Hexokinase Is an Innate Immune Receptor for the Detection of Bacterial Peptidoglycan.

Degradation of Gram-positive bacterial cell wall peptidoglycan in macrophage and dendritic cell phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates processing and secretion of interleukin (IL)-1ß and IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl dipeptide component in the cytosol by NOD2, we report here that NLRP3 inflammasome activation is caused by release of N-acetylglucosamine that is detected in the cytosol by the glycolytic enzyme hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria outer membranes, and we found that this is sufficient to activate the NLRP3 inflammasome. In addition, we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and localization induce inflammasome activation. While previous studies have demonstrated that signaling by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic enzyme can act as a pattern recognition receptor. PAPERCLIP.

Universal Signal Switch Based on a Mesostructured Silica Xerogel-Confined ECL Polymer for Epigenetic Quantification.

The development of a highly accurate electrochemiluminescence (ECL) signal switch to avoid nonspecific stimulus responses is currently a significant and challenging task. Here, we constructed a universal signal switch utilizing a luminophore-quencher pair of mesostructured silica xerogel-confined polymer and gold nanoparticles (Au NPs) that can accurately detect low-abundance epigenetic markers in complex sample systems. Notably, the ECL polymer encapsulated in mesostructured silica xerogel acts as a luminophore, which demonstrated a highly specific dependence on the Au NPs-mediated energy transfer quenching. To demonstrate the feasibility, we specifically labeled the 5-hydroxymethylcytosine (5hmC) site on the random sequence using a double-stranded (dsDNA) tag that was skillfully designed with the CRISPR/Cas12a activator and recombinant polymerase amplification (RPA) template. After amplification by RPA, a large amount of dsDNA tag was generated as the activator to initiate the trans-cleavage activity of CRISPR/Cas12a and subsequently activate the signal switch, allowing for precise quantification of 5hmC. The ECL signal switch improves the stability of the luminophore and prevents nonspecific stimulus responses, providing a new paradigm for constructing high-precision biosensors.

AND Logic Gate-Regulated DNAzyme Nanoflower for Monitoring the Activity of Multiple DNA Repair Enzymes.

Despite the progress that has been made in diverse DNA-based nanodevices to in situ monitor the activity of the DNA repair enzymes in living cells, the significance of improving both the sensitivity and specificity has remained largely neglected and understudied. Herein, we propose a regulatable DNA nanodevice to specifically monitor the activity of DNA repair enzymes for early evaluation of cancer mediated by genomic instability. Concretely, an AND logic gate-regulated DNAzyme nanoflower was rationally designed by the self-assembly of the DNA duplex modified with both apurinic/apyrimidinic (AP) site and methyl lesion site. The DNAzyme nanoflower could be reconfigured under the repair of AP sites and O6-methylguanine sites by apurinic/apyrimidinic endonuclease 1 (APE1) and O6-methylguanine methyltransferase (MGMT) to produce a fluorescent signal, realizing the sensitive monitoring of the activity of APE1 and MGMT. Compared to the free DNAzyme duplex, the fluorescent response of the DNAzyme nanoflower increased by 60%, due to the effective enrichment of the DNA probes by the nanoflower structure. More importantly, we have demonstrated that the dual-enzyme activated strategy allows imaging of specific cancer cells in the AND logic gate manner using MCF-7 as a cancer cell model, improving the specificity of cancer cell imaging. This AND logic gate-regulated multifunctional DNAzyme nanoflower provides a simple tool for simultaneously visualizing multiple DNA repair enzymes, holding great potential in early clinical diagnosis and drug discovery.

Synthetic MRI and amide proton transfer-weighted MRI for differentiating between benign and malignant sinonasal lesions.

