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Background: A prototype of a noninvasive glucometer combining skin excitation by a mid-infrared quantum cascade laser with photothermal detection was evaluated in glucose correlation tests including 100 volunteers (41 people with diabetes and 59 healthy people). Methods: Invasive reference measurements using a clinical glucometer and noninvasive measurements at a finger of the volunteer were simultaneously recorded in five-minute intervals starting from fasting glucose values for healthy subjects (low glucose values for diabetes patients) over a two-hour period. A glucose range from >50 to Results: 98.8% (full data set) and 99.1% (improved algorithm) of glucose results were within Zones A and B of the grid, indicating the highest accuracy level. Less than 1% of the data were in Zone C, and none in Zone D or E. The mean and median percent differences between the invasive as a reference and the noninvasive method were 12.1% and 6.5%, respectively, for the full data set, and 11.3% and 6.4% with the improved algorithm. Conclusions: Our results demonstrate that noninvasive blood glucose analysis combining mid-infrared spectroscopy and photothermal detection is feasible and comparable in accuracy with minimally invasive glucometers and finger pricking devices which use test strips. As a next step, a handheld version of the present device for diabetes patients is being developed.

CYBASC proteins are ascorbate (AscH-) reducible, diheme b containing membrane integral cytochrome b561 proteins (cytb561), which are proposed to be involved in AscH- recycling and facilitation of i...

Der Begriff „Bio“, ob bei Lebensmitteln, beim Bauen oder bei Kraftstoffen, verspricht gesundere Produkte, eine umweltschonende Produktion, oder eine artgerechte Tierhaltung. Ist das wirklich so? Sind „Bio“-Produkte wirklich besser und gesunder? Sind Bio-Kraftstoffe eine umweltschonende Alternative? Es lohnt sich, genauer hinzuschauen. Oft ist das „Bio-“ oder „Oko“-Label ein Mythos. In vielen Fallen hofft der Konsument, der sich fur ein Bioprodukt entscheidet, mit dem Kauf auch gleich ein gutes Gewissen zu erwerben und das Gefuhl zu haben, etwas fur sich selbst und seine Gesundheit, aber auch etwas fur die Umwelt oder das Tierwohl getan zu haben.

Ionisierende Strahlung und Radioaktivitat sind Teil unseres Alltags. Wir unterliegen einer naturlichen Belastung durch Hohenstrahlung aus dem Weltall, aus Gestein im Boden, und aus der aufgenommenen Nahrung. Zusatzlich erfahren wir menschengemachte Strahlenbelastung durch Rontgenaufnahmen in der Medizin oder durch Diagnostik und Therapie mit radioaktiven Stoffen. Hinzu kommt eine mogliche Gefahrdung durch Unfalle in kerntechnischen Anlagen. Dieses Kapitel hilft dabei, die Einflusse von ionisierender Strahlung zu verstehen und mogliche Gefahren einzuschatzen und zu relativieren.

Elektrische und magnetische Felder sind Bestandteil unseres Alltags. Atmospharische Elektrizitat, z. B. bei einem Gewitter, kennt jeder. Das Erdmagnetfeld ist ebenso eine naturliche Grose, die wir z. B. mit dem Kompass nutzen. Mit der Industrialisierung und mit Entwicklungen in der Medizin sind menschengemachte elektrische und magnetische Felder hinzugekommen. Der Streit um die mogliche Gefahrdung durch elektrische und magnetische Felder entzundet sich vor allem am Bau von leistungsfahigen Stromleitungen, insbesondere als Konsequenz aus der Energiewende. Hochspannungsmasten und -leitungen sind hasslich, aber sind sie auch gefahrlich? Wir gehen hier der Frage nach, ob elektrische und magnetische Felder eine Gefahr fur die Gesundheit darstellen konnen, und vergleichen menschengemachte Felder mit Feldern, die naturlich in biologischen Strukturen vorkommen.

Gentechnik gehort zu den Themen, die bei uns sehr kontrovers und leider oft sehr unsachlich diskutiert werden. Ob in der Landwirtschaft, in der Pharmaindustrie oder in der Medizin: gentechnische Verfahren werden kritisch hinterfragt und oft abgelehnt oder sogar bekampft. Was sind die Vorteile, was die Risiken gentechnischer Verfahren? Welche Chancen bietet die Gentechnik? Dieses Kapitel geht diesen Fragen anhand von Beispielen aus der Landwirtschaft und der Pharmaindustrie nach.

