Michael Holtkamp

Organic cation transporter 2 mediates cisplatin-induced oto- and nephrotoxicity and is a target for protective interventions.

The use of the effective antineoplastic agent cisplatin is limited by its serious side effects, such as oto- and nephrotoxicity. Ototoxicity is a problem of special importance in children, because deafness hampers their language and psychosocial development. Recently, organic cation transporters (OCTs) were identified in vitro as cellular uptake mechanisms for cisplatin. In the present study, we investigated in an in vivo model the role of OCTs in the development of cisplatin oto- and nephrotoxicity. The functional effects of cisplatin treatment on kidney (24 hours excretion of glucose, water, and protein) and hearing (auditory brainstem response) were studied in wild-type and OCT1/2 double-knockout (KO) mice. No sign of ototoxicity and only mild nephrotoxicity were observed after cisplatin treatment of knockout mice. Comedication of wild-type mice with cisplatin and the organic cation cimetidine protected from ototoxicity and partly from nephrotoxicity. For the first time we showed that OCT2 is expressed in hair cells of the cochlea. Furthermore, cisplatin-sensitive cell lines from pediatric tumors showed no expression of mRNA for OCTs, indicating the feasibility of therapeutic approaches aimed to reduce cisplatin toxicities by competing OCT2-mediated cisplatin uptake in renal proximal tubular and cochlear hair cells. These findings are very important to establish chemotherapeutical protocols aimed to maximize the antineoplastic effect of cisplatin while reducing the risk of toxicities.

The Chloroplast Calcium Sensor CAS Is Required for Photoacclimation in Chlamydomonas reinhardtii

Reverse genetics approaches were conducted to elucidate the function of the plant-specific calcium binding protein CAS (calcium sensor) that is localized in chloroplast thylakoid membranes. Our findings demonstrate that CAS and Ca2+ are critically involved in the regulation of the high-light response and particularly in the regulation of LHCSR3 expression in green alga Chlamydomonas reinhardtii. The plant-specific calcium binding protein CAS (calcium sensor) has been localized in chloroplast thylakoid membranes of vascular plants and green algae. To elucidate the function of CAS in Chlamydomonas reinhardtii, we generated and analyzed eight independent CAS knockdown C. reinhardtii lines (cas-kd). Upon transfer to high-light (HL) growth conditions, cas-kd lines were unable to properly induce the expression of LHCSR3 protein that is crucial for nonphotochemical quenching. Prolonged exposure to HL revealed a severe light sensitivity of cas-kd lines and caused diminished activity and recovery of photosystem II (PSII). Remarkably, the induction of LHCSR3, the growth of cas-kd lines under HL, and the performance of PSII were fully rescued by increasing the calcium concentration in the growth media. Moreover, perturbing cellular Ca2+ homeostasis by application of the calmodulin antagonist W7 or the G-protein activator mastoparan impaired the induction of LHCSR3 expression in a concentration-dependent manner. Our findings demonstrate that CAS and Ca2+ are critically involved in the regulation of the HL response and particularly in the control of LHCSR3 expression.

Bacteria tracking by in vivo magnetic resonance imaging

BackgroundDifferent non-invasive real-time imaging techniques have been developed over the last decades to study bacterial pathogenic mechanisms in mouse models by following infections over a time course. In vivo investigations of bacterial infections previously relied mostly on bioluminescence imaging (BLI), which is able to localize metabolically active bacteria, but provides no data on the status of the involved organs in the infected host organism. In this study we established an in vivo imaging platform by magnetic resonance imaging (MRI) for tracking bacteria in mouse models of infection to study infection biology of clinically relevant bacteria.ResultsWe have developed a method to label Gram-positive and Gram-negative bacteria with iron oxide nano particles and detected and pursued these with MRI. The key step for successful labeling was to manipulate the bacterial surface charge by producing electro-competent cells enabling charge interactions between the iron particles and the cell wall. Different particle sizes and coatings were tested for their ability to attach to the cell wall and possible labeling mechanisms were elaborated by comparing Gram-positive and -negative bacterial characteristics. With 5-nm citrate-coated particles an iron load of 0.015 ± 0.002 pg Fe/bacterial cell was achieved for Staphylococcus aureus. In both a subcutaneous and a systemic infection model induced by iron-labeled S. aureus bacteria, high resolution MR images allowed for bacterial tracking and provided information on the morphology of organs and the inflammatory response.ConclusionLabeled with iron oxide particles, in vivo detection of small S. aureus colonies in infection models is feasible by MRI and provides a versatile tool to follow bacterial infections in vivo. The established cell labeling strategy can easily be transferred to other bacterial species and thus provides a conceptual advance in the field of molecular MRI.

