A Methylation-Gated DNAzyme Circuit for Spatially Controlled Imaging of MicroRNA in Cells and Animals.

Epigenetic modification plays an indispensable role in regulating routine molecular signaling pathways, yet it is rarely used to modulate molecular self-assembly networks. Herein, we constructed a bioorthogonal demethylase-stimulated DNA circuitry (DSC) system for high-fidelity imaging of microRNA (miRNA) in live cells and mice by eliminating undesired off-site signal leakage. The simple and robust DSC system is composed of a primary cell-specific circuitry regulation (CR) module and an ultimate signal-transducing amplifier (SA) module. After the modularly designed DSC system was delivered into target live cells, the DNAzyme of the CR module was site-specifically activated by endogenous demethylase to produce fuel strands for the subsequent miRNA-targeting SA module. Through the on-site and multiply guaranteed molecular recognitions, the lucid yet efficient DSC system realized the reliably amplified in vivo miRNA sensing and enabled the in-depth exploration of the demethylase-involved signal pathway with miRNA in live cells. Our bioorthogonally on-site-activated DSC system represents a universal and versatile biomolecular sensing platform via various demethylase regulations and shows more prospects for more different personalized theragnostics.

Direct reduction of NO into N2 catalyzed by fullerene-supported rhodium clusters.

Catalytic conversion of NO has long been a focus of atmospheric pollution control and diesel vehicle exhaust treatment. Rhodium is one of the most effective metals for catalyzing NO reduction, and understanding the nature of the active sites and underlying mechanisms can help improve the design of Rh-based catalysts towards NO reduction. In this work, we investigated the detailed catalytic mechanisms for the direct reduction of NO to N2 by fullerene-supported rhodium clusters, C60Rh4+, with density functional theory calculations. We found that the presence of C60 facilitates the smooth reduction of NO into N2 and O2, as well as their subsequent desorption, recovering the catalyst C60Rh4+. Such a process fails to be completed by free Rh4+, emphasizing the critical importance of C60 support. We attribute the novel performance of C60Rh4+ to the electron sponge effect of C60, providing useful guidance for designing efficient catalysts for the direct reduction of NO.

Scaling up of a Self-Confined Catalytic Hybridization Circuit for Robust microRNA Imaging.

The precise regulation of cellular behaviors within a confined, crowded intracellular environment is highly amenable in diagnostics and therapeutics. While synthetic circuitry system through a concatenated chemical reaction network has rarely been reported to mimic dynamic self-assembly system. Herein, a catalytic self-defined circuit (CSC) for the hierarchically concatenated assembly of DNA domino nanostructures is engineered. By incorporating pre-sealed symmetrical fragments into the preying hairpin reactants, the CSC system allows the hierarchical DNA self-assembly via a microRNA (miRNA)-powered self-sorting catalytic hybridization reaction. With minimal strand complexity, this self-sustainable CSC system streamlined the circuit component and achieved localization-intensified cascaded signal amplification. Profiting from the self-adaptively concatenated hybridization reaction, a reliable and robust method has been achieved for discriminating carcinoma tissues from the corresponding para-carcinoma tissues. The CSC-sustained self-assembly strategy provides a comprehensive and smart toolbox for organizing various hierarchical DNA nanostructures, which may facilitate more insights for clinical diagnosis and therapeutic assessment.

Lung microbiome: new insights into the pathogenesis of respiratory diseases.

The lungs were long thought to be sterile until technical advances uncovered the presence of the lung microbial community. The microbiome of healthy lungs is mainly derived from the upper respiratory tract (URT) microbiome but also has its own characteristic flora. The selection mechanisms in the lung, including clearance by coughing, pulmonary macrophages, the oscillation of respiratory cilia, and bacterial inhibition by alveolar surfactant, keep the microbiome transient and mobile, which is different from the microbiome in other organs. The pulmonary bacteriome has been intensively studied recently, but relatively little research has focused on the mycobiome and virome. This up-to-date review retrospectively summarizes the lung microbiome's history, composition, and function. We focus on the interaction of the lung microbiome with the oropharynx and gut microbiome and emphasize the role it plays in the innate and adaptive immune responses. More importantly, we focus on multiple respiratory diseases, including asthma, chronic obstructive pulmonary disease (COPD), fibrosis, bronchiectasis, and pneumonia. The impact of the lung microbiome on coronavirus disease 2019 (COVID-19) and lung cancer has also been comprehensively studied. Furthermore, by summarizing the therapeutic potential of the lung microbiome in lung diseases and examining the shortcomings of the field, we propose an outlook of the direction of lung microbiome research.

