Crystal structure of a tetrameric RNA G-quadruplex formed by hexanucleotide repeat expansions of C9orf72 in ALS/FTD.

The abnormal GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause the fatal neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal dementia. The transcribed RNA HREs, short for r(G4C2)n, can form toxic RNA foci which sequestrate RNA binding proteins and impair RNA processing, ultimately leading to neurodegeneration. Here, we determined the crystal structure of r(G4C2)2, which folds into a parallel tetrameric G-quadruplex composed of two four-layer dimeric G-quadruplex via 5'-to-5' stacking in coordination with a K+ ion. Notably, the two C bases locate at 3'- end stack on the outer G-tetrad with the assistance of two additional K+ ions. The high-resolution structure reported here lays a foundation in understanding the mechanism of neurological toxicity of RNA HREs. Furthermore, the atomic details provide a structural basis for the development of potential therapeutic agents against the fatal neurodegenerative diseases ALS/FTD.

Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence.

The hexanucleotide repeat expansion, GGGGCC (G4C2), within the first intron of the C9orf72 gene is known to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 repeat expansions, either DNA or RNA, are able to form G-quadruplexes which induce toxicity leading to ALS/FTD. Herein, we report a novel crystal structure of d(G4C2)2 that self-associates to form an eight-layer parallel tetrameric G-quadruplex. Two d(G4C2)2 associate together as a parallel dimeric G-quadruplex which folds into a tetramer via 5'-to-5' arrangements. Each dimer consists of four G-tetrads connected by two CC propeller loops. Especially, the 3'-end cytosines protrude out and form C·C+•C·C+/ C·C•C·C+ quadruple base pair or C•C·C+ triple base pair stacking on the dimeric block. Our work sheds light on the G-quadruplexes adopted by d(G4C2) and yields the invaluable structural details for the development of small molecules to tackle neurodegenerative diseases, ALS and FTD.

Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA.

The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

Penetration of Arsenic and Deactivation of a Honeycomb V2O5-WO3/TiO2 Catalyst in a Glass Furnace.

Deactivation of honeycomb V2O5-WO3/TiO2 catalysts by arsenic has been studied widely in coal-fired power plants but rarely in glass furnaces. In this paper, deactivated catalysts that had been used for more than 4000 h were analyzed. We maintained the catalysts in their original monolith shape to retain their adhered substance and used appropriate methods to strip the substance layer by layer. With various characterization techniques, it was determined that the adhered substance was composed almost entirely of Na2SO4 and CaSO4. We also quantified the penetration depth of arsenic visually, which was more than 370 µm. A three-stage penetration and deactivation process induced by arsenic was proposed. It was pointed out that molten and volatile As2O3 played a key role in the deactivation process, while substances in the solid state had little impact on the deep bulk of the catalyst. In this study, we proposed an integrated deactivation process consisting of adhesion, penetration, and deactivation in a honeycomb V2O5-WO3/TiO2 catalyst by arsenic in a glass furnace. Finally, we also provided guidance on alleviating the deactivation caused by arsenic. The key is to convert molten and volatile As2O3 to solid-state substances before it contacts the catalyst.

The crystal structure of an antiparallel chair-type G-quadruplex formed by Bromo-substituted human telomeric DNA.

Human telomeric guanine-rich DNA, which could adopt different G-quadruplex structures, plays important roles in protecting the cell from recombination and degradation. Although many of these structures were determined, the chair-type G-quadruplex structure remains elusive. Here, we present a crystal structure of the G-quadruplex composed of the human telomeric sequence d[GGGTTAGG8GTTAGGGTTAGG20G] with two dG to 8Br-dG substitutions at positions 8 and 20 with syn conformation in the K+ solution. It forms a novel three-layer chair-type G-quadruplex with two linking trinucleotide loops. Particularly, T5 and T17 are coplanar with two water molecules stacking on the G-tetrad layer in a sandwich-like mode through a coordinating K+ ion and an A6•A18 base pair. While a twisted Hoogsteen A12•T10 base pair caps on the top of G-tetrad core. The three linking TTA loops are edgewise and each DNA strand has two antiparallel adjacent strands. Our findings contribute to a deeper understanding and highlight the unique roles of loop and water molecule in the folding of the G-quadruplex.

