Structural insights into the diversity and DNA cleavage mechanism of Fanzor.

Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz's mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts.

Flexible scaffold-based cheminformatics approach for polypharmacological drug design.

Effective treatments for complex central nervous system (CNS) disorders require drugs with polypharmacology and multifunctionality, yet designing such drugs remains a challenge. Here, we present a flexible scaffold-based cheminformatics approach (FSCA) for the rational design of polypharmacological drugs. FSCA involves fitting a flexible scaffold to different receptors using different binding poses, as exemplified by IHCH-7179, which adopted a "bending-down" binding pose at 5-HT2AR to act as an antagonist and a "stretching-up" binding pose at 5-HT1AR to function as an agonist. IHCH-7179 demonstrated promising results in alleviating cognitive deficits and psychoactive symptoms in mice by blocking 5-HT2AR for psychoactive symptoms and activating 5-HT1AR to alleviate cognitive deficits. By analyzing aminergic receptor structures, we identified two featured motifs, the "agonist filter" and "conformation shaper," which determine ligand binding pose and predict activity at aminergic receptors. With these motifs, FSCA can be applied to the design of polypharmacological ligands at other receptors.

Structural insights into the human D1 and D2 dopamine receptor signaling complexes.

The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-Gs and D2R-Gi signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.

GPCRs steer Gi and Gs selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

Serotonin (or 5-hydroxytryptamine, 5-HT) is an important neurotransmitter that activates 12 different G protein-coupled receptors (GPCRs) through selective coupling of Gs, Gi, or Gq proteins. The structural basis for G protein subtype selectivity by these GPCRs remains elusive. Here, we report the structures of the serotonin receptors 5-HT4, 5-HT6, and 5-HT7 with Gs, and 5-HT4 with Gi1. The structures reveal that transmembrane helices TM5 and TM6 alternate lengths as a macro-switch to determine receptor's selectivity for Gs and Gi, respectively. We find that the macro-switch by the TM5-TM6 length is shared by class A GPCR-G protein structures. Furthermore, we discover specific residues within TM5 and TM6 that function as micro-switches to form specific interactions with Gs or Gi. Together, these results present a common mechanism of Gs versus Gi protein coupling selectivity or promiscuity by class A GPCRs and extend the basis of ligand recognition at serotonin receptors.

Fanzor is a eukaryotic programmable RNA-guided endonuclease.

RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have a central role in biological processes in both prokaryotes and eukaryotes. For example, the prokaryotic CRISPR-Cas systems provide adaptive immunity for bacteria and archaea against foreign genetic elements. Cas effectors such as Cas9 and Cas12 perform guide-RNA-dependent DNA cleavage1. Although a few eukaryotic RNA-guided systems have been studied, including RNA interference2 and ribosomal RNA modification3, it remains unclear whether eukaryotes have RNA-guided endonucleases. Recently, a new class of prokaryotic RNA-guided systems (termed OMEGA) was reported4,5. The OMEGA effector TnpB is the putative ancestor of Cas12 and has RNA-guided endonuclease activity4,6. TnpB may also be the ancestor of the eukaryotic transposon-encoded Fanzor (Fz) proteins4,7, raising the possibility that eukaryotes are also equipped with CRISPR-Cas or OMEGA-like programmable RNA-guided endonucleases. Here we report the biochemical characterization of Fz, showing that it is an RNA-guided DNA endonuclease. We also show that Fz can be reprogrammed for human genome engineering applications. Finally, we resolve the structure of Spizellomyces punctatus Fz at 2.7 Å using cryogenic electron microscopy, showing the conservation of core regions among Fz, TnpB and Cas12, despite diverse cognate RNA structures. Our results show that Fz is a eukaryotic OMEGA system, demonstrating that RNA-guided endonucleases are present in all three domains of life.

Recognition of methamphetamine and other amines by trace amine receptor TAAR1.

