Multifunctional lipid droplet probes for observing the polarity change of ferroptosis, inflammation, fatty liver and evaluating the efficacy of drugs.

BACKGROUND: Lipid droplets (LDs) polarity is intricately linked to diverse biological processes and diseases. The visualization of LDs-polarity is of vital importance but challenging due to the lack of high-specificity, high-sensitivity and large-Stokes shift probes for real-time tracking LDs-polarity in biological systems. RESULTS: Four D-π-A based fluorescent probes (TPA-TCF1-TPA-TCF4) have been developed by combining tricyanofuran (an electron acceptor, A) and triphenylamine (an electron donor, D) derivatives with different terminal groups. Among them, TPA-TCF1 and TPA-TCF4 exhibit excellent polar sensitivity, large Stokes shift (≥182 nm in H2O), and efficient LDs targeting ability. In particular, TPA-TCF4 is capable of monitoring the change of LDs-polarity during ferroptosis, inflammation, apoptosis of cancer cell, and fatty liver. SIGNIFICANCE: All these features render TPA-TCF4 a versatile tool for pharmacodynamic evaluation of anti-cancer drugs, in-depth understanding of the biological effect of LDs on ferroptosis, and medical diagnosis of LDs-polarity related diseases.

Near-infrared lipid droplets polarity fluorescent probe for early diagnosis of nonalcoholic fatty liver disease.

Nowadays, it has been proven that lipid droplets (LDs) not only maintain the fundamental cellular functions, but also play an essential role in the pathogenesis of numerous diseases. Non-alcoholic fatty liver disease (NAFLD) is among these diseases. In this work, we designed two polarity sensitive fluorescent probes TST and TSO with D-π-A-D structure by introducing different electron acceptor groups according to the low polarity of LDs. The experimental discovered that probe TST exhibited the characteristics of near-infrared emission, high selectivity towards polarity, large Stokes shift, rapid targeting ability of LDs, and robust wash-free biological imaging capability. Confocal images illustrated that probe TST has been successfully applied in monitoring LDs polarity during ferroptosis, as well as visualizing changes in LDs polarity at both tissue and organ levels in fatty liver conditions. With these exceptional properties, probe TST was anticipated to make further contributions to the field of LDs research.

A wash-free fluorescent probe with a large Stokes shift for the identification of NAFL through tracing the change of lipid droplets.

As one of the important organelles in cells, lipid droplets (LDs) are involved in various physiological processes, especially affecting the occurrence and progression of non-alcoholic fatty liver (NAFL). Therefore, it is of great significance to develop LD-specific probes with excellent biocompatibility, deep penetration and bright fluorescence. Herein, a fluorescent probe LD-HWZ was designed and synthesized based on triphenylamine and the dicyanoisophorone group. It is found that probe LD-HWZ has a large Stokes shift (Δλ = 160 nm in DMSO) and exhibits bright fluorescence in a lipid environment. In addition, biological experiments showed that LD-HWZ can localize in lipid droplets, which can be used to detect the dynamic changes of LDs. Importantly, LD-HWZ has been successfully used to discriminate NAFL tissues from normal livers. The excellent properties of probe LD-HWZ in this work are expected to shed new light on the design of lipid droplet probes for the study of fatty liver diagnosis.

Noncoding RNAs-based high KIF26B expression correlates with poor prognosis and tumor immune infiltration in colon cancer.

