Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT.

CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11's nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHATNTD). Our work demonstrates that DiTPR-CHATNTD can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system.

Progressive insights into fibrosarcoma diagnosis and treatment: leveraging fusion genes for advancements.

Fibrosarcoma, originating from fibroblast cells, represents a malignant neoplasm that can manifest across all genders and age groups. Fusion genes are notably prevalent within the landscape of human cancers, particularly within the subtypes of fibrosarcoma, where they exert substantial driving forces in tumorigenesis. Many fusion genes underlie the pathogenic mechanisms triggering the onset of this disease. Moreover, a close association emerges between the spectrum of fusion gene types and the phenotypic expression of fibrosarcoma, endowing fusion genes not only as promising diagnostic indicators for fibrosarcoma but also as pivotal foundations for its subcategorization. Concurrently, an increasing number of chimeric proteins encoded by fusion genes have been substantiated as specific targets for treating fibrosarcoma, consequently significantly enhancing patient prognoses. This review comprehensively delineates the mechanisms behind fusion gene formation in fibrosarcoma, the lineage of fusion genes, methodologies employed in detecting fusion genes within fibrosarcoma, and the prospects of targeted therapeutic interventions driven by fusion genes within the fibrosarcoma domain.

Cryo-EM structures of human GPR34 enable the identification of selective antagonists.

GPR34 is a functional G-protein-coupled receptor of Lysophosphatidylserine (LysoPS), and has pathogenic roles in numerous diseases, yet remains poorly targeted. We herein report a cryo-electron microscopy (cryo-EM) structure of GPR34 bound with LysoPS (18:1) and Gi protein, revealing a unique ligand recognition mode with the negatively charged head group of LysoPS occupying a polar cavity formed by TM3, 6 and 7, and the hydrophobic tail of LysoPS residing in a lateral open hydrophobic groove formed by TM3-5. Virtual screening and subsequent structural optimization led to the identification of a highly potent and selective antagonist (YL-365). Design of fusion proteins allowed successful determination of the challenging cryo-EM structure of the inactive GPR34 complexed with YL-365, which revealed the competitive binding of YL-365 in a portion of the orthosteric binding pocket of GPR34 and the antagonist-binding-induced allostery in the receptor, implicating the inhibition mechanism of YL-365. Moreover, YL-365 displayed excellent activity in a neuropathic pain model without obvious toxicity. Collectively, this study offers mechanistic insights into the endogenous agonist recognition and antagonist inhibition of GPR34, and provides proof of concept that targeting GPR34 represents a promising strategy for disease treatment.

Advances of Osteosarcoma Models for Drug Discovery and Precision Medicine.

The management of osteosarcoma (OS) patients presents a significant clinical challenge. Despite progress in conventional and targeted therapies, the survival rate of OS patients remains limited largely due to therapy resistance and the high metastatic potential of the disease. OS models that accurately reflect the fundamental characteristics are vital to the innovation and validation of effective therapies. This review provides an insight into the advances and challenges in OS drug development, focusing on various preclinical models, including cell lines, 3D culture models, murine models, and canine models. The relevance, strengths, and limitations of each model in OS research are explored. In particular, we highlight a range of potential therapeutics identified through these models. These instances of successful drug development represent promising pathways for personalized OS treatment.

Structural basis of BAM-mediated outer membrane ß-barrel protein assembly.

The outer membrane structure is common in Gram-negative bacteria, mitochondria and chloroplasts, and contains outer membrane ß-barrel proteins (OMPs) that are essential interchange portals of materials1-3. All known OMPs share the antiparallel ß-strand topology4, implicating a common evolutionary origin and conserved folding mechanism. Models have been proposed for bacterial ß-barrel assembly machinery (BAM) to initiate OMP folding5,6; however, mechanisms by which BAM proceeds to complete OMP assembly remain unclear. Here we report intermediate structures of BAM assembling an OMP substrate, EspP, demonstrating sequential conformational dynamics of BAM during the late stages of OMP assembly, which is further supported by molecular dynamics simulations. Mutagenic in vitro and in vivo assembly assays reveal functional residues of BamA and EspP for barrel hybridization, closure and release. Our work provides novel insights into the common mechanism of OMP assembly.

Identification and validation of a CCL18-related signature for prediction of overall survival in patients with uveal melanoma.

