The catalytic triad of rice NARROW LEAF1 involves H234.

NARROW LEAF1 (NAL1) exerts a multifaceted influence on leaf morphology and crop yield. Recent crystal study proposed that histidine 233 (H233) is part of the catalytic triad. Here we report that unlike suggested previously, H234 instead of H233 is a component of the catalytic triad alongside residues D291 and S385 in NAL1. Remarkably, residue 233 unexpectedly plays a pivotal role in regulating NAL1's proteolytic activity. These findings establish a strong foundation for utilizing NAL1 in breeding programs aimed at improving crop yield.

Structural insights into the N-terminal APHB domain of HrpA: mediating canonical and i-motif recognition.

RNA helicases function as versatile enzymes primarily responsible for remodeling RNA secondary structures and organizing ribonucleoprotein complexes. In our study, we conducted a systematic analysis of the helicase-related activities of Escherichia coli HrpA and presented the structures of both its apo form and its complex bound with both conventional and non-canonical DNAs. Our findings reveal that HrpA exhibits NTP hydrolysis activity and binds to ssDNA and ssRNA in distinct sequence-dependent manners. While the helicase core plays an essential role in unwinding RNA/RNA and RNA/DNA duplexes, the N-terminal extension in HrpA, consisting of three helices referred to as the APHB domain, is crucial for ssDNA binding and RNA/DNA duplex unwinding. Importantly, the APHB domain is implicated in binding to non-canonical DNA structures such as G-quadruplex and i-motif, and this report presents the first solved i-motif-helicase complex. This research not only provides comprehensive insights into the multifaceted roles of HrpA as an RNA helicase but also establishes a foundation for further investigations into the recognition and functional implications of i-motif DNA structures in various biological processes.

Stimulation of ATP Hydrolysis by ssDNA Provides the Necessary Mechanochemical Energy for G4 Unfolding.

The G-quadruplex (G4) is a distinct geometric and electrophysical structure compared to classical double-stranded DNA, and its stability can impede essential cellular processes such as replication, transcription, and translation. This study focuses on the BsPif1 helicase, revealing its ability to bind independently to both single-stranded DNA (ssDNA) and G4 structures. The unfolding activity of BsPif1 on G4 relies on the presence of a single tail chain, and the covalent continuity between the single tail chain and the G4's main chain is necessary for efficient G4 unwinding. This suggests that ATP hydrolysis-driven ssDNA translocation exerts a pull force on G4 unwinding. Molecular dynamics simulations identified a specific region within BsPif1 that contains five crucial amino acid sites responsible for G4 binding and unwinding. A "molecular wire stripper" model is proposed to explain BsPif1's mechanism of G4 unwinding. These findings provide a new theoretical foundation for further exploration of the G4 development mechanism in Pif1 family helicases.

Exploring mechanism of hypolipidemic effect of total Ligustrum robustum (Roxb. ) Blume on hyperlipidemic golden hamsters based on intestinal flora / 中国药理学通报

Aim To evaluate the hypolipidemic effect of the total phenylpropanoid glycosides extracted from Ligustrum robustum (Roxb.) Blume (LRTPG) on hyperlipidemic golden hamsters and explore its regulatory effect on intestinal flora. Methods Sixty hamsters were randomly divided into a control group, a model group, a positive drug group, LRTPG-L group, LRTPG-M group, and LRTPG-H group. After the successful induction of the model by high-fat diet, the animals were continuously administered for four weeks, and their blood lipids and liver lipids were detected. The formed feces from the colorectal region of the hamsters in the control group, model group and LRTPG-H group were collected for 16S rDNA sequencing. Results LRTPG reduced serum TG, TC, LDL-C and liver TG, TC concentrations significantly in hyperlipidemic hamsters. The results of the intestinal microbiota sequencing showed that compared to the control group, LRTPG significantly decreased the relative abundance of the phylum Firmicutes and increased the relative abundance of the phylum Bacteroidetes and Verrucomicrobia (P < 0.01) at the phylum level. At the family level, LRTPG significantly increased the relative abundance of Christensenellaceae, Peptococcaceae, and Verrucomicrobiaceae (P < 0.05 or P < 0.01). At the genus level, LRTPG significantly increased the relative abundance of Oscillospira, Oscillibacter, Flavonifractor and Akkermansiaceae (P < 0.05 or P < 0.01). These changes in the flora were beneficial to the hypolipidemic effect of LRTPG. Conclusion LRTPG may exert its hypolipidemic effect by improving the intestinal flora disorder caused by a high-fat diet in golden hamsters.

