Nitrogen-doped orange emitting carbon dots for ß-carotene detection and lysosomal imaging.

ß-Carotene is a natural antioxidant that has an indispensable effect on the growth and immunity of the human body. For intracellular and in vitro detection of ß-carotene, N-doped carbon quantum dots (O-CDs) were prepared by co-heating carbonization of 1,5-naphthalenediamine and nitric acid in ethanol solvent for 2 h at 200 °C. O-CDs have longer wavelength orange light emission, with an optimal excitation peak of 470 nm and an optimal emission peak of 590 nm. According to the principle of the internal filtering effect on which the detection system is based, O-CDs present a good linear relationship with ß-carotene within a wide range of 0-2000 µM, and the R2 coefficient of the linear regression equation is 0.999. In addition, O-CDs showed targeting of lysosomes in cell imaging and could be used to detect intracellular lysosomal movement. These experiments show that O-CDs can be used for in vivo and in vitro detection of ß-carotene and can serve as a potential substitute to commercial lysosome targeting probes.

Preparation and application of high-brightness red carbon quantum dots for pH and oxidized L-glutathione dual response.

Carbon dots (CDs) with red fluorescence emission are highly desirable for use in bioimaging and trace- substance detection, with potential applications in biotherapy, photothermal therapy, and tumor visualization. Most CDs emit green or blue fluorescence, thus limiting their applicability. We report a novel fluorescent detection platform based on high-brightness red fluorescence emission carbon dots (R-CDs) co-doped with nitrogen and bromine, which exhibit pH and oxidized L-glutathione (GSSG) dual-responsive characteristics. The absolute quantum yield of the R-CDs was as high as 11.93%. We discovered that the R-CDs were able to detect acidic pH in live cells and zebrafish owing to protonation and deprotonation. In addition, GSSG was detected in vitro over a broad linear range (8-200 µM) using the R-CDs with excitation-independent emission. Furthermore, cell imaging and bioimaging experiments demonstrated that the R-CDs were highly cytocompatible and could be used as fluorescent probes to target lysosomes and nucleolus. These studies highlight the promising prospects of R-CDs as biosensing tools for bioimaging and trace-substance detection applications.

Roll-to-Roll DNA Nanomachine for Ultrasensitive Electrochemical Determination of miRNA.

MicroRNAs (miRNAs) are a family of endogenous noncoding RNAs with the functions of gene regulation, which serve as promising markers for a range of diseases such as diabetic foot ulcers, cancers, etc. In this work, we engineered a roll-to-roll DNA nanomachine for highly sensitive electrochemical detection of miRNA. A dumbbell-structured DNA probe could be transitioned to be wheel-structured conformation upon target recognition, which rolls around track strands on the surface of gold nanoparticles (AuNPs) in the presence of nicking endonuclease. The resulting single strands on AuNPs are activated for the second round of rolling at the DNA-modified electrode interface, leading to the variation of electrochemical responses. The roll-to-roll amplification behavior allows a wide detection range with a limit of detection as low as 10 aM. The practicability is also demonstrated by the application in human serum samples with satisfactory results. It is expected that the proposed electrochemical method offers a new paradigm to develop miRNA assays based on DNA nanotechnology.

Red-emissive carbon nanodots for highly sensitive ferric(III) ion sensing and intracellular imaging.

Ferric(III) ions (Fe3+) are one of the most abundant metal ions in environmental and biological systems. The determination of Fe3+ has attracted great attention for healthcare concerns. In this work, we have developed a novel fluorescence method for the sensing and intracellular imaging of Fe3+ based on the prepared red-emissive carbon nanodots. The nanoprobes are synthesized via a microwave method using ammonium fluoride and o-phenylenediamine as carbon precursors, which exhibit excellent optical properties and low toxicity. More importantly, the carbon nanodots show high selectivity towards Fe3+ against other interfering ions. The sensitivity is also high with the limit of detection as low as 0.05 µM. Meanwhile, the carbon nanodots have been successfully used for fluorescence imaging of cells and could be quenched by intracellular Fe3+. These results suggest that the red-emissive carbon nanodots have diverse potential utilities in biomedical fields.

Superior reducing carbon dots from proanthocyanidin for free-radical scavenging and for cell imaging.

