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Proteins capable of switching between distinct active states in response to biochemical cues are ideal for sensing and controlling biological processes. Activatable CRISPR‐Cas systems are significant in precise genetic manipulation and sensitive molecular diagnostics, yet directly controlling Cas protein function remains challenging. Herein, we explore anti‐CRISPR (Acr) proteins as modules to create synthetic Cas protein switches (CasPSs) based on computational chemistry‐directed rational protein interface engineering. Guided by molecular fingerprint analysis, electrostatic potential mapping, and binding free energy calculations, we rationally engineer the molecular interaction interface between Cas12a and its cognate Acr proteins (AcrVA4 and AcrVA5) to generate a series of orthogonal protease‐responsive CasPSs. These CasPSs enable the conversion of specific proteolytic events into activation of Cas12a function with high switching ratios (up to 34.3‐fold). These advancements enable specific proteolysis‐inducible genome editing in mammalian cells and sensitive detection of viral protease activities during virus infection. This work provides a promising strategy for developing CRISPR‐Cas tools for controllable gene manipulation and regulation and clinical diagnostics. [ABSTRACT FROM AUTHOR]

The microstructure and tensile failure evolution of AA5754O aluminum alloy CMT joints were investigated in this study. First, the microstructure and properties of aluminum alloy were observed using a hardness test and metallographic test. The microstructure and tensile failure evolution of AA5754O aluminum alloy CMT joints were studied using in situ CT tests. The defects in the heat-affected zone were mainly composed of pores with large sphericity. The softening failure was mainly due to the decrease in the effective bearing area due to the increase in the number of defects. There were a large number of shrinkage pores with sphericity less than 0.6 in the fusion zone defects. The softening failure was mainly due to the continuous growth and combination of shrinkage pores, which led to a decrease in the effective bearing area. Meanwhile, the variation process of the mean radii of the meso-defects in the heat-affected zone and fusion zone were analyzed. The material constants αRT and αRTm were 1.87 and 6.20 in the heat-affected zone and 7.21 and 5.31 in the fusion zone, respectively, which were found using the Rich and Tracey model and the improved Rich and Tracey model. [ABSTRACT FROM AUTHOR]

Selective modulation of ligand–receptor interaction is essential in targeted therapy. In this study, we design an intelligent "scan and unlock" DNA automaton (SUDA) system to equip a native protein‐ligand with cell‐identity recognition and receptor‐mediated signaling in a cell‐type‐specific manner. Using embedded DNA‐based chemical reaction networks (CRNs) on the cell surface, SUDA scans and evaluates molecular profiles of cell‐surface proteins via Boolean logic circuits. Therefore, it achieves cell‐specific signal modulation by quickly unlocking the protein‐ligand in proximity to the target cell‐surface to activate its cognate receptor. As a proof of concept, we non‐genetically engineered hepatic growth factor (HGF) with distinct logic SUDAs to elicit target cell‐specific HGF signaling and wound healing behaviors in multiple heterogeneous cell types. Furthermore, the versatility of the SUDA strategy was shown by engineering tumor necrotic factor‐α (TNFα) to induce programmed cell death of target cell subpopulations through cell‐specific modulation of TNFR1 signaling. [ABSTRACT FROM AUTHOR]

The comprehensive understanding of the mechanisms underlying the interaction of cells with their membrane microenvironment is of great value for fundamental biological research; however, tracking biomolecules on cell surfaces with high temporal and spatial resolution remains a challenge. Herein, a modular strategy is presented for the construction of cell surface DNA‐based sensors by engineering DNA motifs and synthetic cofactors. In this strategy, a stimuli‐reactive organic molecule is employed as the cofactor for the DNA motif, and the self‐assembly of them forms a FRET‐based holo DNA‐based sensor. With the use of the DNA‐based sensors, the versatility of this modular strategy has been demonstrated in the ratiometric imaging of the cellular extrusion process of endogenous signaling molecules, including sulfur dioxide derivatives and nitric oxide. [ABSTRACT FROM AUTHOR]