Surfactin inhibits Fusarium graminearum by accumulating intracellular ROS and inducing apoptosis mechanisms.

Fusarium graminearum, a devastating fungal pathogen, is the main pathogen of Fusarium head blight (FHB) in wheat globally; it results in significant yield loss and mycotoxin contamination that severely threatens global wheat production and food safety. However, despite ongoing efforts, controlling this pathogen still remains a major challenge. Surfactin, primarily synthesized by Bacillus sp. via non-ribosomal peptide synthetases, exhibits potent surfactant and antibacterial properties, but its antifungal mechanism has yet to be fully elucidated. We found that the EC50 of surfactin against hyphal growth of F. graminearum was 102.1 µg/mL, and control efficacy against wheat FHB under field conditions achieved 86.38% in wheat cultivar Huaimai 40 and 81.60% in wheat cultivar Zhoumai 36, indicating that surfactin has potential antifungal activity against F. graminearum. Accumulated intracellular ROS, decreased mitochondrial membrane potential (MMP), activated metacaspase activity and condensed chromatin, were induced by surfactin in F. graminearum hyphae, suggesting that growth inhibition of fungus is mainly caused by apoptosis-like cell death. Furthermore, accumulated intracellular ROS was evidenced to act as a key mediator of surfactin-induced apoptosis. Broad-spectrum caspase inhibitor Z-VAD-FMK treatment indicated that surfactin induces caspase-independent apoptosis in F. graminearum. Collectively, this study provides evidence that surfactin induces a ROS-mediated mitochondrial apoptosis in F. graminearum hyphae, and may exert its antifungal activity against F. graminearum by activating apoptosis. This study demonstrates the potential of surfactin as an antifungal agent for FHB biocontrol, provides a new perspective on the antifungal mechanism of surfactin against filamentous fungi, and contributes to the application of surfactin-producing microbes in the biocontrol of plant diseases.

Zero-Watermarking for Vector Maps Combining Spatial and Frequency Domain Based on Constrained Delaunay Triangulation Network and Discrete Fourier Transform.

With its lossless properties, zero-watermarking has attracted a lot of attention in the field of copyright protection for vector maps. However, the common zero-watermarking algorithm puts too much emphasis on mining for global features, making it vulnerable to cropping attacks, and the robustness is not comprehensive enough. This study provides a vector map zero-watermarking scheme that utilizes spatial statistical information and frequency domain transformation methods in an effort to solve the aforementioned issue. In order to make the scheme more resistant to cropping and compression, it is constructed on the basis of feature point extraction and point constraint blocking of the original vector map. Within each sub-block, feature points are used to build constraint Delaunay triangulation networks (CDTN), and the angular values within the triangle networks are then extracted as spatial statistics. The angle value sequence is further transformed by discrete Fourier transform (DFT), and the binarized phase sequence is used as the final feature information to build a zero watermark by executing an exclusive disjunction operation with the encrypted copyright watermark image, both of which contribute to the scheme's robustness and security. The results of the attack experiments show that the proposed vector map zero-watermarking can restore identifiable copyright images under common geometric attacks, cropping attacks, and coordinate system transformations, demonstrating a high level of robustness. The theoretical basis for the robustness of this watermarking scheme is the stability of CDTN and the geometric invariance of DFT coefficients, and both theory and experiment validate the method's validity.

Nonstructural N- and C-tails of Dbp2 confer the protein full helicase activities.