The presence of excessive ROS can cause much harm to the human body and can even cause diseases. Therefore, it is important to detect and remove ROS, but there is no ideal method available for this at present. In this research, using procyanidins, a type of plant extract with strong reducibility, as raw materials, fluorescent carbon dots (CDs) were prepared by a hydrothermal method. The proanthocyanidin-based carbon dots (PCDs) emit a light-green colored light under UV irradiation. The PCDs retain the strong reducibility of procyanidins and are highly water-soluble compared with procyanidins. The PCDs, in addition to having good biocompatibility, also have the superior properties of radical scavenging activity and cell imaging. In in vitro experiments, 1,1-diphenyl-2-picrylhydrazyl (DPPH; 100 µM) was reduced by 30% when PCDs were added up to a concentration of 87.5 µg mL-1. At the same time, the fluorescence quenching correlates with the concentration of hypochlorite and hydrogen peroxide and has a good linearity in the range of 250-2250 nM and 60-180 µM with a detection limit of 3.676 nM and 0.602 µM, respectively. Based on the previously described advantages, PCDs have potential as a biomedicine.

High-Mobility Group Box 1 From Hypoxic Trophoblasts Promotes Endothelial Microparticle Production and Thrombophilia in Preeclampsia.

OBJECTIVE: Thrombophilia is a major complication in preeclampsia, a disease associated with placental hypoxia and trophoblast inflammation. Preeclampsia women are known to have increased circulating microparticles that are procoagulant, but the underlying mechanisms remain unclear. In this study, we sought to understand the mechanism connecting placental hypoxia, circulating microparticles, and thrombophilia. APPROACH AND RESULTS: We analyzed protein markers on plasma microparticles from preeclampsia women and found that the increased circulating microparticles were mostly from endothelial cells. In proteomic studies, we identified HMGB1 (high-mobility group box 1), a proinflammatory protein, as a key factor from hypoxic trophoblasts in stimulating microparticle production in human umbilical vein endothelial cells. Immunodepletion or inhibition of HMGB1 in the conditioned medium from hypoxic human trophoblasts abolished the endothelial microparticle-stimulating activity. Conversely, recombinant HMGB1 stimulated microparticle production in cultured human umbilical vein endothelial cells. The microparticles from recombinant HMGB1-stimulated human umbilical vein endothelial cells promoted blood coagulation and neutrophil activation in vitro. Injection of recombinant HMGB1 in pregnant mice increased plasma endothelial microparticles and promoted blood coagulation. In preeclampsia women, elevated placental HMGB1 expression was detected and high levels of plasma HMGB1 correlated with increased plasma endothelial microparticles. CONCLUSIONS: Our results indicate that placental hypoxia-induced HMGB1 expression and release from trophoblasts are important mechanism underlying increased circulating endothelial microparticles and thrombophilia in preeclampsia.

The interaction of MMP-2/B7-H3 in human osteoporosis.

The immune costimulatory molecule B7-H3 has been shown to be involved in the regulation of murine bone formation. However, the role of B7-H3 in bone metabolic diseases remains unknown. In our study, matrix metalloproteinase 2 (MMP-2) and soluble B7-H3 (sB7-H3) were found to be correlatively up-regulated in the sera of osteoporosis patients. Furthermore, our results showed that MG63 cells treated with MMP-2 inhibitors produced lower amounts of sB7-H3 while cells with recombinant MMP-2 had an increased membrane B7-H3 (mB7-H3) shedding. Therefore, elevated MMP-2 levels resulted in an elevation of serum sB7-H3 and reduction of osteoblastic mB7-H3. B7-H3 knockdown in MG63 cells significantly decreased osteoblastic markers and substantially decreased the number of mineralized nodules after 21days. Thus, B7-H3-deficient MG63 cells exhibited impaired bone formation. These results suggest that mB7-H3 is required for the later phases of osteoblast differentiation and that MMP-2/B7-H3 plays a negative regulatory role in osteoporosis.

Correlation between B7-H3 expression and rheumatoid arthritis: A new polymorphism haplotype is associated with increased disease risk.

CD276 (B7-H3) is a costimulatory molecule that plays a potent role in T cell responses, however, the role of B7-H3 in autoimmune diseases has not been elucidated. We analyzed B7-H3 expression in rheumatoid arthritis (RA) for the first time and found B7-H3 was significantly up-regulated on monocytes in RA patients, while the levels of soluble B7-H3 in serum were lower than in controls (P < 0.0001). These differences correlated with clinical and laboratory disease parameters and informatory factor TNF-α. Through in vitro experiments, we demonstrated that B7-H3 promoted TNF-α secretion. In addition, a new polymorphism variant, B7-H3-T-A-C-T, was identified and shown to be associated with the incidence of RA and the decreased release of sB7-H3. These results suggest that B7-H3 may be a promising biomarker associated with the pathogenesis of RA. Notably, the new B7-H3-T-A-C-T polymorphism variant is associated with RA risk and might be associated with the release of soluble B7-H3.

