In Vitro Selection of M2+-Independent, Fast-Responding Acidic Deoxyribozymes for Bacterial Detection.

We report on the first efforts to isolate acidic RNA-cleaving DNAzymes (aRCDs) from a random-sequence DNA pool by in vitro selection that are activated by a microbe Escherichia coli (E. coli), at pH 5.3. Importantly, these E. coli-responsive aRCDs only require monovalent metal ions as cofactors for cleaving a fluorogenic chimeric DNA/RNA substrate. Such characteristics can be used to efficiently protect RCDs from both intrinsic chemical instability and external enzymatic degradation. One remarkable DNAzyme, aRCD-EC1, is specific for E. coli, and its target is likely a protein. Furthermore, truncated aRCD-EC1 had significantly improved catalytic activity with an observed rate constant (kobs) of 1.18 min-1, making it the fastest bacteria-responding RCD reported to date. Clinical evaluation of this aRCD-based fluorescent assay using 40 patient urine samples demonstrated a diagnostic sensitivity of 100% and a specificity of 100% at a total analysis time of 50 min without a bacterial culture. This work can expand the repertoire of DNAzymes that are active under nonphysiological conditions, thus facilitating the development of diverse DNAzyme-based biosensors in clinical diagnosis.

Integration of CRISPR/Cas13a and V-Shape PCR for Rapid, Sensitive, and Specific Genotyping of CYP2C19 Gene Polymorphisms.

Rapid detection of single nucleotide polymorphisms (SNPs) in the CYP2C19 gene is of great significance for clopidogrel-accurate medicine. CRISPR/Cas systems have been increasingly used in SNP detection due to their single-nucleotide mismatch specificity. PCR, as a powerful amplification tool, has been incorporated into the CRISPR/Cas system to improve the sensitivity. However, the complicated three-step temperature control of the conventional PCR impeded rapid detection. The "V" shape PCR can shorten about 2/3 of the amplification time compared with conventional PCR. Herein, we present a new system termed the "V" shape PCR-coupled CRISPR/Cas13a (denoted as VPC) system, achieving the rapid, sensitive, and specific genotyping of CYP2C19 gene polymorphisms. The wild- and mutant-type alleles in CYP2C19*2, CYP2C19*3, and CYP2C19*17 genes can be discriminated by using the rationally programmed crRNA. A limit of detection (LOD) of 102 copies/µL was obtained within 45 min. In addition, the clinical applicability was demonstrated by genotyping SNPs in CYP2C19*2, CYP2C19*3, and CYP2C19*17 genes from clinical blood samples and buccal swabs within 1 h. Finally, we conducted the HPV16 and HPV18 detections to validate the generality of the VPC strategy.

Nucleic Acid Enzyme-Activated CRISPR-Cas12a With Circular CRISPR RNA for Biosensing.

clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are increasingly used in biosensor development. However, directly translating recognition events for non-nucleic acid targets by CRISPR into effective measurable signals represents an important ongoing challenge. Herein, it is hypothesized and confirmed that CRISPR RNAs (crRNAs) in a circular topology efficiently render Cas12a incapable of both site-specific double-stranded DNA cutting and nonspecific single-stranded DNA trans cleavage. Importantly, it is shown that nucleic acid enzymes (NAzymes) with RNA-cleaving activity can linearize the circular crRNAs, activating CRISPR-Cas12a functions. Using ligand-responsive ribozymes and DNAzymes as molecular recognition elements, it is demonstrated that target-triggered linearization of circular crRNAs offers great versatility for biosensing. This strategy is termed as "NAzyme-Activated CRISPR-Cas12a with Circular CRISPR RNA (NA3C)." Use of NA3C for clinical evaluation of urinary tract infections using an Escherichia coli-responsive RNA-cleaving DNAzyme to test 40 patient urine samples, providing a diagnostic sensitivity of 100% and specificity of 90%, is further demonstrated.

Functions of the Zinc-Sensing Receptor GPR39 in Regulating Intestinal Health in Animals.

