). In humans, the innate immune receptor cyclic GMP-AMP synthase (cGAS) detects viral infection to produce the nucleotide second messenger cyclic GMP-AMP (cGAMP), which initiates stimulator of interferon genes (STING)-dependent antiviral signalling1. Bacteria encode evolutionary predecessors of cGAS called cGAS/DncV-like nucleotidyltransferases2 (CD-NTases), which detect bacteriophage infection and produce diverse nucleotide second messengers3. How bacterial CD-NTase activation is controlled remains unknown. Here we show that CD-NTase-associated protein 2 (Cap2) primes bacterial CD-NTases for activation through a ubiquitin transferase-like mechanism. A cryo-electron microscopy structure of the Cap2-CD-NTase complex reveals Cap2 as an all-in-one ubiquitin transferase-like protein, with distinct domains resembling eukaryotic E1 and E2 proteins. The structure captures a reactive-intermediate state with the CD-NTase C terminus positioned in the Cap2 E1 active site and conjugated to AMP. Cap2 conjugates the CD-NTase C terminus to a target molecule that primes the CD-NTase for increased cGAMP production. We further demonstrate that a specific endopeptidase, Cap3, balances Cap2 activity by cleaving CD-NTase-target conjugates. Our data demonstrate that bacteria control immune signalling using an ancient, minimized ubiquitin transferase-like system and provide insight into the evolution of the E1 and E2 machinery across domains of life.

Chromatin binding by HORMAD proteins regulates meiotic recombination initiation.

The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase and is essential for fertility. In S. cerevisiae, the HORMAD protein Hop1 mediates the enrichment of axis proteins at nucleosome-rich islands through a central chromatin-binding region (CBR). Here, we use cryoelectron microscopy to show that the Hop1 CBR directly recognizes bent nucleosomal DNA through a composite interface in its PHD and winged helix-turn-helix domains. Targeted disruption of the Hop1 CBR-nucleosome interface causes a localized reduction of axis protein binding and meiotic DNA double-strand breaks (DSBs) in axis islands and leads to defects in chromosome synapsis. Synthetic effects with mutants of the Hop1 regulator Pch2 suggest that nucleosome binding delays a conformational switch in Hop1 from a DSB-promoting, Pch2-inaccessible state to a DSB-inactive, Pch2-accessible state to regulate the extent of meiotic DSB formation. Phylogenetic analyses of meiotic HORMADs reveal an ancient origin of the CBR, suggesting that the mechanisms we uncover are broadly conserved.

Architecture and self-assembly of the jumbo bacteriophage nuclear shell.

Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR-Cas systems1. In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors2-4. However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation.

A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage.

To protect themselves from the constant threat of bacteriophage (phage) infection, bacteria have evolved diverse immune systems including restriction-modification, CRISPR-Cas, and many others. Here, we describe the discovery of a two-protein transcriptional regulator module associated with hundreds of CBASS immune systems and demonstrate that this module drives the expression of its associated CBASS system in response to DNA damage. We show that the helix-turn-helix transcriptional repressor CapH binds the promoter region of its associated CBASS system to repress transcription until it is cleaved by the metallopeptidase CapP. CapP is activated in vitro by single-stranded DNA, and in cells by DNA-damaging drugs. Together, CapH and CapP drive increased expression of their associated CBASS system in response to DNA damage. We identify CapH- and CapP-related proteins associated with diverse known and putative bacterial immune systems including DISARM and Pycsar antiphage operons. Overall, our data highlight a mechanism by which bacterial immune systems can sense and respond to a universal signal of cell stress, potentially enabling multiple immune systems to mount a coordinated defensive response against an invading pathogen.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

Cost-effectiveness analysis of CYP2C19 genotype-guided antiplatelet therapy for patients with acute minor ischemic stroke and high-risk transient ischemic attack in China.