Etwa ein Drittel bis ein Viertel aller Mediziner in Deutschland verschreiben hauptsachlich oder erganzend homoopathische Arzneimittel. Fur deren Herstellung werden Grundstoffe verwendet, deren therapeutische Wirkung meist auserst fragwurdig ist oder die sogar hochgiftig sind. Diese Stoffe werden dann in verschiedensten Verdunnungsstufen zubereitet, oft so verdunnt, dass vom ursprunglichen Grundstoff kein Atom oder Molekul im Arzneiflaschchen mehr vorhanden ist. Eine der von Homoopathen verbreitete pseudonaturwissenschaftliche Erklarungen fur die Wirkung ist die Struktur des Losungsmittels (z. B. Wasser, Alkohol oder Milchzucker), die sich den Wirkstoff „gemerkt“ hat. Dieses Kapitel geht diesen Vorstellungen auf den Grund und entlarvt sie als Humbug.

Wir haben in den letzten 20 Jahren eine endlose Debatte um Luftschadstoffe, vor allem um Feinstaub und um Stickoxide, erlebt. In dieser Debatte mischen viele mit: Das Umweltbundesamt und die Landerbehorden, die Deutsche Umwelthilfe, Epidemologen und andere. Sachlich begrundet ist diese Debatte durch die Schadstoffe, vor allem bedingt durch den Verkehr in unseren Stadten. In der Auseinandersetzung ging es nicht immer sachlich und naturwissenschaftlich begrundet zu, und es wurden viele vorschnelle Entscheidungen getroffen. Dieses Kapitel befasst sich mit Feinstaub, seiner Charakterisierung, seiner Messung und der gesundheitlichen Gefahrdung, sowie mit Stickoxiden, ihrer Herkunft und ihren Quellen. Sind Umweltzonen fur unsere Stadte die richtige Losung fur die Feinstaubbelastung? Losen Fahrverbote das Problem der Stickoxidbelastung? Ist ein Verbot des Dieselmotors gerechtfertigt?

Wie kommt man zu naturwissenschaftlichen Erkenntnissen? Welche Fehlerquellen gibt es; welche Interpretationen sind moglich? Leider werden auch oft pseudowissenschaftliche Erklarungen verwendet, um Sachverhalte zur verzerren und Fehlinformationen zu verbreiten. Das geht bis hin zum Betrug. Wir gehen hier der Frage nach, wie man zu objektiven und verlasslichen Informationen kommt, um damit eventuelle Risiken und Gefahren richtig einschatzen zu konnen.

Wasser ist unser wichtigstes Lebensmittel und unentbehrlich fur Mensch und Tier, Natur, Landwirtschaft und Industrie. So einfach das Wassermolekul auf den ersten Blick auch erscheint: Es hat einige besondere Eigenschaften. Leider hat sich rund um das Wasser auch eine ganze Esoterik-Industrie angesiedelt, die mit pseudowissenschaftlichen Heilsversprechen eine Qualitatsverbesserung von Wasser anpreist. Wir blicken hier auf die physikalischen Eigenschaften von Wasser und entlarven die Heilsversprechungen der „Wasserjunger“.

CYBASC proteins are ascorbate (AscH

We have recently reported infrared spectroscopy of human skin in vivo using quantum cascade laser excitation and photoacoustic or photothermal detection for non-invasive glucose measurement . Here, we analyze the IR light diffusely reflected from skin layers for spectral contributions of glucose. Excitation of human skin by an external cavity tunable quantum cascade laser in the spectral region from 1000 to 1245 cm− 1, where glucose exhibits a fingerprint absorption, yields reflectance spectra with some contributions from glucose molecules. A simple three-layer model of skin was used to calculate the scattering intensities from the surface and from shallow and deeper layers using the Boltzmann radiation transfer equation. Backscattering of light at wavelengths around 10 μm from the living skin occurs mostly from the Stratum corneum top layers and the shallow layers of the living epidermis. The analysis of the polarization of the backscattered light confirms this calculation. Polarization is essentially unchanged; only a very small fraction (

The performance of solar energy conversion into alternative energy sources in artificial systems highly depends on the thermostability of photosystem I (PSI) complexes Terasaki et al. (2007), Iwuchukwu et al. (2010), Kothe et al. (2013) . To assess the thermostability of PSI complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus heating induced perturbations on the level of secondary structure of the proteins were studied. Changes were monitored by Fourier transform infrared (FT-IR) spectra in the mid-IR region upon slow heating (1°C per minute) of samples in D2O phosphate buffer (pD 7.4) from 20°C to 100°C. These spectra showed distinct changes in the Amide I region of PSI complexes as a function of the rising temperature. Absorbance at the Amide I maximum of PSI monomers (centered around 1653cm-1), gradually dropped in two temperature intervals, i.e. 60-75 and 80-90°C. In contrast, absorbance at the Amide I maximum of PSI trimers (around 1656cm-1) dropped only in one temperature interval 80-95°C. The thermal profile of the spectral shift of α-helices bands in the region 1656-1642cm-1 confirms the same two temperature intervals for PSI monomers and only one interval for trimers. Apparently, the observed absorbance changes at the Amide I maximum during heating of PSI monomers and trimers are caused by deformation and unfolding of α-helices. The absence of absorbance changes in the interval of 20-65°C in PSI trimers is probably caused by a greater stability of protein secondary structure as compared to that in monomers. Upon heating above 80°C a large part of α-helices both in trimers and monomers converts to unordered and aggregated structures. Spectral changes of PSI trimers and monomers heated up to 100°C are irreversible due to protein denaturation and non-specific aggregation of complexes leading to new absorption bands at 1618-1620cm-1. We propose that monomers shield the denaturation sensitive sides at the monomer/monomer interface within a trimer, making the oligomeric structure more stable against thermal stress.

