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Im Feldversuch OptiMoor wird im Rahmen eines Erprobungs- und Entwicklungsvorhabens auf entwassertem Hochmoorgrunland im Hankhauser Moor (Niedersachsen) in sieben Varianten (je 8 m × 24 m) inklusive einer Referenzflache erprobt, welche Masnahmen am erfolgversprechendsten fur die Wiederherstellung von Hochmoorokosystemen in Hinblick auf Natur- und Klimaschutz sind. Im Fokus dieses Beitrags steht, wie sich Wiedervernassung, Oberbodenabtrag und Torfmoosbeimpfung auf die Vegetationsentwicklung der ersten drei Jahre auswirken. Hierzu wurden je Variante drei Dauerquadrate und ein Transekt eingerichtet, um die raumliche Heterogenitat moglichst gut abzubilden. Auserdem wurden die Diasporenpotenziale der unmittelbaren Umgebung und des Spendermaterials untersucht. Die Wiedervernassung hat in den Varianten ohne Oberbodenabtrag zu einer Ausbreitung der Nasse- und Feuchtezeiger gefuhrt. Durch den Oberbodenabtrag wurden konkurrenzarme Bedingungen geschaffen. Die z. T. nur sparlich angesiedelten Arten entstammen dabei v. a. der Diasporenbank von Oberbodenresten und der unmittelbaren Umgebung. Auf den beimpften Varianten haben sich die ubertragenen moortypischen Arten etabliert und es konnte bereits ein nennenswertes Torfmooshohenwachstum beobachtet werden.

As photovoltaic systems grow in size, there has been an increasing desire to automate the detection of arc faults. Any automated system for arc detection must be as fast and accurate as possible: delayed detection of electrical arcs can lead to fire and considerable system damage, while false positives that cause preventative system shutdown are associated with a significant financial cost. In this article, we present a novel approach to detect arcs in dc microgrids via their high-frequency (HF) spectral pattern using ideas from compressed sensing. The acquisition and analysis of HF signals using analog-to-digital converter technology typically requires costly hardware and is not feasible for on-site installation at power plants. However, sparsifying the signal by filtering everything but a narrow HF band enables the use of a modulated wideband converter to sample the arc signature at sub-Nyquist frequencies. We then calculate a characteristic band power within the selected spectrum slice over time and show that it can be used to reliably detect arc events via simple thresholding. We have evaluated our methods on both simulated and experimentally generated arc signals. Finally, we perform statistical analysis of power distributions using linear discriminant analysis in order to identify the frequency range best suited for arc detection.

Many peatlands in Central Europe are under unsustainable drainage-based land use with high greenhouse gas emissions counteracting the aims of the Paris Agreement. After decades of drained and intensive land use many peat bogs are in pitiful state. Rewetting can stop the carbon dioxide (CO2) source function but may result in high methane (CH4) emissions and eutrophication. Further, lack of diaspores my hamper the establishment of typical bog species. Restoration measures like topsoil removal (TSR) or spreading target vegetation propagules are known to improve restoration success in fen peatlands or after peat extraction. However, experience on restoration of bogs after previous agricultural use is scarce and the climate effects of these restoration measures including carbon losses from TSR are unknown.We installed a field trial in a drained bog in North-West Germany to explore the effect of TSR and Sphagnum spreading on greenhouse (GHG) emissions. The trial consists of seven plots (~8 x 24 m each) representing the status quo—intensive grassland use—and six different restoration approaches. Two approaches are rewetting on the original surface with or without regular biomass harvesting. The remaining four represent TSR prior rewetting where two of the four were inoculated with Sphagnum spp. On all plots we measured GHG fluxes fortnightly using closed chambers to obtain two-year GHG budgets. We assessed the climate effects of the status quo and the six restoration approaches by applying a radiative forcing model to the GHG budgets and to published emission factors while incorporating the effect of TSR through different depletion scenarios of the exported topsoil carbon.Compared to the status quo, rewetting alone reduced CO2 emissions by ~75% but substantially increased CH4 emissions, which were much higher than published emission factors for a similar peatland category. After TSR, on-site CO2 emissions were close to 0 or—with Sphagnum spreading—net negative while CH4 emissions remained very low. Based on our GHG budgets, TSR quickly becomes less climate warming than keeping the status quo and rewetting at the original surface. In contrast, based on emission factors, rewetting at the original surface is initially the least climate warming option. In general, the climatic effect of TSR is likely lowest when removing only as much topsoil as necessary to implement nutrient-poor and acidic conditions thereby ensuring rapid establishment of a Sphagnum carpet and by conserving the removed topsoil as long as possible. Here, the climate warming effect of TSR of ~30 cm in combination with rewetting roughly corresponds to the climate warming of rewetted nutrient-rich temperate peatlands without TSR. Therefore, from a climate perspective, we can recommend a shallow TSR of up to 30 cm for peat bog restoration given that the goal is to re-establish typical bog habitats.