OBJECTIVES: To explore the value of the synthetic MRI (SyMRI), combined with amide proton transfer-weighted (APTw) MRI for quantitative and morphologic assessment of sinonasal lesions, which could provide relative scale for the quantitative assessment of tissue properties. METHODS: A total of 80 patients (31 malignant and 49 benign) with sinonasal lesions, who underwent the SyMRI and APTw examination, were retrospectively analyzed. Quantitative parameters (T1, T2, proton density (PD)) and APT % were obtained through outlining the region of interest (ROI) and comparing the two groups utilizing independent Student t test or a Wilcoxon test. Receiver operating characteristic curve (ROC), Delong test, and logistic regression analysis were performed to assess the diagnostic efficiency of one-parameter and multiparametric models. RESULTS: SyMRI-derived mean T1, T2, and PD were significantly higher and APT % was relatively lower in benign compared to malignant sinonasal lesions (p < 0.05). The ROC analysis showed that the AUCs of the SyMRI-derived quantitative (T1, T2, PD) values and APT % ranged from 0.677 to 0.781 for differential diagnosis between benign and malignant sinonasal lesions. The T2 values showed the best diagnostic performance among all single parameters for differentiating these two masses. The AUCs of combined SyMRI-derived multiple parameters with APT % (AUC = 0.866) were the highest than that of any single parameter, which was significantly improved (p < 0.05). CONCLUSION: The combination of SyMRI and APTw imaging has the potential to reflect intrinsic tissue characteristics useful for differentiating benign from malignant sinonasal lesions. CLINICAL RELEVANCE STATEMENT: Combining synthetic MRI with amide proton transfer-weighted imaging could function as a quantitative and contrast-free approach, significantly enhancing the differentiation of benign and malignant sinonasal lesions and overcoming the limitations associated with the superficial nature of endoscopic nasal sampling. KEY POINTS: • Synthetic MRI and amide proton transfer-weighted MRI could differentiate benign from malignant sinonasal lesions based on quantitative parameters. • The diagnostic efficiency could be significantly improved through synthetic MRI + amide proton transfer-weighted imaging. • The combination of synthetic MRI and amide proton transfer-weighted MRI is a noninvasive method to evaluate sinonasal lesions.

The tightest self-assembled ruthenium metal-organic framework combined with proximity hybridization for ultrasensitive electrochemiluminescence analysis of paraquat.

Metal-organic frameworks (MOFs), as porous materials, have great potential for exploring high-performance electrochemiluminescence (ECL) probes. However, the constrained applicability of MOFs in the realm of ECL biosensing is primarily attributed to their inadequate water stability, which consequently impairs the overall ECL efficiency. Herein, we developed a competitive ECL biosensor based on a novel tightest structural ruthenium-based organic framework emitter combining the proximity hybridization-induced catalytic hairpin assembly (CHA) strategy and the quenching effect between the Ru-MOF and ferrocene for detecting paraquat (PQ). Through a simple hydrothermal synthesis strategy, ruthenium and 2,2'-bipyrimidine (bpm) are head-to-head self-assembled to obtain a novel tightest structural Ru-MOF. Due to the metal-ligand charge-transfer (MLCT) effect between ruthenium and the bpm ligand and the connectivity between the internal chromophore units, the Ru-MOF exhibits strong ECL emissions. Meanwhile, the coordination-driven Ru-MOF utilizes strong metal-organic coordination bonds as building blocks, which effectively solves the problem of serious leakage of chromophores caused by water solubility. The sensitive analysis of PQ is realized in the range of 1 pg/mL to 1 ng/mL with a detection limit of 0.352 pg/mL. The tightest structural Ru-MOF driven by the coordination of ruthenium and bridging ligands (2,2'-bipyrimidine, bpm) provides new horizons for exploring high-performance MOF-based ECL probes for quantitative analysis of biomarkers.

GATA6 is a regulator of sinus node development and heart rhythm.