Human DDX5 and its yeast ortholog Dbp2 are ATP-dependent RNA helicases that play a key role in normal cell processes, cancer development, and viral infection. The crystal structure of the RecA1-like domain of DDX5 is available but the global structure of DDX5/Dbp2 subfamily proteins remains to be elucidated. Here, we report the first X-ray crystal structures of the Dbp2 helicase core alone and in complex with ADP at 3.22 Å and 3.05 Å resolutions, respectively. The structures of the ADP-bound post-hydrolysis state and apo-state demonstrate the conformational changes that occur when the nucleotides are released. Our results showed that the helicase core of Dbp2 shifted between open and closed conformation in solution but the unwinding activity was hindered when the helicase core was restricted to a single conformation. A small-angle X-ray scattering experiment showed that the disordered amino (N) tail and carboxy (C) tails are flexible in solution. Truncation mutations confirmed that the terminal tails were critical for the nucleic acid binding, ATPase, and unwinding activities, with the C-tail being exclusively responsible for the annealing activity. Furthermore, we labeled the terminal tails to observe the conformational changes between the disordered tails and the helicase core upon binding nucleic acid substrates. Specifically, we found that the nonstructural terminal tails bind to RNA substrates and tether them to the helicase core domain, thereby conferring full helicase activities to the Dbp2 protein. This distinct structural characteristic provides new insight into the mechanism of DEAD-box RNA helicases.

Structural Studies of Pif1 Helicases from Thermophilic Bacteria.

Pif1 proteins are DNA helicases belonging to Superfamily 1, with 5' to 3' directionality. They are conserved from bacteria to human and have been shown to be particularly important in eukaryotes for replication and nuclear and mitochondrial genome stability. However, Pif1 functions in bacteria are less known. While most Pif1 from mesophilic bacteria consist of the helicase core with limited N-terminal and C-terminal extensions, some Pif1 from thermophilic bacteria exhibit a C-terminal WYL domain. We solved the crystal structures of Pif1 helicase cores from thermophilic bacteria Deferribacter desulfuricans and Sulfurihydrogenibium sp. in apo and nucleotide bound form. We show that the N-terminal part is important for ligand binding. The full-length Pif1 helicase was predicted based on the Alphafold algorithm and the nucleic acid binding on the Pif1 helicase core and the WYL domain was modelled based on known crystallographic structures. The model predicts that amino acids in the domains 1A, WYL, and linker between the Helicase core and WYL are important for nucleic acid binding. Therefore, the N-terminal and C-terminal extensions may be necessary to strengthen the binding of nucleic acid on these Pif1 helicases. This may be an adaptation to thermophilic conditions.

The Dual Role of Non-coding RNAs in the Development of Periodontitis / 生物医学与环境科学（英文）

This review aims to sum up how Non-coding RNAs (ncRNAs) regulate the development of periodontitis and provides a new perspective for understanding the pathogenesis of periodontitis. We explored the ncRNA's dual role in the development of periodontitis by summarizing evidence from previous in vivo and in vitro studies as well as clinical samples. In our review, the downregulation of 18 miRNAs, 22 lncRNAs and 10 circRNAs demonstrates protective roles in periodontitis. In contrast, the expression of other 11 miRNAs, 7 lncRNAs and 6 circRNAs are upregulated in periodontitis, which promote the progression of periodontitis. These dysregulated ncRNAs exert their protective or destructive roles by mainly influencing cell proliferation, differentiation and apoptosis via cross-talking with various molecules or signaling pathways. Our findings suggested which and how ncRNAs promote or delay the progression of periodontitis, which may greatly contribute to diagnose and therapy development of periodontitis based on ncRNAs in the future.

Research Progress on Effects of Different Treatments on Prostate Cancer with Atrial Fibrillation and Prevention Strategies / 肿瘤防治研究

Atrial fibrillation (AF) is one of the most common clinical arrhythmias. As the population ages, there is an upward trend in its prevalence. The risk factors associated with increased risk of AF include old age, diabetes, hypertension, and cancer. Studies have shown that in all age groups, the risk of death, hospitalization expenses, and hospitalization time of cancer patients with AF were higher than that without AF. Thus, increased systemic inflammation, electrolyte abnormalities, and neurohormonal changes in patients with prostate cancer (PCa) lead to a significantly higher incidence of AF than other cancers. However, the treatment of prostate cancer, including surgery, chemotherapy and radiotherapy, may also increase the risk of AF. In this review, relevant literatures are collected to understand the mechanism of AF in patients with PCa, determine the relationship between PCa and AF and its effect on hospitalized prognosis, and provide strategies for the prevention and treatment of AF in patients with PCa.

