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Common challenges in cryogenic electron microscopy, such as orientation bias, conformational diversity, and 3D misclassification, complicate single particle analysis and lead to significant resource expenditure. We previously introduced an in silico method using the maximum Feret diameter distribution, the Feret signature, to characterize sample heterogeneity of disc-shaped samples. Here, we expanded the Feret signature methodology to identify preferred orientations of samples containing arbitrary shapes with only about 1000 particles required. This method enables real-time adjustments of data acquisition parameters for optimizing data collection strategies or aiding in decisions to discontinue ineffective imaging sessions. Beyond detecting preferred orientations, the Feret signature approach can serve as an early-warning system for inconsistencies in classification during initial image processing steps, a capability that allows for strategic adjustments in data processing. These features establish the Feret signature as a valuable auxiliary tool in the context of single particle analysis, significantly accelerating the structure determination process.
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Microscopía por Crioelectrón , Procesamiento de Imagen Asistido por Computador , Flujo de Trabajo , Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Algoritmos , Imagenología Tridimensional/métodosRESUMEN
Actin filament assembly and the regulation of its mechanical properties are fundamental processes essential for eukaryotic cell function. Residue E167 in vertebrate actins forms an inter-subunit salt bridge with residue K61 of the adjacent subunit. Saccharomyces cerevisiae actin filaments are more flexible than vertebrate filaments and have an alanine at this position (A167). Substitution of this alanine for a glutamic acid (A167E) confers Saccharomyces cerevisiae actin filaments with salt-dependent stiffness similar to vertebrate actins. We developed an optimized cryogenic electron microscopy workflow refining sample preparation and vitrification to obtain near-atomic resolution structures of wild-type and A167E mutant Saccharomyces cerevisiae actin filaments. The difference between these structures allowed us to pinpoint the potential binding site of a filament-associated cation that controls the stiffness of the filaments in vertebrate and A167E Saccharomyces cerevisiae actins. Through an analysis of previously published high-resolution reconstructions of vertebrate actin filaments, along with a newly determined high-resolution vertebrate actin structure in the absence of potassium, we identified a unique peak near residue 167 consistent with the binding of a magnesium ion. Our findings show how magnesium can contribute to filament stiffening by directly bridging actin subunits and allosterically affecting the orientation of the DNase-I binding loop of actin, which plays a regulatory role in modulating actin filament stiffness and interactions with regulatory proteins.
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AIM: To report a one-year clinical outcomes of low-dose laser cycloplasty (LCP) among malignant glaucoma patients. METHODS: In this prospective, multicenter, non-comparative clinical study, participants with malignant glaucoma were recruited and underwent LCP at eight ophthalmic centers in China. Patients were followed up at 1wk, 1, 3, 6, and 12mo. Intraocular pressure (IOP), number of glaucoma medications, anterior chamber depth (ACD), and complications were recorded. Anatomical success was defined as the reformation of the anterior chamber based on slit-lamp biomicroscopy. Recurrence was defined by the presence of a shallow or ï¬at anterior chamber after initial recovery from treatment. RESULTS: A total of 34 eyes received LCP. Mean IOP and medications decreased from 36.1±11.5 mm Hg with 3.3±1.5 glaucoma medications pre-treatment to 20.9±9.8 mm Hg (P<0.001) with 2.9±1.6 medications (P=0.046) at 1d, and 17.4±6.7 mm Hg (P<0.001) with 1.3±1.7 medications (P<0.001) at 12mo. The ACD increased from 1.1±0.8 mm at baseline to 1.7±1.0 mm and to 2.0±0.5 mm at 1d and 12mo, respectively. A total of 32 (94.1%) eyes achieved initial anatomical success. During follow-up, 2 (5.9%) eyes failed and 8 (23.5%) eyes relapsed, yielding a 12-month anatomical success rate of 64.3%. Complications including anterior synechia (8.82%), choroidal/ciliary detachment (5.88%) and hypopyon (2.94%) were observed within 1wk. CONCLUSION: LCP is simple, safe, and effective in reforming the anterior chamber in malignant glaucoma.
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In this study, [1+2+2] cyclization of tryptamine-derived isocyanides with 3-ylideneoxindoles was systematically investigated. A series of structurally complex spiro-oxindole derivatives were obtained. Characteristic dynamic covalent chemistry was observed and confirmed by experiments and density functional theory calculation. Through the regulation of the solvent, temperature, and time, the precise and stereodivergent synthesis of spiro-oxindoles was achieved.
