Carbon Nanomaterials for Bioanalytical Sensing and Multicolor Cell Imaging

The hydrothermal preparation of N-doped C-dots from starch and L-tryptophan which was developed and optimized represents a simple and sustainable synthetic method to obtain a highly interesting carbon nanomaterial for bioanalytical applications. These particles exhibit a size distribution between 0.5 and 4.5 nm with an average size of (1.6 ±0.8) nm. They furthermore show a high functionalization with oxygen containing-groups, remnants from the precursor molecules, which renders them highly water-dispersible. This is in accordance to the elemental composition of 44.9% C, 6.1% H, 1.1% N and 47.9% O. The experimental evidence suggests that mainly hydroxyl and carboxyl groups cover the C-dots surface, resulting in a slightly negative charged surface with a zeta potential of (-9.3 ± 3.5) mV at physiological pH. Based on the functionalization with different chemical groups on the surface, the C-dots are incorporated into mammalian cells in a time range of minutes via endocytosis. Probably, the particles are internalized via unspecific pinocytosis due to their ultra-small size, inducing vesicle-mediated incorporation. Receptor-mediated endocytosis cannot be excluded, but is not suggested due to the high selectivity of the receptors. In addition to this, hints indicating further uptake mechanisms such as simple diffusion across the membrane have been gathered. The ultra-small dimensions of these nanoparticles, together with their unique surface may be responsible for the high translocation rates through cell layers observed for several mammalian cell lines. The high penetration rates and the ultra-fast uptake into cells outline the applicability of these C-dots as probes or contrast agents for imaging studies. Biocompatibility is a necessary criterion for the utilization of this novel nanomaterial in cell imaging. The verification of this quality presented a mixed picture for the C-dots. Thereby, cytotoxicity evaluations clearly showed a “critical concentration” of about 1.1 mg/mL for the long-term incubation of NRK cells over days. Higher particle concentrations induced apoptosis among the cells, while lower concentrations left the NRK cells mostly unaffected. Furthermore, cell adhesion, proliferation and migration were dose-dependently delayed in permanent presence of the particles. However, the staining and loading of NRK cells as necessary for the developed applications requires only shortest incubation times in the range of minutes. Thus C-dot stained NRK cells did not show any sign of cytotoxicity. Additionally, migration, as example among impairments of cell behavior, showed only marginal affection for such loaded NRK cells. Accordingly, C-dots may be considered as biocompatible, based on the right choice of concentration and incubation time (typically 1.0 mg/mL for 30 – 60 min) for the respective application. A thorough investigation of the C-dots optical properties revealed unique features of this novel material. Aqueous C-dot suspensions typically exhibit quantum yields of (25.1 ± 2)%. This is about thirty-fold higher than quantum yields of undoped C-dots from starch and may be attributed to the N-doping through the addition of Trp. Since the addition of Trp inducing the formation of larger particles, the amino acid most likely also enables the formation of highly emissive species. Low concentrated suspensions of the N-doped C-dots have their excitation maximum at about 370 nm and exhibit bright blue luminescence with an emission maximum at about 450 nm. Fluorescence is heavily concentration dependent and the excitation wavelength can be varied presumably leading to emission from different C-dot species. Increasing particle concentration in aqueous suspension is suggested to induce reabsorption of emitted light and/or self-quenching among the emitting species. This results in red-shifted fluorescence emission for higher C-dot concentration. The studies of the particles fluorescence at the excitation wavelengths of the CLSM laser wavelengths 405, 488 and 543 nm showed that this phenomenon might be used for the tracking of local concentrations of C-dots via imaging. Bright blue emission is observed for low concentrations at 405 nm excitation. High particle concentrations result in an emission-shift from blue to green with excitation at 405 nm. Furthermore, the green emission upon excitation at 488 nm becomes most prominent. These spectral characteristics can be documented nicely by, utilizing C-dots as fluorescence contrast agent for the imaging of mammalian cells via CLSM. Thereby, local accumulation of C-dots was concluded from the red-shifted fluorescence emission of the particles in suspension at higher concentrations. Similarly to the studies in suspension, the emission shifts from blue to green upon particle accumulations for excitation at 405 nm. Furthermore, the emission intensities rise for higher particle amounts upon excitation at 488 and 543 nm. Living cells were shown to take up C-dots in low concentration mostly via endocytosis, resulting in a blue luminescent cytoplasm upon 405 nm laser excitation, with most of the particles entrapped in vesicles. Dead cells by contrast, exhibited green luminescent nuclei with highest intensities under excitation with the 488 nm laser. This may be attributed to the accumulation of C-dots in the cell nuclei through binding to DNA, resulting in the emission shift. Cells that have lost plasma membrane integrity and underwent cell death enable the direct contact between their nuclei and the bulk phase. As such, C-dots, being not entrapped in vesicles may freely diffuse through the nuclear pore complex and accumulate therein. Furthermore, the binding between C-dots and the DNA is suggested to change the emission properties of the particles, which also contributes to the different coloration. Utilizing both lasers, the 405 nm laser for the blue luminescence of living cells cytoplasm and the 488 nm laser for the dead cells nuclei, allowed the application of C-dots within a Live-Dead-Assay. This gives the same information as commercial available staining kits to distinguish between living and dead cells via imaging. Having