Aims: To understand the roles of the RhoA/ROCK and mitogen-activated protein kinase (MAPK) pathways in high glucose (HG)-induced apoptosis and oxidative stress in cardiomyocytes.Materials and Methods: Neonatal rat cardiomyocytes were cultured in Dulbecco's modified Eagle's medium, supplemented with 5.5 or 30 mmol/L D-glucose, in the presence or absence of fasudil (50 or 100 μM), SB203580, SP600125, or PD98059 (10 μM, respectively). The percentage of early apoptotic cardiomyocytes was evaluated using flow cytometry. The superoxide dismutase activity and malondialdehyde contents in the cellular supernatants were measured. The Bax and Bcl-2 mRNA levels were determined by quantitative real-time PCR. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1), p38MAPK, JNK, and ERK as well as the protein levels of Bax, Bcl-2, and cleaved caspase-3 was analysed by Western blot.Results: Fasudil, SB203580, and SP600125 effectively inhibited the HG-induced early apoptosis increase and decreased Bax mRNA expression, the Bax/Bcl-2 protein expression ratio, and cleaved caspase-3 protein levels in the cardiomyocytes; this was accompanied by upregulation of the Bcl-2 mRNA. Moreover, fasudil markedly increased the superoxide dismutase activity level and suppressed the elevation in HG-induced malondialdehyde content and the phosphorylation of MYPT1, p38MAPK and JNK.Conclusions: The RhoA/ROCK pathway mediates HG-induced cardiomyocyte apoptosis via oxidative stress and activation of p38MAPK and JNK in neonatal rats in vitro. Fasudil effectively ameliorates HG-induced cardiomyocyte apoptosis by suppressing oxidative stress and the p38MAPK and JNK pathways. [ABSTRACT FROM AUTHOR]

A charge designable and tunable green fluorescent protein (GFP)-based protein delivery strategy was proposed. The acquired His29GFP selectively permeates the cell membrane at a target pH of 6.5 and escapes from the endosome efficiently. The delivered RNase A caused substantial mRNA degradation in HeLa cells, and proliferation inhibition in different cell lines and a 3D tumor model at pH 6.5. [ABSTRACT FROM AUTHOR]

Macrophages are key responders of inflammation and are closely related with oxidative stress. Activated macrophages can enhance oxygen depletion, which causes an overproduction of reactive oxygen species (ROS) and leads to further excessive inflammatory response and tissue damage. Agmatine, an endogenous metabolite of L-arginine, has recently been shown to have neuroprotective effects based on its antioxidant properties. However, the antioxidant effects of agmatine in peripheral tissues and cells, especially macrophages, remain unclear. In this study we explored the role of agmatine in mediating antioxidant effects in RAW 264.7 cells and studied its antioxidant mechanism. Our data demonstrate that agmatine is an activator of Nrf2 signaling that markedly enhances Nrf2 nuclear translocation, increases nuclear Nrf2 protein level, up-regulates the expression of the Nrf2 downstream effector HO-1, and attenuates ROS generation induced by Lipopolysaccharide (LPS). We further demonstrated that the agmatine-induced activation of Nrf2 is likely through the PI3K/Akt pathway. LY294002, a specific PI3K/Akt inhibitor, abolished agmatine-induced HO-1 up-regulation and ROS suppression significantly. Inhibiting HO-1 pathway significantly attenuated the antioxidant effect of agmatine which the products of HO-1 enzymatic activity contributed to. Furthermore, the common membrane receptors of agmatine were evaluated, revealing that α2-adrenoceptor, I1-imidazoline receptor or I2-imidazoline receptor are not required by the antioxidant properties of agmatine. Taken together, our findings revealed that agmatine has antioxidant activity against LPS-induced ROS accumulation in RAW 264.7 cells involving HO-1 expression induced by Nrf2 via PI3K/Akt pathway activation. [ABSTRACT FROM AUTHOR]