Uveal melanoma (UM), the most frequent primary intraocular tumor in adults, has poor prognosis. High C-C motif chemokine ligand 18 (CCL18) has been detected in various tumors and is closely correlated with patients' clinicopathological characteristics. However, the essential role of CCL18 in UM remains unclear. Therefore, this study aimed to explore the prognostic value of CCL18 in UM. Uveal melanoma cells (M17) were transfected with pcDNA3.1-CCL18 si-RNA using Lipofectamine™ 2000. Cell growth and invasion abilities were measured through Cell Counting Kit-8 assay and invasion assay. RNA expression data and clinical and histopathological details were downloaded from the UM in The Cancer Genome Atlas (TCGA-UM) and GSE22138 datasets, which were defined as the training and validation cohorts, respectively. Univariate and multivariate Cox regression analyses were performed to identify significant prognostic biomarkers. The coefficients of these significant biomarkers generated by multivariate Cox proportional hazard regression analysis were used to establish a risk score formula. Functional enrichment analyses were also carried out. We found that downregulated CCL18 inhibits M17 cell growth and invasion in vitro. CCL18 may affect UM progression by altering C-C motif receptor 8 related pathways. Higher CCL18 expression was associated with worse clinical outcomes and tumor-specific death in the TCGA-UM dataset. Based on the coefficients obtained from the Cox proportional hazard regression analysis, a CCL18-related prognostic signature formula was constructed as follows: risk score = 0.05590 × age +2.43437 × chromosome 3 status +0.39496 × ExpressionCCL18. Notably, in this formula, the normal chromosome 3 was coded as 0, whereas the chromosome 3 loss was coded as 1. Each patient was assigned to either low-risk or high-risk groups using the median cut-off in the training cohort. High-risk patients survived for a shorter time than low-risk patients. The time-dependent and multivariate receiver operating characteristic curves showed promising diagnostic efficacy. Multivariate Cox regression analysis demonstrated the potential of this CCL18-related signature as an independent prognostic indicator. These results were validated using the GSE22138 dataset. In addition, in both TCGA-UM and GSE22138 datasets, stratification of clinical correlations and survival analyses based on this signature indicated the involvement of clinical progression and survival outcome in UM. In the high-risk group, Gene Ontology analyses mainly indicated the enrichment of immune response pathways, such as the T cell activation, response to interferon-gamma, antigen processing and presentation, interferon-gamma-mediated signaling pathway, MHC protein complex, MHC class II protein complex, antigen binding, and cytokine binding. Meanwhile, Kyoto Encyclopedia of Genes and Genomes analyses showed enrichments of pathways in cancer, cell adhesion, cytokine-cytokine receptor interaction, chemokine signaling pathway, Th1 and Th2 cell differentiation, and chemokine signaling pathway. Moreover, single-sample gene set enrichment analysis demonstrated the enrichment of almost all immune cells and immune functions in the high-risk group. In summary, a new prognostic CCL18-related signature was successfully established using the TCGA-UM dataset and validated using the GSE22138 dataset with meaningful predictive and diagnostic efficacies. This signature could serve as an independent and promising prognostic biomarker for patients with UM.

Complete Genome Sequence of Bacillus velezensis TH-1, a Candidate Biocontrol Bacterium from China.

Bacillus velezensis TH-1 is a plant growth-promoting rhizobacteria with biocontrol potential that was isolated from the rhizosphere of Sophora tonkinensis Radix. Our previous results showed that strain TH-1 demonstrated effective biocontrol activity against root rot of Sophora tonkinensis Radix and bacterial wilt of ginger. Currently, only a few whole-genome sequences of biocontrol strains isolated from the rhizosphere of medicinal plants are available. We report, here, the complete genome sequence of B. velezensis TH-1. The size of TH-1 genome is 3,929,846 bp that consists of 3,900 genes with a total GC content of 46.48%. The strain TH-1 genome has 3,661 coding genes, 86 transfer RNAs, 27 ribosomal RNAs, and 16 small RNAs. Moreover, we identified nine gene clusters coding for the biosynthesis of antimicrobial compounds. The genomic information of TH-1 will provide resources for the study of biological control mechanisms and plant-microbe interactions. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Clinical analysis of bronchoscope diagnosis and treatment for airway foreign body removal in pediatric patients.