proven the independency of the C-dot based readout from the cell death inducing toxicant, the C-dots can furthermore be utilized for the determination of EC50values for a selected toxicant via CLSM imaging. Thereby, the ratio between the green and blue fluorescence intensities at the respective laser excitation could be used as measure for cytotoxicity. The method may be applied on cytotoxicity evaluations for several mammalian cell lines. For NRK cells treated with tBuOOH, for example, the green/blue intensity ratio changed from about 0.1 for healthy cells to about 1.5 for completely dead cells. Additionally, it could be shown that the utilization of the C-dot staining of living and dead cells is not limited on CLSM, but can also be analyzed via FACS. Besides cytotoxicity evaluations, C-dots might be used for the imaging of living and dead cells in MCS as tissue models. As such, also the application of the particles in tissues seems promising. Besides luminescence, the C-dots were found to effectively generate reactive oxygen species (ROS) upon long-term photo-excitation. Thereby, the particles combine every necessary requirement for a photodynamic therapy agent: good water-dispersibility, fast internalization into cells and efficient ROS generation. This was used to selectively subject NRK cells to photodynamic treatment through the photo-excitation of both, C-dots in the bulk phase and internalized particles. It was demonstrated that besides near-UV, also the blue light irradiation may cause phototoxicity. As such, irradiation conditions of applications using C-dots as photodynamic agents are variable. The photogeneration of ROS by internalized C-dots is suggested to result in oxidation of the phospholipids of the vesicles surrounding the incorporated particles. As such, C-dots are suggested to be released from endosomes and generate ROS throughout the whole cell. This induces cell death via apoptosis, which was proven through Annexin V binding studies. Interestingly, released cell-internal C-dots were observed to stain the nuclei of dying cells from the inside even before Annexin V bound to those cells membranes. In a “theranostic” approach with C-dots, the particles’ ability to stain dead cells’ nuclei from the inside together with cell death inducing ROS generation upon photo-excitation was combined. NRK cells were loaded with C-dots through short-term incubation. Target cells were subjected to controlled photodamage via photo-excitation of internalized C-dots for several minutes. This could be performed on large cell areas via epiluminescence microscopy with UV excitation (330 – 380 nm) and on single cell level via CLSM laser excitation at 405 nm. The subsequent analysis of the cells via CLSM allows the explicit control of the treatment through the changed optical properties of the internalized C-dots for dead cells. Thereby, the internalized C-dots undergo the same red-shift through binding and accumulation in the cells’ nuclei DNA as described for the C-dot staining from the bulk phase. Non-treated NRK cells exhibit a blue luminescent cytoplasm, upon 405 nm excitation, while the nuclei of those cells subjected to the photodynamic therapy appear green with 488 nm excitation. Accordingly, the C-dots do not only allow the active therapy of target cells, but also diagnosis on the effort of the treatment – theranostic. Studies on MCF-7 MCS as breastcancer tumor models showed that this approach may also be applied on such large cell aggregates. Therefore, C-dots have to be considered as promising theranostic agents in cancer therapy of living organisms. In side projects of this work, the C-dots were embedded into other nano-sized structures and chemically modified in order to demonstrate possible applications of the particles. Among these, the embedding into agarose nanoparticles was utilized for the optical observation of osmolarity changes based on fluorescence self-quenching in confined volumes. Thereby, fluorescence intensity increases of 15% were observed for the addition of 1 M osmolyte. Although this represents a poor sensitivity, the experiments show a fundamental possibility for future applications of the particles. Additionally, liposomes were investigated as transport vehicles for C-dots into NRK cells. Thereby, internalization of the particles was observed, most likely through a different uptake-mechanism, compared to free C-dots. These studies may be considered as first steps towards the incorporation into target cells. Also the chemical modification of the C-dots is meant to show principle application possibilities for the particles. The modification with oleic acid drastically changed the physicochemical and optical properties of the C-dots. As such, the binding to DNA, for example, is unlikely and not observed. Although, the applied thionyl chloride chemistry may be considered as harsh, this modification route may be utilized for promising surface modifications of the particles. Concerning the inconsistencies in literature, several differences between the bottom-up prepared C-dots and the top-down prepared GQDs with respect to physicochemical and optical properties have been elaborated, to clearly differentiate between both materials. At first, morphology of both materials was found to be highly diverse. As such, C-dots are quasi-spherical in shape with only a few nanometers in diameter and suggested to be mostly amorphous. By contrast, GQDs exhibit flake-like structures with similar heights, but in lateral dimensions of 5 – 25 nm. Furthermore, GQDs are suggested to mostly consist of graphitic carbon, since the precursor material is graphite. Both materials have shown to exhibit fluorescence emission shifts with variation of the excitation wavelengths. However, C-dots seem to have only a few major emissive species dominating their optical properties. By contrast, GQDs exhibit a direct correlation between excitation an emission. As such, GQDs may contain an infinite variety of emissive sites. In conclusion, both materials are closely related to each other concerning composition and functionalities, but clearly have their differences. Accordingly, a differentiation between both materials, C-dots and GQDs, is advisable