UV-VIS absorption spectroscopy is used in almost every spectroscopy laboratory for routine analysis or research. All spectroscopists rely on the Lambert-Beer Law but many of them are less aware of its limitations. This tutorial discusses typical problems in routine spectroscopy that come along with technical limitations or careless selection of experimental parameters. Simple rules are provided to avoid these problems.

Enzymatically-induced degradation of bovine serum albumin (BSA) by serine proteases (trypsin and α-chymotrypsin) in various concentrations was monitored by means of Fourier transform infrared (FT-IR) and ultraviolet circular dichroism (UV-CD) spectroscopy. In this study, the applicability of both spectroscopies to monitor the proteolysis process in real time has been proven, by tracking the spectral changes together with secondary structure analysis of BSA as proteolysis proceeds. On the basis of the FTIR spectra and the changes in the amide I band region, we suggest the progression of proteolysis process via conversion of α-helices (1654 cm(-1)) into unordered structures and an increase in the concentration of free carboxylates (absorption of 1593 and 1402 cm(-1)). For the first time, the correlation between the degree of hydrolysis and the concentration of carboxylic groups measured by FTIR spectroscopy was revealed as well. The far UV-CD spectra together with their secondary structure analysis suggest that the α-helical content decreases concomitant with an increase in the unordered structure. Both spectroscopic techniques also demonstrate that there are similar but less spectral changes of BSA for the trypsin attack than for α-chymotrypsin although the substrate/enzyme ratio is taken the same.

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.

Time-resolved studies were performed for tryptic proteolysis of β-casein in media containing 10–40 % (v/v) ethanol at 37 °C and pH 7.9. The peptide bond demasking, the process which implies the removal of steric obstacles shielding polypeptide sites against enzymatic attack, was quantitatively evaluated with fluorescence spectroscopy by monitoring the exposure of Trp residues to the aqueous polar medium. This process obeys a first-order kinetic law that allowed us the determination of the rate constants of demasking k d . The fraction of initially masked bonds m, being able to convert during proteolysis to the demasked ones, and the fraction of unhydrolysable bonds n were calculated within the framework of a two-step model with consecutive demasking and hydrolysis steps. Parameters m and n were shown to decrease and increase, respectively, with the addition of ethanol.

Immunosurveillance of tumor cells depends on NKp30, a major activating receptor of human natural killer (NK) cells. The human BCL2-associated athanogene 6 (BAG-6, also known as BAT3; 1126 amino acids) is a cellular ligand of NKp30. To date, little is known about the molecular details of this receptor ligand system. Within the current study, we have located the binding site of NKp30 to a sequence stretch of 250 amino acids in the C-terminal region of BAG-6 (BAG-6686–936). BAG-6686–936 forms a noncovalent dimer of 57–59 kDa, which is sufficient for high affinity interaction with NKp30 (KD < 100 nm). As our most important finding, BAG-6686–936 inhibits NKp30-dependent signaling, interferon-γ release, and degranulation of NK cells in the presence of malignantly transformed target cells. Based on these data, we show for the first time that BAG-6686–936 comprises a subdomain of BAG-6, which is sufficient for receptor docking and inhibition of NKp30-dependent NK cell cytotoxicity as part of a tumor immune escape mechanism. These molecular insights provide an access point to restore tumor immunosurveillance by NK cells and to increase the efficacy of cellular therapies.

Time-resolved studies were performed for tryptic proteolysis of β-casein. The accumulation and decay of the proteolysis products was shown to proceed in the form of nanoscale aggregates that exhibited considerable scattering cross sections for visible light. Monitoring the time course of degradation of these aggregates by static light scattering allowed the determination of the rate constants of aggregate degradation. Additionally, another set of rate constants was determined with fluorescence spectroscopy by monitoring the exposure of Trp residues to the aqueous polar medium. These constants were interpreted as the rate constants of peptide bond demasking, the process that implies the removal of steric obstacles shielding polypeptide sites against enzymatic attack. Both methods gave comparable values for the rate constants. It was concluded that for the trypsinolysis of β-casein, demasking proceeds in parallel with the degradation of aggregates that arise at the initial stage of proteolysis.