G protein-coupled receptor 39 (GPR39) is a zinc-sensing receptor (ZnR) that can sense changes in extracellular Zn2+, mediate Zn2+ signal transmission, and participate in the regulation of numerous physiological activities in living organisms. For example, GPR39 activates the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and phosphatidylinositol3-kinase/protein kinase B (PI3K/AKT) signaling pathways upon Zn2+ stimulation, enhances the proliferation and differentiation of colonic cells, and regulates ion transport, as well as exerting other functions. In recent years, with the increased attention to animal gut health issues and the intensive research on GPR39, GPR39 has become a potential target for regulating animal intestinal health. On the one hand, GPR39 is involved in regulating ion transport in the animal intestine, mediating the Cl- efflux by activating the K+/Cl- synergistic protein transporter, and relieving diarrhea symptoms. On the other hand, GPR39 can maintain the homeostasis of the animal intestine, promoting pH restoration in colonic cells, regulating gastric acid secretion, and facilitating nutrient absorption. In addition, GPR39 can affect the expression of tight junction proteins in intestinal epithelial cells, improving the barrier function of the animal intestinal mucosa, and maintaining the integrity of the intestine. This review summarizes the structure and signaling transduction processes involving GPR39 and the effect of GPR39 on the regulation of intestinal health in animals, with the aim of further highlighting the role of GPR39 in regulating animal intestinal health and providing new directions and ideas for studying the prevention and treatment of animal intestinal diseases.

Bacterial and Viral Co-Infection in the Intestine: Competition Scenario and Their Effect on Host Immunity.

Bacteria and viruses are both important pathogens causing intestinal infections, and studies on their pathogenic mechanisms tend to focus on one pathogen alone. However, bacterial and viral co-infections occur frequently in clinical settings, and infection by one pathogen can affect the severity of infection by another pathogen, either directly or indirectly. The presence of synergistic or antagonistic effects of two pathogens in co-infection can affect disease progression to varying degrees. The triad of bacterial-viral-gut interactions involves multiple aspects of inflammatory and immune signaling, neuroimmunity, nutritional immunity, and the gut microbiome. In this review, we discussed the different scenarios triggered by different orders of bacterial and viral infections in the gut and summarized the possible mechanisms of synergy or antagonism involved in their co-infection. We also explored the regulatory mechanisms of bacterial-viral co-infection at the host intestinal immune interface from multiple perspectives.

Zinc is an important inter-kingdom signal between the host and microbe.

Zinc (Zn) is an essential trace element in living organisms and plays a vital role in the regulation of both microbial virulence and host immune responses. A growing number of studies have shown that zinc deficiency or the internal Zn concentration does not meet the needs of animals and microbes, leading to an imbalance in zinc homeostasis and intracellular signalling pathway dysregulation. Competition for zinc ions (Zn2+) between microbes and the host exists in the use of Zn2+ to maintain cell structure and physiological functions. It also affects the interplay between microbial virulence factors and their specific receptors in the host. This review will focus on the role of Zn in the crosstalk between the host and microbe, especially for changes in microbial pathogenesis and nociceptive neuron-immune interactions, as it may lead to new ways to prevent or treat microbial infections.

Research progress on Toll-like receptor signal transduction and its roles in antimicrobial immune responses.

When microorganisms invade a host, the innate immune system first recognizes the pathogen-associated molecular patterns of these microorganisms through pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are known transmembrane PRRs existing in both invertebrates and vertebrates. Upon ligand recognition, TLRs initiate a cascade of signaling events; promote the pro-inflammatory cytokine, type I interferon, and chemokine expression; and play an essential role in the modulation of the host's innate and adaptive immunity. Therefore, it is of great significance to improve our understanding of antimicrobial immune responses by studying the role of TLRs and their signal molecules in the host's defense against invading microbes. This paper aims to summarize the specificity of TLRs in recognition of conserved microbial components, such as lipoprotein, lipopolysaccharide, flagella, endosomal nucleic acids, and other bioactive metabolites derived from microbes. This set of interactions helps to elucidate the immunomodulatory effect of TLRs and the signal transduction changes involved in the infectious process and provide a novel therapeutic strategy to combat microbial infections.

Zinc uptake system ZnuACB is essential for maintaining pathogenic phenotype of F4ac+ enterotoxigenic E. coli (ETEC) under a zinc restricted environment.

Zinc is the second trace element of living organisms after iron. Given its crucial importance, mammalian hosts restrict the bioavailability of Zinc ions (Zn2+) to bacterial pathogens. As a countermeasure, pathogens utilize high affinity Zn2+ transporters, such as ZnuACB to compete with the host for zinc. It is essential for bacteria to maintain zinc homeostasis and thus maintain their physiology and pathogenesis. In an attempt to uncover the zinc transporter in F4+ enterotoxigenic E. coli (ETEC) C83902, we analyzed two RNA-seq data sets of bacteria samples when different zinc treatments (restriction or abundance) were applied. Considering data revealing that the high affinity zinc uptake system ZnuACB acts as the main transporter in ETEC C83902 to resist zinc deficiency, we deleted znuACB genes to study the role of them in ETEC C83902. The deletion of znuACB genes results in growth perturbation and a sharp decrease in the ability of biofilm formation and adhesion of bacteria in vitro. Taking the data together, this study demonstrates that the ZnuACB system is required for ETEC C83902 to acquire zinc, which highly contributes to ETEC pathogenicity as well.