Activation of transient receptor potential vanilloid 1 ameliorates tau accumulation-induced synaptic damage and cognitive dysfunction via autophagy enhancement.

AIMS: The autophagy-lysosomal pathway is important for maintaining cellular proteostasis, while dysfunction of this pathway has been suggested to drive the aberrant intraneuronal accumulation of tau protein, leading to synaptic damage and cognitive impairment. Previous studies have demonstrated that the activation of transient receptor potential vanilloid 1 (TRPV1) by capsaicin has a positive impact on cognition and AD-related biomarkers. However, the effect and mechanism of TPRV1 activation on neuronal tau homeostasis remain elusive. METHODS: A mouse model of tauopathy was established by overexpressing full-length human tau in the CA3 area. Mice were fed capsaicin diet (0.0125%) or normal diet for 9 weeks. The cognitive ability, synaptic function, tau phosphorylation levels, and autophagy markers were detected. In vitro, capsaicin-induced alterations in cellular autophagy and tau degradation were characterized using two cell models. Besides, various inhibitors were applied to validate the role of TRPV1-mediated autophagy enhancement in tau clearance. RESULTS: We observed that TRPV1 activation by capsaicin effectively mitigates hippocampal tau accumulation-induced synaptic damages, gliosis, and cognitive impairment in vivo. Capsaicin promotes the degradation of abnormally accumulated tau through enhancing autophagic function in neurons, which is dependent on TRPV1-mediated activation of AMP-activated protein kinase (AMPK) and subsequent inhibition of the mammalian target of rapamycin (mTOR). Blocking AMPK activation abolishes capsaicin-induced autophagy enhancement and tau degradation in neurons. CONCLUSION: Our findings reveal that capsaicin-induced TRPV1 activation confers neuroprotection by restoring neuronal tau homeostasis via modulating cellular autophagy and provides additional evidence to support the potential of TRPV1 as a therapeutic target for tauopathies.

Quantitative Proteomic and Phosphoproteomic Analyses Reveal a Role of Death-Associated Protein Kinase 1 in Regulating Hippocampal Synapse.

Death-associated protein kinase 1 (DAPK1) is a stress-responsive calcium/calmodulin (CaM)-regulated serine/threonine protein kinase that is actively involved in stress-induced cell death. The dysregulation of DAPK1 has been established in various neurological disorders such as epilepsy, Alzheimer's disease (AD), and Parkinson's disease (PD). Recent research indicates a synaptic localization of DAPK1 in neurons, suggesting a potential role of DAPK1 in modulating synaptic structure and function. However, the key molecules and pathways underlying the influence of DAPK1 on synapses remain elusive. We utilized quantitative proteomic and phosphoproteomic analyses to compare the differences in protein expression and phosphorylation in hippocampal tissues of wild-type (WT) and DAPK1-knockout (KO) mice. Bioinformatic analysis of differentially expressed proteins and phosphoproteins revealed a preferential enrichment of proteins involved in regulating synaptic function, cytoskeletal structure, and neurotransmission. Gene set enrichment analysis (GESA) highlighted altered presynaptic functions including synaptic vesicle priming and glutamate secretion in KO mice. Besides, we observed that proteins with potential phosphorylation motifs of ERK and DAPK1 were overrepresented among the differential phosphoproteins and were highly enriched in neuronal function-related pathways. Furthermore, Western blot analysis validated differences in the expression of several proteins closely associated with presynaptic organization, dendrites and calcium transmembrane transport between KO and WT mice, further corroborating the potential involvement of DAPK1 in the regulation of synaptic functions. Overall, our data provide molecular evidence to elucidate the physiological links between DAPK1 and neuronal functions and help clarify the role of DAPK1 in the pathogenesis of neurodevelopmental and neurodegenerative diseases.