The dispersion releaser (DR) is a dialysis-based setup for the analysis of the drug release from nanosized drug carriers. It is mounted into dissolution apparatus2 of the United States Pharmacopoeia. The present study evaluated the DR technique investigating the drug release of the model compound flurbiprofen from drug solution and from nanoformulations composed of the drug and the polymer materials poly (lactic acid), poly (lactic-co-glycolic acid) or Eudragit®RSPO. The drug loaded nanocarriers ranged in size between 185.9 and 273.6nm and were characterized by a monomodal size distribution (PDI

Infrared spectroscopy in combination with a specially developed attenuated total reflection (ATR) flow cell and multivariate analysis was used for the quantitative analysis of beer and other beverages. IR spectra of samples were obtained in the range from below 1000 cm-1 to 4000 cm-1 and subjected to a multivariate analysis based on calibration sets with laboratory reference standards. In the case of beer, this calibration set included 240 beer samples spanning the entire range of ethanol content, extract and CO2. Based on this calibration, an infrared and UV/Vis spectroscopy-based sensor for the quick and quantitative quality control of beer was developed and subjected to extensive tests in breweries. This sensor meets and exceeds all requirements from brewers for the routine control in the production and bottling. Its use for other beverages, for example wine, juices or apple wine, requires only another set of calibration data for the specific beverage.

The E-pathway of transmembrane proton transfer has been demonstrated previously to be essential for catalysis by the diheme-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes. Two constituents of this pathway, Glu-C180 and heme b(D) ring C (b(D)-C-) propionate, have been validated experimentally. Here, we identify further constituents of the E-pathway by analysis of molecular dynamics simulations. The redox state of heme groups has a crucial effect on the connectivity patterns of mobile internal water molecules that can transiently support proton transfer from the b(D)-C-propionate to Glu-C180. The short H-bonding paths formed in the reduced states can lead to high proton conduction rates and thus provide a plausible explanation for the required opening of the E-pathway in reduced QFR. We found evidence that the b(D)-C-propionate group is the previously postulated branching point connecting proton transfer to the E-pathway from the quinol-oxidation site via interactions with the heme b(D) ligand His-C44. An essential functional role of His-C44 is supported experimentally by site-directed mutagenesis resulting in its replacement with Glu. Although the H44E variant enzyme retains both heme groups, it is unable to catalyze quinol oxidation. All results obtained are relevant to the QFR enzymes from the human pathogens Campylobacter jejuni and Helicobacter pylori.

The pH dependent opening and closure of Escherichia coli OmpG is driven by the formation and breaking of hydrogen bridges in β-strands S11-S13. We have investigated the in situ secondary structural changes of OmpG with ATR-FTIR difference spectroscopy in order to detect the signals associated with the newly established interactions. Curve-fitting of OmpG in two pH conditions revealed the splitting and shifting of β-sheet signals upon opening of the channel. Besides secondary structure changes, there are also amino acid side chain signals that play active role in opening/closing of the channel. An interaction among positively charged arginines and negatively charged aspartic and glutamic acid residues is suggested upon closure of the channel while this interaction is abolished when the channel opens at higher pH.

A method for monitoring hemodialysis based on quantitative infrared spectroscopic determination of the molecules dialyzed from patient blood is reported. The measurements are reagent-free and aim at real-time and in-line monitoring of the hemodialysis patient. A flow cell using attenuated total reflection infrared spectroscopy is coupled downstream of the dialysis filter unit. A calibration model has been developed from real hemodialysis samples analyzed by chemical reference analysis and from artificially mixed dialysis samples. The infrared monitoring of hemodialysis includes quantitative determination of urea as the lead substance, as well as glucose, lactate, and creatinine, all at a precision only limited by the chemical reference analysis. The flow cell can be fitted to all standard hemodialysis systems. Preliminary tests with hemodialysis patients have demonstrated that detoxification can be clearly monitored. Furthermore, these experiments demonstrate that a wide, real-time control of the patient's physiological parameters is possible with this method, which could lead to increased patient safety.

Cytochrome (cyt) b(561) proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b(561)-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b(561) paralogs from Arabidopsis thaliana (Acytb(561)-A, Acytb(561)-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb(561)-A resembles the best characterised member of the CYBASC family, the cytochrome b(561) from adrenomedullary chromaffin vesicles, and that Acytb(561)-B is atypical compared to other CYBASC proteins. Haem oxidation-reduction midpoint potential (E(M)) values were found to be fully consistent with ascorbate oxidation activities and Fe(3+)-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b(561) from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E(M) values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E(M) values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe(3+)-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E(M) values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b(561) paralogs exist as homodimers.