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Bromate is receiving increased attention as a typical disinfection by-product in aquatic environments, but bromate toxicity tests on invertebrate such as Brachionus calyciflorus rotifer are inadequate. In the present study, the long-term toxicity tests on B. calyciflorus were performed during 21 days under the exposure of different bromate concentrations and two algal density conditions. Furthermore, we evaluated the feeding behaviors of the rotifers under the impact of bromate. The maximum population density of rotifers was significantly reduced at 100 and 200 mg/L bromate exposure at the two algal density conditions. However, we observed that the maximum population density and population growth rate of rotifers were higher at 3.0 × 106 cells/mL algal density than those at 1.0 × 106 cells/mL under the same conditions of bromate exposure. These results suggest that higher food density may have alleviated the negative effects of bromate on rotifers. Meanwhile, the ingestion rate at an algal density of 3.0 × 106 cells/mL was higher than that at 1.0 × 106 cells/mL. The present study provides a basic reference to comprehensively evaluate the toxic effects of bromate on aquatic organisms.
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Rotíferos , Contaminantes Químicos del Agua , Animales , Bromatos/toxicidad , Alimentos , Ingestión de Alimentos , Crecimiento Demográfico , Contaminantes Químicos del Agua/toxicidadRESUMEN
Embryogenic cultures of longan (Dimocarpus longan Lour.) contain various metabolites with pharmacological properties that may function in the regulation of somatic embryogenesis (SE). In this study, based on widely targeted metabolomics, 501 metabolites were obtained from the embryogenic calli, incomplete compact proembryogenic cultures, and globular embryos during early SE of longan, among which 41 flavonoids were differentially accumulated during the SE. Using RNA sequencing, 36 flavonoid-biosynthesis-related genes and 43 MYB and 52 bHLH transcription factors were identified as differentially expressed genes. Furthermore, Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the flavonoid metabolism-related pathways were significantly enriched during the early SE. These results suggested that the changes in flavonoid levels in the embryogenic cultures of longan were mediated by MYBs and bHLHs via regulating flavonoid-biosynthesis-related genes, thus potentially regulating early SE. The identified metabolites in the embryogenic cultures of longan can be used to develop pharmaceutical ingredients.
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Sapindaceae , Transcriptoma , Flavonoides/metabolismo , Perfilación de la Expresión Génica , Sapindaceae/genética , Sapindaceae/metabolismo , Desarrollo Embrionario , Regulación de la Expresión Génica de las PlantasRESUMEN
A cobalt(II)-catalyzed coupling-cyclization cascade reaction between tryptamine-derived isocyanides and iodonium ylides is investigated, which allowed for the synthesis of different types of spiroindoline compounds by variation of substituents at the N1- and C2-positions in the indole skeleton. More interesting is that the spiroindoline products could undergo despirocyclization in the presence of amines, enabling efficient construction of enamine compounds.
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Background: Deep vein thrombosis (DVT) in lower extremities as a common complication of acute ischemic stroke (AIS) has long been studied. However, as the therapeutic options for AIS continue to advance, the pathogenic mechanisms behind DVT may change. Endovascular thrombectomy (EVT) has replaced intravenous thrombolysis and become the preferred treatment for AIS patients with large vessel occlusions. Therefore, it is important to update our understanding of DVT and its management. This study aimed to determine the prevalence and risk factors of DVT in AIS patients following EVT. Methods: In this retrospective study, 245 AIS patients who had received EVT were recruited between January 2020 and December 2021. Within 10 days (median 4 days) of thrombectomy, DVT was diagnosed by ultrasonography. Demographic characteristics, clinical findings, and therapeutic procedures were compared between patients with and without DVT using univariate analysis. Cutoff points were defined for EVT time and plasma D-dimer concentration. Multivariable logistic regression was then used to determine the independent risk factors for DVT and evaluate their predictive power. Results: The prevalence of DVT in AIS patients after EVT was 27.3%. Multifactorial logistic regression analysis showed that age (OR 1.036, 95% CI 1.001-1.073; P = 0.045), female sex (OR 3.015, 95% CI 1.446-6.289; P = 0.003), lower limb muscle strength less than grade three (OR 7.015, 95% CI 1.887-26.080; P = 0.004), longer EVT time (OR 1.012, 95% CI 1.004-1.020; P = 0.003), and higher D-dimer levels (OR 1.350, 95% CI 1.150-1.585; P < 0.001) were independently associated with higher DVT risk in AIS patients following EVT. The cutoff points for operative time of EVT and plasma D-dimer were 65.5 min and 1.62 mg/L, respectively, above which the risk for DVT was dramatically increased with OR > 4 in AIS patients. Conclusion: AIS patients are at increased risk of developing DVT following EVT particularly if they have undergone prolonged thrombectomy procedures and exhibit high plasma levels of D-dimers. However, the results of our study need to be validated by a multicenter prospective study with a larger population of stroke patients.