Molecular basis of the general base catalysis of an α/ß-hydrolase catalytic triad.

The serine-histidine-aspartate triad is well known for its covalent, nucleophilic catalysis in a diverse array of enzymatic transformations. Here we show that its nucleophilicity is shielded and its catalytic role is limited to being a specific general base by an open-closed conformational change in the catalysis of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (or MenH), a typical α/ß-hydrolase fold enzyme in the vitamin K biosynthetic pathway. This enzyme is found to adopt an open conformation without a functional triad in its ligand-free form and a closed conformation with a fully functional catalytic triad in the presence of its reaction product. The open-to-closed conformational transition involves movement of half of the α-helical cap domain, which causes extensive structural changes in the α/ß-domain and forces the side chain of the triad histidine to adopt an energetically disfavored gauche conformation to form the functional triad. NMR analysis shows that the inactive open conformation without a triad prevails in ligand-free solution and is converted to the closed conformation with a properly formed triad by the reaction product. Mutation of the residues crucial to this open-closed transition either greatly decreases or completely eliminates the enzyme activity, supporting an important catalytic role for the structural change. These findings suggest that the open-closed conformational change tightly couples formation of the catalytic triad to substrate binding to enhance the substrate specificities and simultaneously shield the nucleophilicity of the triad, thus allowing it to expand its catalytic power beyond the nucleophilic catalysis.

Development of stapled helical peptides to perturb the Cdt1-Mcm6 interaction.

Six all-hydrocarbon-stapled Cdt1 MBD-derived peptides have been designed and synthesized to perturb the Cdt1-Mcm6 interaction, which is involved in DNA replication. Inconsistency between the helicity of the obtained peptidomimetics and their binding affinity has been observed. The helicity of 13-amino acid stapled peptides increased, while their binding to Mcm6 was decreased. On the other hand, the 30-amino acid stapled peptides exhibited decreased helicity but increased binding affinity.

Structural basis for homeodomain recognition by the cell-cycle regulator Geminin.

Homeodomain-containing transcription factors play a fundamental role in the regulation of numerous developmental and cellular processes. Their multiple regulatory functions are accomplished through context-dependent inputs of target DNA sequences and collaborating protein partners. Previous studies have well established the sequence-specific DNA binding to homeodomains; however, little is known about how protein partners regulate their functions through targeting homeodomains. Here we report the solution structure of the Hox homeodomain in complex with the cell-cycle regulator, Geminin, which inhibits Hox transcriptional activity and enrolls Hox in cell proliferative control. Side-chain carboxylates of glutamates and aspartates in the C terminus of Geminin generate an overall charge pattern resembling the DNA phosphate backbone. These residues provide electrostatic interactions with homeodomain, which combine with the van der Waals contacts to form the stereospecific complex. We further showed that the interaction with Geminin is homeodomain subclass-selective and Hox paralog-specific, which relies on the stapling role of residues R43 and M54 in helix III and the basic amino acid cluster in the N terminus. Interestingly, we found that the C-terminal residue Ser184 of Geminin could be phosphorylated by Casein kinase II, resulting in the enhanced binding to Hox and more potent inhibitory effect on Hox transcriptional activity, indicating an additional layer of regulation. This structure provides insight into the molecular mechanism underlying homeodomain-protein recognition and may serve as a paradigm for interactions between homeodomains and DNA-competitive peptide inhibitors.

Structural insights into the Cdt1-mediated MCM2-7 chromatin loading.

Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2-7 (MCM2-7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410-440)/MCM6(708-821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2-7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2-7, our results suggest that the MCM2-7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2-7 chromatin loading.

Boundary sample-based class-weighted semi-supervised learning for malignant tumor classification of medical imaging.