The sinus node (SAN) is the primary pacemaker of the human heart, and abnormalities in its structure or function cause sick sinus syndrome, the most common reason for electronic pacemaker implantation. Here we report that transcription factor GATA6, whose mutations in humans are linked to arrhythmia, is highly expressed in the SAN and its haploinsufficiency in mice results in hypoplastic SANs and rhythm abnormalities. Cell-specific deletion reveals a requirement for GATA6 in various SAN lineages. Mechanistically, GATA6 directly activates key regulators of the SAN genetic program in conduction and nonconduction cells, such as TBX3 and EDN1, respectively. The data identify GATA6 as an important regulator of the SAN and provide a molecular basis for understanding the conduction abnormalities associated with GATA6 mutations in humans. They also suggest that GATA6 may be a potential modifier of the cardiac pacemaker.

Ultraflat Graphene Oxide Membranes with Newton-Ring Prepared by Vortex Shear Field for Ion Sieving.

The wrinkles on graphene oxide (GO) membranes have unique properties; however, they interfere with the mass transfer of interlayer channels, posing a major challenge in the development of wrinkle-free GO membranes with smooth channels. In this study, the wrinkles on GO were flattened using vortex shear to tightly stack them into ultraflat GO membranes with Newton's ring interference pattern, causing hydrolysis of the lipid bonds in the wrinkles and an increase in the number of oxygen-containing groups. With increasing flatness, the interlayer spacing of the GO membranes decreased, improving the stability of the interlayer structure, the flow resistance of water through the ultraflat interlayer decreased, and the water flux increased 3-fold. Importantly, the selectivity for K+/Mg2+ reached approximately 379.17 in a real salt lake. A novel concept is proposed for the development of new membrane preparation methods. Our findings provide insights into the use of vortex shearing to flatten GO.

Insights of Life Molecules' Dynamic Distribution in Live Cells via Sequence-Structure Bispecific Fluorescent RNA.

Life molecules' distributions in live systems construct the complex dynamic reaction networks, whereas it is still challenging to demonstrate the dynamic distributions of biomolecules in live systems. Herein, we proposed a dynamic analysis strategy via sequence-structure bispecific RNA with state-adjustable molecules to monitor the dynamic concentration and spatiotemporal localization of these biomolecules in live cells based on the new insight of fluorescent RNA (FLRNA) interactions and their mechanism of fluorescence enhancement. Typically, computer-based nucleic acid-molecular docking simulation and molecular theoretical calculation have been proposed to provide a simple and straightforward method for guiding the custom-design of FLRNA. Impressively, a novel FLRNA with sequence and structure bispecific RNA named as a structure-switching aptamer (SSA) was introduced to monitor the real-time concentration and spatiotemporal localization of biomolecules, contributing to a deeper insight of the dynamic monitoring and visualization of biomolecules in live systems.

Engineering Molecular Emission Centers of Carbon Dots to Boost the Electrochemiluminescence for the Detection of Cancer Cells.

Despite the fact that electrochemiluminescent (ECL) performance of carbon dots (CDs) could be improved by modulating their surface defects, they are still restricted by inferior controllability and poor reproducibility. In this work, we disclosed a new approach for synthesizing luminescent groups rich in CDs (Lu-CDs) by engineering the luminol as molecular emission centers into the CDs, which exhibited an 80-fold stronger ECL intensity at an ECL onset potential of 0.6 V than the CDs without pre-implanted luminol. Different from the significant deviation between the ECL and fluorescence emission of other surface state-dominated CDs, the ECL emission of Lu-CDs was nearly consistent with its fluorescence emission at 465 nm, which was defined as the molecular emission dominated-ECL CDs herein. To prove this principle, the Lu-CDs were employed to construct an ECL biosensor for MCF-7 cell analysis based on the cell direct recognition and amplification strategy, which made the MCF-7 cells as nanomachines via specific binding with aptamer signal probes on the DNA triangular scaffold. The proposed biosensor displayed a wide detection range from 101 to 104 cell mL-1 and a low detection limit of 8.91 cells mL-1. Overall, this work not only presents a new strategy for preparing CDs with high controllability and excellent reproducibility but also provides a platform for tumor cell sensing.

