Recognition of Oligonucleotide C by Polydopamine-Coated Solid-State Nanopores.

Selective recognition of short oligonucleotides at the single-molecule level is particularly important for early disease detection and treatment. In this work, polydopamine (PDA)-coated nanopores were prepared via self-polymerization as a solid-state nanopore sensing platform for the recognition of oligonucleotide C (PolyC). The PDA coating possesses abundant active sites, such as indole, amino, carboxyl, catechol, and quinone structures, which had interactions with short oligonucleotides to slow down the translocation rate. PDA-coated nanopores selectively interact with PolyC20 by virtue of differences in hydrogen bonding forces, generating a larger blocking current, while polyA and polyT demonstrated very small blockings. At the same time, PDA-coated nanopores can sensitively distinguish PolyC with different lengths, such as 20, 14, and 10 nt. The functionalization of PDA on the solid-state nanopore provides an opportunity for the rational design of the recognition surface for biomolecules.

[Mechanisms of mucus hypersecretion in airway of rats induced by synergies between cold air and cigarette smoke inhalation and intervention effects of drugs].

OBJECTIVE: To explore the mechanisms of mucus hypersecretion in airway of rats induced by the synergies between cold air and cigarette smoke inhalation and understand the intervention effects of saussurea and budesonide in this process. METHODS: A total of 70 SD rats were randomly divided into 7 groups. Group A: control; Group B: cold stimulation group receiving cold air inhalation for 3 h daily for 40 d; Group C: cigarette smoke inhalation group receiving cigarette smoke inhalation for 0.5 h daily for 40 d; Group D: cigarette smoke inhalation + cold stimulation group; Group E: cigarette smoke inhalation + cold stimulation + saussurea (0.8 mg/kg saussurea intraperitoneally injected once daily for 40 d); Group F: cigarette smoke inhalation + cold stimulation + inhaled budesonide (0.5 mg/kg inhaled once daily for 40 d); Group G: cigarette smoke inhalation + cold stimulation + saussurea + inhaled budesonide. The relative quantities of TRPM8 mRNA within bronchial epithelium of each group were detected by real-time polymerase chain reaction (PCR) and TRPM8 protein was detected by immunohistochemical assay and Western blot. The levels of mucin (MUC) 5AC, interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) in bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay. RESULTS: TRPM8 mRNA of groups A-G were 1.00 ± 0.00, 0.98 ± 0.07, 2.27 ± 0.29, 2.31 ± 0.30, 1.55 ± 0.38, 1.66 ± 0.40 and 1.31 ± 0.23; TRPM8 protein 0.16 ± 0.05, 0.16 ± 0.04, 0.22 ± 0.06, 0.25 ± 0.05, 0.17 ± 0.04, 0.18 ± 0.03, 0.15 ± 0.05, 0.25 ± 0.04, 0.24 ± 0.03, 0.58 ± 0.06, 0.56 ± 0.09, 0.41 ± 0.09, 0.39 ± 0.07 and 0.20 ± 0.06 respectively. TRPM8 mRNA and protein of groups C and D were significantly higher than those of group A. And groups E, F and G were significantly lower than those of group D (all P < 0.05). In BALF of groups A-G, MUC5AC were (57 ± 6), (69 ± 5), (66 ± 4), (185 ± 43), (142 ± 30), (147 ± 36) and (60 ± 11) µg/mg, IL-8 (58 ± 14), (146 ± 38), (134 ± 29), (379 ± 101), (262 ± 67), (294 ± 70) and (81 ± 27) ng/L, TNF-α(153 ± 28), (208 ± 90), (274 ± 64), (600 ± 113), (458 ± 96), (498 ± 84) and (169 ± 65) ng/L respectively. The values of groups B, C and D were significantly higher than those of group A (all P < 0.05) while groups E, F and G were significantly lower than those of group D (all P < 0.05). Cigarette smoke inhalation and cold stimulation synergistically enhanced the expression of MUC5AC, IL-8 and TNF-α. Saussurea and inhaled budesonide synergistically inhibited the expression of MUC5AC, IL-8 and TNF-α. CONCLUSIONS: Cold air inhalation evokes the release of proinflammatory cytokines and MUC5AC via activated TRPM8 channel up-regulated by cigarette smoke inhalation. Saussurea and inhaled budesonide synergistically inhibits the above mentioned process.

