Photocatalytic Chemical Crosslinking for Profiling RNA-Protein Interactions in Living Cells.

The dynamic interactions between RNAs and proteins play crucial roles in regulating diverse cellular processes. Proteome-wide characterization of these interactions in their native cellular context remains desirable but challenging. Herein, we developed a photocatalytic crosslinking (PhotoCAX) strategy coupled with mass spectrometry (PhotoCAX-MS) and RNA sequencing (PhotoCAX-seq) for the study of the composition and dynamics of protein-RNA interactions. By integrating the blue light-triggered photocatalyst with a dual-functional RNA-protein crosslinker (RP-linker) and the phase separation-based enrichment strategy, PhotoCAX-MS revealed a total of 2044 RBPs in human HEK293 cells. We further employed PhotoCAX to investigate the dynamic change of RBPome in macrophage cells upon LPS-stimulation, as well as the identification of RBPs interacting directly with the 5' untranslated regions of SARS-CoV-2 RNA.

Bioorthogonally Activatable Base Editing for On-Demand Pyroptosis.

Pyroptosis is an inflammatory cell death form triggered by protease-mediated truncation and release of the N-terminal pore-forming domain of the gasdermin (GSDM) family proteins in various cell types. We report a Bioorthogonally ACtivatable Base editor (BaseBAC) for in situ and on-demand initiation of cell-type-specific pyroptosis. We first made the enzymatic activity of a cytosine base editor (CBE) switchable by establishing a bioorthogonal blockage on the PAM-interacting residue to control its DNA-binding ability. The resulting BaseBAC allowed in situ control of base editing on the GSDME gene that switched to the truncated expression of its N-terminal domain to activate pyroptosis. BaseBAC offers a general method for on-demand awakening of functional domains of self-inhibiting proteins and the corresponding cellular processes with high specificity in living systems.

A far-red hybrid voltage indicator enabled by bioorthogonal engineering of rhodopsin on live neurons.

Membrane potential is a key aspect of cellular signalling and is dynamically regulated by an array of ion-selective pumps and channels. Fluorescent voltage indicators enable non-invasive optical recording of the cellular membrane potential with high spatial resolution. Here, we report a palette of bright and sensitive hybrid voltage indicators (HVIs) with fluorescence intensities sensitive to changes in membrane potential via electrochromic Förster resonance energy transfer. Enzyme-mediated site-specific incorporation of a probe, followed by an inverse-electron-demand Diels-Alder cycloaddition, was used to create enhanced voltage-sensing rhodopsins with hybrid dye-protein architectures. The most sensitive indicator, HVI-Cy3, displays high voltage sensitivity (-39% ΔF/F0 per 100 mV) and millisecond response kinetics, enabling optical recording of action potentials at a sampling rate of 400 Hz over 10 min across a large neuronal population. The far-red indicator HVI-Cy5 could be paired with optogenetic actuators and green/red-emitting fluorescent indicators, allowing an all-optical investigation of neuronal electrophysiology.

Slot-slot waveguide with negative large and flat dispersion covering C+L+U waveband for on-chip photonic networks.

A novel, to the best of our knowledge, dual-core slot-slot waveguide with extreme high dispersion is proposed. The high dispersion value at the desired wavelength is obtained based on strong resonance coupling between two slot-waveguide modes. The properties of dispersion magnitude and bandwidth are numerically analyzed by using the finite-difference time-domain method with a perfectly matched layer boundary. All numerical simulation results reveal that for the optimized geometrical parameters of H1=350 nm, L1=569 nm, S1=31.3 nm, La=1062.39 nm, H2=427 nm, L2=137.4 nm, and S2=63.5 nm, the maximum dispersion of negative 3.645×105 ps·nm-1·km-1 and dispersion full width at half-maximum of 6.3 nm at 1550 nm have been obtained. By cascading the slot-slot waveguides with varying width and height, a large and flattened dispersion of -3.5×105 ps·nm-1·km-1 covering the C+L+U waveband is obtained. Dispersion compensation of a 100 Gbit/s return-to-zero on-off-keying optical time-division multiplexing signal after 50 km full spectrum single-mode optical fiber transmission with five different central wavelengths is demonstrated through simulation for the first time. In addition, fabrication tolerance of the proposed slot-slot waveguide is analyzed. Such a waveguide is compatible with complementary metal-oxide-semiconductor technology and has potential applications in next-generation large-scale photonic integrated circuits.