Medical image classification plays a pivotal role within the field of medicine. Existing models predominantly rely on supervised learning methods, which necessitate large volumes of labeled data for effective training. However, acquiring and annotating medical image data is both an expensive and time-consuming endeavor. In contrast, semi-supervised learning methods offer a promising approach by harnessing limited labeled data alongside abundant unlabeled data to enhance the performance of medical image classification. Nonetheless, current methods often encounter confirmation bias due to noise inherent in self-generated pseudo-labels and the presence of boundary samples from different classes. To overcome these challenges, this study introduces a novel framework known as boundary sample-based class-weighted semi-supervised learning (BSCSSL) for medical image classification. Our method aims to alleviate the impact of intra- and inter-class boundary samples derived from unlabeled data. Specifically, we address reliable confidential data and inter-class boundary samples separately through the utilization of an inter-class boundary sample mining module. Additionally, we implement an intra-class boundary sample weighting mechanism to extract class-aware features specific to intra-class boundary samples. Rather than discarding such intra-class boundary samples outright, our approach acknowledges their intrinsic value despite the difficulty associated with accurate classification, as they contribute significantly to model prediction. Experimental results on widely recognized medical image datasets demonstrate the superiority of our proposed BSCSSL method over existing semi-supervised learning approaches. By enhancing the accuracy and robustness of medical image classification, our BSCSSL approach yields considerable implications for advancing medical diagnosis and future research endeavors.

Effect of gillnet mesh size on the capture probability and capture patterns in the Asian paddle crab (Charybdis japonica) fishery.

In the Asian paddle crab (Charybdis japonica) gillnet fishery in the Yellow Sea, China, the minimum mesh size (MMS) regulation has been of a major importance due to high bycatch rates of undersized crabs. In this study, we evaluated how gillnet mesh size can affect the capture probability of C. japonica and capture patterns in this fishery by comparing the performance of gillnets with four different mesh sizes (60, 70, 80, and 90 mm). Our results showed that changes in gillnet mesh size significantly affect the capture probability of different sizes of crabs. Specifically, increased mesh size decreased the capture probability of undersized crabs and their fraction in the catches decreased from 64 % to 24 % when mesh size was increased from 60 mm to 90 mm. In contrast, gillnets with larger mesh sizes significantly improved the capture probability of legal-sized crabs. Moreover, no significant differences were observed for the species catch composition between gillnets of different mesh sizes. Based on these results, we recommend 90 mm as the MMS in gillnets to improve sustainability in C. japonica fishery.

Ghost fishing efficiency in swimming crab (Portunus trituberculatus) pot fishery.

Abandoned, lost, or otherwise discarded fishing gear (ALDFG) is a global challenge that negatively affects marine environment through plastic pollution and continued capture of marine animals, so-called "ghost fishing". In different pot fisheries, ghost fishing related to ALDFG is of concern, including pot fishery targeting swimming crab (Portunus trituberculatus). This study quantified the ghost fishing efficiency by comparing it to the catch efficiency of actively fished pots of the commercial fishery. The results showed that the ghost fishing affects both target and bycatch species. On average, the ghost fishing pots captured 12.53 % (confidence intervals: 10.45 %-15.00 %) undersized crab and 15.70 % (confidence intervals: 12.08 %-20.74 %) legal-sized crab compared to the actively fished pots. Few individuals of several bycatch species were also captured by ghost fishing pots. The results of this study emphasized the need to develop new management strategies for reducing marine pollution by ALDFG and associated negative effects in this pot fishery.

TAT-W61 peptide attenuates neuronal injury through blocking the binding of S100b to the V-domain of Rage during ischemic stroke.

Ischemic stroke is a devastative nervous system disease associated with high mortality and morbidity rates. Unfortunately, no clinically effective neuroprotective drugs are available now. In ischemic stroke, S100 calcium-binding protein b (S100b) binds to receptor for advanced glycation end products (Rage), leading to the neurological injury. Therefore, disruption of the interaction between S100B and Rage can rescue neuronal cells. Here, we designed a peptide, termed TAT-W61, derived from the V domain of Rage which can recognize S100b. Intriguingly, TAT-W61 can reduce the inflammatory caused by ischemic stroke through the direct binding to S100b. The further investigation demonstrated that TAT-W61 can improve pathological infarct volume and reduce the apoptotic rate. Particularly, TAT-W61 significantly improved the learning ability, memory, and motor dysfunction of the mouse in the ischemic stroke model. Our study provides a mechanistic insight into the abnormal expression of S100b and Rage in ischemic stroke and yields an invaluable candidate for the development of drugs in tackling ischemic stroke. KEY MESSAGES: S100b expression is higher in ischemic stroke, in association with a high expression of many genes, especially of Rage. S100b is directly bound to the V-domain of Rage. Blocking the binding of S100b to Rage improves the injury after ischemic stroke.