Death-associated protein kinase 1 phosphorylates MDM2 and inhibits its protein stability and function.

Breast cancer is one of the major malignancies in women, and most related deaths are due to recurrence, drug resistance, and metastasis. The expression of the mouse double minute 2 (MDM2) oncogene is upregulated in breast cancer; however, its regulatory mechanism has yet to be fully elucidated. Herein, we identified the tumor suppressor death-associated protein kinase 1 (DAPK1) as a novel MDM2 regulator by unbiased peptide library screening. DAPK1 is directly bound to MDM2 and phosphorylates it at Thr419. DAPK1-mediated MDM2 phosphorylation promoted its protein degradation via the ubiquitin-proteasome pathway, resulting in upregulated p53 expression. DAPK1 overexpression, but not its kinase activity-deficient form, decreased colony formation and increased doxorubicin-induced cell death; however, DAPK1 knockdown produced the opposite effects in human breast cancer cells. In a xenograft tumorigenesis assay, DAPK1 overexpression significantly reduced tumor formation, whereas inhibition of DAPK1 kinase activity reduced its antitumorigenic effect. Finally, DAPK1 expression was negatively correlated with MDM2 levels in human breast cancer tissues. Thus, these results suggest that DAPK1-mediated MDM2 phosphorylation and its protein degradation may contribute to its antitumorigenic function in breast cancer.

Efficient Intracellular MicroRNA Imaging Based on the Localized and Self-Sustainable Catalytic DNA Assembly Circuit.

Building dynamic biological networks, especially DNA circuits, has provided a powerful prospect for exploring the intrinsic regulation processes of live cells. Nevertheless, for efficient intracellular microRNA analysis, the available multi-component circuits are constrained by their limited operating speed and efficiency due to the free diffusion of reactants. Herein, we developed an accelerated Y-shaped DNA catalytic (YDC) circuit for high-efficiency intracellular imaging of microRNA. By grafting the catalytic hairpin assembly (CHA) reactants into an integrated Y-shaped scaffold, the CHA probes were concentrated in a compact space, thus achieving high signal amplification. Profiting from the spatially confined reaction and the self-sustainably assembled DNA products, the YDC system facilitated reliable and in situ microRNA imaging in live cells. Compared with the homogeneously dispersed CHA reactants, the integrated YDC system could efficiently promote the reaction kinetics as well as the uniform delivery of CHA probes, thus providing a robust and reliable analytical tool for disease diagnosis and monitoring.

A Smart Deoxyribozyme-Programmable Catalytic DNA Circuit for High-Contrast MicroRNA Imaging.

Synthetic catalytic DNA circuits have been recognized as a promising signal amplification toolbox for sensitive intracellular imaging, yet their selectivity and efficiency are always constrained by uncontrolled off-site signal leakage and inefficient on-site circuitry activation. Thus, the endogenously controllable on-site exposure/activation of DNA circuits is highly desirable for achieving the selective imaging of live cells. Herein, an endogenously activated DNAzyme strategy was facilely integrated with a catalytic DNA circuit for guiding the selective and efficient microRNA imaging in vivo. To prevent the off-site activation, the circuitry constitute was initially caged without sensing functions, which could be selectively liberated by DNAzyme amplifier to guarantee the high-contrast microRNA imaging in target cells. This intelligent on-site modulation strategy can tremendously expand these molecularly engineered circuits in biological systems.

Bioartificial liver with reprogrammed hepatocytes ready for prime time.

In this issue, Wang et al.1 provide evidence of the pre-clinical as well as the clinical utility of in vitro-generated directly reprogrammed human hepatocytes in bioartificial liver. This approach will help offer patients a more curative surgical therapy for liver cancer and improve survival rates.

Ablation of Death-Associated Protein Kinase 1 Changes the Transcriptomic Profile and Alters Neural-Related Pathways in the Brain.

