A DNA circuit that records molecular events.

Characterizing the relative onset time, strength, and duration of molecular signals is critical for understanding the operation of signal transduction and genetic regulatory networks. However, detecting multiple such molecules as they are produced and then quickly consumed is challenging. A MER can encode information about transient molecular events as stable DNA sequences and are amenable to downstream sequencing or other analysis. Here, we report the development of a de novo molecular event recorder that processes information using a strand displacement reaction network and encodes the information using the primer exchange reaction, which can be decoded and quantified by DNA sequencing. The event recorder was able to classify the order at which different molecular signals appeared in time with 88% accuracy, the concentrations with 100% accuracy, and the duration with 75% accuracy. This simultaneous and highly programmable multiparameter recording could enable the large-scale deciphering of molecular events such as within dynamic reaction environments, living cells, or tissues.

The Impact of Oxygen Concentration on the Postcuring of 3D-Printed Dental Resin.

PURPOSE: This study investigated the impact of reducing the oxygen concentration via nitrogen injection during the postcuring process of 3D-printed dental materials. MATERIALS AND METHODS: Resin specimens for dental crown and bridge (15-mm diameter, both 1-mm and 2-mm heights) were 3D-printed and rinsed. Subsequently, the postcuring process was conducted on nine groups categorized according to atmospheric conditions within the curing device (20% [control], 10%, and 5% oxygen) and curing times (10, 15, and 20 minutes). Surface roughness was measured using a gloss meter. Surface polymerization was confirmed through Fourier-transform infrared spectroscopy (FT-IR) analysis, and the flexural strength and elastic modulus of the specimens were measured using a universal testing machine. Water absorption and solubility were determined according to Inernational Organization for Standardization (ISO) standards. All evaluation criteria were statistically analyzed using one-way ANOVA and Tukey's post hoc test based on oxygen concentration. RESULTS: The elastic modulus did not show statistically significant differences in all groups. However, compared to the control group, the flexural strength, degree of conversion, and gloss significantly increased in the groups with decreased oxygen concentrations. Conversely, water solubility and water absorption significantly decreased in a few groups with reduced oxygen concentration. CONCLUSIONS: Reducing oxygen concentration through nitrogen injection during the postcuring process of 3D printing enhances the suitability of the dental prosthetic materials. The significant increase in flexural strength can particularly enhance the utility of these materials in dental prosthetics.

High-Resolution NMR Structures of Intrastrand Hairpins Formed by CTG Trinucleotide Repeats.

The CAG and CTG trinucleotide repeat expansions cause more than 10 human neurodegenerative diseases. Intrastrand hairpins formed by trinucleotide repeats contribute to repeat expansions, establishing them as potential drug targets. High-resolution structural determination of CAG and CTG hairpins poses as a long-standing goal to aid drug development, yet it has not been realized due to the intrinsic conformational flexibility of repetitive sequences. We herein investigate the solution structures of CTG hairpins using nuclear magnetic resonance (NMR) spectroscopy and found that four CTG repeats with a clamping G-C base pair was able to form a stable hairpin structure. We determine the first solution NMR structure of dG(CTG)4C hairpin and decipher a type I folding geometry of the TGCT tetraloop, wherein the two thymine residues form a T·T loop-closing base pair and the first three loop residues continuously stack. We further reveal that the CTG hairpin can be bound and stabilized by a small-molecule ligand, and the binding interferes with replication of a DNA template containing CTG repeats. Our determined high-resolution structures lay an important foundation for studying molecular interactions between native CTG hairpins and ligands, and benefit drug development for trinucleotide repeat expansion diseases.

Monodispersed and Monofunctionalized DNA-Caged Au Nano-Clusters with Enhanced Optical Properties for STED Imaging.

