Dephosphorylation Switch DNAzyme-RCA Circuit: A Robust Strategy for the Homogeneous and Reliable Detection of FTO Demethylase.

Fat mass and obesity-associated protein (FTO) plays a crucial role in regulating the dynamic modification of N6-methyladenosine (m6A) in eukaryotic mRNA. Sensitive detection of the FTO level and efficient evaluation of the FTO demethylase activity are of great importance to early cancer diagnosis and anticancer drug discovery, which are currently challenged by limited sensitivity/precision and low throughput. Herein, a robust strategy based on the dephosphorylation switch DNAzyme-rolling circle amplification (RCA) circuit, termed DSD-RCA, is developed for highly sensitive detection of FTO and inhibitor screening. Initially, the catalytic activity of DNAzyme is silenced by engineering with an m6A modification in its catalytic core. Only in the presence of target FTO can the methyl group on DNAzyme be eliminated, resulting in the activation of the catalytic activity of DNAzyme and thus cleaving the hairpin substrate to release numerous primers. Different from the conventional methods that use the downstream cleavage primer with the original 3'-hydroxyl end directly as the RCA primer with the problem of high background signal, which should be compensated by additional separation and wash steps in heterogeneous format, our DSD-RCA assay uses the upstream cleavage primer with a 2',3'-cyclic phosphate terminus at the 3'-end serving as an intrinsically blocked 3' end. Only after a dephosphorylation reaction mediated by T4 polynucleotide kinase can the upstream cleavage primers with a resultant 3'-hydroxyl end be extended by RCA. With the high signal-to-noise ratio and homogeneous property, the proposed platform can sensitively detect FTO with a limit of detection of 31.4 pM, and the relative standard deviations (RSDs %) ranging from 0.8 to 2.0% were much lower than the heterogeneous methods. The DSD-RCA method was applied for analyzing FTO in cytoplasmic lysates from different cell lines and tissues of breast cancer patients and further used for screening FTO inhibitors without the need for separation or cleaning, providing an opportunity for achieving high throughput and demonstrating the potential applications of this strategy in disease diagnostics, drug discovery, and biological applications.

Aptasensor based on gold nanostructure-decorated 2D Cu metal-organic framework nanosheets for highly sensitive and specific electrochemical lipopolysaccharide detection.

Detecting lipopolysaccharide (LPS) using electrochemical methods is significant because of their exceptional sensitivity, simplicity, and user-friendliness. Two-dimensional metal-organic framework (2D-MOF) that merges the benefits of MOF and 2D nanostructure has exhibited remarkable performance in constructing electrochemical sensors, notably surpassing traditional 3D-MOFs. In this study, Cu[tetrakis(4-carboxylphenyl)porphyrin] (Cu-TCPP) and Cu(tetrahydroxyquinone) (Cu-THQ) 2D nanosheets were synthesized and applied on a glassy carbon electrode (GCE). The 2D-MOF nanosheets, which serve as supporting layers, exhibit improved electron transfer and electronic conductivity characteristics. Subsequently, the modified electrode was subjected to electrodeposition with Au nanostructures, resulting in the formation of Au/Cu-TCPP/GCE and Au/Cu-THQ/GCE. Notably, the Au/Cu-THQ/GCE demonstrated superior electrochemical activity because of the 2D morphology, redox ligand, dense Cu sites, and improved deposition of flower-like Au nanostructure based on Cu-THQ. The electron transfer specific surface area was increased by the improved deposition of Au nanostructures, which facilitates enriched binding of LPS aptamer and significantly improved the detection performance of Apt/Au/Cu-THQ/GCE electrochemical aptasensor. The limit of detection for LPS reached 0.15 fg/mL with a linear range of 1 fg/mL - 100 pg/mL. The proposed aptasensor demonstrated the ability to detect LPS in serum samples with satisfactory accuracy, indicating significant potential for clinical diagnosis.

On-Site-Activated Transmembrane Logic DNA Nanodevice Enables Highly Specific Imaging of Cancer Cells by Targeting Tumor-Related Nucleolin and Intracellular MicroRNA.

