Mechanisms for HNH-mediated target DNA cleavage in type I CRISPR-Cas systems.

The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently identified genome editing tools. However, the underlying working mechanisms remain unknown. Here, structures of type I-FHNH and I-EHNH Cascade complexes at different states are reported. In type I-FHNH Cascade, Cas8fHNH loosely attaches to Cascade head and is adjacent to the 5' end of the target single-stranded DNA (ssDNA). Formation of the full R-loop drives the Cascade head to move outward, allowing Cas8fHNH to detach and rotate ∼150° to accommodate target ssDNA for cleavage. In type I-EHNH Cascade, Cas5eHNH domain is adjacent to the 5' end of the target ssDNA. Full crRNA-target pairing drives the lift of the Cascade head, widening the substrate channel for target ssDNA entrance. Altogether, these analyses into both complexes revealed that crRNA-guided positioning of target DNA and target DNA-induced HNH unlocking are two key factors for their site-specific cleavage of target DNA.

Molecular mechanism for target recognition, dimerization, and activation of Pyrococcus furiosus Argonaute.

The Argonaute nuclease from the thermophilic archaeon Pyrococcus furiosus (PfAgo) contributes to host defense and represents a promising biotechnology tool. Here, we report the structure of a PfAgo-guide DNA-target DNA ternary complex at the cleavage-compatible state. The ternary complex is predominantly dimerized, and the dimerization is solely mediated by PfAgo at PIWI-MID, PIWI-PIWI, and PAZ-N interfaces. Additionally, PfAgo accommodates a short 14-bp guide-target DNA duplex with a wedge-type N domain and specifically recognizes 5'-phosphorylated guide DNA. In contrast, the PfAgo-guide DNA binary complex is monomeric, and the engagement of target DNA with 14-bp complementarity induces sufficient dimerization and activation of PfAgo, accompanied by movement of PAZ and N domains. A closely related Argonaute from Thermococcus thioreducens adopts a similar dimerization configuration with an additional zinc finger formed at the dimerization interface. Dimerization of both Argonautes stabilizes the catalytic loops, highlighting the important role of Argonaute dimerization in the activation and target cleavage.

Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition.

The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for precise DNA insertion. To reveal the mechanism underlying target DNA recognition, we determined a cryoelectron microscopy (cryo-EM) structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a single guide RNA (sgRNA) and a double-stranded target DNA. Coupled with mutagenesis and in vitro DNA transposition assay, our results revealed mechanisms for the recognition of the GGTT protospacer adjacent motif (PAM) sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition. These structural and mechanistic insights should aid in the development of type V-K CRISPR-transposon systems as tools for genome editing.

Structures of rhodopsin in complex with G-protein-coupled receptor kinase 1.

G-protein-coupled receptor (GPCR) kinases (GRKs) selectively phosphorylate activated GPCRs, thereby priming them for desensitization1. Although it is unclear how GRKs recognize these receptors2-4, a conserved region at the GRK N terminus is essential for this process5-8. Here we report a series of cryo-electron microscopy single-particle reconstructions of light-activated rhodopsin (Rho*) bound to rhodopsin kinase (GRK1), wherein the N terminus of GRK1 forms a helix that docks into the open cytoplasmic cleft of Rho*. The helix also packs against the GRK1 kinase domain and stabilizes it in an active configuration. The complex is further stabilized by electrostatic interactions between basic residues that are conserved in most GPCRs and acidic residues that are conserved in GRKs. We did not observe any density for the regulator of G-protein signalling homology domain of GRK1 or the C terminus of rhodopsin. Crosslinking with mass spectrometry analysis confirmed these results and revealed dynamic behaviour in receptor-bound GRK1 that would allow the phosphorylation of multiple sites in the receptor tail. We have identified GRK1 residues whose mutation augments kinase activity and crosslinking with Rho*, as well as residues that are involved in activation by acidic phospholipids. From these data, we present a general model for how a small family of protein kinases can recognize and be activated by hundreds of different GPCRs.

Dynamic chromatin accessibility tuning by the long noncoding RNA ELDR accelerates chondrocyte senescence and osteoarthritis.