The performance of Stimulated Emission Depletion (STED) microscopy depends critically on the fluorescent probe. Ultrasmall Au nanoclusters (Au NCs) exhibit large Stokes shift, and good stimulated emission response, which are potentially useful for STED imaging. However, Au NCs are polydispersed in size, sensitive to the surrounding environment, and difficult to control surface functional group stoichiometry, which results in reduced density and high heterogeneity in the labeling of biological structures. Here, this limitation is overcome by developing a method to encapsulate ultrasmall Au NCs with DNA cages, which yielded monodispersed, and monofunctionalized Au NCs that are long-term stable. Moreover, the DNA-caging also greatly improved the fluorescence quantum yield and photostability of Au NCs. In STED imaging, the DNA-caged Au NCs yielded ≈40 nm spatial resolution and are able to resolve microtubule line shapes with good labeling density and homogeneity. In contrast, without caging, the Au NCs-DNA conjugates only achieved ≈55 nm resolution and yielded spotted, poorly resolved microtubule structures, due to the presence of aggregates. Overall, a method is developed to achieve precise surface functionalization and greatly improve the monodispersity, stability, as well as optical properties of Au NCs, providing a promising class of fluorescent probes for STED imaging.

Structural investigation of pathogenic RFC1 AAGGG pentanucleotide repeats reveals a role of G-quadruplex in dysregulated gene expression in CANVAS.

An expansion of AAGGG pentanucleotide repeats in the replication factor C subunit 1 (RFC1) gene is the genetic cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), and it also links to several other neurodegenerative diseases including the Parkinson's disease. However, the pathogenic mechanism of RFC1 AAGGG repeat expansion remains enigmatic. Here, we report that the pathogenic RFC1 AAGGG repeats form DNA and RNA parallel G-quadruplex (G4) structures that play a role in impairing biological processes. We determine the first high-resolution nuclear magnetic resonance (NMR) structure of a bimolecular parallel G4 formed by d(AAGGG)2AA and reveal how AAGGG repeats fold into a higher-order structure composed of three G-tetrad layers, and further demonstrate the formation of intramolecular G4s in longer DNA and RNA repeats. The pathogenic AAGGG repeats, but not the nonpathogenic AAAAG repeats, form G4 structures to stall DNA replication and reduce gene expression via impairing the translation process in a repeat-length-dependent manner. Our results provide an unprecedented structural basis for understanding the pathogenic mechanism of AAGGG repeat expansion associated with CANVAS. In addition, the high-resolution structures resolved in this study will facilitate rational design of small-molecule ligands and helicases targeting G4s formed by AAGGG repeats for therapeutic interventions.

Insulin-like Growth Factor 2-Tagged Aptamer Chimeras (ITACs) Modular Assembly for Targeted and Efficient Degradation of Two Membrane Proteins.

Overexpression of pathogenic membrane proteins drives abnormal proliferation and invasion of tumor cells. Various strategies to durably knockdown membrane proteins with heterobifunctional degraders have been successfully developed, including LYTAC, KineTAC, and AbTAC. However, challenges including complicated synthetic procedures and the inability to simultaneously degrade multiple pathogenic proteins still exist. Herein, we developed insulin-like growth factor 2 (IGF2)-tagged aptamer chimeras (ITACs) that link the cell-surface lysosome-targeting receptor IGF2R and membrane proteins of interest (POIs) based on specific recognition of aptamers to the POIs and high-affinity binding of IGF2 to IGF2R. We demonstrated that ITACs exhibit robust degradation efficiency of various membrane proteins in multiple cell lines. Furthermore, systematic studies revealed that a moderate cell-surface IGF2R level is responsible for the excellent degradation performance of ITACs. Importantly, we further established a modular assembly strategy that allows assembly of one IGF2 with two aptamers with precise stoichiometry (dITACs), enabling cooperative and simultaneous degradation of two membrane proteins. This work provides an efficient and facile target membrane protein degradation platform and will shed light on the treatment of diseases related to the overexpression of membrane proteins.

Covalent LYTAC Enabled by DNA Aptamers for Immune Checkpoint Degradation Therapy.