The ability to specifically image cancer cells is essential for cancer diagnosis; however, this ability is limited by the false positive associated with single-biomarker sensors and off-site activation of "always active" nucleic acid probes. Herein, we propose an on-site, activatable, transmembrane logic DNA (TLD) nanodevice that enables dual-biomarker sensing of tumor-related nucleolin and intracellular microRNA for highly specific cancer cell imaging. The TLD nanodevice is constructed by assembling a tetrahedral DNA nanostructure containing a linker (L)-blocker (B)-DNAzyme (D)-substrate (S) unit. AS-apt, a DNA strand containing an elongated segment and the AS1411 aptamer, is pre-anchored to nucleolin protein, which is specifically expressed on the membrane of cancer cells. Initially, the TLD nanodevice is firmly sealed by the blocker containing an AS-apt recognition zone, which prevents off-site activation. When the nanodevice encounters a target cancer cell, AS-apt (input 1) binds to the blocker and unlocks the sensing ability of the nanodevice for miR-21 (input 2). The TLD nanodevice achieves dual-biomarker sensing from the cell membrane to the cytoplasm, thereby ensuring cancer cell-specific imaging. This TLD nanodevice represents a promising strategy for the highly reliable analysis of intracellular biomarkers and a promising platform for cancer diagnosis and related biomedical applications.

DNAzyme-Amplified Cascade Catalytic Hairpin Assembly Nanosystem for Sensitive MicroRNA Imaging in Living Cells.

Sensitive imaging of microRNAs (miRNAs) in living cells is significant for accurate cancer clinical diagnosis and prognosis research studies, but it is challenged by inefficient intracellular delivery, instability of nucleic acid probes, and limited amplification efficiency. Herein, we engineered a DNAzyme-amplified cascade catalytic hairpin assembly (CHA)-based nanosystem (DCC) that overcomes these challenges and improves the imaging sensitivity. This enzyme-free amplification nanosystem is based on the sequential activation of DNAzyme amplification and CHA. MnO2 nanosheets were used as nanocarriers for the delivery of nucleic acid probes, which can resist the degradation by nucleases and supply Mn2+ for the DNAzyme reaction. After entering into living cells, the MnO2 nanosheets can be decomposed by intracellular glutathione (GSH) and release the loaded nucleic acid probes. In the presence of target miRNA, the locking strand (L) was hybridized with target miRNA, and the DNAzyme was released, which then cleaved the substrate hairpin (H1). This cleavage reaction resulted in the formation of a trigger sequence (TS) that can activate CHA and recover the fluorescence readout. Meanwhile, the DNAzyme was released from the cleaved H1 and bound to other H1 for new rounds of DNAzyme-based amplification. The TS was also released from CHA and involved in the new cycle of CHA. By this DCC nanosystem, low-abundance target miRNA can activate many DNAzyme and generate numerous TS for CHA, resulting in sensitive and selective analysis of miRNAs with a limit of detection of 5.4 pM, which is 18-fold lower than that of the traditional CHA system. This stable, sensitive, and selective nanosystem holds great potential for miRNA analysis, clinical diagnosis, and other related biomedical applications.

Fluorescence Biosensor for One-Step Simultaneous Detection of Mycobacterium tuberculosis Multidrug-Resistant Genes Using nanoCoTPyP and Double Quantum Dots.