Oxymatrine inhibits lipopolysaccharide-induced inflammation by down-regulating Toll-like receptor 4/nuclear factor-kappa B in macrophages.

Oxymatrine (OMT) is the quinolizidine alkaloid extracted from the Chinese herb Sophora flavescens Ait. that has many pharmacological effects and is used for the treatment of some inflammatory diseases. In this study, RAW264.7 cells and THP-1 differentiated macrophages were pretreated with various concentrations of OMT at 2 h prior to treatment with lipopolysaccharide (LPS) (1.0 µg/mL) for different durations. We detected the anti-inflammatory effect of OMT in LPS-stimulated macrophages and investigated the molecular mechanism. We showed that OMT pretreatment significantly inhibited the LPS-induced secretion of nitric oxide (NO), interleukin-1 beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α) in supernatant, attenuated the mRNA levels of inducible nitric oxide synthase (iNOS), IL-1ß, TNF-α, and Toll-like receptor 4 (TLR4), increased TLR4 and phosphorylation of inhibitor of kappa B-alpha (p-IBα) in cytosol, and decreased the nuclear level of nuclear factor-κB (NF-κB) p65 in macrophages. In conclusion, OMT exerts anti-inflammatory properties in LPS-stimulated macrophages by down-regulating the TLR4/NF-κB pathway.

Water-soluble near-infrared AgInS2 quantum dots for Ca2+ detection and bioimaging.

Calcium ions (Ca2+) are key players in intracellular signaling as second messengers and play a pivotal role in various physiological processes. In this study, near-infrared water-soluble AgInS2 quantum dots (AIS QDs) for Ca2+ detection were synthesized by a one-step hydrothermal method. The fluorescence quantum yield (PL QY) of the quantum dots was as high as 23.99 %. With low cytotoxicity and good fluorescence properties, as well as short reaction time, the ternary AIS QDs have excellent synthesis efficiency and quantum yield, which are advantageous for Ca2+ detection and bioimaging applications. The fluorescence quenching of the quantum dots showed a clear linear relationship with calcium ion concentration in the range of 0-250 µM (detection limit: 0.65 µM). Confocal imaging experiments demonstrate the excellent biofluorescence imaging capability of AIS QDs. By tuning the Ag/In molar ratio, AIS QDs can achieve fluorescence emission in the near-infrared wavelength band (620-700 nm), and the near-infrared fluorescence imaging has deeper tissue penetration, less tissue absorption and photodamage, and lower interference of spontaneous fluorescence, which further expands the potential of QDs for bioimaging applications.

EsxA might as a virulence factor induce antibodies in patients with Staphylococcus aureus infection.

Staphylococcus aureus (S. aureus) is an important human pathogen, which commonly causes the acquired infectious diseases in the hospital and community. Effective and simple antibiotic treatment against S. aureus-related disease becomes increasingly difficult. Developing a safe and effective vaccine against S. aureus has become one of the world's hot spots once again. The key issue of developing the vaccine of S. aureus is how to find an ideal key pathogenic gene of S. aureus. It was previously suggested that EsxA might be a very important factor in S. aureus abscess formation in mice, but clinical experimental evidence was lacking. We therefore expressed EsxA protein through prokaryotic expression system and purified EsxA protein by Ni-affinity chromatography. ELISA was used to detect the anti-EsxA antibodies in sera of 78 patients with S. aureus infection and results showed that the anti-EsxA antibodies were positive in the sera of 19 patients. We further analyzed the EsxA positive antibodies related strains by antimicrobial susceptibility assay and found that all of the corresponding strains were multi-drug resistant. Among those multi-drug resistant strains, 73.7% were resistant to MRSA. The results indicated EsxA is very important in the pathogenesis of S. aureus. We suggested that the EsxA is very valuable as vaccine candidate target antigens for prevention and control of S. aureus infection.

Establishment of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay to Measure Soluble B7-H5 in Patients with Cancer.