Immune checkpoint blockade (ICB) therapy, while achieving tremendous clinical successes, still suffers from a low objective response rate in clinical cancer treatment. As a proof-of-concept study, we propose a new immune checkpoint degradation (ICD) therapy relying on lysosome-targeting chimera (LYTAC) to deplete immune checkpoint programmed death ligand-1 (PD-L1) on the tumor cell surface. Our designed chimeric aptamer on one side targets lysosome-trafficking receptor, and on the other side allows biorthogonal covalent-conjugation-reinforced specific binding of PD-L1. This covalent LYTAC is able to hijack PD-L1 for lysosomal degradation with greatly improved efficiency over its noncovalent counterpart in complex in vivo environment. Beyond abolishing the PD-1/PD-L1 axis associated immune resistance, we demonstrate for the first time that LYTAC-triggered PD-L1 degradation could directly cause immunogenic apoptosis of tumor cells to elicit tumor-specific immune responses, offering unparalleled advantages over ICB antibody therapy. Remarkably, ICD therapy with covalent LYTAC achieves comparable or higher antitumor efficacy while causing significantly less inflammatory injury compared to antibody-based ICB therapy. Moreover, covalent LYTAC can serve as a general platform for specifically degrading other membrane-associated proteins, making it a promising tool for future applications. Our work presents a novel molecular tool for effective LYTAC in complex environments, offering valuable insights in pushing DNA-based LYTAC drugs toward in vivo and clinical applications.

FINDER: A Fluidly Confined CRISPR-Based DNA Reporter on Living Cell Membranes for Rapid and Sensitive Cancer Cell Identification.

The accurate, rapid, and sensitive identification of cancer cells in complex physiological environments is significant in biological studies, personalized medicine, and biomedical engineering. Inspired by the naturally confined enzymes on fluid cell membranes, a fluidly confined CRISPR-based DNA reporter (FINDER) was developed on living cell membranes, which was successfully applied for rapid and sensitive cancer cell identification in clinical blood samples. Benefiting from the spatial confinement effect for improved local concentration, and membrane fluidity for higher collision efficiency, the activity of CRISPR-Cas12a was, for the first time, found to be significantly enhanced on living cell membranes. This new phenomenon was then combined with multiple aptamer-based DNA logic gate for cell recognition, thus a FINDER system capable of accurate, rapid and sensitive cancer cell identification was constructed. The FINDER rapidly identified target cells in only 20 min, and achieved over 80 % recognition efficiency with only 0.1 % of target cells presented in clinical blood samples, indicating its potential application in biological studies, personalized medicine, and biomedical engineering.

Fungal-Algal Association Drives Lichens' Mutualistic Symbiosis: A Case Study with Trebouxia-Related Lichens.

Biotic and abiotic factors influence the formation of fungal-algal pairings in lichen symbiosis. However, the specific determinants of these associations, particularly when distantly related fungi are involved, remain poorly understood. In this study, we investigated the impact of different drivers on the association patterns between taxonomically diverse lichenized fungi and their trebouxioid symbiotic partners. We collected 200 samples from four biomes and identified 41 species of lichenized fungi, associating them with 16 species of trebouxioid green algae, of which 62% were previously unreported. The species identity of both the fungal and algal partners had the most significant effect on the outcome of the symbiosis, compared to abiotic factors like climatic variables and geographic distance. Some obviously specific associations were observed in the temperate zone; however, the nestedness value was lower in arid regions than in cold, polar, and temperate regions according to interaction network analysis. Cophylogenetic analyses revealed congruent phylogenies between trebouxioid algae and associated fungi, indicating a tendency to reject random associations. The main evolutionary mechanisms contributing to the observed phylogenetic patterns were "loss" and "failure to diverge" of the algal partners. This study broadens our knowledge of fungal-algal symbiotic patterns in view of Trebouxia-associated fungi.

Microbial communities and their correlation with flavor compound formation during the mechanized production of light-flavor Baijiu.

Light-flavor Baijiu fermentation is a typical spontaneous solid-state fermentation process fueled by a variety of microorganisms. Mechanized processes have been increasingly employed in Baijiu production to replace traditional manual operation processes, however, the microbiological and physicochemical dynamics in mechanized processes remain largely unknown. Here, we investigated the microbial community succession and flavor compound formation during a whole mechanized fermentation process of light-flavor Baijiu using the conventional dilution plating method, PacBio single-molecule real-time (SMRT) sequencing and headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. The results showed that largely different fungal and bacterial communities were involved in the soaking and fermentation processes. A clear succession from Pantoea agglomerans to Bacillus (B.) smithii and B. coagulans in dominant bacterial species and from Cladosporium exasperatum to Saccharomyces cerevisiae and Lichtheimia ramosa in dominant fungal species occurred in the soaking processes. In the fermentation process, the most dominant bacterial species was shifted from Pantoea agglomerans to Lactobacillus (La.) acetotolerans and the most dominant fungal species were shifted from Lichtheimia ramose and Rhizopus arrhizus to Saccharomyces cerevisiae. The bacterial and fungal species positively associated with acidity and the formation of ethanol and different flavor compounds were specified. The microbial species exhibited strong co-occurrence or co-exclusion relationships were also identified. The results are helpful for the improvement of mechanized fermentation process of light-flavor Baijiu production.