The diagnosis of multidrug-resistant tuberculosis (MDR-TB) is crucial for the subsequent drug guidance to improve therapy and control the spread of this infectious disease. Herein, we developed a novel florescence biosensor for simultaneous detection of Mycobacterium tuberculosis (Mtb) multidrug-resistant genes (rpoB531 for rifampicin and katG315 for isoniazid) by using our synthesized nanocobalt 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine (nanoCoTPyP) and double quantum dots (QDs). Several nanoCoTPyPs with different charges and morphology were successfully prepared via the surfactant-assisted method and their quenching ability and restoring efficiency for DNA detection were systematically analyzed. It was found that spherical nanoCoTPyP with positive charge exhibited excellent quenching effect and sensing performance for the two DNAs' detection due to its affinity differences towards single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). ssDNA attached on QDs (QDs-ssDNA) was specifically hybridized with targets to form QDs-dsDNA, resulting in fluorescence recovery due to the disruption of the interactions between nanoCoTPyP and ssDNA. Two drug-resistant genes could be simultaneously quantified in a single run and relatively low limits of detection (LODs) were obtained (24 pM for T1 and 20 pM for T2). Furthermore, the accuracy and reliability of our method were verified by testing clinical samples. This simple and low-cost approach had great potential to be applied in clinical diagnosis of MDR-TB.

Highly Efficient Isolation and Sensitive Detection of Small Extracellular Vesicles Using a Paper-Based Device.

Small extracellular vesicles (sEVs) play important roles in mediating intercellular communication and regulating biological processes. Facile sEV isolation is the essential and preliminary issue for their function investigation and downstream biomedical applications, while the traditional methods are challenged by tedious procedures, low purity, low yield, and potential damage. In this work, we developed an sEV isolation paper-based device (sEV-IsoPD) based on a three-dimensional (3D) paper chip, which is composed of a porous membrane for size exclusion and a metal-organic framework (MOF)/antibody-modified paper for immunoaffinity capture. In combination with a peristaltic pump-driven flow system, the sEV-IsoPD can efficiently isolate EV from cell culture medium and serum. Compared with the ultracentrifugation method, sEV-IsoPD exhibited a 5.1 times higher yield (1.7 × 109 mL-1), 1.6 times higher purity (1.6 × 1011 mg-1), and 7.5 times higher recovery (77.3%) with only 8.3% of the time (30 min) and 1.0% of the instrument cost ($710). Moreover, sEV concentration can be visually detected in a quantitative manner with this paper-based device with a linear range from 3.0 × 106 to 3.0 × 1010 mL-1 and a detection limit of 2.2 × 106 mL-1. The sEV-IsoPD provides an efficient and practical approach for the rapid isolation and visible detection of sEVs, which are promising for the preparation of sEVs and diagnosis of disease.

Highly Sensitive Fluorescence Detection of Global 5-Hydroxymethylcytosine from Nanogram Input with Strongly Emitting Copper Nanotags.

Quantitative analysis of 5-hydroxymethylcytosine (5hmC) has remarkable clinical significance to early cancer diagnosis; however, it is limited by the requirement in current assays for large amounts of starting material and expensive instruments requring expertise. Herein, we present a highly sensitive fluorescence method, termed hmC-TACN, for global 5hmC quantification from several nanogram inputs based on terminal deoxynucleotide transferase (TdT)-assisted formation of fluorescent copper (Cu) nanotags. In this method, 5hmC is labeled with click tags by T4 phage ß-glucosyltransferase (ß-GT) and cross-linked with a random DNA primer via click chemistry. TdT initiates the template-free extension along the primer at the modified 5hmC site and then generates a long polythymine (T) tail, which can template the production of strongly emitting Cu nanoparticles (CuNPs). Consequently, an intensely fluorescent tag containing numerous CuNPs can be labeled onto the 5hmC site, providing the sensitive quantification of 5hmC with a limit of detection (LOD) as low as 0.021% of total nucleotides (S/N = 3). With only a 5 ng input (∼1000 cells) of genomic DNA, global 5hmC levels were accurately determined in mouse tissues, human cell lines (including normal and cancer cells of breast, lung, and liver), and urines of a bladder cancer patient and healthy control. Moreover, as few as 100 cells can also be distinguished between normal and cancer cells. The hmC-TACN method has great promise of being cost effective and easily mastered, with low-input clinical utility, and even for the microzone analysis of tumor models.

Resistant starch intake alleviates collagen-induced arthritis in mice by modulating gut microbiota and promoting concomitant propionate production.