Upregulation of Long Noncoding RNA (lncRNA) X-Inactive Specific Transcript (XIST) is Associated with Cisplatin Resistance in Non-Small Cell Lung Cancer (NSCLC) by Downregulating MicroRNA-144-3p.

BACKGROUND Patients with advanced non-small cell lung cancer (NSCLC) treated with cisplatin, also termed cis-diamminedichloroplatinum (CDDP) or diamminedichloroplatinum (DDP), may develop chemoresistance. This study aimed to investigate the role of long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) and multidrug resistance-1 (MDR1) in tumor tissue samples and the chemoresistant human NSCLC cell lines, H460/DDP and A549/DDP, and in a murine A549/DDP tumor xenograft. MATERIAL AND METHODS Tissue samples were from patients with NSCLC who responded cisplatin (DDP-sensitive) (n=24), patients with NSCLC unresponsive to cisplatin (DDP-resistant) (n=30), and normal lung tissue (n=25). In H460/DDP and A549/DDP cells, expression of XIST, microRNA (miR)-144-3p, MDR1, and multidrug resistance-associated protein 1 (MRP1) were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The MTT assay measured cell survival and proliferation, a transwell assay evaluated cell migration, and flow cytometry measured apoptosis. Luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays examined the relationship between XIST and miR-144-3p. Tumor xenografts from A549/DDP cells were studied in BALB/c nude mice. RESULTS In tissue from patients with DDP-resistant NSCLC and the mouse A549/DDP tumor xenograft, lncRNA-XIST expression was upregulated and miR-144-3p expression was inhibited. In A549/DDP and H460/DDP cells, down-regulation of lncRNA-XIST and upregulation of miR-144-3p reduced cell survival, proliferation, migration, induced apoptosis and suppressed MDR1 and MRP1 expression. CONCLUSIONS Upregulation of lncRNA-XIST was associated with cisplatin resistance in NSCLC by downregulating miRNA-144-3p in H460/DDP and A549/DDP cells, a murine A549/DDP tumor xenograft, and human tumor tissues from patients with cisplatin-resistant NSCLC.

A high-fidelity DNAzyme-assisted CRISPR/Cas13a system with single-nucleotide resolved specificity.

A CRISPR/Cas system represents an innovative tool for developing a new-generation biosensing and diagnostic strategy. However, the off-target issue (i.e., mistaken cleavage of nucleic acid targets and reporters) remains a great challenge for its practical applications. We hypothesize that this issue can be overcome by taking advantage of the site-specific cleavage ability of RNA-cleaving DNAzymes. To test this idea, we propose a DNAzyme Operation Enhances the Specificity of CRISPR/Cas13a strategy (termed DOES-CRISPR) to overcome the problem of relatively poor specificity that is typical of the traditional CRISPR/Cas13a system. The key to the design is that the partial hybridization of the CRISPR RNA (crRNA) with the cleavage fragment of off-target RNA was not able to activate the collateral cleavage activity of Cas13a. We showed that DOES-CRISPR can significantly improve the specificity of traditional CRISPR/Cas13a-based molecular detection by up to ∼43-fold. The broad utility of the strategy is illustrated through engineering three different systems for the detection of microRNAs (miR-17 and let-7e), CYP2C19*17 gene, SARS-Cov-2 variants (Gamma, Delta, and Omicron) and Omicron subtypes (BQ.1 and XBB.1) with single-nucleotide resolved specificity. Finally, clinical evaluation of this assay using 10 patient blood samples demonstrated a clinical sensitivity of 100% and specificity of 100% for genotyping CYP2C19*17, and analyzing 20 throat swab samples provided a diagnostic sensitivity of 95% and specificity of 100% for Omicron detection, and a clinical sensitivity of 92% and specificity of 100% for XBB.1 detection.

A DNAzymes-in-droplets assay for Burkholderia gladioli pathovar cocovenenans with single-bacterium sensitivity.