M2 factor of controllable dark-hollow beams through a multi-apertured ABCD optical system.

Under an optical system with multiple hard-edged apertures in a cylindrical coordinate system, the recurrence propagation expression is derived for the controllable dark-hollow beams (CDHBs) by expanding the hard-aperture function into a finite sum of complex Gaussian functions. Given the recurrence propagation expression, we deduce the approximate analytical expressions of the beam propagation factor M2 in terms of the generalized truncated second-order moments. This provides a fast algorithm for the evaluation of the beam propagation quality for CDHBs through complicated optical trains with a series of apertures. The propagation of CDHBs through a two-aperture-lens ABCD optical system serves as the special case of multi-apertured ABCD optical systems. Our numerical results suggest that a one-aperture-lens optical system reduces the beam propagation quality of CDHBs, and a two-aperture-lens optical system improves the beam propagation quality of CDHBs by selecting appropriate beam parameters and aperture parameters.

Genetically Encoded Chemical Decaging in Living Bacteria.

We report the genetically encoded chemical decaging strategy for protein activation in living bacterial cells. In contrast to the metabolically labile photocaging groups inside Escherichia coli, our chemical decaging strategy that relies on the inverse electron-demand Diels-Alder (iDA) reaction is compatible with the intracellular environment of bacteria, which can be a general tool for gain-of-function study of a given protein in prokaryotic systems. By applying this strategy for in situ activation of the indole-producing enzyme TnaA, we built an orthogonal and chemically inducible indole production pathway inside E. coli cells, which revealed the role of indole in bacterial antibiotic tolerance.

Mass spectrometry-based proteomic approaches to study pathogenic bacteria-host interactions.

Elucidation of molecular mechanisms underlying host-pathogen interactions is important for control and treatment of infectious diseases worldwide. Within the last decade, mass spectrometry (MS)-based proteomics has become a powerful and effective approach to better understand complex and dynamic host-pathogen interactions at the protein level. Herein we will review the recent progress in proteomic analyses towards bacterial infection of their mammalian host with a particular focus on enteric pathogens. Large-scale studies of dynamic proteomic alterations during infection will be discussed from the perspective of both pathogenic bacteria and host cells.

[Gene cloning and bioinformatics analysis of SABATH methyltransferase in Lonicera japonica var. chinensis].

OBJECTIVE: To clone SABATH methyltransferase (rLjSABATHMT) gene in Lonicera japonica var. chinensis, and compare the gene expression and intron sequence of SABATH methyltransferase orthologous in L. japonica with L. japonica var. chinensis. It provide a basis for gene regulate the formation of L. japonica floral scents. METHOD: The cDNA and genome sequences of LjSABATHMT from L. japonica var. chinensis were cloned according to the gene fragments in cDNA library. The LjSABATHMT protein was characterized by bioinformatics analysis. SABATH family phylogenetic tree were built by MEGA 5.0. The transcripted level of SABATHMT orthologous were analyzed in different organs and different flower periods of L. japonica and L. japonica var. chinensis using RT-PCR analysis. Intron sequences of SABATHMT orthologous were also analyzied. RESULT: The cDNA of LjSABATHMT was 1 251 bp, had a complete coding frame with 365 amino acids. The protein had the conservative SABATHMT domain, and phylogenetic tree showed that it may be a salicylic acid/benzoic acid methyltransferase. Higher expression of SABATH methyltransferase orthologous was found in flower. The intron sequence of L. japonica and L. japonica var. chinensis had rich polymorphism, and two SNP are unique genotype of L. japonica var. chinensis. The motif elements in two orthologous genes were significant differences. CONCLUSION: The intron difference of SABATH methyltransferase orthologous could be inducing to difference of gene expression between L. japonica and L. japonica var. chinensis. These results will provide important base on regulating active compounds of L. japonica.