Simple and rapid quantification of gadolinium in urine and blood plasma samples by means of total reflection X-ray fluorescence (TXRF)

A simple and rapid method to determine gadolinium (Gd) concentrations in urine and blood plasma samples by means of total reflection X-ray fluorescence (TXRF) was developed. With a limit of detection (LOD) of 100 μg L(-1) in urine and 80 μg L(-1) in blood plasma and a limit of quantification (LOQ) of 330 μg L(-1) in urine and 270 μg L(-1) in blood plasma, it allows analyzing urine samples taken from magnetic resonance imaging (MRI) patients during a period of up to 20 hours after the administration of Gd-based MRI contrast agents by means of TXRF. By parallel determination of the urinary creatinine concentration, it was possible to monitor the excretion kinetics of Gd from the patients body. The Gd concentration in blood plasma samples, taken immediately after an MRI examination, could be determined after rapid and easy sample preparation by centrifugation. All measurements were validated with inductively coupled plasma mass spectrometry (ICP-MS). TXRF is considered to be an attractive alternative for fast and simple Gd analysis in human body fluids during daily routine in clinical laboratories.

Dendrimer-Encapsulated Palladium Nanoparticles for Continuous-Flow Suzuki–Miyaura Cross-Coupling Reactions

Generation three, four, and five (G3, G4, and G5) poly(amidoamine) dendrimers were used for the encapsulation of palladium nanoparticles (Pd NPs) and their covalent anchoring within glass microreactors. G3‐encapsulated Pd NPs showed the highest activity for a model Suzuki–Miyaura cross‐coupling (SMC) reaction of the three different encapsulated Pd NPs tested, as compared to G4 and G5. A kinetic study indicated a role of the nanoparticle as a procatalyst, from which molecular species are formed with an induction time of approximately 1 min. The dendrimer–nanoparticle catalytic platform exhibited excellent reactivity (high turnover frequencies and numbers) compared to other Pd NP flow reactors and dendrimer‐encapsulated Pd NPs at batch scale. Moreover, the Pd microreactor exhibited good stability, as witnessed by running the SMC reaction for more than 7 days with a low Pd leaching of 1.2 ppm. The covalently attached dendrimers may play a crucial role in stabilizing the Pd NPs, a critical feature in flow SMC reactions.

Nitrogen purged TXRF for the quantification of silver and palladium

Total reflection X-ray fluorescence (TXRF) analysis is a powerful technique for simultaneous multi-element analysis. Benefits include limits of detection in the microgram per litre range, limited sample preparation for liquid samples and low operating costs. However, the determination of elements such as silver and palladium suffers from interferences, e.g. if an X-ray tube with a molybdenum anode is used, which normally offers the lowest detection limits for most of the elements of the periodic table. Argon emits its X-ray fluorescence radiation in the same energy range as these metals and therefore hinders their detection and subsequent quantification. Since argon is abundant in the atmosphere with approx. 0.93% and is easily activated by X-rays in a TXRF instrument, this interference is always present under routine measurement conditions. We have developed a new method for the analysis of silver and palladium by TXRF. A commercially available TXRF system was modified for being operated under a nitrogen purged atmosphere by surrounding it with an airtight and water-cooled box, which still allows accessing the main hardware components, changing the sample discs or performing long term measurements without temperature drift effects. An easy removal of the box is possible for a rapid change between the nitrogen purged and the common mode under ambient atmosphere. After flushing the system with nitrogen, the signal of argon decreased significantly, allowing the determination of silver and palladium with limits of detection for silver and palladium of 6.6 μg L−1 and 16.6 μg L−1 respectively.

Age-related bone deterioration is diminished by disrupted collagen sensing in integrin α2β1 deficient mice.