Demasking of peptide bonds during proteolysis of β-casein and β-lactoglobulin by trypsin was monitored by the measurement of the overall spectral shift of intrinsic protein fluorescence. A clear shift of the apparent emission maxima from approximately 340–345 nm to 355–360 nm during proteolysis was observed, with a time course, which follows protein degradation and structural opening. In contrast to procedures using extrinsic fluorescence labels, this label-free procedure does not bear the risk of structural alterations. It is easy to perform, fast, and has a relatively high accuracy of determination. Proteolysis was modelled as simple two-step process with consecutive demasking and hydrolysis stages. It was shown that the fluorescence shift can be attributed to the demasking stage. Formally, kinetics of the peptide bond demasking obeys a first-order kinetic law. Both the theoretical simulations and experiment are in accordance giving the similar dependences of the hydrolysis degree on the degree of peptide bond demasking.

Fourier transform infrared (FT-IR)- and UV-circular dichroism (UV-CD) spectroscopy have been used to study real-time proteolytic digestion of β-lactoglobulin (β-LG) and β-casein (β-CN) by trypsin at various substrate/enzyme ratios in D2O-buffer at 37 °C. Both techniques confirm that protein substrate looses its secondary structure upon conversion to the peptide fragments. This perturbation alters the backbone of the protein chain resulting in conformational changes and degrading of the intact protein. Precisely, the most significant spectral changes which arise from digestion take place in the amide I and amide II regions. The FT-IR spectra for the degraded β-LG show a decrease around 1634 cm−1, suggesting a decrease of β-sheet structure in the course of hydrolysis. Similarly, the intensity around the 1654 cm−1 band decreases for β-CN digested by trypsin, indicating a reduction in the α-helical part. On the other hand, the intensity around ∼1594 cm−1 and ∼1406 cm−1 increases upon enzymatic breakdown of both substrates, suggesting an increase in the antisymmetric and symmetric stretching modes of free carboxylates, respectively, as released digestion products. Observation of further H/D exchange in the course of digestion manifests the structural opening of the buried groups and accessibility to the core of the substrate. On the basis of the UV-CD spectra recorded for β-LG and β-CN digested by trypsin, the unordered structure increases concomitant with a decrease in the remaining structure, thus, revealing breakdown of the intact protein into smaller fragments. This model study in a closed reaction system may serve as a basis for the much more complex digestion processes in an open reaction system such as the stomach.

Purpose To develop a liver-mimicking MRI gel phantom for use in the development of temperature mapping and coagulation progress visualization tools needed for the thermal tumor ablation methods, including laser-induced interstitial thermotherapy (LITT) and radiofrequency ablation (RFA). Methods A base solution with an acrylamide concentration of 30 vol. % was prepared. Different components were added to the solution; among them are bovine hemoglobin and MR signal-enhancing contrast agents (Magnevist as T1 and Lumirem as T2 contrast agent) for adjustment of the optical absorption and MR relaxation times, respectively. The absorption was measured in samples with various hemoglobin concentrations (0%-7.5%) at different temperatures (25-80 degrees C) using the near-infrared spectroscopy, measuring the transmitted radiation through the sample. The relaxation times were measured in samples with various concentrations of T1 (0.025%-0.325%) and T2 (0.4%-1.6%) contrast agents at different temperatures (25-75 degrees C), through the MRI technique, acquiring images with specific sequences. The concentrations of the hemoglobin and contrast agents of the gel were adjusted so that its absorption coefficient and relaxation times are equivalent to those of liver. To this end, the absorption and relaxation times of the gel samples were compared to reference values, measured in an ex vivo porcine liver at different temperatures through the same methods used for the gel. For validation of the constructed phantom, the absorption and relaxation times were measured in samples containing the determined amounts of the hemoglobin and contrast agents and compared with the corresponding liver values. To qualitatively test the heat resistance of the phantom, it was heated with the LITT method up to approximately 120 degrees C and then was cut to find out if it has been melted. Results In contrast to liver, where the absorption change with temperature showed a sigmoidal form with a jump at T approximately equal 45 degrees C, the absorption of the gel varied slightly over the whole temperature range. However, the gel absorption presented a linear increase from approximately 1.8 to approximately 2.2 mm(-1) with the rising hemoglobin concentration. The gel relaxation times showed a linear decrease with the rising concentrations of the respective contrast agents. Conversely, with the rising temperature, both T1 and T2 increased linearly and showed almost the same trends as in liver. The concentrations of hemoglobin and T1 and T2 contrast agents were determined as 3.92 +/- 0.42 vol. %, 0.098 +/- 0.023 vol. %, and 2.980 +/- 0.067 vol. %, respectively. The measured ex vivo liver T1 value increased from approximately 300 to approximately 530 ms and T2 value from approximately 45 to approximately 52 ms over the temperature range. The phantom validation experiments resulted in absorption coefficients of 2.0-2.1 mm(-1) with variations of 1.5%-2.95% compared to liver below 50 degrees C, T1 of 246.6-597.2 ms and T2 of 40.8-67.1 ms over the temperature range of 25-75 degrees C. Using the Bland-Altman analysis, a difference mean of -6.1/1.9 ms was obtained for T1/T2 between the relaxation times of the phantom and liver. After heating the phantom with LITT, no evidence of melting was observed. Conclusions The constructed phantom is heat-resistant and MR-compatible and can be used as an alternative to liver tissue in the MR-guided thermal ablation experiments with laser to develop clinical tools for real-time monitoring and controlling the thermal ablation progress in liver.