Identification and Quantification of 5-Methylcytosine and 5-Hydroxymethylcytosine on Random DNA Sequences by a Nanoconfined Electrochemiluminescence Platform.

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are two of the most abundant epigenetic marks in mammalian genomes, and it has been proven that these dual epigenetic marks give a more accurate prediction of recurrence and survival in cancer than the individual mark. However, due to the similar structure and low expression of 5mC and 5hmC, it is challenging to distinguish and quantify the two methylation modifications. Herein, we employed the ten-eleven translocation family dioxygenases (TET) to convert 5mC to 5hmC via a specific labeling process, which realized the identification of the two marks based on a nanoconfined electrochemiluminescence (ECL) platform combined with the amplification strategy of a recombinase polymerase amplification (RPA)-assisted CRISPR/Cas13a system. Benefiting from the TET-mediated conversion strategy, a highly consistent labeling pathway was developed for identifying dual epigenetic marks on random sequence, which reduced the system error effectively. The ECL platform was established via preparing a carbonized polymer dot embedded SiO2 nanonetwork (CPDs@SiO2), which exhibited higher ECL efficiencies and more stable ECL performance compared to those of the scattered emitters due to the nanoconfinement-enhanced ECL effect. The proposed bioanalysis strategy could be employed for the identification and quantification of 5mC and 5hmC in the range from 100 aM to 100 pM, respectively, which provides a promising tool for early diagnosis of diseases associated with abnormal methylation.

Epigenetic Quantification of 5-Hydroxymethylcytosine Signatures via Regulatable DNAzyme Motor Triggered by Strand Displacement Amplification.

DNA methylation predominantly occurs within the CpG dinucleotide, which is the main epigenetic form of gene expression regulation in mammals. Genomic DNA with CpG sites has different sequence lengths and complex secondary structures, resulting in the complexity and diversity of the samples. Therefore, highly efficient quantification of DNA methylation in complex samples remains challenging. Herein, the regulatable DNAzyme motor triggered by strand displacement amplification (SDA) was designed to quantify 5-hydroxymethylcytosine (5hmC) signatures as a model. Briefly, the 5hmC sites as primary target were specifically labeled with DNA primers and converted into a large number of single-stranded DNA (secondary target) via the SDA reaction which could activate the DNAzyme motor. With the increase of secondary target, the DNAzyme motor gradually recovered its activity and could continuously cleave the track strands labeled quenching probes, causing electrochemiluminescence signal recovery and detection limit down to 0.49 fM for 5hmC. This strategy provides a new route to quantify natural base modifications in DNA and would hold promising potential for the early diagnosis of cancer and other diseases related to 5hmC.

Identifying 5-Hydroxymethylcytosine without Sequence Specificity Using MOF-Derived MnOxSy Nanoflowers for Boosting Electrochemiluminescence.

It is universally recognized that the quantification of DNA hydroxymethylation at random gene sequences still remains challenging. Herein, the highly sensitive identifying strategy of 5-hydroxymethylcytosine (5-hmC) without sequence specificity was achieved with a novel electrochemiluminescence (ECL) biosensor, which deftly integrated metal-organic framework (MOF)-derived amorphous MnOxSy nanoflowers (MnOxSy NFs) as a bifunctional co-reaction accelerator and cross-shaped DNA tracks as a well-regulated signal switch. Specifically, the target recognition process of 5-hmC was performed through specific chemical modification, where the hydroxymethyl sites were first aminated and then labeled with a 5'-carboxyl-functioned DNA walker, thus forming the target labeled DNA walker (5-ghmC-walker). Subsequently, the cross-shaped DNA tracks were ingeniously designed to endow the 5-ghmC-walker with continuous mechanical motion due to the long periodic linear alignment structure and well-regulated highly ordered interfaces. Furthermore, as a bifunctional co-reaction accelerator synthesized by in situ Mn-MOF template-sacrificing strategy, the MnOxSy NFs could promote the reduction of both dissolved O2 and S2O82-, remarkably boosting the ECL intensity of a peroxydisulfate (S2O82-) solution by 5.2 times compared to the pure S2O82- solution. Benefiting from specific target recognition and a dual-pathway strategy for boosting ECL, the proposed ECL platform can quantify 5-hmC with a wide linear range of 1 fM-1 nM and a low detection limit of 0.29 fM. This simple, highly sensitive strategy without sequence specificity provides a powerful platform for 5-hmC detection in the epigenetic study and disease pathogenesis.