Epigenetic reprogramming plays a critical role in chondrocyte senescence during osteoarthritis (OA) pathology, but the underlying molecular mechanisms remain to be elucidated. Here, using large-scale individual datasets and genetically engineered (Col2a1-CreERT2;Eldrflox/flox and Col2a1-CreERT2;ROSA26-LSL-Eldr+/+ knockin) mouse models, we show that a novel transcript of long noncoding RNA ELDR is essential for the development of chondrocyte senescence. ELDR is highly expressed in chondrocytes and cartilage tissues of OA. Mechanistically, exon 4 of ELDR physically mediates a complex consisting of hnRNPL and KAT6A to regulate histone modifications of the promoter region of IHH, thereby activating hedgehog signaling and promoting chondrocyte senescence. Therapeutically, GapmeR-mediated silencing of ELDR in the OA model substantially attenuates chondrocyte senescence and cartilage degradation. Clinically, ELDR knockdown in cartilage explants from OA-affected individuals decreased the expression of senescence markers and catabolic mediators. Taken together, these findings uncover an lncRNA-dependent epigenetic driver in chondrocyte senescence, highlighting that ELDR could be a promising therapeutic avenue for OA.

Structural and biochemical characterization of the key components of an auxin degradation operon from the rhizosphere bacterium Variovorax.

Plant-associated bacteria play important regulatory roles in modulating plant hormone auxin levels, affecting the growth and yields of crops. A conserved auxin degradation (iad) operon was recently identified in the Variovorax genomes, which is responsible for root growth inhibition (RGI) reversion, promoting rhizosphere colonization and root growth. However, the molecular mechanism underlying auxin degradation by Variovorax remains unclear. Here, we systematically screened Variovorax iad operon products and identified 2 proteins, IadK2 and IadD, that directly associate with auxin indole-3-acetic acid (IAA). Further biochemical and structural studies revealed that IadK2 is a highly IAA-specific ATP-binding cassette (ABC) transporter solute-binding protein (SBP), likely involved in IAA uptake. IadD interacts with IadE to form a functional Rieske non-heme dioxygenase, which works in concert with a FMN-type reductase encoded by gene iadC to transform IAA into the biologically inactive 2-oxindole-3-acetic acid (oxIAA), representing a new bacterial pathway for IAA inactivation/degradation. Importantly, incorporation of a minimum set of iadC/D/E genes could enable IAA transformation by Escherichia coli, suggesting a promising strategy for repurposing the iad operon for IAA regulation. Together, our study identifies the key components and underlying mechanisms involved in IAA transformation by Variovorax and brings new insights into the bacterial turnover of plant hormones, which would provide the basis for potential applications in rhizosphere optimization and ecological agriculture.

Molecular mechanism for target RNA recognition and cleavage of Cas13h.

RNA-targeting type VI CRISPR-Cas effectors are widely used in RNA applications. Cas13h is a recently identified subtype of Cas13 ribonuclease, with strong RNA cleavage activity and robust in vivo RNA knockdown efficiency. However, little is known regarding its biochemical properties and working mechanisms. Biochemical characterization of Cas13h1 indicated that it lacks in vitro pre-crRNA processing activity and adopts a central seed. The cleavage activity of Cas13h1 is enhanced by a R(G/A) 5'-PFS, and inhibited by tag:anti-tag RNA pairing. We determined the structures of Cas13h1-crRNA binary complex at 3.1 Å and Cas13h1-crRNA-target RNA ternary complex at 3.0 Å. The ternary complex adopts an elongated architecture, and encodes a nucleotide-binding pocket within Helical-2 domain to recognize the guanosine at the 5'-end of the target RNA. Base pairing between crRNA guide and target RNA disrupts Cas13h1-guide interactions, leading to dramatic movement of HEPN domains. Upon target RNA engagement, Cas13h1 adopts a complicated activation mechanism, including separation of HEPN catalytic residues and destabilization of the active site loop and NTD domain, to get activated. Collectively, these insights expand our understanding into Cas13 effectors.

Structural basis for the concerted antiphage activity in the SIR2-HerA system.

