Petr Cigler

Boosting nanodiamond fluorescence: towards development of brighter probes

A novel approach for preparation of ultra-bright fluorescent nanodiamonds (fNDs) was developed and the thermal and kinetic optimum of NV center formation was identified. Combined with a new oxidation method, this approach enabled preparation of particles that were roughly one order of magnitude brighter than particles prepared with commonly used procedures.

CONTRIBUTION TO UNDERSTANDING THE MECHANISM OF TITANIUM ACTION IN PLANT

The biological effects of titanium (Ti) in the form of Ti(IV)-ascorbate on oats (Avena sativa L. cv. Zlaták) were studied in hydroponic experiments on defined nutrient solutions, determining the influence (i) of the chemical form of nitrogen (N) in nutrient solution on the Ti effects on plants (nitrate and an ammonium salt (acetate) were the only N species used) and (ii) of various Ti concentrations in nutrient solution while changing the magnesium (Mg) concentration on the Ti, Mg, iron (Fe), and potassium (K) content in tops and roots, top and root dry weights, chlorophyll a and b content, top height, and root length. It was found that (i) Ti was beneficial for plants grown on the nitrate-containing nutrient solutions as compared to the ammonium-containing nutrient solutions where Ti results in inhibitory effects on plant health status (decrease of top and root dry weights, chlorophyll a and b contents, top height, and root length), therefore, it was deducted that the increase of nitrate reductase activity was mainly responsible for the Ti beneficial effect on plants and (ii) Ti increased the Fe, Mg, and Ti content in plant tissues and this effect was independent of N form in the nutrient solution, chlorophyll a and b content or plant health status. It is suggested that the biological effects of Ti (the synthesis of the Ti chelating α -hydroxy carboxylic acids (citric and malic), and ascorbic acid, the increase of the Fe and Mg contents in plant tissues, the increased nitrate reductase activity, the increased chlorophyll a and b biosynthesis, and the effects on the other enzymatic activities) are the defense mechanisms of the plant against Ti replacing some essential elements from their binding sites (probably mainly from the phosphate-based ones). These defense mechanisms are, in the case of the usual Ti application doses, much stronger than adequate for the elimination of the Ti toxic effects (this ‘‘paradox’’ effect called hormesis has already been described for lead (Pb) and its effect on the hemoglobin content in blood of animals and humans).

Elicitation of pharmacologically active substances in an intact medical plant.

The quality of medical plants used for the production of galenics or pharmacologically useful compounds is usually assessed by the content of biologically active compounds. Because most of these plants are grown in fields, this study focused on stimulation of active compounds by in vivo elicitation. Foliar application of elicitors on the immunostimulating medical plant purple coneflower ( Echinacea purpurea L. Moench.) grown on soil was used to increase the content of biologically active phenolics. Natural plant stress mediators and their derivatives (acetylsalicylic acid, salicylic acid, and methyl salicylate) as well as newly introduced biocompatible metal elicitor [titanium(IV) ascorbate] were chosen as active components of foliar sprays. A tremendous increase of phenolics (up to 10 times compared to control) and stimulation of the biomass yield were achieved. Tuning of organ specificity by modulation of the concentration of elicitor was also observed. This methodology represents a convenient alternative to cell suspension or hydroponic cultures being applicable in wide agricultural practice.

Structure-aided design of novel inhibitors of HIV protease based on a benzodiazepine scaffold.

HIV protease is a primary target for the design of virostatics. Screening of libraries of non-peptide low molecular weight compounds led to the identification of several new compounds that inhibit HIV PR in the low micromolar range. X-ray structure of the complex of one of them, a dibenzo[b,e][1,4]diazepinone derivative, showed that two molecules of the inhibitor bind to the PR active site. Covalent linkage of two molecules of such a compound by a two-carbon linker led to a decrease of the inhibition constant of the resulting compound by 3 orders of magnitude. Molecular modeling shows that these dimeric inhibitors form two crucial hydrogen bonds to the catalytic aspartates that are responsible for their improved activity compared to the monomeric parental building blocks. Dibenzo[b,e][1,4]diazepinone analogues might represent a potential new class of HIV PIs.

Mechanism of physiological effects of titanium leaf sprays on plants grown on soil.

Titanium (Ti) has significant biological effects on plants, being beneficial at low and toxic at higher concentrations. From results of our hydroponical experiment with oats, we have recently proposed that the effect called hormesis is the mechanism of Ti action in plants. Here, we present the experiment with oats (Avena sativa L. cv. Zlat’ák) grown on soil where Ti was applied using leaf sprays. Two different soils, three different concentrations of Ti(IV) citrate spray solution (0, 20, and 50 mg Ti/kg), and three different Mg concentrations in each soil were tested. Some physiological parameters (dry and raw weights, top heights, chlorophyll content) and element contents (Mg, Fe, Zn, Mn) were determined. Ti showed considerable effects on all physiological parameters and the element’s contents were determined. Differences between the two different soil types used was only in the strength of the effect of Ti; the trends remained unchanged. Generally, the effect of Ti is considerably weaker if Ti is applied on leaves than if being added to the nutrient solution. Thus, we confirm here that the action of Ti on plants could be explained by the hormesis effect.