Solid-state Nanopores: Chemical Modifications, Interactions, and Functionalities.

Nanopore technology is a burgeoning detection technology for single-molecular sensing and ion rectification. Solid-state nanopores have attracted more and more attention because of their higher stability and tunability than biological nanopores. However, solid-state nanopores still suffer the drawbacks of low signal-to-noise ratio and low resolution, which hinder their practical applications. Thus, developing operatical and useful methods to overcome the shortages of solid-state nanopores is urgently needed. Here, we summarize the recent research on nanopore modification to achieve this goal. Modifying solid-state nanopores with different coating molecules can improve the selectivity, sensitivity, and stability of nanopores. The modified molecules can introduce different functions into the nanopores, greatly expanding the applications of this novel detection technology. We hope that this review of nanopore modification will provide new ideas for this field.

[Effects of transient receptor potential melastatin 8 cation channels on inflammatory reaction induced by cold temperatures in human airway epithelial cells].

OBJECTIVE: To explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) in cold-induced production of inflammatory factors in airway epithelial cells and related signal transduction mechanism. METHODS: The 16HBE human airway epithelial cells were stimulated with cold temperature (18°C). In intervention experiments, cells were pretreated with TRPM8 channel antagonist BCTC, protein kinase C (PKC) specific inhibitor calphostin C and transfected with TRPM8 shRNA or control shRNA respectively, and thereafter cold stimulation was applied. Cells were divided into 6 groups: a control group (incubated at 37°C), a cold stimulation group, a cold stimulation + BCTC group, a cold stimulation + TRPM8 shRNA group, a cold stimulation + control shRNA group, a cold stimulation + calphostin C group. Western blot was performed to show the extent of knockdown in TRPM8 protein expression in the TRPM8 shRNA transfected cells. Dynamics of relative concentration of intracellular Ca(2+) in the former 5 groups were measured by calcium imaging techniques. Images were taken at one frame per 10 seconds. The levels of interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α mRNA and protein were detected by real-time PCR and ELISA respectively. RESULTS: The highest relative concentration of intracellular calcium in cold stimulation group (2.36 ± 0.24) was higher than that of control group (1.01 ± 0.02) (t = 12.52, P < 0.01). BCTC and TRPM8 shRNA reduced intracellular calcium (1.05 ± 0.09, 1.08 ± 0.09), compared with single cold stimulation group (t = 6.69 and 9.12, all P < 0.01). IL-6, IL-8, TNF-α mRNA and protein in cold stimulation group[0.66 ± 0.16, 0.77 ± 0.15, 0.73 ± 0.09 and (92 ± 13) ng/L, (125 ± 22) ng/L, (88 ± 12) ng/L ] were significantly higher than those in control group [0.37 ± 0.08, 0.32 ± 0.07, 0.48 ± 0.10 and (52 ± 8) ng/L, (50 ± 9) ng/L, (61 ± 8) ng/L] (t = 3.20 - 6.26, all P < 0.05). IL-6 mRNA, IL-8 mRNA, TNF-α mRNA and protein in cold stimulation + BCTC group [0.42 ± 0.09, 0.52 ± 0.13, 0.52 ± 0.12 and (72 ± 8) ng/L, (92 ± 14) ng/L, (68 ± 11) ng/L], cold stimulation + TRPM8 shRNA group [0.41 ± 0.10, 0.49 ± 0.08, 0.50 ± 0.08 and (60 ± 12) ng/L, (89 ± 14) ng/L, (68 ± 11) ng/L] and cold stimulation + calphostin C group [0.40 ± 0.07, 0.44 ± 0.09, 0.47 ± 0.08 and (69 ± 9) ng/L, (86 ± 15) ng/L, (61 ± 10) ng/L] were significantly lower than those in cold stimulation group (t = 2.47 - 4.21, all P < 0.05). IL-6 mRNA, IL-8 mRNA, TNF-α mRNA and protein in cold stimulation + control shRNA group [0.61 ± 0.10, 0.69 ± 0.11, 0.64 ± 0.13 and (89 ± 13) ng/L, (118 ± 20) ng/L, (79 ± 13) ng/L] showed no significant change, compared with cold stimulation group (t = 0.35 - 1.12, all P > 0.05). CONCLUSION: Cold temperature may induce Ca(2+) influx and up-regulate IL-6, IL-8, and TNF-α expression in 16HBE cells by activating the TRPM8 ion channels, and this is via a signaling pathway involving PKC.