Gut dysbiosis precedes clinic symptoms in rheumatoid arthritis (RA) and has been implicated in the initiation and persistence of RA. The early treatment of RA is critical to better clinical outcome especially for joint destruction. Although dietary interventions have been reported to be beneficial for RA patients, it is unclear to whether diet-induced gut microbiome changes can be a preventive strategy to RA development. Here, we investigated the effect of a high fiber diet (HFD) rich with resistant starch (RS) on collagen-induced arthritis (CIA) and gut microbial composition in mice. RS-HFD significantly reduced arthritis severity and bone erosion in CIA mice. The therapeutic effects of RS-HFD were correlated with splenic regulatory T cell (Treg) expansion and serum interleukin-10 (IL-10) increase. The increased abundance of Lactobacillus and Lachnoclostridium genera concomitant with CIA were eliminated in CIA mice fed the RS-HFD diet. Notably, RS-HFD also led to a predominance of Bacteroidetes, and increased abundances of Lachnospiraceae_NK4A136_group and Bacteroidales_S24-7_group genera in CIA mice. Accompanied with the gut microbiome changes, serum levels of the short-chain fatty acid (SCFA) acetate, propionate and isobutyrate detected by GC-TOFMS were also increased in CIA mice fed RS-HFD. While, addition of ß-acids from hops extract to the drinking water of mice fed RS-HFD significantly decreased serum propionate and completely eliminated RS-HFD-induced disease improvement, Treg cell increase and IL-10 production in CIA mice. Moreover, exogenous propionate added to drinking water replicated the protective role of RS-HFD in CIA including reduced bone damage. The direct effect of propionate on T cells in vitro was further explored as at least one mechanistic explanation for the dietary effects of microbial metabolites on immune regulation in experimental RA. Taken together, RS-HFD significantly reduced CIA and bone damage and altered gut microbial composition with concomitant increase in circulating propionate, indicating that RS-rich diet might be a promising therapy especially in the early stage of RA.

DNA nanotetrahedron linked dual-aptamer based voltammetric aptasensor for cardiac troponin I using a magnetic metal-organic framework as a label.

An ultrasensitive voltammetric aptasensor was constructed to analyze cardiac troponin I (cTnI). It is based on DNA nanotetrahedron (NTH) linked dual-aptamer (dAPT) and magnetic metal organic frameworks (mMOFs) of type Fe3O4@UiO-66. Firstly, the DNA NTH linked dAPT (Tro4 and Tro6) were immobilized on a gold electrode for improving the capture efficiency of cTnI. The novel mMOFs Fe3O4@UiO-66 was then decorated by Au@Pt nanoparticles (Au@PtNPs), horseradish peroxidase (HRP), G-quadruplex/hemin (GQH) DNAzyme, and two types of aptamers to form signaling nanoprobes. In the presence of cTnI, an aptamer-protein-nanoprobe sandwich-type structure is formed. Afterward, the nanoprobes including enzyme, GQH DNAzyme and Fe3O4@UiO-66/Au@PtNP were utilized to catalyze the oxidation of hydroquinone by hydrogen peroxide for the electrochemical signals amplification, typically at a working potential of -0.1 V (vs. Ag/AgCl). The voltammetric signal increases linearly in the 0.01 to 100 ng·mL-1 cTnI concentration range, and the detection limit is 5.7 pg·mL-1. Graphical abstract An ultrasensitive voltammetric aptasensor was constructed to analyze cardiac troponin I (cTnI) based on DNA nanotetrahedron linked dual-aptamer and magnetic metal organic frameworks of type Fe3O4@UiO-66. The results indicated the aptasensor has a wide linear response range (0.01 to 100 ng/mL) and low detection limit (5.74 pg/mL) for cTnI. GE: gold electrode; MCH: 6-Mmercapto-1-hexanol; HRP: horseradish peroxidase; HQ: hydroquinone; BQ: benzoquinone.

Overexpression of NgAUREO1, the gene coding for aurechrome 1 from Nannochloropsis gaditana, into Saccharomyces cerevisiae leads to a 1.6-fold increase in lipid accumulation.