AIMS: The study aimed to estimate the cost-effectiveness of CYP2C19 genotype-guided antiplatelet therapy using cilostazol and ticagrelor as an alternative to clopidogrel, compared to conventional antiplatelet therapy with clopidogrel and aspirin. METHODS: A 90-day decision tree and 30-year Markov model were employed to assess the costs and quality-adjusted life years (QALYs) of personalized antiplatelet therapy for patients with minor ischemic stroke and high-risk transient ischemic attack, compared to conventional antiplatelet therapy in the Chinese healthcare system. The primary outcome was the incremental cost-effectiveness ratio (ICER). The data sources included clinical trials, published literature, official documents and local prices. One-way sensitivity analysis and probabilistic sensitivity analysis were performed to confirm the robustness of the findings. RESULTS: The base-case analysis indicated that the CYP2C19 genotype-guided antiplatelet strategy was cost-effective, and cilostazol group and ticagrelor group yielded an ICER of 3327.40 US dollars (USD)/QALY and 3426.92 USD/QALY, respectively, which were less than threshold. The one-way sensitivity analysis showed the results were robust, where the most sensitive parameter was the disability distribution in the modified Rankin scale 3-5. The probabilistic analysis showed that the CYP2C19 genotype-guided antiplatelet therapy with either cilostazol or ticagrelor was 100% cost-effective under the willingness-to-pay threshold. CONCLUSIONS: CYP2C19 genotype-guided antiplatelet therapy using cilostazol and ticagrelor as an alternative to clopidogrel appeared to be more cost-effective than conventional antiplatelet therapy for acute minor ischemic stroke and high-risk transient ischemic attack patients over 30 years in China.

Control of bacterial immune signaling by a WYL domain transcription factor.

Bacteria use diverse immune systems to defend themselves from ubiquitous viruses termed bacteriophages (phages). Many anti-phage systems function by abortive infection to kill a phage-infected cell, raising the question of how they are regulated to avoid cell killing outside the context of infection. Here, we identify a transcription factor associated with the widespread CBASS bacterial immune system, that we term CapW. CapW forms a homodimer and binds a palindromic DNA sequence in the CBASS promoter region. Two crystal structures of CapW suggest that the protein switches from an unliganded, DNA binding-competent state to a ligand-bound state unable to bind DNA. We show that CapW strongly represses CBASS gene expression in uninfected cells, and that phage infection causes increased CBASS expression in a CapW-dependent manner. Unexpectedly, this CapW-dependent increase in CBASS expression is not required for robust anti-phage activity, suggesting that CapW may mediate CBASS activation and cell death in response to a signal other than phage infection. Our results parallel concurrent reports on the structure and activity of BrxR, a transcription factor associated with the BREX anti-phage system, suggesting that CapW and BrxR are members of a family of universal defense signaling proteins.

Mechanistic insights into histone deposition and nucleosome assembly by the chromatin assembly factor-1.

Eukaryotic chromatin is a highly dynamic structure with essential roles in virtually all DNA-dependent cellular processes. Nucleosomes are a barrier to DNA access, and during DNA replication, they are disassembled ahead of the replication machinery (the replisome) and reassembled following its passage. The Histone chaperone Chromatin Assembly Factor-1 (CAF-1) interacts with the replisome and deposits H3-H4 directly onto newly synthesized DNA. Therefore, CAF-1 is important for the establishment and propagation of chromatin structure. The molecular mechanism by which CAF-1 mediates H3-H4 deposition has remained unclear. However, recent studies have revealed new insights into the architecture and stoichiometry of the trimeric CAF-1 complex and how it interacts with and deposits H3-H4 onto substrate DNA. The CAF-1 trimer binds to a single H3-H4 dimer, which induces a conformational rearrangement in CAF-1 promoting its interaction with substrate DNA. Two CAF-1•H3-H4 complexes co-associate on nucleosome-free DNA depositing (H3-H4)2 tetramers in the first step of nucleosome assembly. Here, we review the progress made in our understanding of CAF-1 structure, mechanism of action, and how CAF-1 contributes to chromatin dynamics during DNA replication.

Bacterial Shedu immune nucleases share a common enzymatic core regulated by diverse sensor domains.

Prokaryotes encode diverse anti-bacteriophage immune systems, including the single-protein Shedu nuclease. Here we reveal the structural basis for activation of Bacillus cereus Shedu. In the inactive homotetramer, a key catalytic residue in Shedu's nuclease domain is sequestered away from the catalytic site. Activation involves a conformational change that completes the active site and promotes assembly of a homo-octamer for coordinated double-strand DNA cleavage. Removal of Shedu's N-terminal domain ectopically activates the enzyme, suggesting that this domain allosterically inhibits Shedu in the absence of infection. Bioinformatic analysis of nearly 8,000 Shedu homologs reveals remarkable diversity in their N-terminal regulatory domains: we identify 79 domain families falling into eight functional classes, including diverse nucleic acid binding, enzymatic, and other domains. Together, these data reveal Shedu as a broad family of immune nucleases with a common nuclease core regulated by diverse N-terminal domains that likely respond to a range of infection-related signals.

Degradation of Sodium Acetate by Catalytic Ozonation Coupled with a Mn-Functionalized Fly Ash: Reaction Parameters and Mechanism.