BACKGROUND: The protein kinesin family member 26B (KIF26B) is aberrantly expressed in various cancers. However, its particular role and association with tumor immune infiltration in colon adenocarcinoma (COAD) remain unclear. METHODS: All original data were downloaded directly from The Cancer Genome Atlas (TCGA), UCSC Xena, and Gene Expression Omnibus (GEO) databases and processed with R 3.6.3. KIF26B expression was analyzed using Oncomine, TIMER, TCGA, GEO databases, and our clinical specimens. KIF26B expression at the protein level was explored with Human Protein Atlas (HPA) database. The upstream miRNAs and lncRNAs were predicted by StarBase and validated using RT-qPCR. Correlation of KIF26B expression with the expression of immune-related or immune checkpoint genes and GSEA analysis of KIF26B-related genes were investigated via R software. Relationship of KIF26B expression with immune biomarkers or tumor immune infiltration levels was studied through GEPIA2 and TIMER databases. RESULTS: KIF26B was upregulated, and its overexpression was closely related to overall survival (OS), disease-specific survival (DSS), progression-free interval (PFI), T stage, N stage, and CEA levels in COAD. MIR4435-2HG/hsa-miR-500a-3p/KIF26B axis was identified as the promising regulatory pathway of KIF26B. KIF26B expression was positively correlated with immune-related genes, tumor immune infiltration, and biomarker genes of immune cells in COAD, and KIF26B-related genes were significantly enriched in macrophage activation-related pathways. Expression of immune checkpoint genes, including PDCD1, CD274, and CTLA4, was also closely related to KIF26B expression. CONCLUSIONS: Our results clarified that ncRNA-based increased KIF26B expression was associated with a worse prognosis and high tumor immune infiltration in COAD.

Red-emissive Dual-state Fluorogenic Probe for Wash-free Imaging of Lipid Droplets in Living Cells and Fatty Liver Tissues.

Lipid droplet (LD) dysfunction can result in various diseases, such as nonalcoholic fatty liver disease. Imaging agents built on dual-state emission (DSE) molecules that fluoresce in both dilute solutions and the aggregated state are receiving attention as this type of probe could provide bright fluorescence signals at variable concentrations, avoiding false signal readout caused by the concentration fluctuation in living systems. Herein, we identified a red emissive molecule featuring DSE, from three newly synthesized molecules, for specific detection of LDs in live cells. The bioimaging abilities have been well confirmed by optical spectroscopies, theoretical calculations, cell experiments, as well as animal studies. The DSE probe is effective for LD detection at concentrations ranging from 1 µM to 100 µM while retaining high brightness and signal fidelity. This study provides a knowledge base for the future design of DSE-active fluorescent probes for understanding LD-related diseases.

The accuracy of using guided endodontics in access cavity preparation and the temperature changes of root surface: An in vitro study.

BACKGROUND: Guided endodontics is a successful technique that has been gradually applied to endodontic therapy in recent years without being affected by the operator's experience. However, the guided bur produces excessive heat during continuous rotation and friction with root canal walls, it is not clear whether the degree of temperature increase may lead to the periodontal ligament and alveolar bone damage. METHODS: A total of 58 teeth were used, of which 40 teeth were not grouped, all used to evaluate the accuracy. 40 single-rooted premolars were scanned using CBCT and an intra-oral scanner, and 3D-printed guided plates were made with the pre-designed access. A custom-made guided bur was used to prepare the access cavities. The postoperative CBCT data and pre-designed pathways were matched to evaluate the deviation between the planned and virtual paths. The other 18 teeth were randomly divided into three groups (ET20 and ProTaper F3 as the control group, guided endodontics as the test group), with 6 teeth in each group. The temperature changes on the root surfaces were inspected with a thermocouple thermometer. RESULTS: The average deviation on the tip and the base of the bur was 0.30 mm and 0.28 mm (mesial/distal), and 0.28 mm and 0.25 mm (buccal/lingual). The average angle deviation was 3.62°. The mean root surface temperature rise of the guided endodontics group was the lowest (5.07 °C) (P < 0.05). CONCLUSIONS: The access cavity preparation performed with guided endodontics has feasible accuracy and low-temperature rise on the root surfaces. Due to the limitations of the study, whether it has high reliability and safety in clinical applications needs to be further studied in vivo.

Polarity-sensitive and lipid droplet-specific red emission fluorophore for identifying fatty liver of living mice through in vivo imaging.

The illustration of the correlation between lipid droplets (LDs) variation and nonalcoholic fatty liver disease (NAFLD) is a challenging and important work in biomedical research. Herein, a red emission fluorophore LD-HW containing donor-π-bridge-acceptor (D-π-A) structure was readily constructed and systematically investigated. It was found that LD-HW could selectively identify polarity variation accompanying with an obvious blue-shift (around 80 nm) in fluorescence spectra, and a sharp enhancement (about 440-fold) in fluorescence quantum yield (QY) over the solvent polarity ranging from water (polarity parameter Δf = 0.3200) to 1,4-dioxane (Δf = 0.0205). In addition, probe LD-HW could precisely light up LDs within a short time (≤5 min) through a wash-free procedure and real-time monitor the dynamic behavior of cellular LDs. More importantly, LD-HW exhibited an excellent performance in differentiating fatty liver through in vivo imaging the change of cellular LDs. The in situ fluorescence spectra of corresponding tissue section proved that polarity level in the liver of NAFLD mice was lower than that in normal mice. Taken together, probe LD-HW presented great potential in non-invasive diagnosis of fatty liver through in vivo imaging.