Microbial Community Affects Daqu Quality and the Production of Ethanol and Flavor Compounds in Baijiu Fermentation.

Daqu is a traditional starter for Baijiu fermentation and is produced by spontaneous fermentation of ground and moistened barley or wheat. The quality of Daqu is traditionally evaluated based on physicochemical and subjective sensory parameters without microbiological analysis. Here, we compared the physicochemical characteristics of qualified (QD) and inferior (ID) Daqu, their microbial communities based on plate counting and PacBio SMRT sequencing of rRNA gene libraries, and their impacts on Baijiu fermentation. The results showed that the glucoamylase and α-amylase activities of QD were significantly higher than those of ID. The counts of yeasts and relative abundances of functional microbes, especially the amylolytic bacterium Bacillus licheniformis and fungi Saccharomycopsis fibuligera and Lichtheimia ramosa, were significantly higher in QD than in ID. The laboratory-scale Baijiu fermentation tests showed that the relative abundances of the amylolytic microbes were higher in the QD than the ID fermentation set, resulting in more efficient fermentation, as indicated by more weight loss and higher moisture content in the former. Consequently, more glycerol, acetic acid, ethanol, and other volatile compounds were produced in the QD than in the ID fermentation set. The results suggest that Daqu quality is determined by, and can be evaluated based on, its microbial community.

Unlocking Genetic Profiles with a Programmable DNA-Powered Decoding Circuit.

Human genetic architecture provides remarkable insights into disease risk prediction and personalized medication. Advances in genomics have boosted the fine-mapping of disease-associated genetic variants across human genome. In healthcare practice, interpreting intricate genetic profiles into actionable medical decisions can improve health outcomes but remains challenging. Here an intelligent genetic decoder is engineered with programmable DNA computation to automate clinical analyses and interpretations. The DNA-based decoder recognizes multiplex genetic information by one-pot ligase-dependent reactions and interprets implicit genetic profiles into explicit decision reports. It is shown that the DNA decoder implements intended computation on genetic profiles and outputs a corresponding answer within hours. Effectiveness in 30 human genomic samples is validated and it is shown that it achieves desirable performance on the interpretation of CYP2C19 genetic profiles into drug responses, with accuracy equivalent to that of Sanger sequencing. Circuit modules of the DNA decoder can also be readily reprogrammed to interpret another pharmacogenetics genes, provide drug dosing recommendations, and implement reliable molecular calculation of polygenic risk score (PRS) and PRS-informed cancer risk assessment. The DNA-powered intelligent decoder provides a general solution to the translation of complex genetic profiles into actionable healthcare decisions and will facilitate personalized healthcare in primary care.

m6 A promotes planarian regeneration.

Regeneration is the regrowth of damaged tissues or organs, a vital process in response to damages from primitive organisms to higher mammals. Planarian possesses active whole-body regenerative capability owing to its vast reservoir of adult stem cells, neoblasts, providing an ideal model to delineate the underlying mechanisms for regeneration. RNA N6 -methyladenosine (m6 A) modification participates in many biological processes, including stem cell self-renewal and differentiation, in particular the regeneration of haematopoietic stem cells and axons. However, how m6 A controls regeneration at the whole-organism level remains largely unknown. Here, we demonstrate that the depletion of m6 A methyltransferase regulatory subunit wtap abolishes planarian regeneration, potentially through regulating genes related to cell-cell communication and cell cycle. Single-cell RNA-seq (scRNA-seq) analysis unveils that the wtap knockdown induces a unique type of neural progenitor-like cells (NP-like cells), characterized by specific expression of the cell-cell communication ligand grn. Intriguingly, the depletion of m6 A-modified transcripts grn, cdk9 or cdk7 partially rescues the defective regeneration of planarian caused by wtap knockdown. Overall, our study reveals an indispensable role of m6 A modification in regulating whole-organism regeneration.