B7-H5, an immune checkpoint molecule, is markedly upregulated in multiple cancers and plays an important role in tumor progression and immune escape. However, the expression and significance of soluble B7-H5 (sB7-H5) in cancer remain unclear. Herein, we generated two novel mouse anti-human B7-H5 monoclonal antibodies (mAbs) 2E5 and 7B10, which had different epitopes. Based on the two mAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) system was developed. Using this ELISA, we found that compared with healthy controls (HCs), sB7-H5 levels were significantly increased in the serum of patients with gastric cancer (GC), colorectal cancer (CRC), and lung cancer (LC) and were associated with TNM stage and metastasis. Receiver operating characteristic (ROC) curve analysis showed that sB7-H5 has diagnostic value for GC, CRC, and LC. Collectively, our findings delineate that sB7-H5 may be used as a predictor for diagnosis of cancer and a potential therapeutic target for cancer treatment.

Injectable Carbon Dots-Based Hydrogel for Combined Photothermal Therapy and Photodynamic Therapy of Cancer.

Hydrogel has been widely used in modern biotherapeutics due to its excellent biocompatibility, degradability, and high drug loading capacity. Among them, the construction of a phototherapy system including photosensitizer and hydrogel has aroused great interest in tumor therapy. Unfortunately, complex modifications are necessary to integrate different photosensitizers into the hydrogel. In this work, an injectable hydrogel was proposed by the Schiff base reaction between HA-CHO and carbon dots (CDs), which can realize PTT and PTT simultaneously. Notably, the CDs with rich -NH2 can be used not only as a photosensitizer but also as an efficient cross-linking agent for the Schiff base reaction to form a hydrogel network. The CD@Hydrogel with outstanding biosafety showed a high antitumor effect after 660 nm laser irradiation in in vitro and in vivo experiments. In summary, the CD@Hydrogel can not only realize PTT and PDT synergistic treatment under one light source but also act as a cross-linking agent to react with HA-CHO to form hydrogel, which is simple and efficient, providing a new strategy for cancer phototherapy.

Non-doped and non-modified carbon dots with high quantum yield for the chemosensing of uric acid and living cell imaging.

Carbon dots without heteroatoms-doping and surface modifications were designed to be a novel chemosensing strategy on the quantitative detection of uric acid (UA) with the aid of uricase-induced enzymatic reaction and Fenton reaction. In this work, ascorbic acid (AA)-derived carbon dots (A-CDs) were prepared in the mixture of ethanol and water via one-step hydrothermal synthesis at a relatively low temperature (120 °C) for 10 h. The resultant A-CDs were proved to be excitation-independent. When excited at the wavelength of 420 nm, the nanodots displayed green fluorescence (535 nm) which was then linearly quenched as UA concentration increased in the range of 0-56 µM, according to which the detection limit was calculated to be 0.49 µM. With regards to the excellent sensitivity and selectivity to UA, real sample assay was performed on the A-CDs detection system, which provided relatively reliable recoveries of UA contained in human serum/urine. Besides, in view of the high quantum yield, the A-CDs were applied to live-cell imaging assay and were considered to become an alternative tracer tool in biomedical imaging.

Riboflavin-based carbon dots with high singlet oxygen generation for photodynamic therapy.

Photodynamic therapy, as an effective treatment for superficial tumors, has attracted more and more attention. The development of safe, biocompatible and in vivo photosensitive materials is helpful to promote photodynamic therapy. Here we report green fluorescent carbon quantum dots prepared from a natural vitamin, riboflavin (VB2), as a photosensitizer. The VB2-based carbon dots have excellent water solubility and biocompatibility, and their singlet oxygen generation ability is much stronger than that of riboflavin itself. Through endocytosis, the carbon dots can easily enter the cells and show bright green fluorescence. In vivo experiments show that after photodynamic therapy the carbon dots can significantly inhibit the growth of tumors, and will not have toxic and side effects on other organs.

Starch-Based Carbon Dots for Nitrite and Sulfite Detection.

Nitrite and sulfite play important roles in human health and environmental science, so it is desired to develop a facile and efficient method to evaluate NO2 - and SO3 2- concentrations. In this article, the use of green alternatives with the potential of multi-functionality has been synthesized to detect nitrite and sulfite based on fluorescent probe. The carbon dots (CDs) with starch as only raw materials show fluorescence turn "on-off-on" response towards NO2 - and SO3 2- with the limits of detection of 0.425 and 0.243 µÐœ, respectively. Once nitrite was present in the solution, the fluorescence of CDs was quenched rapidly due to the charge transfer. When sulfite was introduced, the quenching fluorescence of CDs was effectively recovered because of the redox reaction between NO2 - and SO3 2-, and thus providing a new way for NO2 - and SO3 2- detection. Owing to their excellent analytical characteristics and low cytotoxicity, the "on-off-on" sensor was successfully employed for intracellular bioimaging of NO2 - and SO3 2-.