The enzyme complex IκB kinase (IKK) is an essential activator of NF-κB signaling pathway involved in propagating the cellular response to inflammation. The complex contains two functional subunits IKKαand IKKβ, which are structurally conserved kinases and selective inhibition of them would result in distinct biological effects. However, most existing IKK inhibitors show moderate or high promiscuity for the two homologous kinases. Understanding of the molecular mechanism and biological implication underlying the specific interactions in IKK–ligand recognition is thus fundamentally important for the rational design of selective IKK inhibitors. In the current work, we integrated molecular docking, quantum mechanics/molecular mechanics calculation and Poisson–Boltzmann/surface area analysis to investigate the structural basis and energetic property of the selective binding of small-molecule ligands to IKKαand IKKβ. It was found that the selectivity is primarily determined by the size and topology difference in ATP-binding pocket of IKKαand IKKβkinase domains; bulky inhibitor molecules commonly have, respectively, low and appropriate affinities towards IKKαand IKKβ, and thus exhibit relatively high selectivity for IKKβover IKKα, whereas small ligands can only bind weakly to both the two kinases with low selectivity. In addition, the conformation, arrangement and distribution of residues in IKK pockets are also responsible for constituting the exquisite specificity of ligand binding to KKαand IKKβ. Next, a novel quantitative structure–selectivity relationship model was developed to characterize the relative contribution of each kinase residue to inhibitor selectivity and to predict the selectivity and specificity for a number of known IKK inhibitors. Results showed that the active-site residues contribute significantly to the selectivity by directly interacting with inhibitor ligands, while those protein portions far away from the kinase active sites may also play an important role in determining the selectivity through long-range non-bonded forces and indirect allosteric effect. [ABSTRACT FROM PUBLISHER]

The label-free assay has drawn extensive attention because it does not require a labeling step and enables direct interaction and signal transduction between the sensing unit and target analytes. Herein, we demonstrate a proof-of-principle concept of a label-free and visualized nanoplasmonic strategy for silver ions sensing, where only Ti 3 C 2 MXenes are employed by exploring their excellent adsorption affinity and reductive property toward metal ions. Ag + was adsorbed onto the surface of Ti 3 C 2 MXene nanosheets, followed by the Ti 3 C 2 MXenes mediated in situ silver nanoparticles (Ag NPs) generation without adding any extra stabilizing or reducing agent. The excellent localized surface plasmon resonances at a particular wavelength provide Ag NPs the capability for colorimetric assay with a detection limit of 0.615 μM. With the assistance of a smartphone, RGB analysis exhibited visualized results consistent with the results measured on a UV-vis spectrometer, promising a budget, simple-operating on-site detection. Moreover, the detection of Ag + in real samples was achieved with satisfactory results meeting the analysis demand for the Drinking Water Standards of the World Health Organization (WHO) and the United States Environmental Protection Agency (U.S. EPA). These results reveal that Ti 3 C 2 MXenes possess great potential in building convenient label-free colorimetry nanoplatforms and may evoke more inspirations to explore strategies for the direct sensing of analytes.

A click-type protein-DNA conjugation, named as MnDDC (Mn 2+ -activated DCV-DNA conjunction), is presented, where DCV (rep protein of duck circovirus) and its target DNA work as the modular blocks to rapidly and effectively generate Mn 2+ -dependent and site-specific protein-DNA linkage. On the basis of MnDCC, a fluorescent Mn 2+ biosensor composed of DCV and a molecular beacon, was developed for rapid sensing of Mn 2+ within 2 min with nanomolar sensitivity. Using the proposed biosensor, not only analysis of Mn 2+ in real samples (e.g., serum and food), but also wash-free fluorescent imaging of Mn 2+ in extracellular environment and cytoplasm have been achieved. Moreover, the MnDDC-based sensor was proved to be a powerful tool for visualization of Mn 2+ during exploration of the associated cytotoxicity in living neural cells, which is helpful to reveal the cellular responses toward the disordered homeostasis of Mn 2+ in both extracellular and intracellular microenvironments.

