In Situ Assay of Proteins Incorporated with Unnatural Amino Acids in Single Living Cells by Differenced Resonance Light Scattering Correlation Spectroscopy.

Site-specific incorporation of unnatural amino acids (UAAs) into target proteins (UAA-proteins) provides the unprecedented opportunities to study cell biology and biomedicine. However, it is a big challenge to in situ quantitatively determine the expression level of UAA-proteins due to serious interferences from autofluorescence, background scattering, and different viscosity in living cells. Here, we proposed a novel single nanoparticle spectroscopy method, differenced resonance light scattering correlation spectroscopy (D-RLSCS), to measure the UAA-proteins in single living cells. The D-RLSCS principle is based on the simultaneous measurement of the resonance scattering light fluctuation of a single gold nanoparticle (GNP) in two detection channels irradiated by two coaxial laser beams and then autocorrelation analysis on the differenced fluctuation signals between two channels. D-RLSCS can avoid the interferences from intracellular background scattering and provide the concentration and rotational and translational diffusion information of GNPs in solution or in living cells. Furthermore, we proposed a parameter, the ratiometric diffusion time and found that this parameter is proportional to the square of particle size. The theoretical and experimental results demonstrated that the ratiometric diffusion time was not influenced by the intracellular viscosity. This method was successfully applied for in situ quantification of the UAA-protein within single living cells based on the increase in the ratiometric diffusion time of nanoprobes bound with proteins. Using UAA-EGFP (enhanced green fluorescent protein) as a model, we observed the significant difference in the UAA-protein concentrations at different positions in single living cells.

Efficient Solid-State Electrochemiluminescence from High-Quality Perovskite Quantum Dot Films.

Halide perovskite materials have emerged as a new class of revolutionary photovoltaic and optoelectronic nanomaterials. However, the study on electrochemiluminescence (ECL) from halide perovskite nanomaterials is still in its infancy due to their instability, sensitivity, and difficulties in purification and film formation. Here, we propose a scraping coating method for the fabrication of high-quality halide perovskite quantum dot (QD) film on electrode, which shows dense and uniform packing with minimum grain size. When CsPbBr3 QDs are taken as model materials, highly efficient ECL can be obtained from such perovskite QD film with anhydrous ethyl acetate as both electrolyte and coreactant. The CsPbBr3 QD film displays intense and stable ECL with ultranarrow emission spectrum bandwidth (24 nm). The CsPbBr3 QD film shows an extremely high ECL efficiency which is up to 5 times relative to the standard Ru(bpy)32+/tri-n-propylamine system. This approach is universal and also applies to hybrid organic-inorganic halide perovskite QDs. This work not only extends the properties and applications of halide perovskite materials but also provides a new method for the in-depth study on the structure and properties of these kinds of materials.

Inflammatory factor-specific sumoylation regulates NF-κB signalling in glomerular cells from diabetic rats.

OBJECTIVE: The activation of nuclear factor (NF)-κB by cytokines under hyperglycaemic conditions is a potential mechanism for complications in diabetes. We investigated whether small ubiquitin-like modifier 4 (SUMO4) regulates renal NF-κB signalling in diabetic rats. METHODS: Histological changes in kidney were analysed in diabetic GK rats. The expressions of tumour necrosis factor (TNF)-α, NF-κB (p65), IκBα and SUMO4 in renal tissues were examined by immunohistochemistry and Western blotting. Primary cultured glomerular endothelial cells from rats were stimulated by TNF-α or interleukin (IL)-2. RESULTS: The renal expression of TNF-α, NF-κB (p65), IκBα and SUMO4 was significantly higher in diabetic GK rats than in control rats. In control rats, no nuclear translocation was observed for IκBα or NF-κB (p65). However, in diabetic GK rats, translocation of NF-κB (p65) and IκBα into the nucleus was observed, and the expression of SUMO4 and IκBα was up-regulated in the glomerular endothelial cells. SUMO4 was localised in both the cytoplasm and nucleus, while IκBα was predominantly located in the nucleus after stimulation with TNF-α. In contrast, SUMO4 was localised in the nucleus, and increased cytoplasm SUMO4 localisation was found after stimulation with IL-2. CONCLUSIONS: SUMO4 plays a role in regulating NF-κB signalling in glomerular cells. Cytokines have a unique effect in regulating the sumoylation of NF-κB.

Gut microbiota shapes social dominance through modulating HDAC2 in the medial prefrontal cortex.

Social dominance is a ubiquitous phenomenon among social animals, including humans. To date, individual attributes leading to dominance (after a contest) remain largely elusive. Here, we report that socially dominant rats can be distinguished from subordinates based on their intestinal microbiota. When dysbiosis is induced, rats are predisposed to a subordinate state, while dysbiotic rats reclaim social dominance following microbiota transplantation. Winning hosts are characterized by core microbes, a majority of which are associated with butyrate production, and the sole colonization of Clostridium butyricum is sufficient to restore dominance. Regarding molecular aspects, a histone deacetylase, HDAC2, is responsive to microbial status and mediates competition outcome; however, this occurs only in a restricted population of cells in the medial prefrontal cortex (mPFC). Furthermore, HDAC2 acts by modulating synaptic activity in mPFC. Together, these findings uncover a link between commensals and host dominance, providing insight into the gut-brain mechanisms underlying dominance determination.

Antimicrobial photodynamic inactivation as an alternative approach to inhibit the growth of Cronobacter sakazakii by fine-tuning the activity of CpxRA two-component system.

Cronobacter sakazakii is an opportunistic foodborne pathogen primarily found in powdered infant formula (PIF). To date, it remains challenging to control the growth of this ubiquitous bacterium. Herein, antimicrobial photodynamic inactivation (aPDI) was first employed to inactivate C. sakazakii. Through 460 nm light irradiation coupled with hypocrellin B, the survival rate of C. sakazakii was diminished by 3~4 log. The photokilling effect was mediated by the attenuated membrane integrity, as evidenced by PI staining. Besides, scanning electron microscopy showed the deformed and aggregated cell cluster, and intracellular ROS was augmented by 2~3 folds when light doses increase. In addition to planktonic cells, the biofilm formation of C. sakazakii was also affected, showing an OD590nm decline from 0.85 to 0.25. In terms of molecular aspects, a two-component system called CpxRA, along with their target genes, was deregulated during illumination. Using the knock-out strain of ΔCpxA, the bacterial viability was reduced by 2 log under aPDI, a wider gap than the wildtype strain. Based on the promoted expression of CpxR and OmpC, aPDI is likely to play its part through attenuating the function of CpxRA-OmpC pathway. Finally, the aPDI system was applied to PIF, and C. sakazakii was inactivated under various desiccated or heated storage conditions. Collectively, aPDI serves as an alternative approach to decontaminate C. sakazakii, providing a new strategy to reduce the health risks caused by this prevalent foodborne pathogen.