Highly sensitive detection of microRNA-21 by nitrogen-doped carbon dots-based ratio fluorescent probe via nuclease-assisted rolling circle amplification strategy.

Highly sensitive and selective detection of microRNA-21 (miRNA-21) in biological samples is critical for the disease diagnosis and cancer treatment. In this study, a nitrogen-doped carbon dots (N-CDs)-based ratio fluorescence sensing strategy was constructed for miRNA-21 detection with high sensitivity and excellent specificity. Bright-blue N-CDs (λex/λem = 378 nm/460 nm) were synthesized by facile one-step microwave-assisted pyrolysis method by using uric acid as the single precursor, and the absolute fluorescence quantum yield and fluorescence lifetime of N-CDs were 35.8% and 5.54 ns separately. The padlock probe hybridized with miRNA-21 firstly and then was cyclized by T4 RNA ligase 2 to form a circular template. At the present of dNTPs and phi29 DNA polymerase, the oligonucleotide sequence in miRNA-21 was prolonged to hybridize with the surplus oligonucleotide sequences in circular template, generating long and reduplicated oligonucleotide sequences containing abundant guanine nucleotides. Separate G-quadruplex sequences were generated after the addition of Nt.BbvCI nicking endonuclease, and then hemin bound with G-quadruplex sequence to construct the G-quadruplex DNAzyme. Such G-quadruplex DNAzyme catalyzed the redox reaction of o-phenylenediamine (OPD) with H2O2, finally producing the yellowish-brown 2,3-diaminophenazine (DAP) (λem = 562 nm). Due to the inner filter effect between N-CDs and DAP, the ratio fluorescence signal of DAP with N-CDs was utilized for sensitive detection of miRNA-21 with detection limit of 0.87 pM. Such approach has practical feasibility and excellent specificity for miRNA-21 analysis during highly homological miRNA family in HeLa cell lysates and human serum samples.

Rapid microwave fabrication of red-carbon quantum dots as fluorescent on-off-on probes for the sequential determination of Fe(III) ion and ascorbic acid in authentic samples.

In this study, fluorescent red-carbon quantum dots (R-CQDs) with an ultrahigh fluorescence quantum yield of 45% were rapidly and easily synthesized by thermal pyrolysis of 2,5-diaminotoluene sulfate and 4-hydroxyethylpiperazineethanesulfonic acid using a one-step microwave-assisted hydrothermal approach. R-CQDs possessed the excitation-independent fluorescence property with the optimal emission peak at 607 nm under the excitation wavelength of 585 nm. R-CQDs exhibited excellent fluorescence stability under extremely harsh conditions in a pH range of 2-11, high ionic strength (1.8 M of NaCl), and long UV light irradiation time (160 min). The fluorescence quantum yield of these R-CQDs was as high as 45%, implying their preferable application in chemosensors and biological analysis. Because Fe3+ ion bound with R-CQDs and statically quenched the fluorescence of R-CQDs, the fluorescence intensity of R-CQDs was recovered after the addition of ascorbic acid (AA) via its redox reaction with Fe3+ ion. R-CQDs were developed as highly sensitive fluorescent on-off-on probes for sequentially sensing Fe3+ ions and AA. Under the optimal experimental conditions, the linear range for Fe3+ ion detection was 1-70 µM with a detection limit of 0.28 µM, and the linear range for AA detection was 1-50 µM with a detection limit of 0.42 µM. The successful detection of Fe3+ ions in authentic water samples and the successful sensing of AA in human body fluids and vitamin C tablets further proved the practical application prospects of this efficient strategy in the environmental protection and disease diagnosis fields.

A ratiometric fluorescent probe for simultaneous detection of L-ascorbic acid and alkaline phosphatase activity based on red carbon dots/polydopamine nanocomposite.