Biochemical and functional characterization of an exonuclease from Chaetomium thermophilum.

Exonucleases are often found associated with polymerase or helicase domains in the same enzyme or can function as autonomous entities to maintain genome stability. Here, we uncovered Chaetomium thermophilum RecQ family proteins that also have exonuclease activity in addition to their main helicase function. The novel exonuclease activity is separate from the helical core domain and coexists with the latter two enzymatic activities on the same polypeptide. The CtRecQ121-366 exonuclease region performs independently as an exonuclease. We describe its catalytic mechanism and biological characteristics. We demonstrate unequivocally that CtRecQ121-366 exclusively displays exonuclease activity and that this activity has a 3'-5' polarity that can both hydrolyze ssDNA and cleave dsDNA substrates. The hydrolytic activity of majority exonuclease is driven by bimetal ions, and this appears to be the case for the CtRecQ121-366 exonuclease as well. Additionally, the maximum activity of CtRecQ121-366 was observed at pH 8.0-9.0, low salt with Mg2+. The two helices in the structure, a6 and a7, play significant roles in the execution by anticipating their shape and changing essential amino acids.

DEAD-box RNA helicase Dbp2 binds to G-quadruplex nucleic acids and regulates different conformation of G-quadruplex DNA.

G-quadruplexes (G4s) are important in regulating DNA replication, repair and RNA transcription through interactions with specialized proteins. Dbp2 has been identified as a G4 DNA binding protein from Saccharomyces cerevisiae cell lysates. The majority of G4 motifs in Saccharomyces cerevisiae display 5-50 nt loops, only a few have 1-2 nt loops. Human DDX5 could unfold MycG4 DNA, whether Dbp2 also participates in remodeling G4 motifs with short loops in Saccharomyces cerevisiae remains elusive. Here we find that Dbp2 prefers G-rich substrates and binds MycG4 with a high affinity. Dbp2 possesses a dual function for different conformations of MycG4, destabilizing the folded MycG4 and inducing further folding of the unfolded MycG4. Similarly, DDX5 can unfold MycG4, but it exhibits a weaker MycG4 folding-promoting activity relative to Dbp2. Furthermore, Dbp2 facilitates DNA annealing activity in the absence of ATP, suggesting that Dbp2 can work on DNA substrates and possibly participate in DNA metabolism. Our results demonstrate that Dbp2 plays an important role in regulating the folding and unfolding activities of MycG4.

Economic effects of conversion from county (or county-level city) to municipal district in China.

Administrative division is an important resource to promote the urbanization process and economic growth in China. As an important way of urban spatial expansion, the effect of the removal of counties (county-level cities) into municipal districts(RCD) on economic growth remains to be empirically tested. In this paper, the panel data at the county level from 1998 to 2016 and the differential method were selected to study this problem. The results show that, during the study period, the RCD significantly promoted the economic growth of Chinese cities. The effect of removing counties (county-level cities) from large cities and megacities to set up districts is obviously better than that of small and medium-sized cities. In small and medium-sized cities with small urban permanent population, the RCD has obvious negative impact on economic development. The effect of county (county-level city) reform in eastern and central regions is more significant, while the effect of policy in western and northeast regions is not significant. When the development intensity of the municipal district is between 15%-20%, the effect of the RCD is relatively good, and the administrative division adjustment of the municipal district has a certain optimal window period.

Stereotactic body radiotherapy for recurrent and oligometastatic soft tissue sarcoma.