The N-terminal region of Cdc6 specifically recognizes human DNA G-quadruplex.

Guanine (G)-rich nucleic acid sequences can form diverse G-quadruplex structures located in functionally significant genome regions, exerting regulatory control over essential biological processes, including DNA replication in vivo. During the initiation of DNA replication, Cdc6 is recruited by the origin recognition complex (ORC) to target specific chromosomal DNA sequences. This study reveals that human Cdc6 interacts with G-quadruplex structure through a distinct region within the N-terminal intrinsically disordered region (IDR), encompassing residues 7-20. The binding region assumes a hook-type conformation, as elucidated by the NMR solution structure in complex with htel21T18. Significantly, mutagenesis and in vivo investigations confirm the highly specific nature of Cdc6's recognition of G-quadruplex. This research enhances our understanding of the fundamental mechanism governing the interaction between G-quadruplex and the N-terminal IDR region of Cdc6, shedding light on the intricate regulation of DNA replication processes.

PRDX1 Interfering Peptide Disrupts Amino Acids 70-90 of PRDX1 to Inhibit the TLR4/NF-κB Signaling Pathway and Attenuate Neuroinflammation and Ischemic Brain Injury.

Ischemic stroke ranks among the leading causes of death and disability in humans and is accompanied by motor and cognitive impairment. However, the precise mechanisms underlying injury after stroke and effective treatment strategies require further investigation. Peroxiredoxin-1 (PRDX1) triggers an extensive inflammatory cascade that plays a pivotal role in the pathology of ischemic stroke, resulting in severe brain damage from activated microglia. In the present study, we used molecular dynamics simulation and nuclear magnetic resonance to detect the interaction between PRDX1 and a specific interfering peptide. We used behavioral, morphological, and molecular experimental methods to demonstrate the effect of PRDX1-peptide on cerebral ischemia-reperfusion (I/R) in mice and to investigate the related mechanism. We found that PRDX1-peptide bound specifically to PRDX1 and improved motor and cognitive functions in I/R mice. In addition, pretreatment with PRDX1-peptide reduced the infarct area and decreased the number of apoptotic cells in the penumbra. Furthermore, PRDX1-peptide inhibited microglial activation and downregulated proinflammatory cytokines including IL-1ß, IL-6, and TNF-α through inhibition of the TLR4/NF-κB signaling pathway, thereby attenuating ischemic brain injury. Our findings clarify the precise mechanism underlying PRDX1-induced inflammation after ischemic stroke and suggest that the PRDX1-peptide can significantly alleviate the postischemic inflammatory response by interfering with PRDX1 amino acids 70-90 and thereby inhibiting the TLR4/NF-κB signaling pathway. Our study provides a theoretical basis for a new therapeutic strategy to treat ischemic stroke.

Solution structure of the phosphotyrosine binding (PTB) domain of human tensin2 protein in complex with deleted in liver cancer 1 (DLC1) peptide reveals a novel peptide binding mode.

The protein deleted in liver cancer 1 (DLC1) interacts with the tensin family of focal adhesion proteins to play a role as a tumor suppressor in a wide spectrum of human cancers. This interaction has been proven to be crucial to the oncogenic inhibitory capacity and focal adhesion localization of DLC1. The phosphotyrosine binding (PTB) domain of tensin2 predominantly interacts with a novel site on DLC1, not the canonical NPXY motif. In this study, we characterized this interaction biochemically and determined the complex structure of tensin2 PTB domain with DLC1 peptide by NMR spectroscopy. Our HADDOCK-derived complex structure model elucidates the molecular mechanism by which tensin2 PTB domain recognizes DLC1 peptide and reveals a PTB-peptide binding mode that is unique in that peptide occupies the binding site opposite to the canonical NPXY motif interaction site with the peptide utilizing a non-canonical binding motif to bind in an extended conformation and that the N-terminal helix, which is unique to some Shc- and Dab-like PTB domains, is required for binding. Mutations of crucial residues defined for the PTB-DLC1 interaction affected the co-localization of DLC1 and tensin2 in cells and abolished DLC1-mediated growth suppression of hepatocellular carcinoma cells. This tensin2 PTB-DLC1 peptide complex with a novel binding mode extends the versatile binding repertoire of the PTB domains in mediating diverse cellular signaling pathways as well as provides a molecular and structural basis for better understanding the tumor-suppressive activity of DLC1 and tensin2.

