Werner Mäntele

In vivo noninvasive monitoring of glucose concentration in human epidermis by mid-infrared pulsed photoacoustic spectroscopy.

The noninvasive determination of glucose in the interstitial layer of the human skin by mid-infrared spectroscopy is reported. The sensitivity for this measurement was obtained by combining the high pulse energy from an external cavity quantum cascade laser (EC-QCL) tunable in the infrared glucose fingerprint region (1000-1220 cm(-1)) focused on the skin, with a detection of the absorbance process by photoacoustic spectroscopy in the ultrasound region performed by a gas cell coupled to the skin. This combination facilitates a quantitative measurement for concentrations of skin glucose in the range from <50 mg/dL to >300 mg/dL, which is the relevant range for the glucose monitoring in diabetes patients. Since the interstitial fluid glucose level is representative of the blood glucose level and follows it without significant delay (<10 min), this method could be applied to establish a noninvasive, painless glucose measurement procedure that is urgently awaited by diabetes patients. We report here the design of the photoacoustic experiments, the spectroscopy of glucose in vivo, and the calibration method for the quantitative determination of glucose in skin. Finally, a preliminary test with healthy volunteers and volunteers suffering from diabetes mellitus demonstrates the viability of a noninvasive glucose monitoring for patients based on the combination of infrared QCL and photoacoustic detection.

Structural evolution of C‐terminal domains in the p53 family

The tetrameric state of p53, p63, and p73 has been considered one of the hallmarks of this protein family. While the DNA binding domain (DBD) is highly conserved among vertebrates and invertebrates, sequences C‐terminal to the DBD are highly divergent. In particular, the oligomerization domain (OD) of the p53 forms of the model organisms Caenorhabditis elegans and Drosophila cannot be identified by sequence analysis. Here, we present the solution structures of their ODs and show that they both differ significantly from each other as well as from human p53. CEP‐1 contains a composite domain of an OD and a sterile alpha motif (SAM) domain, and forms dimers instead of tetramers. The Dmp53 structure is characterized by an additional N‐terminal β‐strand and a C‐terminal helix. Truncation analysis in both domains reveals that the additional structural elements are necessary to stabilize the structure of the OD, suggesting a new function for the SAM domain. Furthermore, these structures show a potential path of evolution from an ancestral dimeric form over a tetrameric form, with additional stabilization elements, to the tetramerization domain of mammalian p53.

Dimer-tetramer transition controls RUNX1/ETO leukemogenic activity

RUNX1/ETO, the fusion protein resulting from the chromosomal translocation t(8;21), is one of the most frequent translocation products in acute myeloid leukemia. Several in vitro and in vivo studies have shown that the homo-tetramerization domain of ETO, the nervy homology region 2 (NHR2), is essential for RUNX1/ETO oncogenic activity. We analyzed the energetic contribution of individual amino acids within the NHR2 to RUNX1/ETO dimer-tetramer transition and found a clustered area of 5 distinct amino acids with strong contribution to the stability of tetramers. Substitution of these amino acids abolishes tetramer formation without affecting dimer formation. Similar to RUNX1/ETO monomers, dimers failed to bind efficiently to DNA and to alter expression of RUNX1-dependent genes. RUNX1/ETO dimers do not block myeloid differentiation, are unable to enhance the self-renewal capacity of hematopoietic progenitors, and fail to induce leukemia in a murine transplantation model. Our data reveal the existence of an essential structural motif (hot spot) at the NHR2 dimer-tetramer interface, suitable for a molecular intervention in t(8;21) leukemias.

Characterization of two novel redox groups in the respiratory NADH:ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase is the first of the respiratory chain complexes in many bacteria and mitochondria of most eukaryotes. The bacterial complex consists of 14 different subunits. Seven peripheral subunits bear all known redox groups of complex I, namely one FMN and five EPR-detectable iron-sulfur (FeS) clusters. The remaining seven subunits are hydrophobic proteins predicted to fold into 54 alpha-helices across the membrane. Little is known about their function, but they are most likely involved in proton translocation. The mitochondrial complex contains in addition to the homologues of these 14 subunits at least 29 additional proteins that do not directly participate in electron transfer and proton translocation. A novel redox group has been detected in the Neurospora crassa complex, in an amphipathic fragment of the Escherichia coli complex I and in a related hydrogenase and ferredoxin by means of UV/Vis spectroscopy. This group is made up by the two tetranuclear FeS clusters located on NuoI (the bovine TYKY) which have not been detected by EPR spectroscopy yet. Furthermore, we present evidence for the existence of a novel redox group located in the membrane arm of the complex. Partly reduced complex I equilibrated to a redox potential of -150 mV gives a UV/Vis redox difference spectrum that cannot be attributed to the known cofactors. Electrochemical titration of this absorption reveals a midpoint potential of -80 mV. This group is believed to transfer electrons from the high potential FeS cluster to ubiquinone.

Windowless ultrasound photoacoustic cell for in vivo mid-IR spectroscopy of human epidermis: Low interference by changes of air pressure, temperature, and humidity caused by skin contact opens the possibility for a non-invasive monitoring of glucose in the interstitial fluid

The application of a novel open, windowless cell for the photoacoustic infrared spectroscopy of human skin is described. This windowless cavity is tuned for optimum performance in the ultrasound range between 50 and 60 kHz. In combination with an external cavity tunable quantum cascade laser emitting in the range from ~1000 cm(-1) to 1245 cm(-1), this approach leads to high signal-to-noise-ratio (SNR) for mid-infrared spectra of human skin. This opens the possibility to measure in situ the absorption spectrum of human epidermis in the mid-infrared region at high SNR in a few (~5) seconds. Rapid measurement of skin spectra greatly reduces artifacts arising from movements. As compared to closed resonance cells, the windowless cell exhibits the advantage that the influence of air pressure variations, temperature changes, and air humidity buildup that are caused by the contact of the cell to the skin surface can be minimized. We demonstrate here that this approach can be used for continuous and non-invasive monitoring of the glucose level in human epidermis, and thus may form the basis for a non-invasive monitoring of the glucose level for diabetes patients.