The channel activity of the outer-membrane protein G (OmpG) from Escherichia coli is pH-dependent. To investigate the role of the histidine pair His231/His261 in triggering channel opening and closing, we mutated both histidines to alanines and cysteines. Fourier transform infrared spectra revealed that the OmpG mutants stay—independent of pH—in an open conformation. Temperature ramp experiments indicate that the mutants are as stable as the open state of wild-type OmpG. The X-ray structure of the alanine-substituted OmpG mutant obtained at pH 6.5 confirms the constitutively open conformation. Compared to previous structures of the wild-type protein in the open and closed conformation, the mutant structure shows a difference in the extracellular loop L6 connecting β-strands S12 and S13. A deletion of amino acids 220–228, which are thought to block the channel at low pH in wild-type OmpG, indicates conformational changes, which might be triggered by His231/His261.

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.

Blood constituents such as urea, glucose, lactate, phosphate and creatinine are of high relevance in monitoring the process of detoxification in ambulant dialysis treatment. In the present work, 2 different vibrational spectroscopic techniques are used to determine those molecules quantitatively in artificial dialysate solutions. The goal of the study is to compare the performance of near-infrared (NIR) and mid-infrared (MIR) spectroscopy in hyphenation with partial least squares regression (PLSR) directly by using the same sample set. The results show that MIR spectroscopy is better suited to analyze the analytes of interest. Multilevel multifactor design is used to cover the relevant concentration variations during dialysis. MIR spectroscopy coupled to a multi reflection attenuated total reflection (ATR) cell enables reliable prediction of all target analytes. In contrast, the NIR spectroscopic method does not give access to all 5 components but only to urea and glucose. For both methods, coefficients of determination greater or equal to 0.86 can be achieved in the test-set validation process for urea and glucose. Lactate, phosphate and creatinine perform well in the MIR with R2 ≥ 0.95 using test-set validation.

In this study, structural, functional, and mechanistic properties of the Na(+)/H(+) antiporter MjNhaP1 from Methanococcus jannaschii were analyzed by infrared spectroscopic techniques. Na(+)/H(+) antiporters are generally responsible for the regulation of cytoplasmic pH and Na(+) concentration. MjNhaP1 is active in the pH range between pH 6 and pH 6.5; below and above it is inactive. The secondary structure analysis on the basis of ATR-IR spectra provides the first insights into the structural changes between inactive (pH 8) and active (pH 6) state of MjNhaP1. It results in decreased ordered structural elements with increasing the pH-value i.e. with inactivation of the protein. Analysis of temperature-dependent FTIR spectra indicates that MjNhaP1 in the active state exhibits a much higher unfolding temperature in the spectral region assigned to alpha-helical segments. In contrast, the temperature-induced structural changes for beta-sheet structure are similar for inactive and active state. Consequently, this structure element is not the part of the activation region of the protein. The surface accessibility of the protein was analyzed by following the extent of H/D exchange. Due to higher content of unordered structural elements a higher accessibility for amide protons is observed for the inactive as compared to the active state of MjNhaP1. Altogether, the results present the active state of MjNhaP1 as the state with ordered structural elements which exhibit high thermal stability and increased hydrophobicity.

We present here a study on proteorhodopsin (PR) 2D crystals with analytical ultracentrifugation, circular dichroism and Fourier transform infrared (FTIR) spectroscopy. The aim of our experiments was to test the activity of 2D crystal sample preparations and to gain further insight in PR structure, stability and function with these techniques. Our results demonstrate higher stability compared to detergent-solubilized or reconstituted samples. For different pH values, low pH 2D crystals tend to form bigger aggregates and are less stable than at basic pH. The pH 9 sample shows a sharp phase transition during heat denaturation and there is also evidence for protein-protein interaction due to the close proximity of the proteins in the 2D crystals. In the FTIR measurements at cryogenic temperatures (77 K), we characterized the first step in the PR photocycle. At pH 9, the K intermediate could be observed and the samples showed no orientation effects. At pH 5, we could trap the K/L intermediate, characterized by its negative IR signal at 1741 cm(-1). In rapid-scan FTIR experiments, we could also identify the M intermediate of the photocycle at basic pH. We conclude that the PR 2D crystals exhibit a fully functional photocycle and are therefore well suited for further studies on the proton transport mechanism of PR.