BACKGROUND: Along with the wide spread application and technical development of the flexible and rigid bronchoscopy, the airway foreign body removing method cme to the specific technique for different foreign bodies from the single foreign body forceps. METHODS: Selected 633 children who were diagnosed as airway foreign bodies by the Department of Respiratory Intervention, Children's Hospital affiliated to Shandong University from January 1st, 2018 to December 31st, 2021, and the airway foreign bodies were diagnosed using bronchoscopy. After comprehensive assessment of the foreign body nature in the airway, the foreign bodies were removed by freezing, laser, electrocoagulation, balloon and other techniques, the success rate of the foreign body removed from the airway was observed, the percentage of the foreign body removed using different techniques, the operation time, and the incidence of post-adverse reactions during operation. RESULTS: The success rate using flexible bronchoscope alone to remove foreign bodies in the airway was 99.2%. After flexible bronchoscopy, 19 cases of foreign bodies were removed by vacuum suction alone, 513 cases were removed by foreign body forceps alone, 62 cases were combined with cryotherapy, 2 cases were electrocoagulation, 6 cases were mesh baskets, 3 cases were balloons, 5 cases were laser, and various 18 cases of foreign bodies were invloved by technical combination. 5 cases of flexible bronchoscope combined with rigid bronchoscope combined to remove foreign bodies. The operation time was from 5 min to 1 h, with an average of 20 min. There were 17 cases of hypoxemia (2.7%) during operation, 36 cases (5.7%) of bleeding caused by airway mucosa injury after treatment, and 70 cases (11.2%) of laryngeal edema. The total incidence of adverse reactions was 19.6%, there were no deaths due to foreign bodies and treatment. CONCLUSIONS: According to different properties of airway foreign bodies, it is safe and effective to select appropriate techniques to remove foreign bodies using the flexible bronchoscope, which can increase the removal rate of airway foreign bodies and reduce the occurrence of serious complications.

Enhanced chemo-photodynamic therapy of an enzyme-responsive prodrug in bladder cancer patient-derived xenograft models.

Patient-derived xenograft (PDX) models are powerful tools for understanding cancer biology and drug discovery. In this study, a polymeric nano-sized drug delivery system poly (OEGMA)-PTX@Ce6 (NPs@Ce6) composed of a photosensitizer chlorin e6 (Ce6) and a cathepsin B-sensitive polymer-paclitaxel (PTX) prodrug was constructed. The photochemical internalization (PCI) effect and enhanced chemo-photodynamic therapy (PDT) were achieved via a two-stage light irradiation strategy. The results showed that the NPs@Ce6 had great tumor targeting and rapid cellular uptake induced by PCI, thereby producing excellent anti-tumor effects on human bladder cancer PDX models with tumor growth inhibition greater than 98%. Bioinformatics analysis revealed that the combination of PTX chemotherapy and PDT up-regulated oxidative phosphorylation and reactive oxygen species (ROS) generation, blocked cell cycle and proliferation, and down-regulated the pathways related to tumor progression, invasion and metastasis, including hypoxia, TGF-ß signaling and TNF-α signaling pathways. Western blots analysis confirmed that proteins promoting apoptosis (Bax, Cleaved caspase-3, Cleaved PARP) and DNA damage (γH2A.X) were up-regulated, while those inhibiting apoptosis (Bcl-2) and mitosis (pan-actin and α/ß-tubulin) were down-regulated after chemo-PDT treatment. Therefore, this stimuli-responsive polymer-PTX prodrug-based nanomedicine with combinational chemotherapy and PDT evaluated in the PDX models could be a potential candidate for bladder cancer therapy.

Matrix-Bound Growth Factors are Released upon Cartilage Compression by an Aggrecan-Dependent Sodium Flux that is Lost in Osteoarthritis.