Background: Enhancers can act as cis-regulatory elements (CREs) to control development and cellular function by regulating gene expression in a tissue-specific and ubiquitous manner. However, the regulatory network and characteristic of different types of enhancers (e.g., transcribed/non-transcribed enhancers, tissue-specific/ubiquitous enhancers) across multiple tissues are still unclear. Results: Here, a total of 53,924 active enhancers and 10,307 enhancer-associated RNAs (eRNAs) in 10 tissues (adrenal, brain, breast, heart, liver, lung, ovary, placenta, skeletal muscle and kidney) were identified through the integration of histone modifications (H3K4me1, H3K27ac and H3K4me3) and DNase I hypersensitive sites (DHSs) data. Moreover, 40,101 tissue-specific enhancers (TS-Enh), 1,241 ubiquitously expressed enhancers (UE-Enh) as well as transcribed enhancers (T-Enh), including 7,727 unidirectionally transcribed enhancers (1D-Enh) and 1,215 bidirectionally transcribed enhancers (2D-Enh) were defined in 10 tissues. The results show that enhancers exhibited high GC content, genomic variants and transcription factor binding sites (TFBS) enrichment in all tissues. These characteristics were significantly different between TS-Enh and UE-Enh, T-Enh and NT-Enh, 2D-Enh and 1D-Enh. Furt hermore, the results showed that enhancers obviously upregulate the expression of adjacent target genes which were remarkably correlated with the functions of corresponding tissues. Finally, a free user-friendly tissue-specific enhancer database, TiED (http://lcbb.swjtu.edu.cn/TiED), has been built to store, visualize, and confer these results. Conclusion: Genome-wide analysis of the regulatory network and characteristic of various types of enhancers showed that enhancers associated with TFs, eRNAs and target genes appeared in tissue specificity and function across different tissues., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The enzyme complex IκB kinase (IKK) is an essential activator of NF-κB signaling pathway involved in propagating the cellular response to inflammation. The complex contains two functional subunits IKKα and IKKβ, which are structurally conserved kinases and selective inhibition of them would result in distinct biological effects. However, most existing IKK inhibitors show moderate or high promiscuity for the two homologous kinases. Understanding of the molecular mechanism and biological implication underlying the specific interactions in IKK-ligand recognition is thus fundamentally important for the rational design of selective IKK inhibitors. In the current work, we integrated molecular docking, quantum mechanics/molecular mechanics calculation and Poisson-Boltzmann/surface area analysis to investigate the structural basis and energetic property of the selective binding of small-molecule ligands to IKKα and IKKβ. It was found that the selectivity is primarily determined by the size and topology difference in ATP-binding pocket of IKKα and IKKβ kinase domains; bulky inhibitor molecules commonly have, respectively, low and appropriate affinities towards IKKα and IKKβ, and thus exhibit relatively high selectivity for IKKβ over IKKα, whereas small ligands can only bind weakly to both the two kinases with low selectivity. In addition, the conformation, arrangement and distribution of residues in IKK pockets are also responsible for constituting the exquisite specificity of ligand binding to KKα and IKKβ. Next, a novel quantitative structure-selectivity relationship model was developed to characterize the relative contribution of each kinase residue to inhibitor selectivity and to predict the selectivity and specificity for a number of known IKK inhibitors. Results showed that the active-site residues contribute significantly to the selectivity by directly interacting with inhibitor ligands, while those protein portions far away from the kinase active sites may also play an important role in determining the selectivity through long-range non-bonded forces and indirect allosteric effect.

Fibronectin adsorption on biomaterial surfaces plays a key role in the biocompatibility of biomedical implants. In the current study, the adsorption behavior of the 7-10th type III modules of fibronectin (FN-III7-10) in the presence of hydroxyapatite (HAP) was systematically investigated by using molecular docking approach. It was revealed that the FN-III10 is the most important module among FN-III7-10 in promoting fibronectin binding to HAP by optimizing the interaction energy; the arginine residues were observed to directly interact with the hydroxyl group of HAP through electrostatic forces and hydrogen bonding. Moreover, it was found that the HAP-binding sites on FN-III10 are mainly located at the RGD loop region, which does not affect the interaction between the fibronectin protein and its cognate receptors on the cell surface.