Application of distant live broadcast in clinical anesthesiology teaching.

OBJECTIVE: To explore the application value and effect of distance live broadcast in Clinical Anesthesiology teaching. METHODS: Undergraduate students of year 2017 who majored in Anesthesiology at the Wannan Medical College (China) were chosen as the study subjects. According to the different teaching methods, the students were divided into two groups: 59 in the traditional teaching group (control group) and 61 in the traditional teaching combined with distant live broadcasting teaching group (observation group). The teaching feedback, students' satisfaction, and the theory and skill scores of the course were compared between the two groups. RESULTS: The teaching feedback in the observation group was better than that in the control group (P<0.05). The students' satisfaction rate with teaching and the theory and skill learning score in the observation group were higher than those of the control group (P<0.05). CONCLUSION: Traditional teaching combined with distant live broadcast teaching has achieved good results in clinical anesthesiology teaching, improved the overall quality of teaching, and has high clinical teaching value.

DDX10 promotes the proliferation and metastasis of colorectal cancer cells via splicing RPL35.

BACKGROUND: Colorectal cancer (CRC) has become the second deadliest cancer in the world and severely threatens human health. An increasing number of studies have focused on the role of the RNA helicase DEAD-box (DDX) family in CRC. However, the mechanism of DDX10 in CRC has not been elucidated. METHODS: In our study, we analysed the expression data of CRC samples from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Subsequently, we performed cytological experiments and animal experiments to explore the role of DDX10 in CRC cells. Furthermore, we performed Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network analyses. Finally, we predicted the interacting protein of DDX10 by LC-MS/MS and verified it by coimmunoprecipitation (Co-IP) and qPCR. RESULTS: In the present study, we identified that DDX10 mRNA was extremely highly expressed in CRC tissues compared with normal colon tissues in the TCGA and GEO databases. The protein expression of DDX10 was measured by immunochemistry (IHC) in 17 CRC patients. The biological roles of DDX10 were explored via cell and molecular biology experiments in vitro and in vivo and cell cycle assays. We found that DDX10 knockdown markedly reduced CRC cell proliferation, migration and invasion. Then, we constructed a PPI network with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). GO and KEGG enrichment analysis and gene set enrichment analysis (GSEA) showed that DDX10 was closely related to RNA splicing and E2F targets. Using LC-MS/MS and Co-IP assays, we discovered that RPL35 is the interacting protein of DDX10. In addition, we hypothesize that RPL35 is related to the E2F pathway and the immune response in CRC. CONCLUSIONS: In conclusion, provides a better understanding of the molecular mechanisms of DDX10 in CRC and provides a potential biomarker for the diagnosis and treatment of CRC.

Exosomal hsa_circ_0008925 from Urine Is Related to Chronic Renal Fibrosis.

At present, there is no noninvasive biomarker of renal fibrosis. The potential diagnostic value of urinary exosome-derived circRNAs from glomerular disease patients for renal fibrosis is still uncertain. Here, we first detected the expression of hsa_circ_0008925 in TGF-ß1-cultured HK-2 cell-derived exosomes. Secondly, we collected urine samples from 95 biopsy-proven glomerular disease patients and 34 healthy controls. The expression of hsa_circ_0008925 was analyzed, and the correlation with renal function and pathological changes was calculated. The receiver operating characteristic (ROC) curve for the diagnosis of renal fibrosis was performed. The results showed that in exosomes derived from TGF-ß1-cultured HK-2 cells, the expression of hsa_circ_0008925 was increased compared with normal cultured. Further, the expression level of hsa_circ_0008925 was increased in urinary exosomes from renal fibrosis patients and correlated with serum creatinine, blood urea nitrogen (BUN), estimated glomerular filtration rate, and cystatin C. The level of hsa_circ_0008925 was furthermore correlated with the score of tubulointerstitial fibrosis (TIF) and the score of glomerular sclerosis. The ROC curve showed that hsa_circ_0008925 can diagnose renal fibrosis at a cut-off value of 0.093 with a sensitivity of 52.2% and specificity of 96.4%. In summary, we indicated that urinary exosomal hsa_circ_0008925 could be acted as a noninvasive biomarker for renal fibrosis in glomerular diseases patients.