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Chronic and recurrent inflammatory disorders of the gastrointestinal tract caused by a complex interplay between genetics and intestinal dysbiosis are called inflammatory bowel disease. As a result of the interaction between the liver and the gut microbiota, bile acids are an atypical class of steroids produced in mammals and traditionally known for their function in food absorption. With the development of genomics and metabolomics, more and more data suggest that the pathophysiological mechanisms of inflammatory bowel disease are regulated by bile acids and their receptors. Bile acids operate as signalling molecules by activating a variety of bile acid receptors that impact intestinal flora, epithelial barrier function, and intestinal immunology. Inflammatory bowel disease can be treated in new ways by using these potential molecules. This paper mainly discusses the increasing function of bile acids and their receptors in inflammatory bowel disease and their prospective therapeutic applications. In addition, we explore bile acid metabolism and the interaction of bile acids and the gut microbiota.
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Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , Animales , Humanos , Ácidos y Sales Biliares , Intestinos , Hígado , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Disbiosis , MamíferosRESUMEN
Purpose: This study aimed to evaluate the efficacy and safety of predeposit autologous RBC apheresis (PARA) in patients undergoing multilevel spinal fusion surgery. Methods: A total of 112 patients from January 2020 to June 2022 were divided into two groups according to PARA: the PARA group (n = 51) and the control group (n = 61). The baseline characteristics of the patients, outcomes, transfusion cost, hospitalization cost, length of stay, complications, and changes in hemoglobin and hematocrit levels between the two groups were compared. Results: The baseline characteristics were similar in both groups. No significant differences were found in functional outcomes, including VAS score (p = 0.159), ODI score (p = 0.214), JOA score (p = 0.752), and SF-36 score (p = 0.188) between the PARA and control groups. The amount and rate of intraoperative and perioperative allogeneic RBC transfusion were significantly higher in the control group than in the PARA group (p < 0.001). The postoperative (9.04 ± 3.21 vs. 11.05 ± 3.84, p = 0.004) and total length of stay (15.78 ± 3.79 vs. 17.36 ± 4.08, p = 0.038) in the PARA group were significantly lower than those in the control group, respectively. Despite no difference in hospitalization cost (p = 0.737), the total blood transfusion cost in the PARA group was significantly lower, compared with the control group (p < 0.001). For safety evaluation, there were no significant differences in Hb and Hct levels between the two groups at admission, on postoperative day 1, and postoperative day 3, respectively (p > 0.05). Moreover, the number of postoperative infections in the PARA group was significantly lower than that in the control group (p = 0.038). Conclusion: PARA was a novel, safe, and highly efficient technique for mass autologous blood preparation in a quite short preparation time. This method could significantly reduce the amount of allogeneic blood transfusion and length of stay, which could provide a theoretical basis for following clinical practice about the technique.
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An NBS-induced intramolecular annulation of 3-(1H-indol-3-yl)-N-alkoxypropanamide is described. The reactions proceed well and quickly under mild conditions with the help of a base. It was found that C2-substituents on the indole ring in 3-(1H-indol-3-yl)-N-alkoxypropanamide have a great influence upon the reaction. By using C2-methyl- and C2-phenyl-3-(1H-indol-3-yl)-N-alkoxypropanamide as templates, practical protocols for the divergent synthesis of fused- and spirocyclic indoline compounds were studied and established.