We have investigated the temperature-dependent interaction of the porins OmpF from Paracoccus denitrificans and OmpG from Escherichia coli with lipid molecules after reconstitution in lecithin. Effects of incubation at increased temperatures on activity were tested by functional experiments for OmpG and compared with previously published results of OmpF in order to understand the activity loss of OmpF with monomerization. Protein-lipid interaction was monitored by different reporter groups both from lipid molecules and from protein. OmpF loses its activity by approximately 90% at 50 degrees C while OmpG does not show a temperature-dependent change in activity between room temperature and 50 degrees C. The interaction between OmpF and lipid molecules is severely altered in a two-step mechanism at 55 and approximately 75 degrees C for OmpF. The first step is attributed to changes in the degree of interaction between the aromatic girdle of OmpF and the interfacial region of the lipid bilayer, leading to monomerization of this trimeric porin. The second step at 75 degrees C is attributed to the changes in lipid-porin monomer interaction. Around 90 degrees C, reconstituted porin aggregates. For OmpG, changes in lipid-protein interaction were observed starting from approximately 80 degrees C because of temperature-induced breakdown of its folding. This study provides deeper understanding of porin-lipid bilayer interaction as a function of temperature and can explain the functional breakdown by monomerization while porin secondary structure is still preserved.

As a rapidly growing class of therapeutics, biopharmaceuticals have conquered the global market. Despite the great potential from a therapeutic perspective, such formulations often require frequent injections due to their short half-life. Aiming to establish a parenteral dosage form with prolonged release properties, a biodegradable implant was developed, based on a combination of nanoencapsulation of protein-heparin complexes, creation of a slow release matrix by freeze-drying, and compression using hyaluronan and methylcellulose. In order to investigate this novel delivery system, formulations containing IFN-v-1a and trypsinogen as model proteins were developed. No degradation of the proteins was observed at any stage of the formulation processing. The potential of the delivery system was evaluated in vivo and in vitro after fluorescence-labeling of the biopharmaceuticals. An optimized agarose gel was utilized as in vitro release medium to simulate the subcutaneous environment in a biorelevant manner. In addition, the formulations were administered to female SJL mice and release was innovatively tracked by fluorescence imaging, setting up an in vitro-in vivo correlation. A prolonged time of residence of approximately 12 days was observed for the selected formulation design.

Es wurde eine neue, direkte und einfache Methode zur Bestimmung des Heparinspiegels im Blut entwickelt, die auf der Komplexbildung von Heparin mit Protamin beruht. Heparin bildet mit Protamin im Uberschuss nanoskalige Partikel der Grose von ca. 100–200 nm, die mit Lichtstreuverfahren nachgewiesen werden konnen. Die Lichtstreuung ist proportional zur Partikelkonzentration und damit zum vorliegenden Heparinspiegel. Diese Nachweismethode ist sowohl fur fraktionierte, niedermolekulare Heparine als auch fur unfraktionierte Heparine anwendbar und benotigt nur eine geringe Menge ( 500 I.E./kg KG) eingesetzt werden. Da die Methode spezifisch auf die Heparinmolekule anspricht, konnen durch diesen Test die Nachteile verschiedener Methoden zur Bestimmung der Blutgerinnungsfahigkeit (ACT, Hepcon, Heptest, usw.) vermieden werden.

The Na(+)-coupled betaine symporter BetP senses changes in the membrane state and increasing levels of cytoplasmic K(+) during hyperosmotic stress latter via its C-terminal domain and regulates transport activity according to both stimuli. This intriguing sensing and regulation behavior of BetP was intensively studied in the past. It was shown by several biochemical studies that activation and regulation depends crucially on the lipid composition of the surrounding membrane. In fact, BetP is active and regulated only when negatively charged lipids are present. Recent structural studies have revealed binding of phosphatidylglycerol lipids to functional important parts of BetP, suggesting a functional role of lipid interactions. However, a regulatory role of lipid interactions could only be speculated from the snapshot provided by the crystal structure. Here, we investigate the nature of lipid-protein interactions of BetP reconstituted in closely packed two-dimensional crystals of negatively charged lipids and probed at the molecular level with Fourier transform infrared (FTIR) spectroscopy. The FTIR data indicate that K(+) binding weakens the interaction of BetP especially with the anionic lipid head groups. We suggest a regulation mechanism in which lipid-protein interactions, especially with the C-terminal domain and the functional important gating helices transmembrane helice 3 (TMH3) and TMH12, confine BetP to its down-regulated transport state. As BetP is also activated by changes in the physical state of the membrane, our results point toward a more general mechanism of how active transport can be modified by dynamic lipid-protein interactions.