Herein, efficient red carbon dots (R-CDs) were synthesized by one-step hydrothermal treatment of N-(4-amino phenyl) acetamide and (2,3-difluoro phenyl) boronic acid. The optimal emission peak of R-CDs was at 602 nm (under 520 nm excitation) and the absolute fluorescence quantum yield of R-CDs was 12.9%. Polydopamine, which was formed by the self-polymerization and cyclization of dopamine in alkaline condition, emitted characteristic fluorescence with peak position of 517 nm (under 420 nm excitation) and affected the fluorescence intensity of R-CDs through inner filter effect. L-Ascorbic acid (AA), which was the hydrolysis product of L-ascorbic acid-2-phosphate trisodium salt under the catalytic reaction of alkaline phosphatase (ALP), effectively prevented the polymerization of dopamine. Combined with the ALP-mediated AA production and the AA-mediated polydopamine generation, the ratiometric fluorescence signal of polydopamine with R-CDs was correlated closely with the concentration of both AA and ALP. Under optimal conditions, the detection limits of AA and ALP were 0.28 µM during linear range of 0.5-30 µM and 0.044 U/L with linear range of 0.05-8 U/L, respectively. This ratiometric fluorescence detection platform can efficiently shield the background interference of sophisticated samples by introducing a self-calibration as reference signal in a multi-excitation mode, which can detect AA and ALP in human serum samples with satisfactory results. Such R-CDs/polydopamine nanocomposite provides a steadfast quantitative information and makes R-CDs be excellent candidate for biosensors via combining target recognition strategy.

Fluorine and nitrogen co-doped near-infrared carbon dots for fluorescence "on-off-on" determination of nitrite.

A fluorescence "on-off-on" strategy was established for the determination of nitrite in aqueous solution based on fluorine and nitrogen co-doped near-infrared carbon dots (NIR-CDs). NIR-CDs were prepared via one-step hydrothermal method by using N-(4-aminophenyl)-acetamide and 4,5-difluorobenzene-1,2-diamine as precursors. The photoluminescence quantum yield of NIR-CDs reaches to 17.4%, and the optimal emission peak of NIR-CDs is 675 nm under excitation of 530 nm. The Stokes shift of NIR-CDs (145 nm) is higher than that of some CDs with longer emission wavelengths. The red bathophenanthroline disulfonic acid (BPS)-Fe2+ complex can quench the fluorescence of NIR-CDs via inner filter effect and static quench modes. Nitrite can oxidize Fe2+ to produce Fe3+ in acidic environment, resulting in not only the formation of colorless and unstable BPS-Fe3+ complex but also the fluorescence recovery of NIR-CDs. This fluorescence "on-off-on" phenomenon also comes with the color variation of the mixture, resulting in both the fluorescence and the visual determination of nitrite. Under optimal conditions, this assay exhibits a good linear range from 1 to 50 µM and a low detection limit of 0.056 µM for nitrite determination. The method showed good applicability for nitrite determination in soil extract, human urine, and water samples with acceptable results. A convenient fluorescence "on-off-on" strategy for nitrite detection based on fluorine and nitrogen co-doped near-infrared carbon dots (NIR-CDs) and bathophenanthroline disulfonic acid (BPS)-Fe2+ complex was innovatively established. This probe showed a low detection limit of 0.056 µM for nitrite in authentic samples, which offered a new sight for fluorescent and visual detection of nitrite in environmental protection and human health areas.

Circ_0000228 Promotes Cervical Cancer Progression via Regulating miR-337-3p /TGFBR1 Axis.

Objective: This study aims to investigate the biological function of circular RNA (circRNA) circ_0000228 in the cervical cancer (CC). Materials and Methods: In this experimental study, the GSE113696 dataset was downloaded from the Gene Expression Omnibus (GEO). GEO2R was employed to obtain differentially expressed circRNA between CC tissues and matched paracancerous tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were employed to detect circ_0000228, microRNA-337-3p (miR-337-3p ) and transforming growth factor, beta receptor I (TGFBR1) expression levels in the CC tissues and cells. Following gain-of-function and loss-of-function models establishment, CCK-8 and BrdU tests were conducted to examine cell proliferation. Transwell experiment was executed to examine CC cells migration and invasion. A lung metastasis model was utilized to determine the ability of circ_0000228 on the lung metastasis. Bioinformatics analysis, dual-luciferase reporter experiment and RNA immunoprecipitation (RIP) assay were applied to verify the targeting relationship among miR-337-3p , circ_0000228, and TGFBR1. Results: Circ_0000228 expression in the CC tissues and cells was up-modulated. Circ_0000228 overexpression markedly enhanced cell proliferation, migration, and invasion, while knocking down circ_0000228 remarkably repressed cell proliferation, migration, and invasion. MiR-337-3p could be adsorbed by circ_0000228. TGFBR1 was identified as a target gene of miR-337-3p that indirectly and positively modulated bycirc_0000228 in the CC cells. Conclusion: Circ_0000228 up-modulates TGFBR1 by targeting miR-337-3p to enhance CC cell proliferation, migration and invasion. Also, Circ_0000228 is a promising therapeutic target for the CC.