Aureochrome-1 (AUREO1) is a transcription factor that is induced by blue light and controls branching of Vaucheria frigida. We have cloned the gene, NgAUREO1, coding for AUREO1 from Nannochloropsis gaditana, and report that the lipid content in recombinant Saccharomyces cerevisiae was 1.6-fold more than in wild-type S. cerevisiae (6.3 % lipid increased to 10 %). Over-expression of AUREO1 in S. cerevisiae up-regulated the expression of acetyl-CoA carboxylase and acyl-CoA:diacylglycerol acyl-transferase but down-regulated the expression of long-chain-acyl CoA synthetase. This enhanced the accumulation of lipid. This study highlights a novel function of AUREO1 and allows a better understanding of the regulation mechanism of fatty acid metabolism.

Psychological capital and music performance anxiety: the mediating role of self-esteem and flow experience.

Background: This study aimed to explore the correlation between psychological capital and music performance anxiety among Chinese college students majoring in music. Additionally, the study evaluated the chain mediating effects of self-esteem and flow experience. Methods: Three hundred twenty-nine college students pursuing a music major from institutions in Eastern China participated in the study. They were asked to complete the Psychological Capital, Self-Esteem, Flow Experience, and Music Performance Anxiety Scales. Descriptive statistics and correlation analysis were performed, and sophisticated mediation models were created. Results: The results suggest that self-esteem and flow experience play a role in connecting psychological capital and music performance anxiety. Moreover, the ultimate model elucidates significant connections linking psychological capital to self-esteem and flow experience in music performance anxiety. Conclusion: This study offers useful insights for future interventions aimed at mitigating music performance anxiety through the cultivation of students' psychological capital, self-esteem, and flow experiences.

ARMCX1 inhibits lung adenocarcinoma progression by recruiting FBXW7 for c-Myc degradation.

BACKGROUND: Armadillo Repeat Containing X-Linked 1 (ARMCX1), a member of the ARM Repeat X-linked protein family, exerts inhibitory function in various tumors. However, its biological role in lung adenocarcinoma (LUAD) and the underlying molecular mechanisms require further exploration. METHODS: LUAD tissue microarrays and bioinformatic databases were used to evaluate the relationship between ARMCX1 and clinicopathological features. The influence of ARMCX1 on LUAD cell proliferation, migration, and invasion in vitro was determined by colony formation, CCK-8, EdU incorporation, cell cycle, wound healing, and Transwell assays. The impact of ARMCX1 on LUAD cell growth and metastasis in vivo was determined by subcutaneously transplanted tumor and pulmonary metastasis assays. Western blot, immunoprecipitation, immunofluorescence, cycloheximide, and proteasome inhibitor assays were finally conducted to explore the potential underlying molecular mechanisms. RESULTS: ARMCX1 expression was downregulated in clinical LUAD samples due to which patient prognoses were poor. Functional experiments indicated that ARMCX1 overexpression inhibited the growth and metastasis of LUAD cells in vitro and in vivo. The molecular mechanism suggested that ARMCX1 recruits the E3 ubiquitin ligase FBXW7 for mediating ubiquitinated degradation of c-Myc, suppressing its nuclear accumulation, and ultimately inactivating cell cycle and epithelial-mesenchymal transition (EMT) signals. CONCLUSION: ARMCX1 inhibits LUAD cell proliferation and metastasis by interacting with c-Myc and enhancing its ubiquitination and degradation. Consequently, it can act as a tumor suppressor in this disease. These results suggest that ARMCX1 is a potential target in the treatment of LUAD.

Research progress of metal-organic framework nanozymes in bacterial sensing, detection, and treatment.