A spectroscopic method based on attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy has been developed for reagent-free analysis of blood and urine constituents in the clinical laboratory and for point-of-care-applications. Blood plasma, whole blood, and urine were analyzed without any sample preparation, such as drying, concentration, or enrichment. Sample volumes as small as 5 microL (a single drop of blood) can be used. Mathematical models, including partial least-squares regression, were used to construct a prediction model which can calculate the concentration of albumin, cholesterol, glucose, total protein, urea, and triglycerides in whole blood or blood plasma samples and the concentration of urea, uric acid, phosphate and creatinine in urine samples. The absolute precision and reproducibility of the prediction reached is sufficient for routine clinical analysis and is only limited by the precision of the reference analysis used for calibration. This was achieved by use of a large number of calibration samples (approx. 400 for blood samples and approx. 100 for urine samples) carefully selected for physiological and pathological range and for specific disease profiles.

A novel electrochemical reflection cell combining electrochemical techniques and spectroscopy which uses a solid gold working electrode as an optical mirror is described. This cell can be used at path lengths as low as a few micrometers and thus is suitable for ultraviolet/visible (UV/Vis) and infrared spectroscopy even for aqueous solutions and suspensions. The cell was designed for small sample volumes of only a few microliters, thus reducing the effort for sample preparation. Due to the short path length of some micrometers, the entire volume is within the Nernst diffusion layer, hence resulting in fast equilibration. Evaluation of the technique is described with direct electrochemistry of horse heart cytochrome c at the gold electrode modified with 4,4'-dithiodipyridine. Cyclic voltammograms indicate rapid and reversible electrochemistry with the correct midpoint potential (52 mV vs Ag/AgCl/3 M KCl). Chronoamperometry and coulometry confirm rapid and complete oxidation and reduction; the cell volume can be entirely fully reduced within less than 10-20 s. Spectroscopy in the UV/Vis region, with potentials at the working electrode stepped between -390 and 390 mV, show perfect titration of the cytochrome c heme bands. A Nernst fit of the alpha band absorption, with redox potential Em and number of electrons n left as parameters, yields a midpoint potential of 49 mV and n=0.9. The potential of this cell in the investigation of biological electron transfer reactions and in the study of bioenergetic systems is discussed.

We have investigated outer membrane protein porin from Paracoccus denitrificans for its stability against heat and pH. Pathways of unfolding and refolding have been analyzed. Porin incubated at pH 12.5 and above undergoes a slow unfolding into an unordered structure. The unfolded protein could be refolded into a nativelike structure that is functionally active but with distinct deviation from the native protein. This nativelike structure exhibited an entirely different thermal stability. Although aggregation is normally considered a structural "dead-end", the possibility of opening an aggregated porin and forming a functionally active structure was analyzed here. Porin aggregates on heating above 86.2 degrees C. Incubating the heat-aggregated protein at high pH (> or = 12.5) leads to a slow opening of the protein into an unordered structure. It was possible to refold this unordered protein into a trimeric nativelike structure which was capable of forming active pores. However, the thermal stability of the refolded porin was unlike that of the native porin. To understand the basic mechanism behind the unfolding processes, the protein was subjected to heating at various pH values. It was observed that at pH > or = 12.5 the protein does not aggregate upon heating; instead, it opens into an unordered structure. We conclude that at high pH values, the electrostatic interactions of various amino acid residues are perturbed which leads to unfolding into an unordered structure. This study shows for the first time an entirely new unfolding and refolding pathway for porin.

Quinol:fumarate reductase (QFR) is the terminal enzyme of anaerobic fumarate respiration. This membrane protein complex couples the oxidation of menaquinol to menaquinone to the reduction of fumarate to succinate. Although the diheme-containing QFR from Wolinella succinogenes is known to catalyze an electroneutral process, its three-dimensional structure at 2.2 A resolution and the structural and functional characterization of variant enzymes revealed locations of the active sites that indicated electrogenic catalysis. A solution to this apparent controversy was proposed with the so-called "E-pathway hypothesis". According to this, transmembrane electron transfer via the heme groups is strictly coupled to a parallel, compensatory transfer of protons via a transiently established pathway, which is inactive in the oxidized state of the enzyme. Proposed constituents of the E-pathway are the side chain of Glu C180 and the ring C propionate of the distal heme. Previous experimental evidence strongly supports such a role of the former constituent. Here, we investigate a possible heme-propionate involvement in redox-coupled proton transfer by a combination of specific (13)C-heme propionate labeling and Fourier transform infrared (FTIR) difference spectroscopy. The labeling was achieved by creating a W. succinogenes mutant that was auxotrophic for the heme-precursor 5-aminolevulinate and by providing [1-(13)C]-5-aminolevulinate to the medium. FTIR difference spectroscopy revealed a variation on characteristic heme propionate vibrations in the mid-infrared range upon redox changes of the distal heme. These results support a functional role of the distal heme ring C propionate in the context of the proposed E-pathway hypothesis of coupled transmembrane electron and proton transfer.