Let-7b-TLR7 Signaling Axis Contributes to the Anesthesia/Surgery-Induced Cognitive Impairment.

Perioperative neurocognitive disorders (PNDs) are severe and common neurological complications among elderly patients following anesthesia and surgery. As the first line of defense of the innate immune system, Toll-like receptors (TLRs) have been found to be involved in the occurrence of neurodegenerative diseases in recent years. However, the role of TLR7 in the pathology and development of PNDs remains largely unclear. In our current study, we hypothesized that increased microRNA let-7b (let-7b) during anesthesia and surgical operation would activate TLR7 signaling pathways and mediate PNDs. Using a mouse model of PNDs, 18-20 months wild-type (WT) mice were undergoing unilateral nephrectomy, and increased TLR7 and let-7b expression levels were found in the surgery group compared with the Sham group. Of note, increased TLR7 was found to be co-localized with let-7b in the hippocampal area CA1 in the PNDs model. In addition, TLR7 and let-7b inhibition could improve hippocampus-dependent memory and attenuate the production of inflammatory cytokines. Together, our results indicated that TLR7 activation and up-regulation might be triggered by increased let-7b under stressful conditions and initiated the downstream inflammatory signaling, playing a substantial role in the development of PNDs.

Perioperative platelet count in peripheral blood is associated with the early stage of PND after major orthopedic surgery: a prospective observational study.

BACKGROUND: Perioperative neurocognitive disorders (PND) are common complications of major surgery among elderly patients, remarkably decreasing patients' life quality. Platelet count has been proved to be an essential factor in inflammation. However, as far as we know, the relationship between platelet count and PND is not clear yet in the orthopedic area. PND could be a long-term disease, which sometimes lasts for several years, and it is meaningful to find a biomarker of PND at the early stage. Thus, we designed this study to find out the association between perioperative platelet count and occurrence of PND, and determine whether preoperative platelet count could be a biomarker of the early stage of PND. METHODS: A prospective observational study was performed on the patients who would take total knee arthroplasty or total hip arthroplasty. Their peripheral platelets were counted by blood routine examination 1 day before and 3 days after the surgery. And we assessed their neurocognitive functions 1 day before and 3 days after the surgery. These data were recorded and analyzed to find out the relationship between platelet count and the occurrence of PND. RESULTS: Eventually, 70 patients finished the whole process, and 14 of them developed PND. The median preoperative platelet count in the PND group was significantly higher than that in the non-PND group (239 vs 168 × 10^9/L, p = 0.009). Preoperative platelet count was an independent risk factor for PND (odds ratio = 1.014, 95% confidence interval [CI] 1.000-1.027, P = 0.043) in the logistic multivariable regression, while the area under the curve of the receiver operating characteristic curve of the prediction model was 0.796 (95% CI 0.676-0.916). CONCLUSIONS: The higher preoperative and postoperative level of platelet count in the peripheral blood were associated with the early stage of PND, and preoperative platelet count could be a potential predictor of the early stage of PND in patients undergoing major orthopedic surgeries. TRIAL REGISTRATION: Chinese Clinical Trial Registry: ChiCTR2000033001 , registration date: 17 May 2020.

Highly sensitive detection of DNA methyltransferase activity and its inhibitor screening by coupling fluorescence correlation spectroscopy with polystyrene polymer dots.

DNA methylation is a critical part of epigenetics and plays a vital role in maintaining normal cell function, genetic imprinting, and human tumorigenesis. Thus, it is important to develop a sensitive method for the determination of DNA methyltransferase (MTase) activity. Here, we present a simple and sensitive method based on single molecule fluorescence correlation spectroscopy (FCS) and polystyrene polymer dots (PS Pdots) for the quantitative detection of DNA adenine methylation (Dam) MTase activity and its inhibitor screening in homogeneous solution without separation. Its principle is based on the measurement of the characteristic diffusion time (τD) of unmethylated and methylated DNA-fluorescent probes by FCS. A hairpin DNA probe including the 5'-GATC-3' sequence is used by doubly labelling fluorophore Alexa Fluor 488 (Alexa 488) and biotin at the 5'- and 3'-terminus, respectively. Dam MTase catalyzed the methylation of the sequence of 5'-GATC-3', and DpnI cleaved the sequence of 5'-G-Am-TC-3'. Streptavidin conjugated PS Pdots were used to react with DNA probes without methylation to further increase the difference in τD values between methylated and unmethylated DNA-Alexa 488 probes. We used the FCS method to measure the τD values of DNA-Alexa 488 probes and further obtained the activity of Dam MTase. It is found that the τD value of the methylated DNA probe is negatively correlated with the logarithm of Dam MTase concentration in the range from 0.025 U mL-1 to 3 U mL-1. The detection limit is as low as 0.025 U mL-1. Furthermore, we evaluated the inhibition effect of drug-related DNA methylation and the half-maximal inhibitory concentration (IC50) value is consistent with a previous study. The results demonstrated that our proposed method will become a promising platform for the determination of Dam MTase activity and inhibitor screening.

Simultaneously monitoring endogenous MAPK members in single living cells by multi-channel fluorescence correlation spectroscopy.