Articular cartilage is a dense extracellular matrix-rich tissue that degrades following chronic mechanical stress, resulting in osteoarthritis (OA). The tissue has low intrinsic repair especially in aged and osteoarthritic joints. Here, we describe three pro-regenerative factors; fibroblast growth factor 2 (FGF2), connective tissue growth factor, bound to transforming growth factor-beta (CTGF-TGFß), and hepatoma-derived growth factor (HDGF), that are rapidly released from the pericellular matrix (PCM) of articular cartilage upon mechanical injury. All three growth factors bound heparan sulfate, and were displaced by exogenous NaCl. We hypothesised that sodium, sequestered within the aggrecan-rich matrix, was freed by injurious compression, thereby enhancing the bioavailability of pericellular growth factors. Indeed, growth factor release was abrogated when cartilage aggrecan was depleted by IL-1 treatment, and in severely damaged human osteoarthritic cartilage. A flux in free matrix sodium upon mechanical compression of cartilage was visualised by 23Na -MRI just below the articular surface. This corresponded to a region of reduced tissue stiffness, measured by scanning acoustic microscopy and second harmonic generation microscopy, and where Smad2/3 was phosphorylated upon cyclic compression. Our results describe a novel intrinsic repair mechanism, controlled by matrix stiffness and mediated by the free sodium concentration, in which heparan sulfate-bound growth factors are released from cartilage upon injurious load. They identify aggrecan as a depot for sequestered sodium, explaining why osteoarthritic tissue loses its ability to repair. Treatments that restore matrix sodium to allow appropriate release of growth factors upon load are predicted to enable intrinsic cartilage repair in OA. SIGNIFICANCE STATEMENT: Osteoarthritis is the most prevalent musculoskeletal disease, affecting 250 million people worldwide.1 We identify a novel intrinsic repair response in cartilage, mediated by aggrecan-dependent sodium flux, and dependent upon matrix stiffness, which results in the release of a cocktail of pro-regenerative growth factors after injury. Loss of aggrecan in late-stage osteoarthritis prevents growth factor release and likely contributes to disease progression. Treatments that restore matrix sodium in osteoarthritis may recover the intrinsic repair response to improve disease outcome.

Structural basis for bacterial lipoprotein relocation by the transporter LolCDE.

Lipoproteins in the outer membrane of Gram-negative bacteria are involved in various vital physiological activities, including multidrug resistance. Synthesized in the cytoplasm and matured in the inner membrane, lipoproteins must be transported to the outer membrane through the Lol pathway mediated by the ATP-binding cassette transporter LolCDE in the inner membrane via an unknown mechanism. Here, we report cryo-EM structures of Escherichia coli LolCDE in apo, lipoprotein-bound, LolA-bound, ADP-bound and AMP-PNP-bound states at a resolution of 3.2-3.8 Å, covering the complete lipoprotein transport cycle. Mutagenesis and in vivo viability assays verify features of the structures and reveal functional residues and structural characteristics of LolCDE. The results provide insights into the mechanisms of sorting and transport of outer-membrane lipoproteins and may guide the development of novel therapies against multidrug-resistant Gram-negative bacteria.

Structural insights into outer membrane asymmetry maintenance in Gram-negative bacteria by MlaFEDB.

The highly asymmetric outer membrane of Gram-negative bacteria functions in the defense against cytotoxic substances, such as antibiotics. The Mla pathway maintains outer membrane lipid asymmetry by transporting phospholipids between the inner and outer membranes. It comprises six Mla proteins, MlaFEDBCA, including the ABC transporter MlaFEDB, which functions via an unknown mechanism. Here we determine cryo-EM structures of Escherichia coli MlaFEDB in an apo state and bound to phospholipid, ADP or AMP-PNP to a resolution of 3.3-4.1 Å and establish a proteoliposome-based transport system that includes MlaFEDB, MlaC and MlaA-OmpF to monitor the transport direction of phospholipids. In vitro transport assays and in vivo membrane permeability assays combined with mutagenesis identify functional residues that not only recognize and transport phospholipids but also regulate the activity and structural stability of the MlaFEDB complex. Our results provide mechanistic insights into the Mla pathway, which could aid antimicrobial drug development.

Bibliometric analysis of the 100 most cited articles on cervical cancer radiotherapy.

PURPOSE: To identify the 100 most cited research articles on cervical cancer radiotherapy. METHODS: The Web of Science and Scopus databases were searched to identify the 100 most cited articles on cervical cancer radiotherapy as of September 29, 2019. Articles were ranked based on the total citations received from 2 databases. One hundred articles about radiotherapy for cervical cancer were identified. The following important information was extracted: author, journal, year and month of publication, country or region, and radiotherapy technologies. RESULTS: The 100 most cited articles on cervical cancer radiotherapy were published between 1964 and 2016, and the total citations from 2 databases ranged from 3478 to 211, including a total of 49,262 citations as of September 29, 2019. The index of citations per year ranged from 170.4 to 13.1. These articles were from 16 countries or regions, with most publications being from the United States (n = 38), followed by Austria (n = 15), Canada (n = 8), France (n = 8) and the United Kingdom (n = 7). The International Journal of Radiation Oncology, Biology, Physics produced the most articles (n = 42), followed by Radiotherapy and Oncology (n = 13), Cancer (n = 8) and Journal of Clinical Oncology (n = 7). These articles were categorized as original studies (n = 86), recommendations (n = 5), guidelines (n = 5) and reviews (n = 4). Of the 100 most cited articles, intracavitary brachytherapy (n = 50) and 3-dimensional conformal radiotherapy (n = 34) were the most commonly used treatment techniques. CONCLUSION: To the best of our knowledge, this is the first report and analysis of the most cited articles on cervical cancer radiotherapy. This bibliographic study presents the history of technological development in external radiation therapy and brachytherapy. Brachytherapy is an indispensable part of radiotherapy for cervical cancer. The International Journal of Radiation Oncology Biology Physics is the journal with the most publications related to cervical cancer radiotherapy.