Biologic characteristics of the side population of human small cell lung cancer cell line H446.

BACKGROUND AND OBJECTIVE: Recently, the theory of cancer stem cells (CSCs) has presented new targets and orientations for tumor therapy. The major difficulties in researching CSCs include their isolation and purification. The aim of this study is to identify and characterize the side population (SP) cells in small cell lung cancer (SCLC) cell line H446, which lays the foundation for the isolation and purification of CSCs. METHODS: Fluorescence-activated cell sorting (FACS) was used to sort SP and non-SP (NSP) cells from H446. Both subgroups were cultivated to survey the capacity to form into suspended tumor cell spheres. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR were used to evaluate the expression levels of the mRNA of CD133, ABCG2, and nucleostemin in both subgroups. The capacity of proliferation and the differences in drug resistance of both subgroups and unsorted cells were tested by the MTT method. The differentiation ability of both subgroups was determined by FACS. Proliferation was determined by subcutaneous tumor formation in nude mice. RESULTS: The percent of Hoechst 33342 negative cells was about (5.1 +/- 0.2)% in H446 by fluorescence microscopy. The percent of SP cells was (6.3 +/- 0.1)% by flow cytometry. SP cells had a stronger capability of forming into tumor spheres than NSP cells. The mRNA expression levels of ABCG2, CD133, and nucleostemin in SP cells were 21.60 +/- 0.26, 7.10 +/- 0.14, and 1.02 +/- 0.08 folds higher than that in NSP cells (P < 0.01, P < 0.01, and P > 0.05, respectively). In vivo, SP cells showed better proliferative ability and tougher viability when treated with drugs. SP cells can differentiate into NSP cells, but NSP cells cannot differentiate into SP cells. SP cells had a greater ability to form tumors. CONCLUSION: The H446 cell line contained some SP cells with stem cell properties. CD133 and ABCG2 may be cancer stem cell markers of SCLC.

Duplex-specific nuclease assisted miRNA assay based on gold and silver nanoparticles co-decorated on electrode interface.

miRNAs are small non-coding RNAs for gene regulation, which serve as promising biomarkers for the diagnosis of certain diseases. In this contribution, we have proposed a convenient electrochemical biosensing strategy based on the interaction between DNA modified gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs). In principle, citrate capped AuNPs and AgNPs can be co-decorated on the electrode successively. However, with the modification of DNA on AuNPs surface, a strong negative layer is formed. AuNPs@DNA modified electrode could then inhibit subsequent adsorption of AgNPs due to the electrostatic repulsion and steric hindrance effect. As a result, electrochemical response from AgNPs is significantly decreased. On the other hand, in the presence of target miRNA, DNA on AuNPs hybridizes with miRNA and can thus be cyclically digested by duplex-specific nuclease (DSN). Without the shield of DNA, AgNPs can be relaunched at the AuNPs modified electrode. By analyzing the silver stripping peak, highly sensitive detection of miRNA can be achieved. This biosensor exhibits the limit of detection as low as 0.62 fM and a broad linear range from 1 fM to 1 pM. It may hold great potential utility for miRNA assay in the applications of biomedical researches and early clinical diagnosis.

Author Correction: The Transmembrane Serine Protease HAT-like 4 Is Important for Epidermal Barrier Function to Prevent Body Fluid Loss.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Green Synthesis of Lutein-Based Carbon Dots Applied for Free-Radical Scavenging within Cells.

Reactive oxygen species (ROS) in the body play an important role in various processes. It is well known that harmful high levels of ROS can cause many problems in living organisms in a variety of ways. One effective way to remove intracellular ROS is to use reducing materials that can enter the cell. Herein, we developed a strong reducing carbon nano-dot from a natural product, lutein, as an initial raw material. This is a hydrothermal synthesis method with the advantages of simplicity, high yield, mild reaction conditions, and environmental friendliness. The prepared carbon dots exhibit bright blue fluorescence, and have good water solubility and biocompatibility. In particular, the carbon dots can easily enter the cell and effectively remove ROS. Therefore, the carbon dots are thought to protect cells from oxidative damage by high levels of ROS.