Hepatocyte specific expression of an oncogenic variant of β-catenin results in cholestatic liver disease

[Background] The Wnt/β-catenin signaling pathway plays a crucial role in embryonic development, tissue homeostasis, wound healing and malignant transformation in different organs including the liver. The consequences of continuous β-catenin signaling in hepatocytes remain elusive. [Results] Livers of Ctnnb1CA hep mice were characterized by disturbed liver architecture, proliferating cholangiocytes and biliary type of fibrosis. Serum ALT and bile acid levels were significantly increased in Ctnnb1CA hep mice. The primary bile acid synthesis enzyme Cyp7a1 was increased whereas Cyp27 and Cyp8b1 were reduced in Ctnnb1CA hep mice. Expression of compensatory bile acid transporters including Abcb1, Abcb4, Abcc2 and Abcc4 were significantly increased in Ctnnb1CA hep mice while Ntcp was reduced. Accompanying changes of bile acid transporters favoring excretion of bile acids were observed in intestine and kidneys of Ctnnb1CA hep mice. Additionally, disturbed bile acid regulation through the FXR-FGF15-FGFR4 pathway was observed in mice with activated β-catenin. [Materials and Methods] Mice with a loxP-flanked exon 3 of the Ctnnb1 gene were crossed to Albumin-Cre mice to obtain mice with hepatocyte-specific expression of a dominant stable form of β-catenin (Ctnnb1CA hep mice). Ctnnb1CA hep mice were analyzed by histology, serum biochemistry and mRNA profiling. [Conclusion] Expression of a dominant stable form of β-catenin in hepatocytes results in severe cholestasis and biliary type fibrosis

Associations between message features and subjective evaluations of the sensation value of antidrug public service announcements.

In 1998, the U.S. Congress stepped up the nation's focus on drug and alcohol abuse by allocating $1 billion to the Office of National Drug Control Policy for mass media-based prevention campaigns and evaluations. We now know some of the key elements of effective media-based antidrug campaigns, including effec

Genome-Wide and Phase-Specific DNA-Binding Rhythms of BMAL1 Control Circadian Output Functions in Mouse Liver

Temporal mapping during a circadian day of binding sites for the BMAL1 transcription factor in mouse liver reveals genome-wide daily rhythms in DNA binding and uncovers output functions that are controlled by the circadian oscillator

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Recommendations for accelerating open preprint peer review to improve the culture of science

Peer review is an important part of the scientific process, but traditional peer review at journals is coming under increased scrutiny for its inefficiency and lack of transparency. As preprints become more widely used and accepted, they raise the possibility of rethinking the peer-review process. Preprints are enabling new forms of peer review that have the potential to be more thorough, inclusive, and collegial than traditional journal peer review, and to thus fundamentally shift the culture of peer review toward constructive collaboration. In this Consensus View, we make a call to action to stakeholders in the community to accelerate the growing momentum of preprint sharing and provide recommendations to empower researchers to provide open and constructive peer review for preprints

Reduced graphene oxide and graphene composite materials for improved gas sensing at low temperature

Reduced graphene oxide (rGO) was investigated as a material for use in chemiresistive gas sensors. The carbon nanomaterial was transferred onto a silicon wafer with interdigital gold electrodes. Spin coating turned out to be the most reliable transfer technique, resulting in consistent rGO layers of reproducible quality. Fast changes in the electrical resistance at a low operating temperature of 85 °C could be detected for the gases NO2, CH4 and H2. Especially upon adsorption of NO2 the high signal changes allowed a minimum detection of 0.3 ppm (S/N = 3). To overcome the poor selectivity, rGO was chemically functionalized with octadecylamine, or modified by doping with metal nanoparticles such as Pd and Pt, and also metal oxides such as MnO2, and TiO2. The different response patterns for six different materials allowed the discrimination of all of the test gases by pattern recognition based on principal component analysis