Discrimination between Cancer Cells and DNA-Damaged Cells: Pre-miRNA Region Recognition Based on Hyperbranched Hybrid Chain Reaction Amplification for Simultaneous Sensitive Detection and Imaging of miRNA and Pre-miRNA.

Herein, a novel region recognition of precursor microRNA (Pre-miRNA) based on hyperbranched hybrid chain reaction (HB-HCR) amplification was constructed to effectively eliminate the interference of Pre-miRNA to the mature microRNA (miRNA) by establishing the linear mapping relation between the two fluorescence signals produced by the miRNA sequence in the Pre-miRNA and Pre-miRNA residues to first realize simultaneous sensitive detection of Pre-miRNA and miRNA as well as highly sensitive imaging of intracellular Pre-miRNA and miRNA, which solves one main challenge of in vitro tumor disease diagnostics: inaccurate detection of tumor-induced miRNA changes. Impressively, this strategy easily distinguishes cancer cells from normal cells and DNA-damaged cells by the difference in miRNA and Pre-miRNA expression, which provides an innovative approach for accurate clinical diagnosis of cancer and precise treatment of prognosis.

Conductive Covalent Organic Frameworks with Conductivity- and Pre-Reduction-Enhanced Electrochemiluminescence for Ultrasensitive Biosensor Construction.

Covalent organic frameworks (COFs) have attracted widespread attention in the electrochemiluminescence (ECL) field owing to their high load capacity of ECL luminophores and porous structures, but their ECL performance is still limited by the intrinsic poor conductivity (generally <10-8 S m-1). To address this shortcoming, we used 2,3,6,7,10,11-hexaaminotriphenylene (HATP) and 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) to synthesize a conductive COF (HHTP-HATP-COF, conductivity = 3.11 × 10-4 S m-1). Compared with HATP, HHTP, and low-conductive HHTP-DABZ-COF, HHTP-HATP-COF exhibited superior ECL performance, not only because HHTP-HATP-COF possessed massive ECL luminophores but also because its conductive porous framework accelerated charge transport in the whole framework and improved the utilization ratio of ECL luminophores. More interestingly, the ECL intensity of the HHTP-HATP-COF/S2O82- system was further improved after pre-reduction electrolysis due to the accumulation of HHTP-HATP-COF cation radicals. The experimental results showed that the ECL intensity of the HHTP-HATP-COF/S2O82- system after pre-reduction was about 1.64-, 3.96-, 6.88-, and 8.09-fold higher than those of HHTP-HATP-COF/S2O82-, HHTP-DABZ-COF/S2O82-, HHTP/S2O82-, and HATP/S2O82- systems, respectively. Considering the superior ECL property of the HHTP-HATP-COF/S2O82- system after pre-reduction, it was used as a high-efficient ECL beacon together with an aptamer/protein proximity binding-induced three-dimensional bipedal DNA walker to construct an ultrasensitive biosensor for thrombin detection, which displayed broad linearity (100 aM to 1 nM) with a detection limit of 62.1 aM. Overall, the work offered effective ways to increase ECL performance by the enhancement of conductivity and by the pre-reduction, proposing new ideas to design high-efficiency COF-based ECL materials and endowing conductive COFs with ECL biosensor application for the first time.

Ultrasensitive Fluorescence Detection and Imaging of MicroRNA in Cells Based on a Hyperbranched RCA-Assisted Multiposition SDR Signal Amplification Strategy.