The 100 most cited articles in prostate cancer brachytherapy: systematic review and bibliometric analysis.

PURPOSE: The aim of this study was to identify the 100 most cited research articles in prostate cancer brachytherapy (PCB) and to review the characteristics of these citation. MATERIAL AND METHODS: The Web of Science Core Collection was used to identify the 100 most cited articles in PCB as of December 31st, 2019. The following important information was extracted: year and month of publication, title, journal, country of origin, authors, type of article, treatment modality, and topics. RESULTS: The 100 most cited articles in PCB were published between 1999 and 2018, and the number of citations ranged from 455 to 54; these articles had collectively been cited 10,331 times at the time of search. These articles were from 11 countries, with most publications being from the United States (n = 61), followed by Canada (n = 10), the United Kingdom (n = 8), and Germany (n = 5). The "International Journal of Radiation Oncology, Biology, Physics" published the most articles (n = 47), followed by the "Journal of Urology" (n = 11), "Radiotherapy and Oncology" (n = 10), "Cancer" (n = 7), and "Urology" (n = 6). Permanent interstitial brachytherapy (n = 52) was the most widely used treatment modality, followed by temporary brachytherapy (n = 45). Disease control (n = 51) was the most common topic, followed by side effects (n = 44) and quality of life (n = 27). CONCLUSIONS: The bibliometric analysis presents a detailed list of the 100 most cited articles in prostate cancer brachytherapy. There are clear recommendations for treatment with prostate cancer brachytherapy. The goal of prostate cancer brachytherapy is to improve long-term outcomes and quality of life.

Dose-effect response in image-guided adaptive brachytherapy for cervical cancer: A systematic review and meta-regression analysis.

PURPOSE: This study aimed to integrate and update the dose-effect relationship between volumetric dose and local control for cervical cancer brachytherapy. METHODS AND MATERIALS: We identified studies that reported high-risk clinical target volume (HR-CTV) D90 and local control probability by searching PubMed, Web of Science, and the Cochrane Library databases through Oct 27, 2019. The regression analyses were performed using a probit model between HR-CTV D90, D100, intermediate-risk clinical target volume (IR-CTV) D90, and dose to Point A vs. local control probability. Subgroup analyses were performed according to stratification: time of local control, income level of the country or region, stage of cancer, pathology, mean volume of HR-CTV, dose rate, image modality, concurrent chemoradiotherapy proportion, interstitial proportion, and mean overall treatment time. RESULTS: Thirty-three studies encompassing 2893 patients were included. The probit model showed a significant relationship between the HR-CTV D90 value and the local control probability, p < 0.0001. The D90 corresponding to a probability of 90% local control was 83.7 GyEQD2,10 (80.6-87.8 GyEQD2,10). Of the 33 studies included in our analysis, eight studies, including 1172 patients, reported the IR-CTV D90 value, ranging from 59.1 GyEQD2,10 to 72.3 GyEQD2,10. The probit model also showed a significant relationship between the IR-CTV D90 value and the local control probability, p = 0.0464. The 60 GyEQD2,10 for IR-CTV D90 corresponded to an 86.1% local control probability (82.0%-89.8%). CONCLUSIONS: A significant dependence of local control on HR-CTV D90 and IR-CTV D90 was found. A tumor control probability of >90% can be expected at doses >84 GyEQD2,10 and 69 GyEQD2,10, respectively, based on an updated meta-regression analysis.

The 100 most cited articles in cervical cancer brachytherapy.