The mitogen-activated protein kinase (MAPK) pathway is a major module for cellular signal transduction. The dysregulation of the MAPK pathway has been involved in the pathogenesis of multiple diseases ranging from cancers to chronic inflammations. So far, we have not fully understood the influences of external factors and signaling networks on the MAPK pathway due to the lack of in situ methods for simultaneous detection of multiple kinases in the pathway in living cells. Herein, we present a new strategy for in situ and simultaneously monitoring MAPK pathway kinases in single living cells combining multi-channel fluorescence correlation spectroscopy (FCS) with affinity fluorescent probes. We chose rapidly growing fibrosarcoma kinase (RAF), mitogen-activated protein kinase (MEK), and extracellular signal-regulated kinase (ERK) as representative members in the MAPK pathway. We designed and synthesized three fluorescent affinity probes and experimental results demonstrated that the three probes specifically targeted endogenous BRAF, MEK1/2, and ERK1/2 in living cells. Based on the multi-channel FCS system, we studied the influences of biological substances, drugs and oxidative stress on the activities of endogenous MAPK kinases and the cross-talk between the MAPK and PI3K-mTOR pathways. We have found that serum, sorafenib, and hydrogen peroxide can regulate multiple MAPK kinases and the effects of external stimuli can transmit to the MAPK pathway; furthermore, we have observed that the MAPK pathway can be activated by modulating the PI3K-mTOR pathway. Our results illustrated the complexity of a cellular signal network and the necessity of in situ and simultaneous determination of biomolecules in living cells.

Artificial Aquaporin That Restores Wound Healing of Impaired Cells.

Artificial aquaporins are synthetic molecules that mimic the structure and function of natural aquaporins (AQPs) in cell membranes. The development of artificial aquaporins would provide an alternative strategy for treatment of AQP-related diseases. In this report, an artificial aquaporin has been constructed from an amino-terminated tubular molecule, which operates in a unimolecular mechanism. The artificial channel can work in cell membranes with high water permeability and selectivity rivaling those of AQPs. Importantly, the channel can restore wound healing of the cells that contain function-lost AQPs.

In Situ Study of the Drug-Target Protein Interaction in Single Living Cells by Combining Fluorescence Correlation Spectroscopy with Affinity Probes.

The drug-target protein interaction is the basis of drug screening and precise therapy in modern clinical medicine. How to acquire the information about the drug-target protein interaction in single living cell is a great challenge due to the shortage of efficient methods. Here we propose a new strategy for in situ studying the drug-target protein interaction in single living cells based on the competition of candidate drugs to the fluorescent probe-target complexes and fluorescence correlation spectroscopy (FCS) with a microfluidic chip. In this study, we used ABL kinase (target) as a model and synthesized a fluorescent probe (Cy3-dasatinib) with an affinity to the target using ABL inhibitor dasatinib as a precursor. We systematically investigated the association of the probe with targets and the dissociation of the drug-target complexes in the presence of candidate drug. We presented a new parameter IC50 (τD) to assess the inhibitory effect of drugs on the basis of the changes in the characteristic diffusion time (τD) and the binding ratio (y) of fluorescent probes during the drug competition process in living cells. We found a remarkable difference of IC50 (τD) values in living cells and in solutions, suggesting it is quite necessary to evaluate the drug-target interactions in living cells. Compared with current methods, our approach can be used to in situ and real-time study the drug-target interaction in living cells, and it may become a promising and universal tool for in situ drug research at molecular level.

Catalytic Chemiluminescence Polymer Dots for Ultrasensitive In Vivo Imaging of Intrinsic Reactive Oxygen Species in Mice.

Chemiluminescence (CL) is a promising bioimaging method due to no interferences of light source and autofluorescence. However, compared to fluorescent emission, most CL reactions show short emission time and wavelength and weak emission intensity, which limit their applications in in vivo imaging. Here, we report mimic-enzyme catalytic CL polymer dots (hemin-Pdots) consisting of hemin and fluorescent conjugated polymer based on chemiluminescence resonance energy transfer. Hemin-Pdots show about 700× enhanced CL and over 10 h light emission in the presence of CL substrates and H2O2. These properties are mainly due to high-catalytic activity of hemin-Pdots and slow-diffusion-controlled heterogeneous reaction. Hemin-Pdots also possess excellent biocompatibility, good stability, emission wavelength redshift, and ultrasensitive response to reactive oxygen species (ROS), and they were successfully used for real-time imaging ROS levels in the peritoneal cavity and normal and tumor tissues of mice. Hemin-Pdots as new CL probes have wide applications in bioassays, bioimaging, and photodynamic therapy.

In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy.

Protein-protein interactions play a central role in signal transduction, transcription regulations, enzymatic activity, and protein synthesis. The p53 protein is a key transcription factor, and its activity is precisely regulated by the p53-MDM2 interaction. Although the p53-MDM2 interaction has been studied, it is still not clear how p53 structures and external factors influence the p53-MDM2 interaction in living cells. Here, we developed a direct method for monitoring the p53-MDM2 interaction in single living cells using single-molecule fluorescence cross-correlation spectroscopy with a microfluidic chip. First, we labeled p53 and MDM2 proteins with enhanced green fluorescent protein (EGFP) and mCherry, respectively, using lentivirus infection. We then designed various mutants covering the three main domains of p53 (tetramerization, transactivation, and DNA-binding domains) and systematically studied effects of p53 protein primary, secondary, and quaternary structures on p53-MDM2 binding affinity in single living cells. We found that p53 dimers and tetramers can bind to MDM2, that the binding affinity of p53 tetramers is higher than that of p53 dimers, and that the affinity is closely correlated to the helicity of the p53 transactivation domain. The hot-spot mutation R175H in the DNA-binding domain reduced the binding of p53 to MDM2. Finally, we studied effects of inhibitors on p53-MDM2 interactions and dissociation dynamics of p53-MDM2 complexes in single living cells. We found that inhibitors Nutlin 3α and MI773 efficiently inhibited the p53-MDM2 interaction, but RITA did not work in living cells. This study provides a direct way for quantifying the relationship between protein structure and protein-protein interactions and evaluation of inhibitors in living cells.