Supported ozone catalysts usually take alumina, activated carbon, mesoporous molecular sieve, graphene, etc. as the carrier for loading metal oxide via the impregnation method, sol-gel method and precipitation method. In this work, a Mn-modified fly ash catalyst was synthesized to reduce the consumption and high unit price of traditional catalyst carriers like alumina. As a solid waste discharged from coal-fired power plants fueled by coal, fly ash also has porous spherical fine particles with constant surface area and activity, abd is expected to be applied as the main component in the synthesis of ozone catalyst. After the pretreatment process and modification with MnOx, the obtained Mn-modified fly ash exhibited stronger specific surface area and porosity combined with considerable ozone catalytic performance. We used sodium acetate as the contaminant probe, which is difficult to directly decompose with ozone as the end product of ozone oxidation, to evaluate the performance of this Mn-modified fly. It was found that ozone molecules can be transformed to generate ·OH, ·O2- and 1O2 for the further oxidation of sodium acetate. The oxygen vacancy produced via Mn modification plays a crucial role in the adsorption and excitation of ozone. This work demonstrates that fly ash, as an industrial waste, can be synthesized as a potential industrial catalyst with stable physical and chemical properties, a simple preparation method and low costs.

Identification of the bacteriophage nucleus protein interaction network.

In the arms race between bacteria and bacteriophages (phages), some large-genome jumbo phages have evolved a protein shell that encloses their replicating genome to protect it against host immune factors. By segregating the genome from the host cytoplasm, however, the 'phage nucleus' introduces the need to specifically translocate messenger RNA and proteins through the nuclear shell and to dock capsids on the shell for genome packaging. Here, we use proximity labeling and localization mapping to systematically identify proteins associated with the major nuclear shell protein chimallin (ChmA) and other distinctive structures assembled by these phages. We identify six uncharacterized nuclear-shell-associated proteins, one of which directly interacts with self-assembled ChmA. The structure and protein-protein interaction network of this protein, which we term ChmB, suggest that it forms pores in the ChmA lattice that serve as docking sites for capsid genome packaging and may also participate in messenger RNA and/or protein translocation.

A Physiologically Based Pharmacokinetic Approach to Recommend an Individual Dose of Tacrolimus in Adult Heart Transplant Recipients.

Tacrolimus is the principal immunosuppressive drug which is administered after heart transplantation. Managing tacrolimus therapy is challenging due to a narrow therapeutic index and wide pharmacokinetic (PK) variability. We aimed to establish a physiologically based pharmacokinetic (PBPK) model of tacrolimus in adult heart transplant recipients to optimize dose regimens in clinical practice. A 15-compartment full-PBPK model (Simbiology® Simulator, version 5.8.2) was developed using clinical observations from 115 heart transplant recipients. This study detected 20 genotypes associated with tacrolimus metabolism. CYP3A5*3 (rs776746), CYP3A4*18B (rs2242480), and IL-10 G-1082A (rs1800896) were identified as significant genetic covariates in tacrolimus pharmacokinetics. The PBPK model was evaluated using goodness-of-fit (GOF) and external evaluation. The predicted peak blood concentration (Cmax) and area under the drug concentration-time curve (AUC) were all within a two-fold value of the observations (fold error of 0.68-1.22 for Cmax and 0.72-1.16 for AUC). The patients with the CYP3A5*3/*3 genotype had a 1.60-fold increase in predicted AUC compared to the patients with the CYP3A5*1 allele, and the ratio of the AUC with voriconazole to alone was 5.80 when using the PBPK model. Based on the simulation results, the tacrolimus dosing regimen after heart transplantation was optimized. This is the first PBPK model used to predict the PK of tacrolimus in adult heart transplant recipients, and it can serve as a starting point for research on immunosuppressive drug therapy in heart transplant patients.

Identification of the bacteriophage nucleus protein interaction network.

In the arms race between bacteria and bacteriophages (phages), some large-genome jumbo phages have evolved a protein shell that encloses their replicating genome to protect it against DNA-targeting immune factors. By segregating the genome from the host cytoplasm, however, the "phage nucleus" introduces the need to specifically transport mRNA and proteins through the nuclear shell, and to dock capsids on the shell for genome packaging. Here, we use proximity labeling and localization mapping to systematically identify proteins associated with the major nuclear shell protein chimallin (ChmA) and other distinctive structures assembled by these phages. We identify six uncharacterized nuclear shell-associated proteins, one of which directly interacts with self-assembled ChmA. The structure and protein-protein interaction network of this protein, which we term ChmB, suggests that it forms pores in the ChmA lattice that serve as docking sites for capsid genome packaging, and may also participate in mRNA and/or protein transport.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the Chimalliviridae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.