Ultrasensitive ratiometric fluorescent probes for Hg(II) and trypsin activity based on carbon dots and metalloporphyrin via a target recycling amplification strategy.

A convenient and ultrasensitive ratiometric fluorescent probe was innovatively developed for Hg(II) detection and trypsin activity evaluation based on carbon dots (CDs) and tetraphenylporphyrin tetrasulfonic acid (TPPS) using bovine serum albumin (BSA) as the substrate of trypsin. The ratiometric fluorescence signal arises from CDs (λem = 506 nm) and TPPS (λem = 645 nm) via an inner filter effect. Hg2+ can trigger the formation of TPPS-Mn2+ metalloporphyrin for target Hg2+ recycling amplification, while both TPPS-Hg2+ and TPPS-Mn2+ metalloporphyrins do not affect the fluorescence of CDs. Small amino acids and peptide fragments, which are the products of BSA under the digestion of trypsin, bind stronger with Hg2+ than with TPPS. The decomposition of both TPPS-Hg2+ and TPPS-Mn2+ metalloporphyrins leads to a variation in the ratiometric fluorescence signal. Under optimized conditions, this probe provided an inspiring detection limit of 0.086 nM for Hg2+ and 0.013 ng mL-1 for trypsin, which possessed acceptable sensitivity for Hg2+ detection and trypsin activity evaluation in authentic samples. This unprecedented CD-based ratiometric fluorescence proposal for ultrasensitive quantification of Hg2+ concentration and selective assessment of trypsin activity gives a new insight for designing metal ion assays or enzymatic activity bioassays under different enzymatic substrates in the near future.

[Mechanism of carbon monoxide affecting the expression of cellular adhesion molecule under stimulation of inflammatory cytokines to human gingival fibroblasts].

OBJECTIVE: To investigate the mechanism by which carbon monoxide inhibits the expression of adhesion molecules on human gingival fibroblasts (HGF) stimulated with inflammatory cytokines. METHODS: HGF were cultured in vitro, and stimulated with 50 ng x mL tumor necrosis factor-alpha (TNF-alpha) and 10 ng x mL(-1) interleukin-1beta (IL-1beta) concurrently in the presence or absence of carbon monoxide releasing molecule-3 (CORM-3) at 500 micromol x L-1. Expression of phosphorylated extracellular regulated protein kinase (ERK), phosphorylated c-Jun N-terminal kinase (NK) and phosphorylated p38 in mitogen-activated protein kinase(MAPK) pathway was studied by Western blot at 10 min and 20 min, respectively. Nuclear expression of nuclear factor-kappaB (NF-kappaB) was checked by Western blot after 4 h stimulation. In some experiments, cells were prestimulated by 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) for 8 h before cytokine stimulation and the expression of intercellular adhesion molecule-1 (ICAM-1) was checked by Western blot after 24 h. RESULTS: CORM-3 significantly inhibited the phosphorylation of MAPK p38 after 10 min stimulation with cytokines, but had no signifi-cant effect on the phosphorylation of ERK and JNK. CORM-3 significantly inhibited the nuclear expression of NF-KB-p65 on HGF after 4 h stimulation by inflammatory cytokines. The inhibitory effect of CORM-3 on the expression of ICAM-1 was not influenced by guanylate cyclase inhibitor ODQ. CONCLUSION: The inhibitory effect of carbon monoxide on the expression of adhesion molecules might be exerted by its inhibitory effect on the NF-kappaB activity and MAPK p38 phosphorylation.