BACKGROUND: Soft tissue sarcoma (STS) is a malignant tumor of highly heterogeneous mesenchymal origin. STS has a biological pattern and clinical transformation with localized invasive growth and is susceptible to hematogenous metastasis. Local therapeutic strategies may treat recurrent and oligometastatic STS, including surgery and radiation therapy. This study aimed to evaluate the safety and efficacy of stereotactic body radiotherapy (SBRT) for recurrent and oligometastatic STS. METHODS: We retrospectively analyzed 37 recurrent and oligometastatic STS patients with 58 lesions treated with SBRT from 2009 to 2019 at our institution. Oligometastatic is defined as metastatic lesions less than or equal to 3. The primary endpoint was local control (LC); secondary endpoints were survival and toxicity. RESULTS: The median follow-up was 21.0 months (3.0 to 125.0 months). Among 37 patients, 18 were recurrent patients, and 19 were oligometastatic patients. Median LC was 25.0 months (95% CI 20.0-45.0). The 1-, 2-, and 3-year LC rates were 80.2%, 58.3%, and 46.6%, respectively. Median overall survival (OS) was 24.0 months (95% CI 13.0-28.0), and the survival rates after SBRT were 71.5%, 40.0%, and 29.1% at 1, 2, and 3-year, respectively. Median progression-free survival (PFS) was 10.0 months (95% CI 8.0-15.0 months), PFS rate after SBRT was 43.6%, 26.8%, and 18.4% at 1, 2, and 3 years, respectively. Late grade 3 radiation dermatitis was observed in one patient (2.7%). Using univariate and multivariate COX analysis, better OS, PFS, and LC were obtained in the histologic grade 1(G1) group, and tumor size and a number of lesions influenced LC. CONCLUSIONS: SBRT is a safe and effective treatment for patients with recurrent and oligometastatic STS. Histological grade influences local control and survival. SBRT may be a promising treatment option for recurrent and oligometastatic STS.

Recent advances in applying G-quadruplex for SARS-CoV-2 targeting and diagnosis: A review.

The coronavirus SARS-CoV-2 has caused a health care crisis all over the world since the end of 2019. Although vaccines and neutralizing antibodies have been developed, rapidly emerging variants usually display stronger immune escape ability and can better surpass vaccine protection. Therefore, it is still vital to find proper treatment strategies. To date, antiviral drugs against SARS-CoV-2 have mainly focused on proteases or polymerases. Notably, noncanonical nucleic acid structures called G-quadruplexes (G4s) have been identified in many viruses in recent years, and numerous G4 ligands have been developed. During this pandemic, literature on SARS-CoV-2 G4s is rapidly accumulating. Here, we first summarize the recent progress in the identification of SARS-CoV-2 G4s and their intervention by ligands. We then introduce the potential interacting proteins of SARS-CoV-2 G4s from both the virus and the host that may regulate G4 functions. The innovative strategy to use G4s as a diagnostic tool in SARS-CoV-2 detection is also reviewed. Finally, we discuss some key questions to be addressed in the future.

Bloom Syndrome Helicase Compresses Single-Stranded DNA into Phase-Separated Condensates.

Bloom syndrome protein (BLM) is a conserved RecQ family helicase involved in the maintenance of genome stability. BLM has been widely recognized as a genome "caretaker" that processes structured DNA. In contrast, our knowledge of how BLM behaves on single-stranded (ss) DNA is still limited. Here, we demonstrate that BLM possesses the intrinsic ability for phase separation and can co-phase separate with ssDNA to form dynamically arrested protein/ssDNA co-condensates. The introduction of ATP potentiates the capability of BLM to condense on ssDNA, which further promotes the compression of ssDNA against a resistive force of up to 60 piconewtons. Moreover, BLM is also capable of condensing replication protein A (RPA)- or RAD51-coated ssDNA, before which it generates naked ssDNA by dismantling these ssDNA-binding proteins. Overall, our findings identify an unexpected characteristic of a DNA helicase and provide a new angle of protein/ssDNA co-condensation for understanding the genomic instability caused by BLM overexpression under diseased conditions.

Targeting the RNA G-Quadruplex and Protein Interactome for Antiviral Therapy.

In recent years, G-quadruplexes (G4s), types of noncanonical four-stranded nucleic acid structures, have been identified in many viruses that threaten human health, such as HIV and Epstein-Barr virus. In this context, G4 ligands were designed to target the G4 structures, among which some have shown promising antiviral effects. In this Perspective, we first summarize the diversified roles of RNA G4s in different viruses. Next, we introduce small-molecule ligands developed as G4 modulators and highlight their applications in antiviral studies. In addition to G4s, we comprehensively review the medical intervention of G4-interacting proteins from both the virus (N protein, viral-encoded helicases, severe acute respiratory syndrome-unique domain, and Epstein-Barr nuclear antigen 1) and the host (heterogeneous nuclear ribonucleoproteins, RNA helicases, zinc-finger cellular nucelic acid-binding protein, and nucleolin) by inhibitors as an alternative way to disturb the normal functions of G4s. Finally, we discuss the challenges and opportunities in G4-based antiviral therapy.