Recently, a novel two-gene bacterial defense system against phages, encoding a SIR2 NADase and a HerA ATPase/helicase, has been identified. However, the molecular mechanism of the bacterial SIR2-HerA immune system remains unclear. Here, we determine the cryo-EM structures of SIR2, HerA and their complex from Paenibacillus sp. 453MF in different functional states. The SIR2 proteins oligomerize into a dodecameric ring-shaped structure consisting of two layers of interlocked hexamers, in which each subunit exhibits an auto-inhibited conformation. Distinct from the canonical AAA+ proteins, HerA hexamer alone in this antiphage system adopts a split spiral arrangement, which is stabilized by a unique C-terminal extension. SIR2 and HerA proteins assemble into a ∼1.1 MDa torch-shaped complex to fight against phage infection. Importantly, disruption of the interactions between SIR2 and HerA largely abolishes the antiphage activity. Interestingly, binding alters the oligomer state of SIR2, switching from a dodecamer to a tetradecamer state. The formation of the SIR2-HerA binary complex activates NADase and nuclease activities in SIR2 and ATPase and helicase activities in HerA. Together, our study not only provides a structural basis for the functional communications between SIR2 and HerA proteins, but also unravels a novel concerted antiviral mechanism through NAD+ degradation, ATP hydrolysis, and DNA cleavage.

GmEID1 modulates light signaling through the Evening Complex to control flowering time and yield in soybean.

Soybean (Glycine max) morphogenesis and flowering time are accurately regulated by photoperiod, which determine the yield potential and limit soybean cultivars to a narrow latitudinal range. The E3 and E4 genes, which encode phytochrome A photoreceptors in soybean, promote the expression of the legume-specific flowering repressor E1 to delay floral transition under long-day (LD) conditions. However, the underlying molecular mechanism remains unclear. Here, we show that the diurnal expression pattern of GmEID1 is opposite to that of E1 and targeted mutations in the GmEID1 gene delay soybean flowering regardless of daylength. GmEID1 interacts with J, a key component of circadian Evening Complex (EC), to inhibit E1 transcription. Photoactivated E3/E4 interacts with GmEID1 to inhibit GmEID1-J interaction, promoting J degradation resulting in a negative correlation between daylength and the level of J protein. Notably, targeted mutations in GmEID1 improved soybean adaptability by enhancing yield per plant up to 55.3% compared to WT in field trials performed in a broad latitudinal span of more than 24°. Together, this study reveals a unique mechanism in which E3/E4-GmEID1-EC module controls flowering time and provides an effective strategy to improve soybean adaptability and production for molecular breeding.

Deep profiling of potential substrate atlas of porcine epidemic diarrhea virus 3C-like protease.

Coronavirus (CoV) 3C-like protease (3CLpro) is essential for viral replication and is involved in immune escape by proteolyzing host proteins. Deep profiling the 3CLpro substrates in the host proteome extends our understanding of viral pathogenesis and facilitates antiviral drug discovery. Here, 3CLpro from porcine epidemic diarrhea virus (PEDV), an enteropathogenic CoV, was used as a model which to identify the potential 3CLpro cleavage motifs in all porcine proteins. We characterized the selectivity of PEDV 3CLpro at sites P5-P4'. We then compiled the 3CLpro substrate preferences into a position-specific scoring matrix and developed a 3CLpro profiling strategy to delineate the protein substrate landscape of CoV 3CLpro. We identified 1,398 potential targets in the porcine proteome containing at least one putative cleavage site and experimentally validated the reliability of the substrate degradome. The PEDV 3CLpro-targeted pathways are involved in mRNA processing, translation, and key effectors of autophagy and the immune system. We also demonstrated that PEDV 3CLpro suppresses the type 1 interferon (IFN-I) cascade via the proteolysis of multiple signaling adaptors in the retinoic acid-inducible gene I (RIG-I) signaling pathway. Our composite method is reproducible and accurate, with an unprecedented depth of coverage for substrate motifs. The 3CLpro substrate degradome establishes a comprehensive substrate atlas that will accelerate the investigation of CoV pathogenicity and the development of anti-CoV drugs.IMPORTANCECoronaviruses (CoVs) are major pathogens that infect humans and animals. The 3C-like protease (3CLpro) encoded by CoV not only cleaves the CoV polyproteins but also degrades host proteins and is considered an attractive target for the development of anti-CoV drugs. However, the comprehensive characterization of an atlas of CoV 3CLpro substrates is a long-standing challenge. Using porcine epidemic diarrhea virus (PEDV) 3CLpro as a model, we developed a method that accurately predicts the substrates of 3CLpro and comprehensively maps the substrate degradome of PEDV 3CLpro. Interestingly, we found that 3CLpro may simultaneously degrade multiple molecules responsible for a specific function. For instance, it cleaves at least four adaptors in the RIG-I signaling pathway to suppress type 1 interferon production. These findings highlight the complexity of the 3CLpro substrate degradome and provide new insights to facilitate the development of anti-CoV drugs.