Characterization of Green Part of Steel from Metal Injection Molding: An Analysis Using Moldflow.

Metal injection molding (MIM) is a quick manufacturing method that produces elaborate and complex items accurately and repeatably. The success of MIM is highly impacted by green part characteristics. This work characterized the green part of steel produced using MIM from feedstock with a powder/binder ratio of 93:7. Several parameters were used, such as dual gates position, injection temperature of ~150 °C, and injection pressure of ~180 MPa. Analysis using Moldflow revealed that the aformentioned parameters were expected to produce a green part with decent value of confidence to fill. However, particular regions exhibited high pressure drop and low-quality prediction, which may lead to the formation of defects. Scanning electron microscopy, as well as three-dimensional examination using X-ray computed tomography, revealed that only small amounts of pores were formed, and critical defects such as crack, surface wrinkle, and binder separation were absent. Hardness analysis revealed that the green part exhibited decent homogeneity. Therefore, the observed results could be useful to establish guidelines for MIM of steel in order to obtain a high quality green part.

Engineering Circular Aptamer Assemblies with Tunable Selectivity to Cell Membrane Antigens In Vitro and In Vivo.

The strategy of enhancing molecular recognition by improving the binding affinity of drug molecules against targets has generated a lot of successful therapeutic applications. However, one critical consequence of such affinity improvement, generally called "on-target, off-tumor" toxicity, emerged as a major obstacle limiting their clinical usage. Herein, we provide a modular assembly strategy that affords affinity-tunable DNA nanostructures allowing for immobilizing multiple aptamers that bind to the example antigen of EpCAM with different affinities. We develop a theoretical model proving that the apparent affinity of aptamer assemblies to target cells varies with antigen density as well as aptamer valency. More importantly, we demonstrate experimentally that the theoretical model can be used to predict the least valency required for discrimination between EpCAMhigh and EpCAMlow cells in vitro and in vivo. We believe that our strategy will have broad applications in an engineering nucleic acid-based delivery platform for targeted and cell therapy.

Sequential Control of Cellular Interactions Using Dynamic DNA Displacement.

Intercellular interactions play a significant role in various complex biological processes, and their dysregulation promotes disease progression. To reveal the mechanisms of intercellular interactions without destroying basic life processes, it is necessary to mimic multicellular behaviors in vitro. However, the precise control of multicellular systems remains technically challenging owing to dynamic interactions. Here, we used DNA as a molecular lock and key to sequentially assemble and disassemble different cell clusters in a programmed way, regulating intercellular interactions. Tagging the surface of live cells with cholesterol-modified DNA enabled dynamical intercellular assemblies. By consecutively adding corresponding metaphorical locks (attaching DNA strands) and keys (detaching DNA strands), clusters of different cells could be sequentially formed. This strategy improved the capability of natural killer NK-92 cells to target tumor cells, improving the antitumor therapy efficacy. Our suggested approach allows dynamic regulation of intercellular interactions in complex cell systems and increases understanding of intercellular communication networks.

Controlled Self-Assembly of the Catalytic Core of Hydrolases Using DNA Scaffolds.

Precisely organizing functional molecules of the catalytic cores in natural enzymes to promote catalytic performance is a challenging goal in respect to artificial enzyme construction. In this work, we report a DNA-scaffolded mimicry of the catalytic cores of hydrolases, which showed a controllable and hierarchical acceleration of the hydrolysis of fluorescein diacetate (FDA). The results revealed that the efficiency of hydrolysis was greatly increased by the DNA-scaffold-induced proximity of catalytic amino acid residues (histidine and arginine) with up to 4-fold improvement relative to the free amino acids. In addition, DNA-scaffolded one-dimensional and two-dimensional assemblies of multiple catalytic cores could further accelerate the hydrolysis. This work demonstrated that the DNA-guided assembly could be used as a promising platform to build enzyme mimics in a programmable and hierarchical way.

Beautiful seems good, but perhaps not in every way: Linking attractiveness to moral evaluation through perceived vanity.

