Ultra-trace Ag doped carbon quantum dots with peroxidase-like activity for the colorimetric detection of glucose.

Carbon quantum dots (CQDs) have significant applications in nanozymes. However, previous studies have not elucidated the structure-activity relationship and enzyme mechanism. In this study, we employed a one-step microwave method to synthesize ultra-trace Ag-doped carbon quantum dots (Ag-CQDs). In the presence of hydrogen peroxide (H2O2), we used the oxidative coupling reaction of 3,3',5,5'-tetramethylbenzidine (TMB) to evaluate the intrinsic peroxidase-like activity, kinetics, and mechanism of Ag-CQDs. The trace amount of doped Ag (1.64 %) facilitated electron transfer from the CQDs interior to the surface. The electron transfer triggered the peroxide activity of CQDs, producing hydroxyl radical (·OH), which oxidized the colorless TMB to blue-colored TMB (oxTMB). By coupling with glucose oxidase (GOx), the Ag-CQDs/H2O2/TMB system has been used for colorimetric glucose determination. The system demonstrated a low detection limit (0.17 µM), wide linear range (0.5-5.5 µM), and satisfactory results when fruit juice was analyzed. This study reports a feasible method for the colorimetric detection of glucose by synthesizing ultra-trace Ag-doped carbon quantum dots with peroxidase-mimicking activity.

In situ microwave-assisted preparation of NS-codoped carbon dots stabilized silver nanoparticles as an off-on fluorescent probe for trace Hg2+ detection.

An off-on fluorescent probe (NS-CDs-AgNPs) was synthesized based on a one-pot microwave process by utilizing N, S co-doping carbon dots (NS-CDs) and silver nitrate as precursors. The significant peak of NS-CDs-AgNPs at 393 nm in ultraviolet spectrum indicated silver nanoparticle (AgNPs) were successfully synthesized. A faint blue fluorescence emission (442 nm) was displayed when excited NS-CDs-AgNPs at 371 nm. A remarkable fluorescence recovery was observed upon adding of trance Hg2+, whereas the other heavy metal ions did not elicit this response. The reason for this phenomenon was revealed in this work that a spontaneous redox reaction occurred between NS-CDs-AgNPs and Hg2+, which leaded to the formation of NS-CDs-Agn-2NPsHg complexes. On the basis of this mechanism, a new off-on fluorescent analytical method was constructed for Hg2+ detection with linear range of 10-400 nM (R2 = 0.9941), and the detection limit (LOD) of 5.16 nM. Additionally, satisfactory recovery (90.28%-106.13%) and the relative standard deviation (RSD) (RSD＜5.21%) were obtained in water sample detection. More importantly, the NS-CDs-AgNPs exhibited lower cytotoxicity and better biocompatibility, indicating a huge potential in cell imaging and clinical medicine.

GP IIb/IIIa-Mediated Platelet Activation and Its Modulation of the Immune Response of Monocytes Against Candida albicans.

Candida albicans is the most common fungal pathogen in humans, causing invasive disease and even potentially life-threatening systemic infections when tissue homeostasis is disrupted. Previous studies have identified an essential role of platelets in infection and immunity, especially when they are activated. However, it is still unclear whether platelets can be activated by C. albicans, and even less is known about the role of platelets in C. albicans infection. Herein, we showed that C. albicans induced platelet activation in vitro. C. albicans elevated the levels of AKT Ser473 phosphorylation, and inhibition of the PI3K-AKT signaling pathway reversed C. albicans-induced platelet activation. Surprisingly, C. albicans-induced platelet activation occurred in an integrin glycoprotein (GP) IIb/IIIa-dependent manner but was independent of the pattern recognition receptors toll-like receptor (TLR) 2 and TLR4. Interestingly, platelets enhanced the phagocytosis of human monocytes challenged with C. albicans and upregulated the expression of inflammatory cytokines, which were dependent on platelet activation mediated by GP IIb/IIIa. The present work provides new insights into the role of activated platelets in the defense against C. albicans, highlighting the importance of GP IIb/IIIa in the recognition of C. albicans.

Ribosomal L1 domain-containing protein 1 coordinates with HDM2 to negatively regulate p53 in human colorectal Cancer cells.

BACKGROUND: Ribosomal L1 domain-containing protein 1 (RSL1D1) is a nucleolar protein that is essential in cell proliferation. In the current opinion, RSL1D1 translocates to the nucleoplasm under nucleolar stress and inhibits the E3 ligase activity of HDM2 via direct interaction, thereby leading to stabilization of p53. METHODS: Gene knockdown was achieved in HCT116p53+/+, HCT116p53-/-, and HCT-8 human colorectal cancer (CRC) cells by siRNA transfection. A lentiviral expression system was used to establish cell strains overexpressing genes of interest. The mRNA and protein levels in cells were evaluated by qRT-PCR and western blot analyses. Cell proliferation, cell cycle, and cell apoptosis were determined by MTT, PI staining, and Annexin V-FITC/PI double staining assays, respectively. The level of ubiquitinated p53 protein was assessed by IP. The protein-RNA interaction was investigated by RIP. The subcellular localization of proteins of interest was determined by IFA. Protein-protein interaction was investigated by GST-pulldown, BiFC, and co-IP assays. The therapeutic efficacy of RSL1D1 silencing on tumor growth was evaluated in HCT116 tumor-bearing nude mice. RESULTS: RSL1D1 distributed throughout the nucleus in human CRC cells. Silencing of RSL1D1 gene induced cell cycle arrest at G1/S and cell apoptosis in a p53-dependent manner. RSL1D1 directly interacted with and recruited p53 to HDM2 to form a ternary RSL1D1/HDM2/p53 protein complex and thereby enhanced p53 ubiquitination and degradation, leading to a decrease in the protein level of p53. Destruction of the ternary complex increased the level of p53 protein. RSL1D1 also indirectly decreased the protein level of p53 by stabilizing HDM2 mRNA. Consequently, the negative regulation of p53 by RSL1D1 facilitated cell proliferation and survival and downregulation of RSL1D1 remarkably inhibited the growth of HCT116p53+/+ tumors in a nude mouse model. CONCLUSION: We report, for the first time, that RSL1D1 is a novel negative regulator of p53 in human CRC cells and more importantly, a potential molecular target for anticancer drug development.

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade.

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

Development and Optimization of an In Vitro Multienzyme Synthetic System for Production of Kaempferol from Naringenin.

An in vitro multienzyme synthetic system was developed and optimized to efficiently produce kaempferol in a single reaction tube. Two key genes, Atf3h and Atfls1, in the biosynthetic pathway of kaempferol were cloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The recombinant proteins were purified through affinity chromatography and showed activities of flavanone 3-hydroxylase and flavonol synthase, respectively, followed by development of an in vitro synthetic system for producing kaempferol. The system contains 8.2 mM α-ketoglutaric acid, 0.01 mM ferrous ion, 0.4% sodium ascorbate, 25 µg/mL of each recombinant enzyme, and 10% glycerol in 100 mM Tris-HCl (pH 7.2). When the reaction was carried out at 40 °C for 40-50 min, the yield of kaempferol was 37.55 ± 1.62 mg/L and the conversion rate from NRN to KMF was 55.89% ± 2.74%. Overall, this system provides a promising and efficient approach to produce kaempferol economically.