PURPOSE: This study aimed to identify the 100 most cited research articles on cervical cancer brachytherapy. METHODS AND MATERIALS: The Institute for Scientific Information Web of Science was used to identify the 100 most cited articles in cervical cancer brachytherapy as of July 5, 2019. The following important information was extracted: journal, year and month, country of region, author, type of article, type of dose rate, type of radionuclide, and image modality for brachytherapy planning. RESULTS: The 100 most cited articles in cervical cancer brachytherapy were published between 1981 and 2016, and the citations ranged from 858 to 49, which collectively had been cited 11,372 times at the time of searching. The index of citations per year ranged from 63.56 to 1.43. These articles were from 16 countries or regions, with most publications being from the United States (n = 27), followed by Austria (n = 26), Japan (n = 10), France (n = 7), and the Netherlands (n = 7). The International Journal of Radiation Oncology, Biology, Physics produced the most articles (n = 46), followed by Radiotherapy and Oncology (n = 39) and Gynecologic Oncology (n = 5). These articles were categorized as original studies (n = 75), reviews (n = 2), editorials (n = 2), surveys (n = 2), guidelines (n = 3), and recommendations (n = 6). A high dose rate (n = 69) was the most widely used, dose rate followed by a low dose rate (n = 20) and pulsed dose rate (n = 16). CONCLUSIONS: The bibliometric analysis presents a detailed list of the 100 most cited articles in cervical cancer brachytherapy. This analysis provides an insight into historical developments and enables the important advances in this field to be recognized.

The design of an individualized cylindrical vaginal applicator with oblique guide holes using 3D modeling and printing technologies.

High-dose-rate brachytherapy for cervical cancer after subtotal hysterectomy using standardized applicators cannot achieve a good absorbed-dose coverage of the target volume in special tumor morphologies and topographies due to the steep dose gradient. The aim of this pictorial essay is to present an individualized cylindrical vaginal applicator with oblique guide holes using 3D modeling and printing technologies used at the China-Japan Union Hospital of Jilin University for cervical cancer patients. We use images to describe the steps of this method.

Cryo-EM structures of lipopolysaccharide transporter LptB2FGC in lipopolysaccharide or AMP-PNP-bound states reveal its transport mechanism.

Lipopolysaccharides (LPS) of Gram-negative bacteria are critical for the defence against cytotoxic substances and must be transported from the inner membrane (IM) to the outer membrane (OM) through a bridge formed by seven membrane proteins (LptBFGCADE). The IM component LptB2FG powers the process through a yet unclarified mechanism. Here we report three high-resolution cryo-EM structures of LptB2FG alone and complexed with LptC (LptB2FGC), trapped in either the LPS- or AMP-PNP-bound state. The structures reveal conformational changes between these states and substrate binding with or without LptC. We identify two functional transmembrane arginine-containing loops interacting with the bound AMP-PNP and elucidate allosteric communications between the domains. AMP-PNP binding induces an inward rotation and shift of the transmembrane helices of LptFG and LptC to tighten the cavity, with the closure of two lateral gates, to eventually expel LPS into the bridge. Functional assays reveal the functionality of the LptF and LptG periplasmic domains. Our findings shed light on the LPS transport mechanism.

Colorimetric Method for Sensitive Detection of Microcystin-LR Using Surface Copper Nanoparticles of Polydopamine Nanosphere as Turn-On Probe.

A novel, facile sensor was further developed for microcystin-LR (MC-LR) determination by visible spectroscopy. Antibody-functionalized SiO2-coated magnetic nanoparticles (Fe3O4@SiO2) and aptamer-functionalized polydopamine nanospheres decorated with Cu nanoparticles (PDA/CuNPs) recognized specific sites in MC-LR and then the sandwich-type composites were separated magnetically. The Cu in the separated composites was converted to Cu2+ ions in solution and turn-on visible absorption was achieved after reaction with bis(cyclohexanone)oxaldihydrazone (BCO) (λmax = 600 nm). There was a quantitative relationship between the spectral intensity and MC-LR concentration. In addition, under the optimum conditions, the sensor turns out to be a linear relationship from 0.05 to 25 nM, with a limit of detection of 0.05 nM (0.05 µg/L) (S/N = 3) for MC-LR. The sensitivity was dependent on the low background absorption from the off-to-on spectrum and label amplification by the polydopamine (PDA) surface. The sensor had high selectivity, which shows the importance of dual-site recognition by the aptamer and antibody and the highly specific color formed by BCO with Cu2+. The bioassay was complete within 150 min, which enabled quick determination. The sensor was successfully used with real spiked samples. These results suggest it has potential applications in visible detection and could be used to detect other microcystin analogs.