Evaluation of the Oxidative Stress Response of Aging Yeast Cells in Response to Internalization of Fluorescent Nanodiamond Biosensors

Fluorescent nanodiamonds (FNDs) are proposed to be used as free radical biosensors, as they function as magnetic sensors, changing their optical properties depending on their magnetic surroundings. Free radicals are produced during natural cell metabolism, but when the natural balance is disturbed, they are also associated with diseases and aging. Sensitive methods to detect free radicals are challenging, due to their high reactivity and transiency, providing the need for new biosensors such as FNDs. Here we have studied in detail the stress response of an aging model system, yeast cells, upon FND internalization to assess whether one can safely use this biosensor in the desired model. This was done by measuring metabolic activity, the activity of genes involved in different steps and the locations of the oxidative stress defense systems and general free radical activity. Only minimal, transient FND-related stress effects were observed, highlighting excellent biocompatibility in the long term. This is a crucial milestone towards the applicability of FNDs as biosensors in free radical research

The Response of HeLa Cells to Fluorescent NanoDiamond Uptake

Fluorescent nanodiamonds are promising probes for nanoscale magnetic resonance measurements. Their physical properties predict them to have particularly useful applications in intracellular analysis. Before using them in intracellular experiments however, it should be clear whether diamond particles influence cell biology. While cytotoxicity has already been ruled out in previous studies, we consider the non-fatal influence of fluorescent nanodiamonds on the formation of reactive oxygen species (an important stress indicator and potential target for intracellular sensing) for the first time. We investigated the influence of different sizes, shapes and concentrations of nanodiamonds on the genetic and protein level involved in oxidative stress-related pathways of the HeLa cell, an important model cell line in research. The changes in viability of the cells and the difference in intracellular levels of free radicals, after diamond uptake, are surprisingly small. At lower diamond concentrations, the cellular metabolism cannot be distinguished from that of untreated cells. This research supports the claims of non-toxicity and includes less obvious non-fatal responses. Finally, we give a handhold concerning the diamond concentration and size to use for non-toxic, intracellular measurements in favour of (cancer) research in HeLa cells

Targeting Nanodiamonds to the Nucleus in Yeast Cells

Nanodiamonds are widely used for drug delivery, labelling or nanoscale sensing. For all these applications it is highly beneficial to have control over the intracellular location of the particles. For the first time, we have achieved targeting the nucleus of yeast cells. In terms of particle uptake, these cells are challenging due to their rigid cell wall. Thus, we used a spheroplasting protocol to remove the cell wall prior to uptake. To achieve nuclear targeting we used nanodiamonds, which were attached to antibodies. When using non-targeted particles, only 20% end up at the nucleus. In comparison, by using diamonds linked to antibodies, 70% of the diamond particles reach the nucleus

The fate of lipid-coated and uncoated fluorescent nanodiamonds during cell division in yeast

Fluorescent nanodiamonds are frequently used as biolabels. They have also recently been established for magnetic resonance and temperature sensing at the nanoscale level. To properly use them in cell biology, we first have to understand their intracellular fate. Here, we investigated, for the first time, what happens to diamond particles during and after cell division in yeast (Saccharomyces cerevisiae) cells. More concretely, our goal was to answer the question of whether nanodiamonds remain in the mother cells or end up in the daughter cells. Yeast cells are widely used as a model organism in aging and biotechnology research, and they are particularly interesting because their asymmetric cell division leads to morphologically different mother and daughter cells. Although yeast cells have a mechanism to prevent potentially harmful substances from entering the daughter cells, we found an increased number of diamond particles in daughter cells. Additionally, we found substantial excretion of particles, which has not been reported for mammalian cells. We also investigated what types of movement diamond particles undergo in the cells. Finally, we also compared bare nanodiamonds with lipid-coated diamonds, and there were no significant differences in respect to either movement or intracellular fate

Abdominal aortic aneurysm is associated with a variant in low-density lipoprotein receptor-related protein 1