[Expression of high mobility group box 1 in gingival tissues of chronic periodontitis].

OBJECTIVE: To investigate the expression of high mobility group box 1 (HMGB1) in gingival tissues of chronic periodontitis. METHODS: Human peripheral blood mononuclear cells(PBMC) were stimulated with 1 microg x mL(-1) lipopolysaccharide (LPS) for 24 h or 48 h. Expression and release of HMGB1 were checked by immunofluorescence and enzyme-linked immunosorbent assay (ELISA), respectively. PBMC were stimulated with 100 ng x mL(-1) HMGB1 or 50 ng x mL(-1) tumor necrosis factor-alpha (TNF-alpha), the expressions of TNF-alpha and HMGB1 in the supernatant were studied by ELISA. Gingival tissues and gingival crevicular fluids (GCF) were collected from patients and healthy people. Expression of HMGB1 in gingival tissues and GCF was studied using immunofluorescence and ELISA, respectively. RESULTS: HMGB1 was translocated from nucleus to cytosol in PBMC after LPS stimulation for 24 h. The content of HMGB1 in the supernatant from stimulated cells was significantly higher than that from unstimulated cells after 48 h (P < 0.01). HMGB1 was released by PBMC in response to TNF-alpha stimulation, it also stimulated PBMC to release TNF-alpha (P < 0.01). Translocation of HMGB1 from nucleus to cytosol was also found in infiltrated cells in gingival tissues from patients, and HMGB1 in GCF from patients was significantly higher than that from healthy people P < 0.01). CONCLUSION: The results suggest that HMGB1 may play an important role in the pathological progress of chronic periodontitis.

[Influence of carbon monoxide on the expression of adhesion molecules stimulated with tumor necrosis factor-alpha and interleukin-1beta on human gingival fibroblasts].

OBJECTIVE: To investigate the influence of carbon monoxide on the expression of adhesion molecules stimulated by inflammatory cytokines on human gingival fibroblasts. METHODS: Human gingival fibroblasts were stimulated with 50 ng x mL(-1) tumor necrosis factor (TNF)-alpha and 10 ng x mL(-1) interleukin (IL)-1beta concurrently in the presence or absence of 500 micromol x L(-1) carbon monoxide releasing molecule (CORM). Expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 at protein and mRNA level was examined by Western blot and reverse transcription polymerase chain reaction (RT-PCR), respectively. Activity of transcription factor NF-kappaB was evaluated by reporter gene assay. RESULTS: Expression of ICAM-1 and VCAM-1 on human gingival fibroblasts increased dramatically after concurrent stimulation of TNF-alpha and IL-1beta, while CORM inhibited the upregulation of ICAM-1 and VCAM-1. CORM decreased the activity of NF-KB stimulated by TNF-alpha and IL-1beta. CONCLUSION: Carbon monoxide could be a promising way in treating of periodontitis.

Poly[methano-ltetra-kis[µ(3)-5-(4-methoxy-phen-yl)pyrazole-3-carboxyl-ato(2-)]tri-copper(II)disodium(I).

The asymmetric unit of the polymeric title complex, [Cu(3)Na(2)(C(11)H(8)N(2)O(3))(4)(CH(4)O)](n), consists of two Na(I) atoms, three Cu(II) atoms, four 5-(4-methoxy-phen-yl)pyrazole-3-carboxyl-ate ligands and one methanol mol-ecule. The central Cu(II) atom is coordinated by four N atoms from four pyrazole-3-carboxyl-ate ligands, forming a distorted tetra-hedral (CuN(4)) geometry, while each of the other two Cu(II) atoms is coordinated by two O atoms and two N atoms from the two ligands, forming a slightly distorted square-planar (CuN(2)O(2)) geometry. Each of the two Na(I) atoms is coordinated by five O atoms, forming a distorted octahedral geometry; four O atoms are from the carboxyl-ate groups of the three ligands and the remaining O atom is from the meth-oxy group of the ligand or from the methanol mol-ecule.