Cleavage of HDAC6 to dampen its antiviral activity by nsp5 is a common strategy of swine enteric coronaviruses.

HDAC6, a structurally and functionally unique member of the histone deacetylase (HDAC) family, is an important host factor that restricts viral infection. The broad-spectrum antiviral activity of HDAC6 makes it a potent antiviral agent. Previously, we found that HDAC6 functions to antagonize porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with zoonotic potential. However, the final outcome is typically a productive infection that materializes as cells succumb to viral infection, indicating that the virus has evolved sophisticated mechanisms to combat the antiviral effect of HDAC6. Here, we demonstrate that PDCoV nonstructural protein 5 (nsp5) can cleave HDAC6 at glutamine 519 (Q519), and cleavage of HDAC6 was also detected in the context of PDCoV infection. More importantly, the anti-PDCoV activity of HDAC6 was damaged by nsp5 cleavage. Mechanistically, the cleaved HDAC6 fragments (amino acids 1-519 and 520-1159) lost the ability to degrade PDCoV nsp8 due to their impaired deacetylase activity. Furthermore, nsp5-mediated cleavage impaired the ability of HDAC6 to activate RIG-I-mediated interferon responses. We also tested three other swine enteric coronaviruses (transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and swine acute diarrhea syndrome-coronavirus) and found that all these coronaviruses have adopted similar mechanisms to cleave HDAC6 in both an overexpression system and virus-infected cells, suggesting that cleavage of HDAC6 is a common strategy utilized by swine enteric coronaviruses to antagonize the host's antiviral capacity. Together, these data illustrate how swine enteric coronaviruses antagonize the antiviral function of HDAC6 to maintain their infection, providing new insights to the interaction between virus and host.IMPORTANCEViral infections and host defenses are in constant opposition. Once viruses combat or evade host restriction, productive infection is achieved. HDAC6 is a broad-spectrum antiviral protein that has been demonstrated to inhibit many viruses, including porcine deltacoronavirus (PDCoV). However, whether HDAC6 is reciprocally targeted and disabled by viruses remains unclear. In this study, we used PDCoV as a model and found that HDAC6 is targeted and cleaved by nsp5, a viral 3C-like protease. The cleaved HDAC6 loses its deacetylase activity as well as its ability to degrade viral proteins and activate interferon responses. Furthermore, this cleavage mechanism is shared among other swine enteric coronaviruses. These findings shed light on the intricate interplay between viruses and HDAC6, highlighting the strategies employed by viruses to evade host antiviral defenses.

Hyperacetylated microtubules assist porcine deltacoronavirus nsp8 to degrade MDA5 via SQSTM1/p62-dependent selective autophagy.