[Role of transient receptor potential canonical 1 in airway remodeling and effect of budesonide on its pulmonary expression in asthmatic guinea pigs].

OBJECTIVE: To explore the role of transient receptor potential canonical 1 (TRPC1) in airway remodeling and the effect of budesonide intervention on its expression in the lungs of guinea pigs with ovalbumin-induced asthma. METHODS: Fifty male guinea pigs were randomized into 5 equal groups, including a blank control group, ovalbumin group, ovalbumin+TRPC1 siRNA group, ovalbumin+luciferase siRNA group, and ovalbumin+budesonide group. After corresponding treatments, bronchoalveolar lavage was collected from the guinea pigs for eosinophils analysis and detection of IL-5 and IL-13 levels using ELISA. The lung tissues were stained with HE and Masson's trichrome to observe the bronchial wall thickness, smooth muscle hypertrophy, subepithelial collagen deposition, and lung inflammations. Immunohistochemistry and real-time quantitative PCR were performed to detect TRPC1 protein and mRNA expressions in the lungs, respectively. RESULTS: The guinea pig models of ovalbumin-induced asthma showed significantly increased thickness of the bronchial wall, smooth muscle hypertrophy, collagen deposition and inflammatory cell infiltration, but these pathologies were obviously alleviated by treatment with TRPC1 siRNA or budesonide (P/0.05). Immunohistochemstry showed that TRPC1 protein was distributed mainly on the cell membrane and in the nuclei of the basal cells or columnar epithelial cells. CONCLUSION: The up-regulated expression of TRPC1 ion channel is closely associated with the occurrence and progression of airway remodeling and chronic airway inflammation in asthma. Budesonide can partially suppress airway remodeling and inflammation by regulating the expression of TRPC1.

The pathophysiological mechanisms underlying mucus hypersecretion induced by cold temperatures in cigarette smoke-exposed rats.

In a recent study, we demonstrated that transient receptor potential melastatin 8 (TRPM8), a calcium-permeable cation channel that is activated by cold temperatures, is localized in the bronchial epithelium and is upregulated in subjects with chronic obstructive pulmonary disease, which causes them to be more sensitive to cold air. In the present study, we found that exposure to cold temperatures induced ciliary ultrastructural anomalies and mucus accumulation on the epithelial surface. Male Sprague-Dawley rats were exposed to cold temperatures to determine the effects of cold air on ultrastructural changes in cilia and the airway epithelial surface. The rats were also exposed to cigarette smoke and/or cold temperatures to determine the effects of smoke and cold air on TRPM8 expression and the role of cold air in cigarette smoke-induced mucus hypersecretion. Following real-time RT-PCR and western blot analysis, we observed a high expression of TRPM8 mRNA and protein in the bronchial tissue following cigarette smoke inhalation. As shown by ELISA, concurrent cold air enhanced the levels of mucin 5AC (MUC5AC) protein, as well as those of inflammatory factors [tumor necrosis factor (TNF)-α and interleukin (IL)-8] that were induced by cigarette smoke inhalation to a greater extent than stimulation with separate stimuli (cold air and cigarette smoke separately). The results suggest that cold air stimuli are responsible for the ultrastructural abnormalities of bronchial cilia, which contribute to abnormal mucus clearance. In addition, cold air synergistically amplifies cigarette smoke-induced mucus hypersecretion and the production of inflammatory factors through the elevated expression of the TRPM8 channel that is initiated by cigarette smoke inhalation.