The high efficiency and specificity of enzymes make them play an important role in life activities, but the high cost, low stability and high sensitivity of natural enzymes severely restrict their application. In recent years, nanozymes have become convincing alternatives to natural enzymes, finding utility across diverse domains, including biosensing, antibacterial interventions, cancer treatment, and environmental preservation. Nanozymes are characterized by their remarkable attributes, encompassing high stability, cost-effectiveness and robust catalytic activity. Within the contemporary scientific landscape, metal-organic frameworks (MOFs) have garnered considerable attention, primarily due to their versatile applications, spanning catalysis. Notably, MOFs serve as scaffolds for the development of nanozymes, particularly in the context of bacterial detection and treatment. This paper presents a comprehensive review of recent literature pertaining to MOFs and their pivotal role in bacterial detection and treatment. We explored the limitations and prospects for the development of MOF-based nanozymes as a platform for bacterial detection and therapy, and anticipate their great potential and broader clinical applications in addressing medical challenges.

CRISPR/Pepper-tDeg: A Live Imaging System Enables Non-Repetitive Genomic Locus Analysis with One Single-Guide RNA.

CRISPR-based genomic-imaging systems have been utilized for spatiotemporal imaging of the repetitive genomic loci in living cells, but they are still challenged by limited signal-to-noise ratio (SNR) at a non-repetitive genomic locus. Here, an efficient genomic-imaging system is proposed, termed CRISPR/Pepper-tDeg, by engineering the CRISPR sgRNA scaffolds with the degron-binding Pepper aptamers for binding fluorogenic proteins fused with Tat peptide derived degron domain (tDeg). The target-dependent stability switches of both sgRNA and fluorogenic protein allow this system to image repetitive telomeres sensitively with a 5-fold higher SNR than conventional CRISPR/MS2-MCP system using "always-on" fluorescent protein tag. Subsequently, CRISPR/Pepper-tDeg is applied to simultaneously label and track two different genomic loci, telomeres and centromeres, in living cells by combining two systems. Given a further improved SNR by the split fluorescent protein design, CRISPR/Pepper-tDeg system is extended to non-repetitive sequence imaging using only one sgRNA with two aptamer insertions. Neither complex sgRNA design nor difficult plasmid construction is required, greatly reducing the technical barriers to define spatiotemporal organization and dynamics of both repetitive and non-repetitive genomic loci in living cells, and thus demonstrating the large application potential of this genomic-imaging system in biological research, clinical diagnosis and therapy.

Association between oxidative balance score and heart failure in the older adults: Results from the NHANES 2005-2018.

BACKGROUND: Heart failure (HF) imposes a substantial burden on older adults, and healthy diets and lifestyles may bring with benefits. However, quantifiable studies on the dietary and lifestyle risk factors for HF are scant. The Oxidative Balance Score (OBS) reflects the oxidative stress status of dietary components and lifestyle factors, but its relationship with HF risk is unclear. OBJECTIVE: We aims to explore the association between OBS and the prevalence of HF. METHODS: Using data from the National Health and Nutrition Examination Survey (NHANES) 2005-2018, the association between OBS and the HF prevalence was analyzed by weighted logistic regression and restricted cubic splines (RCS). Subgroup and sensitivity analyses assessed the stability of the results. RESULTS: The prevalence of HF in the cohort of 6238 older adults was 5.55 %. Compared to the lowest quintile, the adjusted ORs for HF in the highest quintile of OBS and lifestyle OBS were 0.57 (95 % CI: 0.33,0.97) and 0.21 (95 %CI: 0.09,0.50), respectively. The association between OBS and HF prevalence remained stable across different models and subgroups. RCS revealed a potential inflection point. Sensitivity analysis validated the negative association between OBS and HF prevalence, and the correlation analysis between OBS and serum γ-glutamyltransferase (γ-GGT) confirmed the reliability of the study design. CONCLUSION: The OBS is negatively associated with HF prevalence in older adults, and may help prevent HF in this population.

A Multicomponent Nucleic Acid Enzyme-Cleavable Quantum Dot Nanobeacon for Highly Sensitive Diagnosis of Tuberculosis with the Naked Eye.