Trace amine-associated receptor 1 (TAAR1), the founding member of a nine-member family of trace amine receptors, is responsible for recognizing a range of biogenic amines in the brain, including the endogenous ß-phenylethylamine (ß-PEA)1 as well as methamphetamine2, an abused substance that has posed a severe threat to human health and society3. Given its unique physiological role in the brain, TAAR1 is also an emerging target for a range of neurological disorders including schizophrenia, depression and drug addiction2,4,5. Here we report structures of human TAAR1-G-protein complexes bound to methamphetamine and ß-PEA as well as complexes bound to RO5256390, a TAAR1-selective agonist, and SEP-363856, a clinical-stage dual agonist for TAAR1 and serotonin receptor 5-HT1AR (refs. 6,7). Together with systematic mutagenesis and functional studies, the structures reveal the molecular basis of methamphetamine recognition and underlying mechanisms of ligand selectivity and polypharmacology between TAAR1 and other monoamine receptors. We identify a lid-like extracellular loop 2 helix/loop structure and a hydrogen-bonding network in the ligand-binding pockets, which may contribute to the ligand recognition in TAAR1. These findings shed light on the ligand recognition mode and activation mechanism for TAAR1 and should guide the development of next-generation therapeutics for drug addiction and various neurological disorders.

Structures of the human dopamine D3 receptor-Gi complexes.

The dopamine system, including five dopamine receptors (D1R-D5R), plays essential roles in the central nervous system (CNS), and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders. Here, we report two cryo-EM structures of human D3R in complex with an inhibitory G protein and bound to the D3R-selective agonists PD128907 and pramipexole, the latter of which is used to treat patients with Parkinson's disease. The structures reveal agonist binding modes distinct from the antagonist-bound D3R structure and conformational signatures for ligand-induced receptor activation. Mutagenesis and homology modeling illuminate determinants of ligand specificity across dopamine receptors and the mechanisms for Gi protein coupling. Collectively our work reveals the basis of agonist binding and ligand-induced receptor activation and provides structural templates for designing specific ligands to treat CNS diseases targeting the dopaminergic system.

Hormone- and antibody-mediated activation of the thyrotropin receptor.

Thyroid-stimulating hormone (TSH), through activation of its G-protein-coupled thyrotropin receptor (TSHR), controls the synthesis of thyroid hormone-an essential metabolic hormone1-3. Aberrant signalling of TSHR by autoantibodies causes Graves' disease (hyperthyroidism) and hypothyroidism, both of which affect millions of patients worldwide4. Here we report the active structures of TSHR with TSH and the activating autoantibody M225, both bound to the allosteric agonist ML-1096, as well as an inactivated TSHR structure with the inhibitory antibody K1-707. Both TSH and M22 push the extracellular domain (ECD) of TSHR into an upright active conformation. By contrast, K1-70 blocks TSH binding and cannot push the ECD into the upright conformation. Comparisons of the active and inactivated structures of TSHR with those of the luteinizing hormone/choriogonadotropin receptor (LHCGR) reveal a universal activation mechanism of glycoprotein hormone receptors, in which a conserved ten-residue fragment (P10) from the hinge C-terminal loop mediates ECD interactions with the TSHR transmembrane domain8. One notable feature is that there are more than 15 cholesterols surrounding TSHR, supporting its preferential location in lipid rafts9. These structures also highlight a similar ECD-push mechanism for TSH and autoantibody M22 to activate TSHR, therefore providing the molecular basis for Graves' disease.

Structures of full-length glycoprotein hormone receptor signalling complexes.

Luteinizing hormone and chorionic gonadotropin are glycoprotein hormones that are related to follicle-stimulating hormone and thyroid-stimulating hormone1,2. Luteinizing hormone and chorionic gonadotropin are essential to human reproduction and are important therapeutic drugs3-6. They activate the same G-protein-coupled receptor, luteinizing hormone-choriogonadotropin receptor (LHCGR), by binding to the large extracellular domain3. Here we report four cryo-electron microscopy structures of LHCGR: two structures of the wild-type receptor in the inactive and active states; and two structures of the constitutively active mutated receptor. The active structures are bound to chorionic gonadotropin and the stimulatory G protein (Gs), and one of the structures also contains Org43553, an allosteric agonist7. The structures reveal a distinct 'push-and-pull' mechanism of receptor activation, in which the extracellular domain is pushed by the bound hormone and pulled by the extended hinge loop next to the transmembrane domain. A highly conserved 10-residue fragment (P10) from the hinge C-terminal loop at the interface between the extracellular domain and the transmembrane domain functions as a tethered agonist to induce conformational changes in the transmembrane domain and G-protein coupling. Org43553 binds to a pocket of the transmembrane domain and interacts directly with P10, which further stabilizes the active conformation. Together, these structures provide a common model for understanding the signalling of glycoprotein hormone receptors and a basis for drug discovery for endocrine diseases.