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Azoxy compounds have aroused extensive attention due to their unique biological activities, but the chemical synthesis of these compounds often suffers from limitations due to their requirement for stoichiometric oxidants, high costs, and restricted substrate range. Herein, a series of azoxy compounds were constructed via facile coupling reactions by using cost-effective N-methoxyformamide and nitroso compounds over Cu-based catalysts, affording high product yields with excellent tolerance of functional groups. Significantly, the mesoporous silica nanosphere-encapsulated ultrasmall Cu (Cu@MSN) catalyst was developed via a one-pot synthetic method and first used for the synthesis of azoxy compounds. As compared with copper salt catalysts, the Cu@MSN catalyst exhibited remarkably enhanced catalytic activity and superior recycling stability. Such a Cu@MSN catalyst overcame the inherent drawbacks of low activity, fast deactivation, and difficult recycling of traditional metal salt catalysts in organic reactions. This work provides a green and efficient method for the construction of azoxy compounds and also creates new prospects for the application of nanoporous materials confined metal catalysts in organic synthesis.
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OBJECTIVES: To clarify the adaptability of cancer patients to return to work and explore its influencing factors. DESIGN: A cross-sectional study. SETTINGS/PARTICIPANTS: From March to October 2021, 283 cancer patients in the follow-up period were recruited from the oncology departments of four secondary and above hospitals and cancer friendship associations in Nantong city using self-developed scale of adaptability to return to work for cancer patients by convenience sampling method. METHODS: The contents included general sociodemographic data, disease-related data, cancer patients' readability to work Scale, Medical Coping Style Questionnaire, Social Support Rating Scale, Family Closeness and Readability Scale, General self-efficacy Scale and Social impact Scale. Paper questionnaires were used for face-to-face data collection, and SPSS17.0 was used for statistical analysis. Univariable analyses and multiple linear regression analysis were conducted. RESULTS: The overall score of cancer patients' adaptability to return to work was (87.05±20.255), (22.54±4.234) for the dimension of focused rehabilitation, (32.02±9.013) for the dimension of reconstruction effectiveness, and (32.49±9.023) for the dimension of adjustment planning. Multiple linear regression analysis showed that the current return to full-time work (ß =0.226, P 0.05), the current return to non-full-time work (ß =0.184, P 0.05), yield response (ß = -0.132, P 0.05), and general self-efficacy (ß =0.226, P 0.05) could affect their return to work adaptation. CONCLUSION: The results of status quo and influencing factors showed that the adaptability of cancer patients to return to work was generally higher in this study. Cancer patients who had participated in work, had lower yield coping scores and stigma scores, and higher self-efficacy scores and family adjustment and intimacy scores had better adaptability to return to work again. ETHICAL APPROVAL: It has been approved by the Human Research Ethics Committee of the Affiliated Hospital of Nantong University (Project No.202065).
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Neoplasias , Reinserción al Trabajo , Humanos , Estudios Transversales , Adaptación Psicológica , Encuestas y CuestionariosRESUMEN
A palladium-catalyzed multicomponent reaction involving o-bromobenzaldehydes and two different isocyanides was developed to assemble series of isoindolinones with spiroindolenine or azepinoindole skeletons. This sequential insertion reaction features mild conditions, a wide substrate scope, and high efficiency. Preliminary mechanistic study indicated that the difference in steric hindrance between isocyanide components is crucial when regulating the reaction sequence, whereas the ligand also played an important role during the whole process.
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Nanodiscs have become a popular tool in structure determination of membrane proteins using cryogenic electron microscopy and single particle analysis. However, the structure determination of small membrane proteins remains challenging. When the embedded protein is in the same size range as the nanodisc, the nanodisc can significantly contribute to the alignment and classification during the structure determination process. In those cases, it is crucial to minimize the heterogeneity in the nanodisc preparations to assure maximum accuracy in the classification and alignment steps of single particle analysis. Here, we introduce a new in-silico method for the characterization of nanodisc samples that is based on analyzing the Feret diameter distribution of their particle projection as imaged in the electron microscope. We validated the method with comprehensive simulation studies and show that Feret signatures can detect subtle differences in nanodisc morphologies and composition that might otherwise go unnoticed. We used the method to identify a specific biochemical nanodisc preparation with low size variations, allowing us to obtain a structure of the 23-kDa single-span membrane protein Bcl-xL while embedded in a nanodisc. Feret signature analysis can steer experimental data collection strategies, allowing more efficient use of high-end data collection hardware, as well as image analysis investments in studies where nanodiscs significantly contribute to the total volume of the full molecular species.