Hydroxamic Acids As a Novel Family of Serine Racemase Inhibitors: Mechanistic Analysis Reveals Different Modes of Interaction with the Pyridoxal-5′-phosphate Cofactor

Mammalian serine racemase (SR) is a pyridoxal-5-phosphate (PLP) dependent enzyme responsible for the biosynthesis of the neurotransmitter D-serine, which activates N-methyl-D-aspartate (NMDA) receptors in the CNS. Aberrant regulation of NMDA receptor signaling has been implicated in a variety of neuropathologies, and inhibitors of SR would therefore be a worthwhile tool for further investigation or treatment of such conditions. Here, we identify a series of small aliphatic hydroxamic acids (HAs) that act as potent SR inhibitors. However, specificity studies showed that some of these HAs can act as nonspecific inhibitors of PLP-dependent enzymes. We employed NMR, MS, and UV/vis spectroscopic techniques to reveal that the nonspecific effect is likely due to irreversible interaction of the HA moiety with PLP to form aldoxime species. We also characterize L-aspartic acid beta-hydroxamate as a competitive and selective SR inhibitor that could be used as a scaffold for further inhibitor development.

Optical imaging of localized chemical events using programmable diamond quantum nanosensors

Development of multifunctional nanoscale sensors working under physiological conditions enables monitoring of intracellular processes that are important for various biological and medical applications. By attaching paramagnetic gadolinium complexes to nanodiamonds (NDs) with nitrogen-vacancy (NV) centres through surface engineering, we developed a hybrid nanoscale sensor that can be adjusted to directly monitor physiological species through a proposed sensing scheme based on NV spin relaxometry. We adopt a single-step method to measure spin relaxation rates enabling time-dependent measurements on changes in pH or redox potential at a submicrometre-length scale in a microfluidic channel that mimics cellular environments. Our experimental data are reproduced by numerical simulations of the NV spin interaction with gadolinium complexes covering the NDs. Considering the versatile engineering options provided by polymer chemistry, the underlying mechanism can be expanded to detect a variety of physiologically relevant species and variables.

Triggering HIV polyprotein processing by light using rapid photodegradation of a tight-binding protease inhibitor

HIV protease (PR) is required for proteolytic maturation in the late phase of HIV replication and represents a prime therapeutic target. The regulation and kinetics of viral polyprotein processing and maturation are currently not understood in detail. Here we design, synthesize, validate and apply a potent, photodegradable HIV PR inhibitor to achieve synchronized induction of proteolysis. The compound exhibits subnanomolar inhibition in vitro. Its photolabile moiety is released on light irradiation, reducing the inhibitory potential by 4 orders of magnitude. We determine the structure of the PR-inhibitor complex, analyze its photolytic products, and show that the enzymatic activity of inhibited PR can be fully restored on inhibitor photolysis. We also demonstrate that proteolysis of immature HIV particles produced in the presence of the inhibitor can be rapidly triggered by light enabling thus to analyze the timing, regulation and spatial requirements of viral processing in real time.

Nanodiamonds as Intracellular Probes for Imaging in Biology and Medicine

In recent years, diamond nanoparticles have received a great deal of attention due to their unique photophysical and biological properties. Nanodiamonds (NDs) show low toxicity and are considered to be a highly biocompatible carbon nanomaterial useful in a wide range of applications. Thanks to their ability to accommodate nitrogen-vacancy (N-V) color centers, NDs are a prime example of non-photobleachable fluorescent labels and nanosensors. Here, we present a survey of ND applications in biology and medicine with an emphasis on bio-imaging. We focus on distinguishing the properties of detonation NDs and high-pressure high-temperature (HPHT) NDs and describing their physicochemical properties, structure and possible modifications by small molecules and biomolecules. We summarize and critically evaluate in vitro and in vivo data on ND toxicity and biocompatibility, cellular internalization, localization and targeting by surface-attached ligands. We discuss current achievements in bioimaging using fluorescent NDs and the potential of NDs in diagnostics and drug delivery.

Interactions between iron and titanium metabolism in spinach: a chlorophyll fluorescence study in hydropony.

One of the elements showing strong beneficial effect on plants at low concentrations and toxic effects at higher concentrations is titanium (Ti). We investigated the interconnection between the Fe uptake and the Ti intoxication in model experiment on Fe-deficient spinach (Spinacia oleracea) plants to help to elucidate the mechanism of the biological activity of titanium in plants. The two different Ti (0 and 20 mg L⁻¹) and two different Fe (0 and 1.35 mg L⁻¹) concentrations in hydroponic medium were used in all four possible combinations. We compared chemical analysis of Ti and Fe in roots and shoots with the changes of the in vivo chlorophyll fluorescence. Although Fe and Ti concentration found in shoots of Ti-non-treated Fe-deficient plants was comparable with that in Ti-treated Fe-deficient plants, the soluble form of Ti present in the growth media had a negative effect on photosynthetic activity monitored by chlorophyll fluorescence measurements. The presence of Fe in growth medium significantly decreased the Ti concentration in shoots and increased the photosynthetic activity. Here, we propose that Ti affect components of electron transport chain containing Fe in their structure (particularly photosystem I) and decrease the photosystem II efficiency.