Death-associated protein kinase 1 (DAPK1), a Ca2+/calmodulin-dependent serine/threonine kinase, mediates various neuronal functions, including cell death. Abnormal upregulation of DAPK1 is observed in human patients with neurological diseases, such as Alzheimer's disease (AD) and epilepsy. Ablation of DAPK1 expression and suppression of DAPK1 activity attenuates neuropathology and behavior impairments. However, whether DAPK1 regulates gene expression in the brain, and whether its gene profile is implicated in neuronal disorders, remains elusive. To reveal the function and pathogenic role of DAPK1 in neurological diseases in the brain, differential transcriptional profiling was performed in the brains of DAPK1 knockout (DAPK1-KO) mice compared with those of wild-type (WT) mice by RNA sequencing. We showed significantly altered genes in the cerebral cortex, hippocampus, brain stem, and cerebellum of both male and female DAPK1-KO mice compared to those in WT mice, respectively. The genes are implicated in multiple neural-related pathways, including: AD, Parkinson's disease (PD), Huntington's disease (HD), neurodegeneration, glutamatergic synapse, and GABAergic synapse pathways. Moreover, our findings imply that the potassium voltage-gated channel subfamily A member 1 (Kcna1) may be involved in the modulation of DAPK1 in epilepsy. Our study provides insight into the pathological role of DAPK1 in the regulatory networks in the brain and new therapeutic strategies for the treatment of neurological diseases.

Early Predictors of Infected Pancreatic Necrosis in Severe Acute Pancreatitis: Implications of Neutrophil to Lymphocyte Ratio, Blood Procalcitonin Concentration, and Modified CT Severity Index.

BACKGROUND: Infected pancreatic necrosis (IPN) accounts for 30% mortality in severe acute pancreatitis (SAP). Early prediction of IPN occurrence is critical for prophylactic measures to be taken. This study aimed to evaluate the predicting value for IPN of combined markers at early stages of SAP. METHODS: The clinical records of 324 SAP patients admitted within 48 h after disease onset were retrospectively analyzed. As potential predictors, the neutrophil to lymphocyte ratio (NLR), blood procalcitonin (PCT) concentration on the 1st, 4th, and 7th day post admission, as well as modified computerized tomography severity index (MCTSI) on the 5-7th day post admission, were extracted. Correlations between these features with IPN were analyzed using logistic regression, and predictive values were estimated using the receiver operating characteristic curve analyses. RESULTS: NLR, PCT, body mass index, and MCTSI were significantly higher in the IPN group (p < 0.001) compared to the control, among which NLR, PCT, and MCTSI were identified as independent predictors for IPN in logistic regression model. Combination of these parameters yielded significant predicting values with an area under curve of 0.92, sensitivity of 97.2%, and specificity of 77.2% in receiver operating characteristic curve analysis. CONCLUSION: Combination of NLR, PCT, MCTSI might facilitate the prediction of IPN occurrence in SAP patients.

The compact integration of a cascaded HCR circuit for highly reliable cancer cell discrimination.

The accurate identification of multiple biomarkers involved in disease plays a vital role in effectively distinguishing cancer cells from normal cells, facilitating reliable cancer diagnosis. Motivated by this knowledge, we have engineered a compact and clamped cascaded DNA circuit for specifically discriminating cancer cells from normal cells via the amplified multi-microRNA imaging strategy. The proposed DNA circuit combines the traditional cascaded DNA circuit with multiply localized responsive character through the elaboration of two super-hairpin reactants, thus concurrently streamlining the circuit components and realizing localization-intensified cascaded signal amplification. In parallel, the multiple microRNA-stimulated sequential activations of the compact circuit, combined with a handy logic operation, significantly elevated the cell-discriminating reliability. Applications of the present DNA circuit in vitro and in cellular imaging experiments were executed with expected results, therefore illustrating that our DNA circuit is useful for precise cell discrimination and further clinical diagnosis.

Miniature Hierarchical DNA Hybridization Circuit for Amplified Multiplexed MicroRNA Imaging.