The microtubule (MT) is a highly dynamic polymer that functions in various cellular processes through MT hyperacetylation. Thus, many viruses have evolved mechanisms to hijack the MT network of the cytoskeleton to allow intracellular replication of viral genomic material. Coronavirus non-structural protein 8 (nsp8), a component of the viral replication transcriptional complex, is essential for viral survival. Here, we found that nsp8 of porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with a zoonotic potential, inhibits interferon (IFN)-ß production by targeting melanoma differentiation gene 5 (MDA5), the main pattern recognition receptor for coronaviruses in the cytoplasm. Mechanistically, PDCoV nsp8 interacted with MDA5 and induced autophagy to degrade MDA5 in wild-type cells, but not in autophagy-related (ATG)5 or ATG7 knockout cells. Further screening for autophagic degradation receptors revealed that nsp8 interacts with sequestosome 1/p62 and promotes p62-mediated selective autophagy to degrade MDA5. Importantly, PDCoV nsp8 induced hyperacetylation of MTs, which in turn triggered selective autophagic degradation of MDA5 and subsequent inhibition of IFN-ß production. Overall, our study uncovers a novel mechanism employed by PDCoV nsp8 to evade host innate immune defenses. These findings offer new insights into the interplay among viruses, IFNs, and MTs, providing a promising target to develop anti-viral drugs against PDCoV.IMPORTANCECoronavirus nsp8, a component of the viral replication transcriptional complex, is well conserved and plays a crucial role in viral replication. Exploration of the role mechanism of nsp8 is conducive to the understanding of viral pathogenesis and development of anti-viral strategies against coronavirus. Here, we found that nsp8 of PDCoV, an emerging enteropathogenic coronavirus with a zoonotic potential, is an interferon antagonist. Further studies showed that PDCoV nsp8 interacted with MDA5 and sequestosome 1/p62, promoting p62-mediated selective autophagy to degrade MDA5. We further found that PDCoV nsp8 could induce hyperacetylation of MT, therefore triggering selective autophagic degradation of MDA5 and inhibiting IFN-ß production. These findings reveal a novel immune evasion strategy used by PDCoV nsp8 and provide insights into potential therapeutic interventions.

Cell-surface d-glucuronyl C5-epimerase binds to porcine deltacoronavirus spike protein facilitating viral entry.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus with zoonotic potential. The coronavirus spike (S) glycoprotein, especially the S1 subunit, mediates viral entry by binding to cellular receptors. However, the functional receptor of PDCoV remains poorly understood. In this study, we used the soluble PDCoV S1 protein as bait to capture the S1-binding cellular transmembrane proteins in combined immunoprecipitation and mass spectrometry analyses. A single guide RNA screen identified d-glucuronyl C5-epimerase (GLCE), a heparan sulfate-modifying enzyme, as a proviral host factor for PDCoV infection. GLCE knockout significantly inhibited the attachment and internalization stages of PDCoV infection. We also demonstrated the interaction between GLCE and PDCoV S with coimmunoprecipitation in both an overexpression system and PDCoV-infected cells. GLCE could be localized to the cell membrane, and an anti-GLCE antibody suppressed PDCoV infection. Although GLCE expression alone did not render nonpermissive cells susceptible to PDCoV infection, GLCE promoted the binding of PDCoV S to porcine amino peptidase N (pAPN), acting synergistically with pAPN to enhance PDCoV infection. In conclusion, our results demonstrate that GLCE is a novel cell-surface factor facilitating PDCoV entry and provide new insights into PDCoV infection. IMPORTANCE: The identification of viral receptors is of great significance, potentially extending our understanding of viral infection and pathogenesis. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus with the potential for cross-species transmission. However, the receptors or coreceptors of PDCoV are still poorly understood. The present study confirms that d-glucuronyl C5-epimerase (GLCE) is a positive regulator of PDCoV infection, promoting viral attachment and internalization. The anti-GLCE antibody suppressed PDCoV infection. Mechanically, GLCE interacts with PDCoV S and promotes the binding of PDCoV S to porcine amino peptidase N (pAPN), acting synergistically with pAPN to enhance PDCoV infection. This work identifies GLCE as a novel cell-surface factor facilitating PDCoV entry and paves the way for further insights into the mechanisms of PDCoV infection.

Inhibitory mechanism of CRISPR-Cas9 by AcrIIC4.

CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti-CRISPR (Acr) proteins to inactivate the CRISPR-Cas systems. Recently, AcrIIC4, identified from Haemophilus parainfluenzae phage, has been reported to inhibit the endonuclease activity of Cas9 from Neisseria meningitidis (NmeCas9), but the inhibition mechanism is not clear. Here, we biochemically and structurally investigated the anti-CRISPR activity of AcrIIC4. AcrIIC4 folds into a helix bundle composed of three helices, which associates with the REC lobe of NmeCas9 and sgRNA. The REC2 domain of NmeCas9 is locked by AcrIIC4, perturbing the conformational dynamics required for the target DNA binding and cleavage. Furthermore, mutation of the key residues in the AcrIIC4-NmeCas9 and AcrIIC4-sgRNA interfaces largely abolishes the inhibitory effects of AcrIIC4. Our study offers new insights into the mechanism of AcrIIC4-mediated suppression of NmeCas9 and provides guidelines for the design of regulatory tools for Cas9-based gene editing applications.

Unidirectional Lasing from Mirror-Coupled Dielectric Lattices.

This paper reports how a hybrid system composed of transparent dielectric lattices over a metal mirror can produce high-quality lattice resonances for unidirectional lasing. The enhanced electromagnetic fields are concentrated in the cladding of the periodic dielectric structures and away from the metal. Based on a mirror-image model, we reveal that such high-quality lattice resonances are governed by bound states in the continuum resulting from destructive interference. Using hexagonal arrays of titanium dioxide nanoparticles on a silica-coated silver mirror, we observed lattice resonances with quality factors of up to 2750 in the visible regime. With the lattice resonances as optical feedback and dye solution as the gain medium, we demonstrated unidirectional lasing under optical pumping, where the array size was down to 100 µm × 100 µm. Our scheme can be extended to other spectral regimes to simultaneously achieve strongly enhanced surface fields and high quality factors.

Integrated metagenomic and metabolomic analysis reveals distinctive stage-specific gut-microbiome-derived metabolites in intracranial aneurysms.

OBJECTIVE: Our study aimed to explore the influence of gut microbiota and their metabolites on intracranial aneurysms (IA) progression and pinpoint-related metabolic biomarkers derived from the gut microbiome. DESIGN: We recruited 358 patients with unruptured IA (UIA) and 161 with ruptured IA (RIA) from two distinct geographical regions for conducting an integrated analysis of plasma metabolomics and faecal metagenomics. Machine learning algorithms were employed to develop a classifier model, subsequently validated in an independent cohort. Mouse models of IA were established to verify the potential role of the specific metabolite identified. RESULTS: Distinct shifts in taxonomic and functional profiles of gut microbiota and their related metabolites were observed in different IA stages. Notably, tryptophan metabolites, particularly indoxyl sulfate (IS), were significantly higher in plasma of RIA. Meanwhile, upregulated tryptophanase expression and indole-producing microbiota were observed in gut microbiome of RIA. A model harnessing gut-microbiome-derived tryptophan metabolites demonstrated remarkable efficacy in distinguishing RIA from UIA patients in the validation cohort (AUC=0.97). Gut microbiota depletion by antibiotics decreased plasma IS concentration, reduced IA formation and rupture in mice, and downregulated matrix metalloproteinase-9 expression in aneurysmal walls with elastin degradation reduction. Supplement of IS reversed the effect of gut microbiota depletion. CONCLUSION: Our investigation highlights the potential of gut-microbiome-derived tryptophan metabolites as biomarkers for distinguishing RIA from UIA patients. The findings suggest a novel pathogenic role for gut-microbiome-derived IS in elastin degradation in the IA wall leading to the rupture of IA.

An examination of the metal ion content in the active sites of human endonucleases CPSF73 and INTS11.

Human cleavage and polyadenylation specificity factor (CPSF)73 (also known as CPSF3) is the endoribonuclease that catalyzes the cleavage reaction for the 3'-end processing of pre-mRNAs. The active site of CPSF73 is located at the interface between a metallo-ß-lactamase domain and a ß-CASP domain. Two metal ions are coordinated by conserved residues, five His and two Asp, in the active site, and they are critical for the nuclease reaction. The metal ions have long been thought to be zinc ions, but their exact identity has not been examined. Here we present evidence from inductively coupled plasma mass spectrometry and X-ray diffraction analyses that a mixture of metal ions, including Fe, Zn, and Mn, is present in the active site of CPSF73. The abundance of the various metal ions is different in samples prepared from different expression hosts. Zinc is present at less than 20% abundance in a sample expressed in insect cells, but the sample is active in cleaving a pre-mRNA substrate in a reconstituted canonical 3'-end processing machinery. Zinc is present at 75% abundance in a sample expressed in human cells, which has comparable endonuclease activity. We also observe a mixture of metal ions in the active site of the CPSF73 homolog INTS11, the endonuclease for Integrator. Taken together, our results provide further insights into the role of metal ions in the activity of CPSF73 and INTS11 for RNA 3'-end processing.