Cost-Utility Analysis of CYP2C19 Genotype Detection for Selection of Acid-Suppressive Therapy with Lansoprazole or Vonoprazan for Patients with Reflux Esophagitis in China.

BACKGROUND: The cytochrome P450 (CYP) 2C19 genotype has a profound effect on the efficacy of lansoprazole, with less of an influence on vonoprazan. Both are first-choice drugs for the treatment of reflux esophagitis in China. OBJECTIVE: We aimed to estimate the cost-effectiveness of acid-suppressive treatments in Chinese patients with reflux esophagitis over 1 year from the societal perspective. METHODS: We developed a decision-based Markov model with a 4-week cycle to simulate the economic benefits and quality-adjusted life-years between different treatment strategies for patients with reflux esophagitis: universal lansoprazole, universal vonoprazan, and CYP2C19 genotype-guided strategies. The primary outcome was the incremental cost-effectiveness ratio. Data sources were the published literature, clinical trials, documents, and local charges. We used sensitivity analyses to detect the robustness of the findings and explored subgroup analyses and scenario analyses to make further evaluations. RESULTS: Compared to lansoprazole, vonoprazan and the CYP2C19 genotype-guided strategy were not preferable for Chinese patients with reflux esophagitis, with an incremental cost-effectiveness ratio of 222,387.1316 yuan/quality-adjusted life-year and 349,627.5000 yuan/quality-adjusted life-year, respectively. Sensitivity analyses showed the impact factors were the utility scores and the expenditures for the maintenance stage with lansoprazole and vonoprazan. When the willingness-to-pay threshold was 215,484 yuan/quality-adjusted life-year, 46.20% of the reflux esophagitis population was willing to pay for vonoprazan, compared with 8.30% for the CYP2C19 genotype-guided strategies. Vonoprazan and the CYP2C19 genotype-guided strategy were cost effective in the severe reflux esophagitis population, and in the reduction of the price of vonoprazan. CONCLUSIONS: The health economic evaluations revealed that for Chinese patients with reflux esophagitis, vonoprazan and the CYP2C19 genotype-guided strategy were not cost-effective regimens compared with lansoprazole. However, we found that in certain conditions like a reduction in the price of vonoprazan and in patients with severe reflux esophagitis these could be cost-effective.

Solvent Effect on the Solvothermal Synthesis of Mesoporous NiO Catalysts for Activation of Peroxymonosulfate to Degrade Organic Dyes.

In this work, we successfully prepared three different mesoporous NiO nanostructures with preferential (111) planes using three different solvents-water, a water-ethanol mixture, and a water-ethylene glycol mixture. The NiO nanosheets prepared from the water-ethylene glycol mixture and denoted as NiO-EG showed a nanosheet morphology thinner than 10 nm, whereas the water-ethanol and water samples were 30-40 nm and above 100 nm thick, respectively. The NiO-EG catalyst was found to exhibit a high catalyzing ability to activate peroxymonosulfate (PMS) for decoloring dyes, by which 94.4% of acid orange 7 (AO7) was degraded under the following reaction conditions: AO7 = 50 mg/L, catalyst = 0.2 g/L, PMS = 0.8 g/L, pH = 7, and 30 min reaction time. The dye degradation rate was investigated as a function of the catalyst dosage, pH, and dye concentration. According to quenching experiments, it was found that SO4 •-, HO•, and O2 •- were the dominant radicals for AO7 degradation, and oxygen vacancies played a significant role in the generation of radicals. High surface area, thin flaky structure, rich oxygen vacancies, fast charge transport, and low diffusion impedance all enhanced the catalytic activity of NiO-EG, which exhibited the highest degradation ability due to its abundant accessible active sites for both adsorption and catalysis.

Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay.

Nucleosomes organize the eukaryotic genome into chromatin. In cells, nucleosome assembly relies on the activity of histone chaperones, proteins with high binding affinity to histones. At least a subset of histone chaperones promotes histone deposition in vivo. However, it has been challenging to characterize this activity, due to the lack of quantitative assays. Here we developed a quantitative nucleosome assembly (NAQ) assay to measure the amount of nucleosome formation in vitro. This assay relies on a Micrococcal nuclease (MNase) digestion step that yields DNA fragments protected by the deposited histone proteins. A subsequent run on the Bioanalyzer machine allows the accurate quantification of the fragments (length and amount), relative to a loading control. This allows us to measure nucleosome formation by following the signature DNA length of ~150 bp. This assay finally enables the characterization of the nucleosome assembly activity of different histone chaperones, a step forward in the understanding of the functional roles of these proteins in vivo.