Accurate diagnosis requires the development of multiple-guaranteed DNA circuits. Nevertheless, for reliable multiplexed molecular imaging, existing DNA circuits are limited by poor cell-delivering homogeneity due to their cumbersome and dispersive reactants. Herein, we developed a compact-yet-efficient hierarchical DNA hybridization (HDH) circuit for in situ simultaneous analysis of multiple miRNAs, which could be further exploited for specifically discriminating cancer cells from normal ones. By integrating the traditional hybridization chain reaction and catalytic hairpin assembly reactants into two highly organized hairpins, the HDH circuit is fitted with condensed components and multiple response domains, thus permitting the programmable multiple microRNA-guaranteed sequential activations and the localized cascaded signal amplification. The synergistic multi-recognition and amplification features of the HDH circuit facilitate the magnified detection of multiplex endogenous miRNAs in living cells. The in vitro and cellular imaging experimental results revealed that the HDH circuit displayed a reliable sensing performance with high selective cell-identification capacity. We anticipate that this compact design can provide a powerful toolkit for accurate diagnostics and pathological evolution.

Self-Stacking Autocatalytic Molecular Circuit with Minimal Catalytic DNA Assembly.

Isothermal autocatalytic DNA circuits have been proven to be versatile and powerful biocomputing platforms by virtue of their self-sustainable and self-accelerating reaction profiles, yet they are currently constrained by their complicated designs, severe signal leakages, and unclear reaction mechanisms. Herein, we developed a simpler-yet-efficient autocatalytic assembly circuit (AAC) for highly robust bioimaging in live cells and mice. The scalable and sustainable AAC system was composed of a mere catalytic DNA assembly reaction with minimal strand complexity and, upon specific stimulation, could reproduce numerous new triggers to expedite the whole reaction. Through in-depth theoretical simulations and systematic experimental demonstrations, the catalytic efficiency of these reproduced triggers was found to play a vital role in the autocatalytic profile and thus could be facilely improved to achieve more efficient and characteristic autocatalytic signal amplification. Due to its exponentially high signal amplification and minimal reaction components, our self-stacking AAC facilitated the efficient detection of trace biomolecules with low signal leakage, thus providing great clinical diagnosis and therapeutic assessment potential.

Bioorthogonal Disassembly of Hierarchical DNAzyme Nanogel for High-Performance Intracellular microRNA Imaging.

Rolling circle amplification (RCA) enables the facile construction of compact and versatile DNA nanoassemblies which are yet rarely explored for intracellular analysis. This is might be ascribed to the uncontrollable and inefficient probe integration/activation. Herein, by encoding with tandem allosteric deoxyribozyme (DNA-cleaving DNAzyme), a multifunctional RCA nanogel was established for realizing the efficient intracellular microRNA imaging via the successive activation of the RCA-disassembly module and signal amplification module. The endogenous microRNA stimulates the precise degradation of DNA nanocarriers, thus leading to the efficient exposure of RCA-entrapped DNAzyme biocatalyst for an amplified readout signal. Our bioorthogonal DNAzyme disassembly strategy achieved the robust analysis of intracellular biomolecules, thus showing more prospects in clinical diagnosis.

Overexpression of fibroblast growth factor 13 ameliorates amyloid-ß-induced neuronal damage.

Previous studies have shown that fibroblast growth factor 13 is downregulated in the brain of both Alzheimer's disease mouse models and patients, and that it plays a vital role in the learning and memory. However, the underlying mechanisms of fibroblast growth factor 13 in Alzheimer's disease remain unclear. In this study, we established rat models of Alzheimer's disease by stereotaxic injection of amyloid-ß (Aß1-42)-induced into bilateral hippocampus. We also injected lentivirus containing fibroblast growth factor 13 into bilateral hippocampus to overexpress fibroblast growth factor 13. The expression of fibroblast growth factor 13 was downregulated in the brain of the Alzheimer's disease model rats. After overexpression of fibroblast growth factor 13, learning and memory abilities of the Alzheimer's disease model rats were remarkably improved. Fibroblast growth factor 13 overexpression increased brain expression levels of oxidative stress-related markers glutathione, superoxide dismutase, phosphatidylinositol-3-kinase, AKT and glycogen synthase kinase 3ß, and anti-apoptotic factor BCL. Furthermore, fibroblast growth factor 13 overexpression decreased the number of apoptotic cells, expression of pro-apoptotic factor BAX, cleaved-caspase 3 and amyloid-ß expression, and levels of tau phosphorylation, malondialdehyde, reactive oxygen species and acetylcholinesterase in the brain of Alzheimer's disease model rats. The changes were reversed by the phosphatidylinositol-3-kinase inhibitor LY294002. These findings suggest that overexpression of fibroblast growth factor 13 improved neuronal damage in a rat model of Alzheimer's disease through activation of the phosphatidylinositol-3-kinase/AKT/glycogen synthase kinase 3ß signaling pathway.