The bacterial dioxygenation of mono- or polycyclic aromatic compounds is an intensely studied field. However, only in a few cases has the repeated dioxygenation of a substrate possessing more than a single aromatic ring been described. We previously characterized the aryl-hydroxylating dioxygenase BphA-B4h, an artificial hybrid of the dioxygenases of the biphenyl degraders Burkholderia xenovorans LB400 and Pseudomonas sp. strain B4-Magdeburg, which contains the active site of the latter enzyme, as an exceptionally powerful biocatalyst. We now show that this dioxygenase possesses a remarkable capacity for the double dioxygenation of various bicyclic aromatic compounds, provided that they are carbocyclic. Two groups of biphenyl analogues were examined: series A compounds containing one heterocyclic aromatic ring and series B compounds containing two homocyclic aromatic rings. Whereas all of the seven partially heterocyclic biphenyl analogues were solely dioxygenated in the homocyclic ring, four of the six carbocyclic bis-aryls were converted into ortho,meta-hydroxylated bis-dihydrodiols. Potential reasons for failure of heterocyclic dioxygenations are discussed. The obtained bis-dihydrodiols may, as we also show here, be enzymatically re-aromatized to yield the corresponding tetraphenols. This opens a way to a range of new polyphenolic products, a class of compounds known to exert multiple biological activities. Several of the obtained compounds are novel molecules.

Peatland restoration is seen as an effective contribution to help achieve the aims of the Paris Agreement because currently huge amounts of peatlands in Northern Central Europe are under unsustainable drainage-based land use. If net zero greenhouse gas emissions from peatlands shall be reached by 2050, restoration measures have to be done as soon as possible. However, rewetting drained peatlands that were under intensive grassland use frequently results in high methane (CH4) emissions, which is often seen as a counter-argument against rewetting. To find the source of high CH4 emissions after rewetting and to explore the best possible way of peatland restoration (i.e., low CH4 emissions after rewetting) under near-natural conditions, we installed a field trial in a drained bog in north-western Germany. The trial consists of seven plots (~8 × 24 m2) representing the status quo—intensive grassland use— and six restoration approaches with combinations of rewetting either on the original surface or after topsoil removal (TSR), biomass harvesting or spreading Sphagnum spp. to initiate vegetation succession. On all seven plots we measured CH4 fluxes using closed chambers. In addition, we investigated CH4 production potential by incubating soil samples and determining methanogen abundance by quantitative PCR. Compared to rewetting on the original surface, CH4 emissions were reduced on TSR plots by factor 30 to 400. Spreading of Sphagnum spp. had only little effect on CH4 emissions during the first year of establishment. TSR also reduced CH4 production potential and methanogen abundance. Further, the response of CH4 fluxes to methanogen abundance was lower after TSR. This suggests that both reduction in labile substrate and in methanogen abundance contribute to near-zero CH4 emissions after TSR. These are the first field-scale results that demonstrate the efficiency of removing degraded topsoil to avoid high CH4 emissions after rewetting.