Structural insights into the lipid and ligand regulation of serotonin receptors.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules-particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.

Integrative bioinformatics analysis to identify ferroptosis-related genes in non-obstructive azoospermia.

PURPOSE: The objective of this study was to discern ferroptosis-related genes (FRGs) linked to non-obstructive azoospermia and investigate the associated molecular mechanisms. METHOD: A dataset related to azoospermia was retrieved from the Gene Expression Omnibus database, and FRGs were sourced from GeneCards. Ferroptosis-related differentially expressed genes (FRDEGs) were discerned. Subsequently, these genes underwent analyses encompassing Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, as well as protein-protein interaction (PPI) networks and assessments of functional similarity. Following the identification of hub genes, an exploration of immune infiltration, single-cell expression, diagnostic utility, and interactions involving hub genes, RNA-binding proteins (RBPs), transcription factors (TFs), microRNAs (miRNAs), and drugs was conducted. RESULTS: A total of 35 differentially expressed FRGs were discerned. These genes demonstrated enrichment in functions and pathways associated with ferroptosis. From the PPI network, eight hub genes were selected. Functional similarity analysis highlighted the potential pivotal roles of HMOX1 and GPX4 in azoospermia. Analysis of immune cell infiltration indicated a significant decrease in activated dendritic cells in the azoospermia group, with notable correlations between hub genes, particularly SAT1 and HMGCR, and immune cell infiltration. Unique expression patterns of hub genes across various cell types in the human testis were observed, with GPX4 prominently enriched in spermatid/sperm. Eight hub genes exhibited robust diagnostic value (AUC > 0.75). Lastly, a comprehensive hub gene-miRNA-TF-RBP-drug network was constructed. CONCLUSION: In summary, our investigation unveiled eight FRDEGs associated with azoospermia, which hold potential as biomarkers for the diagnosis and treatment of azoospermia.

LLPSDB v2.0: an updated database of proteins undergoing liquid-liquid phase separation in vitro.

SUMMARY: Emerging evidences have suggested that liquid-liquid phase separation (LLPS) of proteins plays a vital role both in a wide range of biological processes and in related diseases. Whether a protein undergoes phase separation not only is determined by the chemical and physical properties of biomolecule themselves, but also is regulated by environmental conditions such as temperature, ionic strength, pH, as well as volume excluded by other macromolecules. A web accessible database LLPSDB was developed recently by our group, in which all the proteins involved in LLPS in vitro as well as corresponding experimental conditions were curated comprehensively from published literatures. With the rapid increase of investigations in biomolecular LLPS and growing popularity of LLPSDB, we updated the database, and developed a new version LLPSDB v2.0. In comparison of the previously released version, more than double contents of data are curated, and a new class 'Ambiguous system' is added. In addition, the web interface is improved, such as that users can search the database by selecting option 'phase separation status' alone or combined with other options. We anticipate that this updated database will serve as a more comprehensive and helpful resource for users. AVAILABILITY AND IMPLEMENTATION: LLPSDB v2.0 is freely available at: http://bio-comp.org.cn/llpsdbv2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Research on the Error of Global Positioning System Based on Time Series Analysis.

Due to the poor dynamic positioning precision of the Global Positioning System (GPS), Time Series Analysis (TSA) and Kalman filter technology are used to construct the positioning error of GPS. According to the statistical characteristics of the autocorrelation function and partial autocorrelation function of sample data, the Autoregressive (AR) model which is based on a Kalman filter is determined, and the error model of GPS is combined with a Kalman filter to eliminate the random error in GPS dynamic positioning data. The least square method is used for model parameter estimation and adaptability tests, and the experimental results show that the absolute value of the maximum error of longitude and latitude, the mean square error of longitude and latitude and average absolute error of longitude and latitude are all reduced, and the dynamic positioning precision after correction has been significantly improved.

Benzene metabolite hydroquinone promotes DNA homologous recombination repair via the NF-κB pathway.