MiR-191-5p Attenuates Tau Phosphorylation, Aß Generation, and Neuronal Cell Death by Regulating Death-Associated Protein Kinase 1.

Dysregulation of microRNAs has been implicated in diverse diseases, including Alzheimer's disease (AD). MiR-191-5p in plasma/serum has been identified as a novel and promising noninvasive diagnostic biomarker for AD. However, whether miR-191-5p is involved in AD pathogenesis is largely unknown, and its levels in human AD brains are undetermined. Herein, we demonstrated that miR-191-5p downregulated tau phosphorylation at multiple AD-related sites and promoted neurite outgrowth using immunoblotting, immunofluorescence, and neurite outgrowth assays. Moreover, immunoblotting and enzyme-linked immunosorbent assays indicated that miR-191-5p decreased amyloid precursor protein phosphorylation levels and beta-amyloid (Aß) generation. Furthermore, miR-191-5p reduced ceramide-induced neuronal cell death analyzed by trypan blue staining, the in situ cell death detection kit, and Annexin V-FITC/PI flow cytometry. Next, we verified that death-associated protein kinase 1 (DAPK1) was a direct target of miR-191-5p through the dual luciferase reporter assay and confirmed that the effects of miR-191-5p were antagonized by restoration of DAPK1 expression. Finally, the hippocampal miR-191-5p level was found to be decreased in humans with AD compared with controls and was inversely correlated with the DAPK1 expression level. Collectively, these findings suggest that miR-191-5p might exert inhibitory effects on tau phosphorylation, Aß secretion, and neuronal cell death by directly targeting DAPK1, providing an attractive therapeutic option for AD.

Monitoring and modulating a catalytic hybridization circuit for self-adaptive bioorthogonal DNA assembly.

Constructing artificial domino nanoarchitectures, especially dynamic DNA circuits associated with the actuation of biological functions inside live cells, represents a versatile and powerful strategy to regulate the behaviors and fate of various living entities. However, the stepwise operation of conventional DNA circuits always relies on freely diffusing reactants, which substantially slows down their operation rate and efficiency. Herein, a self-adaptive localized catalytic circuit (LCC) is developed to execute the self-sustained bioorthogonal assembly of DNA nanosponges within a crowded intracellular environment. The LCC-generated DNA scaffolds are utilized as versatile templates for realizing the proximity confinement of LCC reactants. Single-molecule-detecting fluorescence correlation spectroscopy (FCS) is used to explore the reaction acceleration of the catalytic circuit. This self-adaptive DNA circuit facilitates the bioorthogonal assembly of highly branched DNA networks for robust and accurate monitoring of miRNA targets. Based on its intriguing and modular design, the LCC system provides a pivotal molecular toolbox for future applications in early disease diagnosis.

Functional Zeolitic Imidazolate Framework for Robust l-Deoxyribozyme-Based Therapy.

Programmable chiral biocatalysis represents a promising therapeutic strategy for its high stereospecific control over various biotransformations (e.g., chiral Aß isomerization) of living entities yet is rarely explored. With an extraordinary resistance to nuclease digestion, the non-natural left-handed deoxyribozyme (l-DNAzyme) therapy is constrained by inefficient delivery/release and insufficient cofactors supply. Herein, an efficient adenosine triphosphate (ATP)-stimulated disassembly of l-histidine (l-His)-integrated ZIF-8 (l-His-ZIF-8) is reported for sustaining the l-DNAzyme-amplified photodynamic therapy. This self-sufficient l-therapeutic platform can intelligently release the l-DNAzyme probe and simultaneously supply l-His DNAzyme cofactors via endogenous ATP. Then, the intrinsic microRNA-21 catalyzes the generation of robust l-DNAzyme via the catalytic hybridization reaction for activating the photosensitizer with multiplied guaranteed therapeutic operation. This l-therapeutic strategy opens up great prospects for more precise diagnosis and customized gene silencing-based therapy.