Abdominal aortic aneurysm (AAA) is a common cause of morbidity and mortality and has a significant heritability. We carried out a genome-wide association discovery study of 1866 patients with AAA and 5435 controls and replication of promising signals (lead SNP with a p value < 1 × 10-5) in 2871 additional cases and 32,687 controls and performed further follow-up in 1491 AAA and 11,060 controls. In the discovery study, nine loci demonstrated association with AAA (p < 1 × 10-5). In the replication sample, the lead SNP at one of these loci, rs1466535, located within intron 1 of low-density-lipoprotein receptor-related protein 1 (LRP1) demonstrated significant association (p = 0.0042). We confirmed the association of rs1466535 and AAA in our follow-up study (p = 0.035). In a combined analysis (6228 AAA and 49182 controls), rs1466535 had a consistent effect size and direction in all sample sets (combined p = 4.52 × 10-10, odds ratio 1.15 [1.10-1.21]). No associations were seen for either rs1466535 or the 12q13.3 locus in independent association studies of coronary artery disease, blood pressure, diabetes, or hyperlipidaemia, suggesting that this locus is specific to AAA. Gene-expression studies demonstrated a trend toward increased LRP1 expression for the rs1466535 CC genotype in arterial tissues; there was a significant (p = 0.029) 1.19-fold (1.04-1.36) increase in LRP1 expression in CC homozygotes compared to TT homozygotes in aortic adventitia. Functional studies demonstrated that rs1466535 might alter a SREBP-1 binding site and influence enhancer activity at the locus. In conclusion, this study has identified a biologically plausible genetic variant associated specifically with AAA, and we suggest that this variant has a possible functional role in LRP1 expression

Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function.

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways

Optical Detection of Intracellular Quantities Using Nanoscale Technologies

Optical probes that can be used to measure certain quantities with subcellular resolution give us access to a new level of information at which physics, chemistry, life sciences, and medicine become strongly intertwined. The emergence of these new technologies is owed to great advances in the physical sciences. However, evaluating and improving these methods to new standards requires a joint effort with life sciences and clinical practice. In this Account, we give an overview of the probes that have been developed for measuring a few highly relevant parameters at the subcellular scale: temperature, pH, oxygen, free radicals, inorganic ions, genetic material, and biomarkers. Luminescent probes are available in many varieties, which can be used for measuring temperature, pH, and oxygen. Since they are influenced by virtually any metabolic process in the healthy or diseased cell, these quantities are extremely useful to understand intracellular processes. Probes for them can roughly be divided into molecular dyes with a parameter dependent fluorescence or phosphorescence and nanoparticle platforms. Nanoparticle probes can provide enhanced photostability, measurement quality, and potential for multiple functionalities. Embedding into coatings can improve biocompatibility or prevent nonspecific interactions between the probe and the cellular environment. These qualities need to be matched however with good uptake properties, colloidal properties and eventually intracellular targeting to optimize their practical applicability. Inorganic ions constitute a broad class of compounds or elements, some of which play specific roles in signaling, while others are toxic. Their detection is often difficult due to the cross-talk with similar ions, as well as other parameters. The detection of free radicals, DNA, and biomarkers at extremely low levels has significant potential for biomedical applications. Their presence is linked more directly to physiological and clinical manifestations. Since existing methods for free radical detection are generally poor in sensitivity and spatiotemporal resolution, new reliable methods that are generally applicable can contribute greatly to advancing this topic in biology. Optical methods that detect DNA or RNA and protein biomarkers exist for intracellular applications, but are mostly relevant for the development of rapid point-of-care sample testing. To elucidate the inner workings of cells, focused multidisciplinary research is required to define the validity and limitations of a nanoparticle probe, in both physical and biological terms. Multifunctional platforms and those that are easily made compatible with conventional research equipment have an edge over other techniques in growing the body of research evidencing their versatility

Nanodiamonds and Their Applications in Cells

Diamonds owe their fame to a unique set of outstanding properties. They combine a high refractive index, hardness, great stability and inertness, and low electrical but high thermal conductivity. Diamond defects have recently attracted a lot of attention. Given this unique list of properties, it is not surprising that diamond nanoparticles are utilized for numerous applications. Due to their hardness, they are routinely used as abrasives. Their small and uniform size qualifies them as attractive carriers for drug delivery. The stable fluorescence of diamond defects allows their use as stable single photon sources or biolabels. The magnetic properties of the defects make them stable spin qubits in quantum information. This property also allows their use as a sensor for temperature, magnetic fields, electric fields, or strain. This Review focuses on applications in cells. Different diamond materials and the special requirements for the respective applications are discussed. Methods to chemically modify the surface of diamonds and the different hurdles one has to overcome when working with cells, such as entering the cells and biocompatibility, are described. Finally, the recent developments and applications in labeling, sensing, drug delivery, theranostics, antibiotics, and tissue engineering are critically discussed