Herein, an innovative fluorescent sensor was courageously empoldered for precise and ultrasensitive detection and imaging of target miRNA-21 through the agency of a dextrous target-motivated polymerization/nicking DNA nanomachineries based on a hyperbranched rolling circle amplification (HB-RCA)-assisted multiposition strand displacement reaction (SDR) signal amplification approach. Impressively, the ingenious technique not only realized target recycling via polymerization/nicking DNA nanomachineries but also involved HB-RCA amplification induced by the released transformation target as the repeated signal amplification. Most importantly, HB-RCA was firstly exploited to remarkably increase the local concentration and collision efficiency of the templates and primers, which could simultaneously generate multiple repeated DNA sequences as initiators to supply substantial banding positions for SDR, removing the massive fluorescence-resonance-energy-transfer (FRET) DNA duplexes from the repeated DNA sequences to remarkably avert the self-quenching of the fluorescence signal due to self-aggregation caused by the winding of the HB-RCA products, thereby leading to a conspicuously improved signal amplification multiplier. As proof of concept, an ingenious technique effectively and accurately distinguished target miRNA-21 even with a tiny change in cells compared to the conventional fluorescence in situ hybridization (FISH) approach. Moreover, the proposed fluorescent method apparently discriminated drug-manipulative miRNA expression level abnormities. Therefore, the proposed cascade nucleic acid amplification strategy could provide an epigamic avenue for ultrasensitive imaging of diverse biomarkers, which help researchers to better study the tumor mechanism, thereby unambiguously increasing cancer cure rates and reducing the risk of recurrence.

Three-in-One System Based on Multi-Path Nucleic Acid Amplification for Bioanalysis of Pre-miRNA/miRNA and Dicer Activity.

Today, a lot of attention is being paid to the pre-miRNAs/miRNAs or activity of Dicer due to their important functions in various physiological processes. Especially, the intrinsic relationship among these associated targets is of significant importance for more in-depth research on the mechanism of disease formation and early diagnosis. Herein, a strategy for simultaneous bioanalysis of miRNAs/pre-miRNAs and Dicer enzyme based on the self-designed multi-path nucleic acid amplification technology was proposed. Typically, in the presence of pre-miRNA-155, it can hybridize with Helper to generate a structure with two new toeholds, one of which could react with H1, H2, and H3, performing a modified CHA reaction with obvious fluorescence responses of FAM, and another of which could hybridize with H4, H5, and H6 to construct the [H4-H5-H6]n DNA nanosphere with obvious fluorescence responses of Cy5. Similarly, miRNA-155 could just hybridize with H1, H2, and H3 to generate the same modified CHA reaction with obvious fluorescence responses of FAM. Due to the successful multi-path nucleic acid amplification, the proposed bioanalysis strategy could be successfully employed for miRNA-155 and pre-miRNA-155 analysis in the range from 500 pM to 100 nM and 1 to 300 nM, respectively. The proposed strategy could be applied to explore another inter-related nucleic acid relationship also, providing great potential in bioanalysis of various nucleic acids.

One-Step Digital Droplet Auto-Catalytic Nucleic Acid Amplification with High-Throughput Fluorescence Imaging and Droplet Tracking Computation.

Digital droplet technology has emerged as a powerful new tool for biomarker analysis. Temperature cycling, enzymes, and off-chip processes are, nevertheless, always required. Herein, we constructed a digital droplet auto-catalytic hairpin assembly (ddaCHA) microfluidic system to achieve digital quantification of single-molecule microRNA (miRNA). The designed continuous chip integrates droplet generation, incubation, and fluorescence imaging on the chip, avoiding the requirement for extra droplet re-collection and heating operations. Clearly, the digital readout was obtained by partitioning miRNA into many individual pL-sized small droplets in which the target molecule is either present ("positive") or absent ("negative"). Importantly, the suggested enzyme-free auto-catalytic hairpin assembly (aCHA) in droplets successfully mitigated the effects of the external environment and thermal cycling on droplets, and its reaction rate is significantly superior to that of traditional CHA. We got excellent sensitivity with a linear correlation from 1 pM to 10 nM and a detection limit of 0.34 pM in the fluorescence spectrum section, as well as high selectivity to other miRNAs. Furthermore, the minimum target concentration could be reduced to 10 fM based on the high-throughput tracking computation of fluorescent droplets with a self-developed Python script, and the fluorescence intensity distribution agreed well with the theoretical value, demonstrating that it is feasible to detect miRNA efficiently and accurately, which has great potential applications in clinical diagnostics and biochemical research.