The convergence of head-on DNA unwinding forks induces helicase oligomerization and activity transition.

Helicases are multifunctional motor proteins with the primary task of separating nucleic acid duplexes. These enzymes often exist in distinct oligomeric forms and play essential roles during nucleic acid metabolism. Whether there is a correlation between their oligomeric state and cellular function, and how helicases effectively perform functional switching remains enigmatic. Here, we address these questions using a combined single-molecule approach and Bloom syndrome helicase (BLM). By examining the head-on collision of two BLM-mediated DNA unwinding forks, we find that two groups of BLM, upon fork convergence, promptly oligomerize across the fork junctions and tightly bridge two independent single-stranded (ss) DNA molecules that were newly generated by the unwinding BLMs. This protein oligomerization is mediated by the helicase and RNase D C-terminal (HRDC) domain of BLM and can sustain a disruptive force of up to 300 pN. Strikingly, onsite BLM oligomerization gives rise to an immediate transition of their helicase activities, from unwinding dsDNA to translocating along ssDNA at exceedingly fast rates, thus allowing for the efficient displacement of ssDNA-binding proteins, such as RPA and RAD51. These findings uncover an activity transition pathway for helicases and help to explain how BLM plays both pro- and anti-recombination roles in the maintenance of genome stability.

Structural mechanism underpinning Thermus oshimai Pif1-mediated G-quadruplex unfolding.

G-quadruplexes (G4s) are unusual stable DNA structures that cause genomic instability. To overcome the potential barriers formed by G4s, cells have evolved different families of proteins that unfold G4s. Pif1 is a DNA helicase from superfamily 1 (SF1) conserved from bacteria to humans with high G4-unwinding activity. Here, we present the first X-ray crystal structure of the Thermus oshimai Pif1 (ToPif1) complexed with a G4. Our structure reveals that ToPif1 recognizes the entire native G4 via a cluster of amino acids at domains 1B/2B which constitute a G4-Recognizing Surface (GRS). The overall structure of the G4 maintains its three-layered propeller-type G4 topology, without significant reorganization of G-tetrads upon protein binding. The three G-tetrads in G4 are recognized by GRS residues mainly through electrostatic, ionic interactions, and hydrogen bonds formed between the GRS residues and the ribose-phosphate backbone. Compared with previously solved structures of SF2 helicases in complex with G4, our structure reveals how helicases from distinct superfamilies adopt different strategies for recognizing and unfolding G4s.

Neoadjuvant Programmed Cell Death Protein 1 Blockade Combined With Stereotactic Body Radiation Therapy for Stage III(N2) Non-Small Cell Lung Cancer: A Case Series.

The addition of radiotherapy in neoadjuvant chemotherapy did not improve event-free or overall survival in resectable non-small cell lung carcinoma (NSCLC). Neoadjuvant immunotherapy produced major pathologic response(MPR) rate of up to 45%. The potential synergy between radiotherapy and immunotherapy has been described in several studies. We reported outcomes of three cases of stage III/N2 NSCLC treated with neoadjuvant immunotherapy and stereotactic body radiation therapy (SBRT) in a single center. This explanatory trial included treatment-naive patients with stage III resectable NSCLC who received two doses of the programmed cell death protein 1 (PD-1) inhibitor toripalimab after 1 week of receiving SBRT for lung lesions. Thereafter, surgery was planned 4-6 weeks after the second dose. The primary endpoints were safety and feasibility, while the secondary endpoint was the pathologic response rate. Toripalimab combined with SBRT as a neoadjuvant treatment had well-tolerable side effects and did not lead to a delay in surgery. Among the included patients, one achieved pathologic complete response (PCR), one achieved MPR, and one with 20% residual tumor did not achieve MPR. There were few side effects of toripalimab combined with SBRT as a neoadjuvant treatment, and the treatment did not cause a delay in surgery. This study preliminarily explored the outcomes of a new neoadjuvant treatment.