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The throughput and fidelity of cryogenic cellular electron tomography (cryo-ET) is constantly increasing through advances in cryogenic electron microscope hardware, direct electron detection devices, and powerful image processing algorithms. However, the need for careful optimization of sample preparations and for access to expensive, high-end equipment, make cryo-ET a costly and time-consuming technique. Generally, only after the last step of the cryo-ET workflow, when reconstructed tomograms are available, it becomes clear whether the chosen imaging parameters were suitable for a specific type of sample in order to answer a specific biological question. Tools for a-priory assessment of the feasibility of samples to answer biological questions and how to optimize imaging parameters to do so would be a major advantage. Here we describe MEPSi (Membrane Embedded Protein Simulator), a simulation tool aimed at rapid and convenient evaluation and optimization of cryo-ET data acquisition parameters for studies of transmembrane proteins in their native environment. We demonstrate the utility of MEPSi by showing how to detangle the influence of different data collection parameters and different orientations in respect to tilt axis and electron beam for two examples: (1) simulated plasma membranes with embedded single-pass transmembrane αIIbß3 integrin receptors and (2) simulated virus membranes with embedded SARS-CoV-2 spike proteins.
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COVID-19 , Proteínas de la Membrana , Humanos , SARS-CoV-2RESUMEN
The dynamic alterations in cell wall (CW) biosynthesis play an essential role in physiological isolation during the plant somatic embryogenesis (SE). However, the mechanisms underlying the functions of cell wall-associated miRNAs (CW-miRNA) remain poorly understood in plant SE. Here, we have identified 36 distinct candidate miRNAs associated with CW biosynthesis from longan third-generation genome as well as miRNA transcriptome, and modified RLM-RACE validated four distinct miRNA, which specifically targeted four CW-related genes. More importantly, we found that the dlo-miR397a-antagomir significantly enhanced DlLAC7 expression and improved laccase activity. Interestingly, inhibition of dlo-miR397a increased CW lignin deposition and promoted the tightening of protodermal cell by miRNA-mimic technology during early SE. Moreover, overexpression of dlo-miR408-3p (dlo-miR408-3p-agomir) markedly decreased DlLAC12 expression. dlo-miR408-3p-agomir activated rapid cell division, thus promoting the globular embryo (GE) development, which might be due to high DNA synthesis activity in protoepidermal cells, rather than affecting lignin synthesis. The subcellular location also indicated that both DlLAC7 and DlLAC12 proteins were primarily localized in CW and regulated CW biosynthesis. Overall, our findings provided new insight on the molecular regulatory networks comprising various miRNAs associated with cell wall, and established that dlo-miR397a and dlo-miR408-3p played differential roles during early SE in longan. The findings also shed some light on the potential role of miRNA target DlLAC regulating in vivo embryonic development of plant.
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MicroARNs , Pared Celular/metabolismo , Desarrollo Embrionario , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Lignina/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Técnicas de Embriogénesis Somática de Plantas , SapindaceaeRESUMEN
By utilizing an underexplored reaction mode of C,N-cyclic azomethine imines, a catalyst-free [1+2+3] cycloaddition/N-N bond cleavage sequential reaction for accessing spiroindolines with syn-stereoselectivity was developed. On the basis of experimental results and DFT calculations, peroxide and ethereal solvent were identified to trigger the hydrogen abstraction of the unstable [1+2+3] cycloaddition adducts, followed by homolytic cleavage of the N-N bond and hydrogen absorption.
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A palladium-catalyzed three-component reaction of isocyanides, 2,2,2-trifluoro-N-(2-iodophenyl)acetimidoyl chlorides, and amines for the one-pot synthesis of 2-(trifluoromethyl)quinazolin-4(3H)-imines was described. The protocol features a wide substrate scope, high efficiency, and readily available raw materials.
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A divergent reaction of isocyanides with o-bromobenzaldehydes for the synthesis of isoindolinone-derived ketenimines and lactams was disclosed. The reaction features readily available reactants, relatively mild conditions, and high yields of products. Ketenimines could be applied in further transformations for access to other functional molecules. A mechanism study showed that the palladium-migration/imine-insertion process was the key step in this reaction.