The Antibiofilm Activity and Mechanism of Nanosilver- and Nanozinc-Incorporated Mesoporous Calcium-Silicate Nanoparticles.

BACKGROUND: Mesoporous calcium-silicate nanoparticles (MCSNs) have good prospects in the medical field due to their great physicochemical characteristics, antibacterial activity and drug delivery capacity. This study was to analyze the antibiofilm activity and mechanisms of silver (Ag) and zinc (Zn) incorporated MCSNs (Ag/Zn-MCSNs) with different percentages of Ag and Zn. METHODS: Ag/Zn(1:9, molar ratio)-MCSNs and Ag/Zn(9:1, molar ratio)-MCSNs were prepared and characterized. Endocytosis of nanoparticles by Enterococcus faecalis (E. faecalis) treated with Ag/Zn-MCSNs was observed using TEM to explore the antibacterial mechanisms. The antibiofilm activity of Ag/Zn-MCSNs with different ratios of Ag and Zn was tested by E. faecalis biofilm model in human roots. The human roots pretreated by different Ag/Zn-MCSNs were cultured with E. faecalis. Then, SEM and CLSM were used to observe the survival of E. faecalis on the root canal wall. Cytotoxicity of the nanoparticles was tested by CCK8 kits. RESULTS: The Ag/Zn-MCSNs release Ag+ and destroy the cell membranes to kill bacteria. The MCSNs containing Ag showed antibacterial activity against E. faecalis biofilms in different degrees, and they can adhere to dentin surfaces to get a continuous antibacterial effect. However, MTA, MCSNs and Zn-MCSNs could not disrupt the bacterial biofilms obviously. MCSNs, Ag/Zn(1:1, molar ratio)-MCSNs and Ag/Zn(1:9)-MCSNs showed no obvious cytotoxicity, while Ag-MCSNs and Ag/Zn(9:1)-MCSNs showed cytotoxicity. Zn-MCSNs can slightly promote cell proliferation. CONCLUSION: Ag/Zn-MCSNs have good antibiofilm activity. They might achieve an appropriate balance between the antibacterial activity and cytotoxicity by adjusting the ratio of Ag and Zn. Ag/Zn-MCSNs are expected to be a new type of root canal disinfectant or sealer for root canal treatment.

Root dentine thickness of danger zone in mesial roots of mandibular first molars.

BACKGROUND: Better understanding of the danger zone anatomy in mesial roots (MRs) of mandibular first molars (MFMs) may serve to decrease the risk of mishaps. This study aimed to measure the minimal distal dentine thicknesses of danger zone in MRs of MFMs in a native Chinese population using cone-beam computed tomography (CBCT). METHODS: CBCT images of 1792 MFMs from 898 Chinese patients were analyzed. The minimal distal dentine thicknesses of the mesiobuccal (MB) and mesiolingual (ML) canals below the furcation 1, 2, 3, 4, 5 mm were measured. The association between the minimal distal dentine thicknesses and the root lengths, patient's age and gender, side were assessed. RESULTS: The minimal distal dentine thicknesses of MB and ML canals are located 3 ∼ 4 mm below the furcation for both men and women. There are no differences between MB and ML canals, while the minimal distal dentine thicknesses of MB and ML canals were higher in men than women (P < 0.05), except at 1 and 3 mm of ML canals (P > 0.05). The minimal distal dentine thicknesses of MB and ML canals increased with age in both men and women at each location (P < 0.05). The minimum distal dentine thickness at every location were significantly different between long teeth and short teeth both in men and women (P < 0.05), with short teeth having the smallest mean values. There are no significant differences between two sides (P > 0.05). CONCLUSIONS: The minimal distal dentine thicknesses of MRs in MSMs have close correlation with root length, patient's age and gender.