Clinical tuberculosis (TB) screening and diagnosis are crucial for controlling the spread of this life-threatening infectious disease. In this work, a novel, rapid, and simple colorimetric detection platform for TB was developed based on a quantum dot-based nanobeacon (QD-NB) and multicomponent nucleic acid enzyme (MNAzyme). In the presence of target DNA (IS1081 gene fragment), the recombinase polymerase amplification (RPA) was performed and the amplicons were chemically DNA-denatured and then subjected to MNAzyme reaction. RNA-cleaving MNAzyme assembly included the recognition of target DNA and hybridization with a QD-NB fluorescence probe. Under the addition of Mg2+, the RNA-containing QD-NB as a cleavable substrate could be broken into two DNA fragments, leading to green fluorescence release due to their departure from a black hole quencher (BHQ2). The TB detection could be achieved with the naked eye under a portable and inexpensive UV flashlight. Our results demonstrated that QD-NB-based MNAzyme colorimetric assays improved the detection sensitivity by 1 order of magnitude compared with the detection using RPA. The limit of detection (LOD) of the visual reading was as low as 2 copies/µL (3.3 amol/L). Excellent specificity and reproducibility could also be achieved. Furthermore, the practical application of the colorimetric method for TB diagnosis was verified by 36 clinical TB patients and 20 healthy individuals. The developed QD-NB-based MNAzyme colorimetric assays provided a rapid, convenient, sensitive, and accurate alternative for clinical TB screening and diagnosis.

Alteration of Gut Microbiota in Individuals at High-Risk for Rheumatoid Arthritis Associated With Disturbed Metabolome and the Initiation of Arthritis Through the Triggering of Mucosal Immunity Imbalance.

OBJECTIVE: In this study, we aimed to decipher the gut microbiome (GM) and serum metabolic characteristic of individuals at high risk for rheumatoid arthritis (RA) and to investigate the causative effect of GM on the mucosal immune system and its involvement in the pathogenesis of arthritis. METHODS: Fecal samples were collected from 38 healthy individuals and 53 high-risk RA individuals with anti-citrullinated protein antibody (ACPA) positivity (Pre-RA), 12 of 53 Pre-RA individuals developed RA within 5 years of follow-up. The differences in intestinal microbial composition between the healthy controls and Pre-RA individuals or among Pre-RA subgroups were identified by 16S ribosomal RNA sequencing. The serum metabolite profile and its correlation with GM were also explored. Moreover, antibiotic-pretreated mice that received GM from the healthy control or Pre-RA groups were then evaluated for intestinal permeability, inflammatory cytokines, and immune cell populations. Collagen-induced arthritis (CIA) was also applied to test the effect of fecal microbiota transplantation (FMT) from Pre-RA individuals on arthritis severity in mice. RESULTS: Stool microbial diversity was lower in Pre-RA individuals than in healthy controls. The bacterial community structure and function significantly differed between healthy controls and Pre-RA individuals. Although there were differences to some extent in the bacterial abundance among the Pre-RA subgroups, no robust functional differences were observed. The metabolites in the serum of the Pre-RA group were dramatically different from those in the healthy controls group, with KEGG pathway enrichment of amino acid and lipid metabolism. Moreover, intestinal bacteria from the Pre-RA group increased intestinal permeability in FMT mice and zonula occludens-1 expression in the small intestine and Caco-2 cells. Moreover, Th17 cells in the mesenteric lymph nodes and Peyer's patches were also increased in mice receiving Pre-RA feces compared to healthy controls. The changes in intestinal permeability and Th17-cell activation prior to arthritis induction enhanced CIA severity in PreRA-FMT mice compared with HC-FMT mice. CONCLUSION: Gut microbial dysbiosis and metabolome alterations already occur in individuals at high risk for RA. FMT from preclinical individuals triggers intestinal barrier dysfunction and changes mucosal immunity, further contributing to the development of arthritis.

A DNAzyme dual-feedback autocatalytic exponential amplification biocircuit for microRNA imaging in living cells.