Foodborne pathogens pose a serious risk to human health, and the simple and rapid detection of such bacteria in complex food matrices remains challenging. Herein, we present the selection and characterization of a novel RNA-cleaving fluorogenic DNAzyme, named RFD-BC1, with exceptional specificity for Burkholderia gladioli pv. cocovenenans (B. cocovenenans), a pathogen strongly associated with fatal food poisoning cases. RFD-BC1 was activated by a protein secreted specifically by whole viable B. cocovenenans and displayed an optimum pH distinct from the selection pH, with a rate constant of approximately 0.01 min-1 at pH 5.0. Leveraging this newly discovered DNAzyme, we developed a novel system, termed DNAzymes-in-droplets (DID), that integrates droplet microfluidics to achieve the rapid and selective detection of live B. cocovenenans with single-cell sensitivity. We believe that the approach described herein holds promise for combating specific bacterial pathogens in food samples, offering significant potential for broader applications in food safety and public health.

Amplification-free detection of Escherichia coli using an acidic deoxyribozyme-based paper device.

We reported a colorimetric paper-based device by integrating the modified acid RNA-cleaving DNAzymes (MaRCD-EC1) for highly sensitive (detection limit = 102 CFU mL-1), and rapid (within 30 min) detection of E. coli without amplification. This device exhibited a clinical sensitivity of 100% and a specificity of 100% in identifying E. coli-associated urinary tract infections (UTIs) using the clinical urine samples.

In vitro selection of N 1-methyladenosine-sensitive RNA-cleaving deoxyribozymes with 105-fold selectivity over unmethylated RNA.

RNA-cleaving DNAzymes (RCDs) are catalytically active DNA molecules that cleave a wide range of RNA targets with extremely high sequence-selectivity, but none is able to faithfully discriminate methylated from unmethylated RNA (typically <30-fold). We report the first efforts to isolate RCDs from a random-sequence DNA pool by in vitro selection that cleave RNA/DNA chimera containing N 1-methyladenosine (m1A), one of the most prevalent RNA modifications that plays important regulatory roles in gene expression and human cancers. A cis-acting deoxyribozyme, RCD1-S2m1A, exhibits an observed rate constant (k obs) of 5.3 × 10-2 min-1, resulting in up to 105-fold faster cleavage of the m1A-modified versus unmethylated RNA. Furthermore, a trans-acting fluorogenic deoxyribozyme was constructed by labeling a fluorophore and a quencher at the 5' and 3' ends of the chimeric substrate, respectively. It permits the synchronization of RNA-cleaving with real-time fluorescence signaling, thus allowing the selective monitoring of ALKBH3-mediated demethylation and inhibitor screening in living cells.

Distance-based paper device coupled with uracil-rich DNA hydrogel for visual quantification of Uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG), an enzyme for repairing uracil-containing DNA damage, is crucial for maintaining genomic stability. Simple and fast quantification of UDG activity is essential for biological assay and clinical diagnosis, since its aberrant level is associated with DNA damage and various diseases. Herein, we developed a fully integrated "sample in-signal out" distance-based paper analytical device (dPAD) for visual quantification of UDG using a flow-controlled uracil-rich DNA hydrogel (URDH). The uracil base sites contained in the DNA hydrogel are mis-incorporated with dUTP by rolling circle amplification (RCA), which simplifies the preparation process of the functionalized hydrogel. In the presence of UDG, the uracil in URDH can be recognized and removed to induce the permeability change of URDH, resulting in the visible distance signal along the paper channel. Using dPAD, as low as 6.4 × 10-4 U/mL of UDG (within 80 min) is visually identified without any instruments and complicated operations. This integrated dPAD is advantageous for its simplicity, cost effectiveness, and ease of use. We envision that it has the great potential for point-of-care testing (POCT) in DNA damage testing, personalized healthcare assessment, and biomedical applications.

GATA-binding protein 4 (GATA-4) and T-cell acute leukemia 1 (TAL1) regulate myogenic differentiation and erythropoietin response via cross-talk with Sirtuin1 (Sirt1).