Applying an ensemble of small models in predicting habitat suitability of invasive M. sallei along the southern coast of China.

A false mussel Mytilopsis sallei has caused serious ecological and economic losses after invading in China. In this research, we first assessed the niche differentiation between its native range and invasive range in China and then predicted the habitat suitability along the southern coast of China under present and future climatic circumstances. Distance to shore and water depth were the first two important factors in affecting the distribution of M. sallei, followed by minimum chlorophyll concentration and salinity. The niche of M. sallei shows significant expansion and unfilling. The ensemble of small models can account for few occurrences and presents high predictive performance. A general reduction and northward movement of suitable areas were found in the southern coast of China in the future. This study furnished significant insights regarding the areas under invasive risks, and provided valuable information for preventing the further invasion of M. sallei in China.

Comparison of physical properties and fishing performance between biodegradable PLA and conventional PA trammel nets in grey mullet (Mugil cephalus) and red-lip mullet (Liza haematocheila) fishery.

Marine plastic pollution and continuous capture of marine animals, so-called "ghost fishing", by abandoned, lost, or otherwise discarded fishing gear (ALDFG) are global concerns. This study investigated whether biodegradable polylactic acid (PLA) monofilaments can be used to replace conventionally used non-biodegradable polyamide (PA) in trammel net fishery for limiting ALDFG associated effects. It evaluated the physical properties of PLA and PA monofilaments and compared fishing performance of PLA and PA trammel nets in a commercial mullet fishery in the Yellow Sea, China. Although PA monofilament exhibited superior physical properties, no significant differences in catch efficiency between PA and PLA trammel nets were observed. Fish of both species were mainly captured by pocketing which can further explain observed similar catch efficiency. These initial results suggest a potential for applying biodegradable materials in trammel net fisheries. Therefore, further long-term testing is encouraged to investigate whether this promising performance is persistent over long-term.

An essential Noc3p dimerization cycle mediates ORC double-hexamer formation in replication licensing.

Replication licensing, a prerequisite of DNA replication, helps to ensure once-per-cell-cycle genome duplication. Some DNA replication-initiation proteins are sequentially loaded onto replication origins to form pre-replicative complexes (pre-RCs). ORC and Noc3p bind replication origins throughout the cell cycle, providing a platform for pre-RC assembly. We previously reported that cell cycle-dependent ORC dimerization is essential for the chromatin loading of the symmetric MCM double-hexamers. Here, we used Saccharomyces cerevisiae separation-of-function NOC3 mutants to confirm the separable roles of Noc3p in DNA replication and ribosome biogenesis. We also show that an essential and cell cycle-dependent Noc3p dimerization cycle regulates the ORC dimerization cycle. Noc3p dimerizes at the M-to-G1 transition and de-dimerizes in S-phase. The Noc3p dimerization cycle coupled with the ORC dimerization cycle enables replication licensing, protects nascent sister replication origins after replication initiation, and prevents re-replication. This study has revealed a new mechanism of replication licensing and elucidated the molecular mechanism of Noc3p as a mediator of ORC dimerization in pre-RC formation.

Cryo-EM structure of human hexameric MCM2-7 complex.

The central step in the initiation of eukaryotic DNA replication is the loading of the minichromosome maintenance 2-7 (MCM2-7) complex, the core of the replicative DNA helicase, onto chromatin at replication origin. Here, we reported the cryo-EM structure of endogenous human single hexameric MCM2-7 complex with a resolution at 4.4 Å, typically an open-ring hexamer with a gap between Mcm2 and Mcm5. Strikingly, further analysis revealed that human MCM2-7 can self-associate to form a loose double hexamer which potentially implies a novel mechanism underlying the MCM2-7 loading in eukaryote. The high-resolution cryo-EM structure of human MCM2-7 is critical for understanding the molecular mechanisms governing human DNA replication, especially the MCM2-7 chromatin loading and pre-replicative complex assembly.