Infrared spectroscopic analysis of human interstitial fluid in vitro and in vivo using FT-IR spectroscopy and pulsed quantum cascade lasers (QCL): Establishing a new approach to non invasive glucose measurement.

Interstitial fluid, i.e. the liquid present in the outermost layer of living cells of the skin between the Stratum corneum and the Stratum spinosum, was analyzed by Fourier transform infrared spectroscopy and by infrared spectroscopy using pulsed quantum cascade infrared lasers with photoacoustic detection. IR spectra of simulated interstitial fluid samples and of real samples from volunteers in the 850-1800cm(-1) range revealed that the major components of interstitial fluid are albumin and glucose within the physiological range, with only traces of sodium lactate if at all. The IR absorbance of glucose in interstitial fluid in vivo was probed in healthy volunteers using a setup with quantum cascade lasers and photoacoustic detection previously described. A variation of blood glucose between approx. 80mg/dl and 250mg/dl in the volunteers was obtained using the standard oral glucose tolerance test (OGT). At two IR wavelengths, 1054cm(-1) and 1084cm(-1), a reasonable correlation between the photoacoustic signal from the skin and the blood glucose value as determined by conventional glucose test sticks using blood from the finger tip was obtained. The infrared photoacoustic glucose signal (PAGS) may serve as the key for a non-invasive glucose measurement, since the glucose content in interstitial fluid closely follows blood glucose in the time course and in the level (a delay of some minutes and a level of approx. 80-90% of the glucose level in blood). Interstitial fluid is present in skin layers at a depth of only 15-50μm and is thus within the reach of mid-IR energy in an absorbance measurement. A non-invasive glucose measurement for diabetes patients based on mid-infrared quantum cascade lasers and photoacoustic detection could replace the conventional measurement using enzymatic test stripes and a drop of blood from the finger tip, thus reducing pain and being a cost-efficient alternative for millions of diabetes patients.

Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochromec oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a α-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochromec yielded a kinetic difference spectrum resembling that of heme a 3. This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from CuA to the binuclear center may not occur via hemea but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Δψ generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.

Similarities and dissimilarities in the structure-function relation between the cytochrome c oxidase from bovine heart and from Paracoccus denitrificans as revealed by FT-IR difference spectroscopy

The redox dependent changes in the cytochrome c oxidase from bovine heart were studied with a combined electrochemical and FT‐IR spectroscopic approach. A direct comparison to the electrochemically induced FT‐IR difference spectra of the cytochrome c oxidase from Paracoccus denitrificans reveals differences in the structure and intensity of vibrational modes. These differences are partially attributed to interactions of subunits influencing the heme and protein modes. In the spectral regions characteristic for ν(C=O) and ν(COO−)s/as modes of protonated and deprotonated Asp and Glu residues, additional signals at 1736, 1602 and 1588 cm−1 are observed. On this basis, the possible involvement of Asp‐51, a residue specifically conserved in mammalian oxidase and previously proposed to show redox depended conformational changes in the respective X‐ray structures, is critically discussed.

Effect of proline to alanine mutation on the thermal stability of the all-β-sheet protein tendamistat

The temperature dependent denaturation of wild-type tendamistat and of the proline-free triple mutant P7A/P9A/P50A was investigated using Fourier-transform infrared (FTIR) spectroscopy. Whereas the temperature-induced unfolding is reversible in the wild type, aggregation was observed for the proline-free tendamistat when studied under the same conditions. The midpoint unfolding temperature T(m) was found as 82.3+/-0.5 degrees C in (2)H2O. The thermal stability of the proline-free mutant is reduced by 15 degrees C as compared to the wild type. Changes in the strength of hydrogen bonding of tyrosine O-H groups upon unfolding and aggregation are reflected in small shifts of the C-C stretching mode of the aromatic ring near 1515 cm(-1). Evaluation of data from different infrared (IR) bands sensitive to changes in secondary structure as well as to changes in tertiary structure strongly supports a two-state model for the unfolding process of wild-type tendamistat.

Assessing the drug release from nanoparticles: Overcoming the shortcomings of dialysis by using novel optical techniques and a mathematical model.

The aim of the present investigation was to develop a reliable method which can be applied to the measurement of in vitro drug release from nanocarriers. Since the limited membrane transport is one major obstacle to the assessment of drug release with dialysis techniques, the determination of this parameter was our objective. Therefore, a novel drug release automatic monitoring system (DREAMS) was designed to conduct continuous measurements during the dialysis process. Moreover, a mathematical model was used for evaluation of the experimental data. This combination of mathematical and analytical tools enabled the quantification of the total amount of free drug in the system. Eudragit(®) RS 100 nanoparticles loaded with the model compound 5,10,15,20-tetrakis(m-hydroxypheny)chlorin (mTHPC) were investigated and the drug release was continuously monitored by using a fluorescence spectrometer that is part of the setup. Free drug and drug-loaded nanoparticles were tested to discriminate between the two formulations. In addition, two types of membranes composed of different materials were evaluated and the kinetics of membrane transport was determined. The data obtained from the apparatus were further treated by a mathematical model, which yielded distinguishable release profiles between samples of different compositions. The method offers a promising option for release testing of nanoparticles.