Working Memory Guides Action Valuation in Model-based Decision-making Strategy.

Humans use both model-free (or habitual) and model-based (or goal-directed) strategies in sequential decision-making. Working memory (WM) is essential for the model-based strategy; however, its exact role in these processes remains elusive. This study investigates the influence of WM processes on decision-making and the underlying cognitive computing mechanisms. Specifically, we used experimental data from two-stage decision tasks and found that delay and load, two WM-specific variables, impact goal-revisiting behaviors. Then, we proposed possible computational mechanisms by which WM participates in information processing and integrated them into the model-based system. The proposed Hybrid-WM model reproduced the observed experimental effects and fit human behavior better than the classic hybrid reinforcement learning model. These results were verified with independent data sets. Furthermore, differences in model parameters explain the age-related difference in sequential decision-making. Overall, this study suggests that WM guides action valuation in model-based strategies, highlighting the contribution of higher cognitive functions to sequential decision-making.

Characterization and gene expression analysis reveal universal stress proteins respond to abiotic stress in Gossypium hirsutum.

BACKGROUND: Universal stress proteins (USPs) are a class of stress-induced proteins that play a crucial role in biotic and abiotic stress responses. These proteins have previously been reported to participate directly in responses to various stress and protect plants against unfavorable environmental conditions. However, there is limited research on USPs in cotton, and systematic characterization of USPs in Gossypium species is lacking. RESULTS: In the present study, the USP genes in Gossypium hirsutum were systematically identified and classified into six distinct subfamilies. The expansion of USPs in Gossypium species is mainly caused by dispersed duplication and whole genome duplication. Notably, the USPs that have expanded through allotetraploidization events are highly conserved in the allotetraploid species. The promoter regions of GhUSPs contain a diverse range of cis-acting elements associated with stress response. The RNA-Seq analysis and RT-qPCR assays revealed a significant induction of numerous GhUSPs expressions in response to various abiotic stresses. The co-expression network of GhUSPs revealed their involvement in stress response. CONCLUSIONS: This study systematically analyzed the biological characteristics of GhUSPs and their response to abiotic stress. These findings serve as a theoretical basis for facilitating the breeding of cotton varieties in future research.

Test-retest reliability of the attention network test from the perspective of intrinsic network organization.

The attention network test (ANT), developed based on the triple-network taxonomy by Posner and colleagues, has been widely used to examine the efficacy of alerting, orienting and executive control in clinical and developmental neuroscience studies. Recent research suggests the imperfect reliability of the behavioural ANT and its variants. However, the classical ANT fMRI task's test-retest reliability has received little attention. Moreover, it remains ambiguous whether the attention-related intrinsic network components, especially the dorsal attention, ventral attention and frontoparietal network, manifest acceptable reliability. The present study approaches these issues by utilizing an openly available ANT fMRI dataset for participants with Parkinson's disease and healthy elderly. The reproducibility of group-level activations across sessions and participant groups and the test-retest reliability at the individual level were examined at the voxel, region and network levels. The intrinsic network was defined using the Yeo-Schaefer atlas. Our results reveal three critical facets: (1) the overlapping of the group-level contrast map between sessions and between participant groups was unsatisfactory; (2) the reliability of alerting, orienting and executive, defined as a contrast between conditions, was worse than estimates of specific conditions. (3) Dorsal attention, ventral attention, visual and somatomotor networks showed acceptable reliability for the congruent and incongruent conditions. Our results suggest that specific condition estimates might be used instead of the contrast map for individual or group-difference studies.