Collagen binding integrins are of essential importance in the crosstalk between cells and the extracellular matrix. Integrin α2β1 is a major receptor for collagen I, the most abundant protein in bone. In this study we show for the first time that integrin α2 deficiency is linked to collagen type I expression in bone. Investigating the femurs of wild type and integrin α2β1 deficient mice, we found that loss of integrin α2 results in altered bone properties. Histomorphometric analysis of integrin α2 long bones displayed more trabecular network compared to wild type bones. During age related bone loss the integrin α2β1 deficient bones retain trabecular structure even at old age. These findings were supported by functional, biomechanical testing, wherein the bones of integrin α2β1 deficient mice do not undergo age-related alteration of biomechanical properties. These results might be explained by the increased presence of collagen in integrin α2β1 deficient bone. Collagen type I could be detected in higher quantities in the integrin α2β1 deficient bones, forming abnormal, amorphous fibrils. This was linked to higher expression levels of collagen type I and other bone formation related proteins as alkaline phosphatase of integrin α2β1 deficient osteoblasts. Osteoclasts of integrin α2β1 deficient mice did not show any differences. Consequently these results indicate that the absence of integrin α2β1 alleviates the effects of age related bone degradation through over-expression of collagen type I and demonstrate a molecular mechanism how collagen binding integrins might directly impact bone aging.

Gallium-containing polymer brush film as efficient supported Lewis acid catalyst in a glass microreactor.

Summary Polystyrene sulfonate polymer brushes, grown on the interior of the microchannels in a microreactor, have been used for the anchoring of gallium as a Lewis acid catalyst. Initially, gallium-containing polymer brushes were grown on a flat silicon oxide surface and were characterized by FTIR, ellipsometry, and X-ray photoelectron spectroscopy (XPS). XPS revealed the presence of one gallium per 2–3 styrene sulfonate groups of the polymer brushes. The catalytic activity of the Lewis acid-functionalized brushes in a microreactor was demonstrated for the dehydration of oximes, using cinnamaldehyde oxime as a model substrate, and for the formation of oxazoles by ring closure of ortho-hydroxy oximes. The catalytic activity of the microreactor could be maintained by periodic reactivation by treatment with GaCl3.

Fast and low sample consuming quantification of manganese in cell nutrient solutions by flow injection ICP-QMS.

Inductively coupled plasma mass spectrometry with quadrupole mass analyzers (ICP-QMS) is one of the most powerful analytical techniques due to its superb limits of detection and the fast, quasi simultaneous quantification of different elements in one single run. However, sample consumption is typically too large for use in biological studies and spectral as well as non-spectral interferences are often hard to compensate for. Hence, a flow injection (FIA) approach for quantification of manganese (Mn) in biologically relevant cell nutrient solutions was developed, extending the sample throughput and versatility of a common system. The investigated cell nutrient solutions are, for example, used in in vitro models of the blood-brain and the blood-liquor barrier and represent a complex matrix, while Mn is of interest due to its potential neurotoxic effects, but shows several challenges in ICP-QMS analysis. Therefore, the aim of the study was not only to devise a system as simple as possible, but also to have a tool allowing the measurement of several hundreds of samples within a short period of time. Furthermore, statistical data treatment was used to evaluate the need for matrix matching and internal standardization for the four different solutions. The calculated lowest detection limits (LODs) were in the low μg L(-1) range due to successful use of a collision/reaction cell, while only 11 μL of sample volume was needed per injection by means of a segment sample loop filling. The analysis of a certified reference material further confirmed the suitability of this approach in biological studies.

Complexation and oxidation strategies for improved TXRF determination of mercury in vaccines

A thorough investigation of strategies to overcome the effect of mercury losses during TXRF analysis was carried out. The vaporisation of mercury on sampling targets, associated with too low concentrations determined, depends on the nature of the respective Hg species and the dwell time of the analytes on the target. To prevent vaporisation, oxidation with ammonium persulfate and complexation with ethylenediamine tetraacetate (EDTA) and dimercaptosuccinic acid (DMSA) were investigated. Whereas both approaches were effective in reducing the Hg losses by vaporisation, the complexation approaches turned out to be particularly efficient. Both EDTA and DMSA retain the different mercury species quantitatively over several hours. Based on this approach, a method for mercury determination by TXRF in liquid samples was developed, successfully validated by inductively coupled plasma optical emission spectrometry and applied to the analysis of a mercury-containing vaccine sample.