Pyrene-Based Hydrogen-Bonded Organic Frameworks as New Emitters with Porosity- and Aggregation-Induced Enhanced Electrochemiluminescence for Ultrasensitive MicroRNA Assay.

Exploring new electrochemiluminescence (ECL) luminophores with strong ECL emission is highly desirable for developing ultrasensitive ECL sensors. Herein, a pyrene-based hydrogen-bonded organic framework (Py-HOF) featuring prominent ECL performance was prepared by utilizing 1,3,6,8-tetrakis(p-benzoic acid) pyrene (H4TBAPy) with an aggregation-induced enhanced emission (AIEE) property as a building block, exhibiting a stronger ECL emission than those of H4TBAPy monomers, H4TBAPy aggregates, the low-porosity Py-HOF-210 °C and Py-HOF-180 °C. We have coined the term "the porosity- and aggregation-induced enhanced ECL (PAIE-ECL)" for this intriguing phenomenon. The Py-HOF displayed superb and stable ECL intensity, not only because the luminophore H4TBAPy was assembled into the Py-HOF via four pairs of O-H···O hydrogen bonds, which constrained the intramolecular movements to reduce nonradiative transition, but also because the H4TBAPy in Py-HOF was stacked in a slipped face-to-face mode to form J-aggregates that benefited the ECL enhancement. Furthermore, the high porosity of Py-HOF allowed the enrichment of coreactants and facilitated the migration of ions, electrons, and coreactants, which made it possible for the inner and outer H4TBAPy to be electrochemically excited. Considering the remarkable ECL performance, Py-HOF was first employed as an ECL probe combined with a 3D DNA nanomachine amplification strategy to assemble a hypersensitive "on-off" ECL sensor for the microRNA-141 assay, presenting a satisfactory linear range (100 aM to 1 nM) with a detection limit of 14.4 aM. The PAIE-ECL manifested by Py-HOF provided a bright avenue for the design and synthesis of outstanding HOF-based ECL materials and offered new opportunities for the development of ECL biosensors with excellent sensitivity.

Intermittent fasting reduces neuroinflammation in intracerebral hemorrhage through the Sirt3/Nrf2/HO-1 pathway.