The involvement of multidrug and toxin extrusion protein 1 in the distribution and excretion of berberine.

1. Berberine (BBR), an isoquinoline alkaloid, has demonstrated multiple clinical pharmacological actions. As a substrate of multiple transporters in the liver, BBR is rarely excreted into the bile but can be found in the urine. The purpose of the present study was to investigate the role of multidrug and toxin extrusion protein 1 (MATE1) in the transport of BBR in the liver and kidney. 2. Using human MATE1 (hMATE1)-transfected HEK293 cells, BBR was shown to be a substrate of hMATE1 (Km = 4.28 ± 2.18 µM). In primary rat hepatocytes, pH-dependent uptake and efflux studies suggested that the transport of BBR was driven by the exchange of H+ and involved Mate1. In rats, we found that pyrimethamine (PYR), an inhibitor of Mate1, increased hepatic and renal distribution of BBR and decreased systematic excretion of BBR. 3. These findings indicated that BBR is a substrate of MATE1 and that hepatic and renal Mate1 promote excretion of BBR into bile and urine, respectively. In conclusion, Mate1 plays a key role in the distribution and excretion of BBR, and we speculate that drug-drug interactions (DDIs) caused by MATE1 may occur between BBR and other co-administered drugs.

Computational design of peptide ligands to target the intermolecular interaction between viral envelope protein and pediatric receptor.

The recognition and binding of viral envelope protein to pediatric receptor subverts the membrane-trafficking apparatus to mediate virion export in young children. Here, we described a successful computational design of peptide ligands to target the intermolecular interaction between the virus large envelope protein (LHB) and adaptin receptor (ADT). Based on the crystal structure of ADT in complex with an oligopeptide segment corresponding to the core binding site of LHB, a sequence-specific amino acid preference profile was determined systematically for the ADT-binding peptides using structural bioinformatics approach. With the information harvested from the profile, a genetic evolution procedure was run to improve the biological potency of a peptide population generated randomly from the LHB. A number of potential hits were obtained from the evolution, and four were measured to interact with ADT at micromolar level. A high-affinity hit peptide was then optimized according to computational structural analysis. It is revealed that a potent peptide can be divided into three regions, i.e. a negatively charged region at N-terminus, a hydrophobic core region in middle, and a small, polar region at C-terminal tail. In addition, the two termini of peptide are partially out of the active pocket of ADT, thus contributing moderately to the peptide binding.

Synthesis and characterization of fluorinated polyacrylate latex emulsified with novel surfactants.

The fluorinated polyacrylate latex were successfully prepared with semi- continuous seeded emulsion polymerization of butyl acrylate (BA), methyl methacrylate (MMA) and hexafluorobutyl methacrylate (HFMA) which was initiated with potassium persulfate (KPS) initiator and emulsified with the novel mixed surfactants of sodium lauryl glutamate (SLG) and alkylphenol ethoxylates (OP-10). The structure of the resultant latex was confirmed by Fourier transform infrared spectroscopy (FTIR). The particle size of the latex was measured by Zetatrac dynamic light scattering detector. The film of latex was tested by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and contact angle (CA). The optimum conditions of preparing the novel fluorinated polyacrylate latex are optimized and the results are as follows: the amount of emulsifiers is 4.0%; mass ratio of SLG to OP-10 is 1:1, the amount of the initiator is 0.6%. The mass ratio of MMA to BA is 1:1 and the amount of HFMA is 7.0%. In this case, the conversion is high and the polymerization stability is good. In addition, the water resistance and thermal properties of the latex films were improved significantly in comparison with the film of the latex prepared without the fluorinated monomer.

[Absorption, distribution, metabolism, excretion and toxicity of biologics and its application in pharmacokinetic modeling].

Recently, more and more attentions of drug development are placed to macromolecules, such as monoclonal antibodies, proteins, etc. It has become one of the most promising areas in drug research and development in 21st Century. In terms of the structure and the ADMET (absorption, distribution, metabolism, excretion and toxicity), macromolecules is different from small molecule drugs, which lead to a distinct modeling strategy. The characterization of biologics ADMET processes and its application in the PK model selection of macromolecules are reviewed in this paper.