Autocatalytic biocircuit are powerful tools for analysing intracellular biomarkers, but these tools are constrained by limitations in amplification capacity and intracellular delivery efficiency. In this work, we developed a DNAzyme-based dual-feedback autocatalytic exponential amplification biocircuit sustained by a honeycomb MnO2 nanosponge (EDA2@hMNS) for live-cell imaging of intracellular low-abundance microRNAs (miRNA). The EDA2 biocircuit comprises a blocked DNAzyme (b-DNAzyme), a Fuel strand and a Substrate strand. In the EDA2 biocircuit, target miRNAs are recycled and feedback for rounds of DNAzymatic amplification, and the DNAzymatic reactions continuously generate target miRNA analogues for dual-feedback to achieve multiple parallel cascade DNAzymatic reactions that improve amplification capacity substantially. In addition, the hMNS ensures high loading and delivery efficiency of biocircuit probes into living cells and also provides sufficient Mn2+ DNAzyme cofactor from in situ decomposition by intracellular glutathione (GSH). The EDA2@hMNS realized a detection limit of 17 pM, which is 288-fold lower than the b-DNAzyme lacking the DNAzymatic amplification. These results demonstrate the great promise for this critical tool in analysing low-abundance biomarkers and cancer diagnostics.

Engineering an endonuclease-assisted rolling circle amplification synergistically catalyzing hairpin assembly mediated fluorescence platform for miR-21 detection.

As one of the earliest miRNAs discovered in the human genome, miRNA-21 can provide vital information for the early diagnosis, drug treatment, and prognosis of cancers. Herein, we construct a fast, time-saving fluorescence detection system for miRNA-21 detection by coalescing the improved endonuclease-mediated rolling circle amplification with catalytic hairpin assembly (RCA-NESA-CHA). Firstly, the target miRNA cyclized the padlock, initiating rolling circle amplification (RCA) and extending a long-concatenated DNA. The modified Protector bonded with the long-strand DNA to generate an endonuclease-specific site and trigger the nicking process. Finally, DNA products with repetitive sequences not only recombined with the padlock to reactivate a new recycle of RCA but also triggered the catalytic hairpin assembly to form the H1-H2 complex, realizing the cooperative amplification of the signal. In this system, RCA-NESA and CHA were integrated into one step, which essentially simplifies the sensing process. Moreover, the introduction of the Protector would inhibit the extension reaction caused by the combination of the padlock and the RCA products, slowing down the non-specific reaction time and improving the sensitivity of the fluorescence detection system. Under the optimal experimental conditions, the fluorescence system achieved a limit-of-detection of 0.025 amol miR-21 in a 40 µL sample and successfully applied to miR-21 detection in serum samples from breast cancer patients, showing good agreement with the results of RT-PCR, which exhibited great potential in biomedical research and clinical diagnosis.

Down-regulating Interleukin-22/Interleukin-22 binding protein axis promotes inflammation and aggravates diet-induced metabolic disorders.

The prevalence of metabolic diseases has become a severe public health problem. Previously, we reported that Interleukin-22 (IL-22) was independently associated with type 2 diabetes mellitus and cardiovascular disease, and could protect endothelial cells from glucose- and lysophosphatidylcholine-induced injury. The activity of IL-22 is strongly regulated by IL-22-binding protein (IL-22BP). The aim of this investigation was to determine the effect of IL-22/IL-22BP axis on glucolipid metabolism. Serum IL-22 and IL-22BP expression in metabolic syndrome (MetS) patients and healthy controls was examined. IL-22BP-knockout (IL-22ra2-/-) and wild-type (WT) mice were fed with control diet (CTD) and high-fat diet (HFD) for 12 weeks. The IL-22 related pathway expression, the glucolipid metabolism, and inflammatory markers in mice were examined. Serum IL-22 and IL-22BP levels were found significantly increased in MetS patients (p < 0.001). IL-22BP deficiency down-regulated IL-22-related pathway, aggravated glucolipid metabolism disorder, and promoted inflammation in mice. Collectively, this work deepens the understanding of the relationship between IL-22/IL-22BP axis and metabolism disorders, and identified that down-regulation of IL-22/IL-22BP axis promotes metabolic disorders in mice.