Benzene, a widespread environmental pollutant, induces DNA double-strand breaks (DSBs) and DNA repair, which may further lead to oncogenic mutations, chromosomal rearrangements and leukemogenesis. However, the molecular mechanisms underlying benzene-induced DNA repair and carcinogenesis remain unclear. The human osteosarcoma cell line (U2OS/DR-GFP), which carries a GFP-based homologous recombination (HR) repair reporter, was treated with hydroquinone, one of the major benzene metabolites, to identify the potential effects of benzene on DSB HR repair. RNA-sequencing was further employed to identify the potential key pathway that contributed to benzene-initiated HR repair. We found that treatment with hydroquinone induced a significant increase in HR. NF-κB pathway, which plays a critical role in carcinogenesis in multiple tumors, was significantly activated in cells recovered from hydroquinone treatment. Furthermore, the upregulation of NF-κB by hydroquinone was also found in human hematopoietic stem and progenitor cells. Notably, the inhibition of NF-κB activity by small molecule inhibitors (QNZ and JSH-23) significantly reduced the frequency of hydroquinone-initiated HR (-1.36- and -1.77-fold, respectively, P < 0.01). Our results demonstrate an important role of NF-κB activity in promoting HR repair induced by hydroquinone. This finding sheds light on the underlying mechanisms involved in benzene-induced genomic instability and leukemogenesis and may contribute to the larger exploration of the influence of other environmental pollutants on carcinogenesis.

A Lithium Ion Highway by Surface Coordination Polymerization: In Situ Growth of Metal-Organic Framework Thin Layers on Metal Oxides for Exceptional Rate and Cycling Performance.

A thin layer of a highly porous metal-organic framework material, ZIF-8, is fabricated uniformly on the surface of nanostructured transition metal oxides (ZnO nanoflakes and MnO2 nanorods) to boost the transfer of lithium ions. The novel design and uniform microstructure of the MOF-coated TMOs (ZIF-8@TMOs) exhibit dramatically enhanced rate and cycling performance comparing to their pristine counterparts. The capacities of ZIF-8@ZnO (nanoflakes) and ZIF-8@MnO2 (nanorods) are 28 % and 31 % higher that of the pristine ones at the same current density. The nanorods of ZIF-8@MnO2 show a capacity of 1067 mAh g-1 after 500 cycles at 1 Ag-1 and without any fading. To further improve the conductivity and capacity, the ZIF-8-coated materials are pyrolyzed at 700 °C in an N2 atmosphere (ZIF-8@TMO-700 N). After pyrolysis, a much higher capacity improvement is achieved: ZIF-8@ZnO-700 N and ZIF-8@MnO2 -700 N have 54 % and 69 % capacity increases compared with the pristine TMOs, and at 1 Ag-1 , the capacity of ZIF-8@MnO2 -700 N is 1060 mAh g-1 after cycling for 300 cycles.

[Combined cytotoxicity mechanism of chlorpyrifos and carbofuran pesticides in vitro].

OBJECTIVE: To evaluate the single and combined effects of chlorpyrifos( CPF) and carbofuran( CF) pesticides on cell lines cultured in vitro, and clarify the pattern of joint action. METHODS: Rat pheochromocytoma( PC12 cells) were treated with different concentrations of CPF( 0, 50, 100, 200 and 400 µmol/L) and CF( 0, 25, 50, 100 and 200 µmol/L) for 12 h separately, the combined effects of two kinds of pesticides should be studied respectively in the low dose( CPF 50 µmol/L, CF 25 µmol/L) and high dose( CPF 200 µmol/L, CF 100 µmol/L) levels. After exposure, detectingacetylcholinesterase( ACh E) activity and using fluorescent probe 2', 7'-dichlorfluorescin diacetate( DCFH-DA), thiobarbituric acid( TBA) method, xanthine oxidation, 5, 5 '-dithio-bis-2-nitrobenzoic acid( DTNB) coloration to detect the intracellular reactive oxygen species( ROS) production, lipid peroxidation production malondialdehyde( MDA), activity of antioxidant enzymes superoxide dismutase( SOD) and glutathione peroxidase( GPx), respectively. RESULTS: Compared with the control group, CPF and CF could decrease the ACh E activity, induce ROS overproduction in a dose-effect way and increase the activity of SOD, GPx( P < 0. 01), but MDA content showed no significant change. Factorial ANOVA revealed that the combined effect of CPF and CF, there was no interaction at lower dose level, but interaction existed at higher dose level( P < 0. 01). The main mode of action was synergistic effect. CONCLUSION: Chlorpyrifos, carbofuran single or combined, has cytotoxicity effect. The main combined effect between chlorpyrifos and carbofuran is synergistic effect, oxidative stress damage may be one of the mechanisms.

A dew-responsive pectin-based herbicide for enhanced photodynamic inactivation.