Erythropoietin (EPO), the cytokine required for erythrocyte production, contributes to muscle progenitor cell proliferation and delay myogenic differentiation. However, the underlying mechanism is not yet fully understood. Here, we report that EPO changes the skeletal myogenic regulatory factor expression program and delays differentiation via induction of GATA-4 and the basic helix-loop-helix TAL1 and that knockdown of both factors promotes differentiation. EPO increases the Sirt1 level, a NAD(+)-dependent deacetylase, and also induces the NAD(+)/NADH ratio that further increases Sirt1 activity. Sirt1 knockdown reduced GATA-4 and TAL1 expression, impaired EPO effect on delayed myogenic differentiation, and the Sirt1 knockdown effect was abrogated when combined with overexpression of GATA-4 or TAL1. GATA-4 interacts with Sirt1 and targets Sirt1 to the myogenin promoter and represses myogenin expression, whereas TAL1 inhibits myogenin expression by decreasing MyoD binding to and activation of the myogenin promoter. Sirt1 was found to bind to the GATA-4 promoter to directly regulate GATA-4 expression and GATA-4 binds to the TAL1 promoter to regulate TAL1 expression positively. These data suggest that GATA-4, TAL1, and Sirt1 cross-talk each other to regulate myogenic differentiation and mediate EPO activity during myogenic differentiation with Sirt1 playing a role upstream of GATA-4 and TAL1. Taken together, our findings reveal a novel role for GATA-4 and TAL1 to affect skeletal myogenic differentiation and EPO response via cross-talk with Sirt1.

Accumulation of pre-let-7g and downregulation of mature let-7g with the depletion of EWS.

EWS functions in RNA splicing and transcription by encoding an RNA binding protein, which results in the chromosomal translocation t(11;22)(q24;q12) found in Ewing sarcoma. EWS interacts with the microprocessor complex involving Drosha and DGCR8, which play roles as the cofactors of primary microRNA processing. However, the role of EWS in microRNA biogenesis has not been investigated. Here, we show that endogenous EWS interacts with endogenous Drosha by IP-western blotting. In addition, EWS knockout mouse decreased the expression of Drosha. The depletion of EWS results in the accumulation of precursor let-7g but down-regulates mature let-7g in U2OS cells. Consistently, mature let 7g was suppressed in both Ewing sarcoma cell and primary Ewing sarcoma. Also, expression levels of Dicer and CCND1 (Cyclin D1), which are known target genes of the let-7 family were upregulated. Our findings suggest that EWS mediates generation of mature let-7g from pre-let-7g.

An Electrochemical Aptasensor Integrating Zeolitic Imidazolate Framework for Highly Selective Detection of Bioaerosols.

Bioaerosols are the biological materials in the air, which may cause a continuous threat to human health. However, there are many challenges in monitoring bioaerosols such as lack of sensitivity and selectivity. Herein, we synthesized a series of nanohybrids containing zeolitic imidazolate frameworks (ZIFs) and covalent organic frameworks (COFs) to construct an electrochemical aptasensor for detecting adenosine triphosphate (ATP), a biomarker for bioaerosols. The synthesized nanohybrids can not only improve the selectivity of aptasensor because of the original crystal and chemical features of ZIF-67, but also boost its sensitivity due to the excellent conductivity of COFs. After optimizing the nanohybrids, the novel developed sensing platform achieved highly selective detection of ATP with an excellent detection limit of 0.11 nM in a wide linear range from 0.1 nM to 100 nM. Furthermore, this assay was applied to detect bioaerosols in real air samples, and the result showed a positive correlation with that of the culturing-based method, suggesting its potential applicability.

Spatially Guided Assembly of Polyoxometalate Superlattices and Their Derivatives as High-Power Sodium-Ion Battery Anodes.

The use of polyoxometalate clusters (POMs) with multitudinous structures and surface properties as building blocks has sparked the development of cluster-assembled materials with many prospective applications. In comparison to classic molecules and assembly processes, control over the steric interactions and linkage of large POMs to achieve superlattices with multiple levels of organization remains a great challenge. This work presents a universal approach to modulate the spatial coordination behavior and configurations, and achieves a class of cluster superlattice architectures formed by linear alignment and two-dimensional arrangement of POM units. The formation mechanism is explained as a stepwise co-assembly pathway in which POMs can intervene and dictate a typical stripping-restacking combination mode with the lamellar mediator. These cluster superlattices with long-range POMs ordering impart distinct merits to their derivatives by sulfuration, for which we demonstrate the substantially promoted power and cycling life of these POM derivatives applied as sodium-ion battery anodes.

Single bacteria detection by droplet DNAzyme-coupled rolling circle amplification.

We described a new system termed droplet DNAzyme-coupled rolling circle amplification (dDRCA) that can selectively detect bacteria from clinical urine samples with single-cell sensitivity within 1.5 h compared with the several hours needed for traditionally used culture-based methods.