For almost 50 years, psychologists have understood that what is beautiful is perceived as good. This simple and intuitively appealing hypothesis has been confirmed in many ways, prompting a wide range of studies documenting the depth and breadth of its truth. Yet, for what is arguably one of the most important forms of "goodness" that there is-moral goodness-research has told a different story. Although greater attractiveness is associated with a host of positive attributes, it has been only inconsistently associated with greater perceived morality (or lesser immorality), and meta-analyses have suggested the total effect of beauty on moral judgment is near zero. The current research documents one plausible reason for this. Across nine experiments employing a variety of methodological and measurement strategies, we show how attractiveness can be perceived as both morally good and bad. We found that attractiveness causally influences beliefs about vanity, which translates into beliefs that more attractive targets are less moral and more immoral. Then, we document a positive association between attractiveness and sociability-the nonmoral component of warmth-and show how sociability exerts a countervailing positive effect on moral judgments. Likewise, we document findings suggesting that vanity and sociability mutually suppress the effects of attractiveness on each other and on moral judgments. Ultimately, this work provides a comprehensive process account of why beauty seems good but can also be perceived as less moral and more immoral, highlighting complex interrelations among different elements of person perception. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

What are the risk factors for recurrent UTI with repeated ESBL-producing Enterobacteriaceae? A retrospective cohort study.

INTRODUCTION: A previous study has shown that two-thirds of patients with urinary tract infections (UTIs) caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae experience recurrence with the same bacteria on subsequent UTI episodes. However, little is known about which patients suffer from UTI due to ESBL-producing Enterobacteriaceae repeatedly. This study aimed to investigate the risk factors for recurrent UTI due to repeated ESBL-producing organism infections. METHODS: This retrospective, single-center, observational cohort study screened all patients with UTI caused by ESBL-producing strains between January 2012 and April 2019. Among the patients who were followed up, patients who experienced UTI recurrence were enrolled and divided into two groups: ESBL recurrence group and non-ESBL recurrence group. Multivariable Cox proportional hazards regression analyses were performed to evaluate the association between patient characteristics and the development of recurrent UTI caused by ESBL-producing Enterobacteriaceae. RESULTS: A total of 330 patients were followed up after the diagnosis of UTI caused by ESBL-producing organisms. Among the patients, 115 (34.8%) experienced UTI recurrence, and 71 (61.7%) of them experienced subsequent recurrent UTI due to ESBL-producing organisms. Patient's age (hazard ratio [HR], 1.02; 95% confidence interval [CI], 1.00-1.04; P = 0.046) and recurrent UTI history (HR, 1.69; 95% CI, 1.05-2.72; P = 0.031) were significantly associated with an increased risk of recurrence with ESBL-producing Enterobacteriaceae. CONCLUSION: These findings showed that a history of previous frequent UTI recurrence is the risk factor for recurrence of UTI due to repeated ESBL producing Enterobacteriaceae infections.

Surface Functionalization of Extracellular Vesicles with Nucleic Acids towards Biomedical Applications.

Extracellular vesicles (EVs) are lipid bilayer-delimited particles secreted by cells and are regarded as a promising class of nanocarriers for biomedical applications such as disease diagnosis, drug delivery, and immunomodulation, as they carry biomarkers from the parental cells and can also transport diverse cargo molecules between cells. Surface functionalization of EVs can help obtain detectable signals for their quantification and also add various properties for EV-based delivery. Aptamers are specific oligonucleotides selected as artificial antibodies that could serve as 'cruise missiles' to target EVs for diagnosis or as navigators to bring EVs to lesions for treatment. DNA logic devices or nanostructures based on aptamers are intelligent designs to endow EVs with additional features, such as multi-target disease diagnosis in one pot and promoting retention of EVs in complex disease microenvironments. Oligonucleotides or DNA nanostructures composed of natural nucleic acids can be easily degraded by nuclease in the biological sample which limits their applications. Thus, the oligonucleotides composed of artificial nucleic acids which are synthesized against degradation would be a potential strategy to improve their stability in vitro or in vivo. Herein, we review the methods for surface functionalization of EVs by nucleic acids and highlight their applications in quantification and targeted delivery towards disease diagnosis and therapy.