5-aminolevulinic acid (5-ALA) has been fully demonstrated as a biodegradable, without resistance, and pollution-free pesticide. However, the lack of targeting and the poor adhesion result in a low utilization rate, limiting its practical application. Herein, a dew-responsive polymer pro-pesticide Pec-hyd-ALA was successfully synthesized by grafting 5-ALA onto the pectin (PEC) backbone via acid-sensitive acylhydrazone bonds. When the pro-pesticide is exposed to acid dew on plant surfaces at night, 5-ALA is released and subsequently converted to photosensitize (Protoporphyrin IX, PpIX)in plant cells, leading to its accumulation and promoting photodynamic inactivation (PDI). An inverted fluorescence microscope has verified the accumulation of tetrapyrrole in plant cells. In addition, the highly bio-adhesive PEC backbone effectively improved the wetting and retention of 5-ALA on leaves. The pot experiment also demonstrated the system's control effect on barnyard grass. This work provides a promising approach to improving the herbicidal efficacy of 5-ALA.

e3SIM: epidemiological-ecological-evolutionary simulation framework for genomic epidemiology.

Infectious disease dynamics are driven by the complex interplay of epidemiological, ecological, and evolutionary processes. Accurately modeling these interactions is crucial for understanding pathogen spread and informing public health strategies. However, existing simulators often fail to capture the dynamic interplay between these processes, resulting in oversimplified models that do not fully reflect real-world complexities in which the pathogen's genetic evolution dynamically influences disease transmission. We introduce the epidemiological-ecological-evolutionary simulator (e3SIM), an open-source framework that concurrently models the transmission dynamics and molecular evolution of pathogens within a host population while integrating environmental factors. Using an agent-based, discrete-generation, forward-in-time approach, e3SIM incorporates compartmental models, host-population contact networks, and quantitative-trait models for pathogens. This integration allows for realistic simulations of disease spread and pathogen evolution. Key features include a modular and scalable design, flexibility in modeling various epidemiological and population-genetic complexities, incorporation of time-varying environmental factors, and a user-friendly graphical interface. We demonstrate e3SIM's capabilities through simulations of realistic outbreak scenarios with SARS-CoV-2 and Mycobacterium tuberculosis, illustrating its flexibility for studying the genomic epidemiology of diverse pathogen types.

Oligosaccharides increased both leaf biomass and steviol glycosides content of Stevia rebaudiana.

Steviol glycosides (SGs) are a variety of important natural sweeteners. They are 200-350 times sweeter than sucrose without calories. Currently, their production is still mainly dependent on extraction from Stevia rebaudiana Bertoni (stevia). Oligosaccharides are environmentally friendly elicitors that promote plant growth and accumulation of secondary metabolites. In the present study, different concentrations of chitosan oligosaccharides (COS) and alginate oligosaccharides (AOS) were applied to stevia to explore their effect on growth and SGs biosynthesis. It was found that both COS and AOS promoted biomass production by increasing the leaf number and photosynthetic efficiency, which may be related to the decreased content of abscisic acid. The content of SGs was significantly increased after 50 mg/L AOS treatment, which not only increased the contents of stevioside (STV) and rebaudioside A (Reb A) significantly, but some important minority glucosides, like Reb E, Reb D, and Reb M. The increased SGs contents were the combined effect of the higher expression of SGs biosynthesis related genes, including KAH, UGT74G1, UGT85C2, and UGT91D2. The geometry changes of stem induced by COS and AOS may help to increase the lodging resistance of stevia. Thus, COS and AOS can be used in the field planting of stevia to increase the yield of SGs for industrial purposes.

Structural basis for motilin and erythromycin recognition by motilin receptor.

Motilin is an endogenous peptide hormone almost exclusively expressed in the human gastrointestinal (GI) tract. It activates the motilin receptor (MTLR), a class A G protein-coupled receptor (GPCR), and stimulates GI motility. To our knowledge, MTLR is the first GPCR reported to be activated by macrolide antibiotics, such as erythromycin. It has attracted extensive attention as a potential drug target for GI disorders. We report two structures of Gq-coupled human MTLR bound to motilin and erythromycin. Our structures reveal the recognition mechanism of both ligands and explain the specificity of motilin and ghrelin, a related gut peptide hormone, for their respective receptors. These structures also provide the basis for understanding the different recognition modes of erythromycin by MTLR and ribosome. These findings provide a framework for understanding the physiological regulation of MTLR and guiding drug design targeting MTLR for the treatment of GI motility disorders.