BACKGROUND: Inflammation contributes to the poor prognosis of intracerebral hemorrhage (ICH). Intermittent fasting (IF) has been shown to be protective against inflammation in multiple pathogenic processes. In the present study, we aimed to investigated the beneficial effects of IF in attenuating neuroinflammation and neurological deficits in a mouse model of ICH and to investigate the underlying mechanism. METHODS: ICH was modeled by intrastriatal injection of autologous blood and IF was modeled by every-other-day feeding in male control mice (C57BL/6), mice with and microglia specific knockout Sirt3f/f;Cx3cr1-Cre (Sirt3 cKO), and Sirt3f/f (wild-type) mice. Brain tissues and arterial blood were harvested at 1, 3, 7 and 28 days after ICH for immunohistochemistry analysis of Iba-1, DARPP-32 and HO-1, morphological analysis by HE staining and inflammatory factor release tests by ELISA. Neurological functions were approached by corner test and cylinder test. Fluorescent double-labeled staining of Iba-1 with CD16, Arg1 or Sirt3 was used to provide direct image of co-expression of these molecules in microglia. TUNEL, cleaved caspase-3 and Nissl staining was performed to evaluate cellular injuries. RESULTS: IF alleviated neurological deficits in both acute and chronic phases after ICH. Morphologically, IF enhanced hematoma clearance, reduced brain edema in acute phase and attenuated striatum atrophy in chronic phase. In addition, IF decreased the numbers of TUNEL+ cells and increased Nissl+ neuron number at day 1, 3 and 7 after ICH. IF suppressed CD16+Iba-1+ microglia activation at day 3 after ICH and reduced inflammatory releases, such as IL-1ß and TNF-α. The above effects of IF were attenuated by microglia Sirt3 deletion partly because of an inhibition of Nrf2/HO-1 signaling pathway. Interestingly, IF increased Iba-1+ microglia number at day 7 which mainly expressed Arg1 while decreased the proinflammatory factor levels. In mice with microglia-specific Sirt3 deletion, the effects of IF on Iba-1+ microglia activation and anti-inflammatory factor expressions were attenuated when compared with wild-type Sirt3f/f mice. CONCLUSIONS: IF protects against ICH by suppressing the inflammatory responses via the Sirt3/Nrf2/HO-1 pathway.

Innate Immune Nod1/RIP2 Signaling Is Essential for Cardiac Hypertrophy but Requires Mitochondrial Antiviral Signaling Protein for Signal Transductions and Energy Balance.

BACKGROUND: Cardiac hypertrophy is a key biological response to injurious stresses such as pressure overload and, when excessive, can lead to heart failure. Innate immune activation by danger signals, through intracellular pattern recognition receptors such as nucleotide-binding oligomerization domain 1 (Nod1) and its adaptor receptor-interacting protein 2 (RIP2), might play a major role in cardiac remodeling and progression to heart failure. We hypothesize that Nod1/RIP2 are major contributors to cardiac hypertrophy, but may not be sufficient to fully express the phenotype alone. METHODS: To elucidate the contribution of Nod1/RIP2 signaling to cardiac hypertrophy, we randomized Nod1-/-, RIP2-/-, or wild-type mice to transverse aortic constriction or sham operations. Cardiac hypertrophy, fibrosis, and cardiac function were examined in these mice. RESULTS: Nod1 and RIP2 proteins were upregulated in the heart after transverse aortic constriction, and this was paralleled by increased expression of mitochondrial proteins, including mitochondrial antiviral signaling protein (MAVS). Nod1-/- and RIP2-/- mice subjected to transverse aortic constriction exhibited better survival, improved cardiac function, and decreased cardiac hypertrophy. Downstream signal transduction pathways that regulate inflammation and fibrosis, including NF (nuclear factor) κB and MAPK (mitogen-activated protein kinase)-GATA4/p300, were reduced in both Nod1-/- and RIP2-/- mice after transverse aortic constriction compared with wild-type mice. Coimmunoprecipitation of extracted cardiac proteins and confocal immunofluorescence microscopy showed that Nod1/RIP2 interaction was robust and that this complex also included MAVS as an essential component. Suppression of MAVS expression attenuated the complex formation, NF κB signaling, and myocyte hypertrophy. Interrogation of mitochondrial function compared in the presence or ablation of MAVS revealed that MAVS serves to suppress mitochondrial energy output and mediate fission/fusion related dynamic changes. The latter is possibly linked to mitophagy during cardiomyocytes stress, which may provide an intriguing link between innate immune activation and mitochondrial energy balance under stress or injury conditions. CONCLUSIONS: We have identified that innate immune Nod1/RIP2 signaling is a major contributor to cardiac remodeling after stress. This process is critically joined by and regulated through the mitochondrial danger signal adapter MAVS. This novel complex coordinates remodeling, inflammatory response, and mitochondrial energy metabolism in stressed cardiomyocytes. Thus, Nod1/RIP2/MAVS signaling complex may represent an attractive new therapeutic approach toward heart failure.