A major reason for the low success rate in current drug development through chemical synthesis has been ascribed to the large fraction of quasi planar candidate molecules. Therefore, an "escape from flatland" strategy has been recommended for the generation of bioactive chemical entities. In a first attempt to test this recommendation, we synthesized a small collection of bridged bicyclic compounds possessing a rigid spherical core structure by combining a group of cyclic dienes with a collection of dienophiles. We started from planar biphenyl analogues and, by enzymatic dioxygenation, transformed them into hydroxylated diene structures. Using a small library of newly synthesized dienophiles, the dienes were converted into bridged bicycles via the Diels-Alder reaction. The resulting collection of 78 structures was first tested for bioactivity in a generic assay based on interference with the proliferation of mammalian cells. A more mechanism-targeted bioactivity profiling method, exploiting cellular impedance monitoring, was subsequently used to obtain suggestions for the mode of action exerted by those compounds that were the most active in the proliferation assay. Proteasome inhibition could be confirmed for 8 of a series of 9 respective candidates. Whilst 7 of these molecules showed relatively weak interference with proteasome activity, one candidate exerted a moderate but distinct inhibition. This result appears remarkable in view of the small size of the compound library, which was synthesized following a few basic considerations. It encourages the application of diverse synthetic approaches to further investigate the role of spherical shape for the success of compound libraries.

Flavonoids are known to possess a multitude of biological activities. Therefore, diversification of the core structures is of considerable interest. One of nature's important tailoring reactions in the generation of bioactive compounds is glycosylation, which is able to influence numerous molecular properties. Here, we examined two non-Leloir glycosyltransferases that use sucrose as an inexpensive carbohydrate donor, glycosyltransferase R from Streptococcus oralis (GtfR) and amylosucrase from Neisseria polysaccharea (Ams), for the glucosylation of flavonoids. Flavones generally were poor substrates. Several inhibited Ams. In contrast, flavanes were well accepted by both enzymes. All glucose attachments occurred via α1 linkages. Comparison of the three available stereoisomers of 3,5,7,3',4'-pentahydroxyflavane revealed significant differences in glycoside formation between them as well as between the two enzymes. The latter were shown to possess largely complementary product ranges. Altogether, three of the four hydroxy substituents of the terminal flavonoid rings were glycosylated. Typically, Ams glucosylated the B ring at position 3', whereas GtfR glucosylated this ring at position 4' and/or the A ring at position 7. In several instances, short carbohydrate chains were attached to the aglycones. These contained α 1-4 linkages when formed by Ams, but α 1-3 bonds when generated by GtfR. The results show that both enzymes are useful catalysts for the glucodiversification of flavanes. In total, more than 16 products were formed, of which seven have previously not been described.

High-resolution frequency domain optical coherence tomography (OCT) stands out amongst a range of novel dermatologic imaging technologies, with its good detection sensitivity around -100 dB, high measurement speeds allowing real-time image acquisition and its ability to acquire high definition cross-sectional and 3D tomograms of regions greater than 1 cm2, providing tissue information comparable to conventional histopathology without the need for any contrast agents. Typical axial and transverse resolutions of state-of-the-art OCT systems range between 1–10 µm and approximately 20 µm, respectively, depending on the employed wavelength region. This review investigates the significant progress accomplished in the field of dermatologic OCT with respect to other in vivo diagnostic methods for pre-excisional imaging of nonmelanoma skin cancers and specifically emphasizes state-of-the-art results achieved in different clinical pilot studies. Further technological extensions of OCT, various multimodal imaging ap...

PURPOSE: To gather sub-surface in situ images of microneedle-treated human skin, in vivo, using optical coherence tomography (OCT). This is the first study to utilise OCT to investigate the architectural changes that are induced in skin following microneedle application. METHODS: Steel, silicon and polymer microneedle devices, with different microneedle arrangements and morphologies, were applied to two anatomical sites in human volunteers following appropriate ethical approval. A state-of-the-art ultrahigh resolution OCT imaging system operating at 800 nm wavelength and

The range of genetic and genomic resources available makes the mouse a powerful model for the genetic dissection of complex traits. Because accurate, high-throughput phenotypic characterisation is crucial to the success of such endeavours, we recently developed an optical coherence tomography (OCT) system with extended depth range scanning capability for measuring ocular component dimensions in mice. In order to test whether the accuracy and reproducibility of our OCT system was sufficient for gene mapping studies, we carried out an experiment designed to estimate the heritability of mouse ocular component dimensions. High-resolution, two dimensional tomograms were obtained for both eyes of 11 pairs of 8 week-old outbred MF1 mice. Subsequently, images were obtained when their offspring were aged 8 weeks. Biometric data were extracted after image segmentation, reconstruction of the geometric shape of each surface, and calculation of intraocular distances. The repeatability of measurements was evaluated for 12 mice scanned on consecutive days. Heritability estimates were calculated using variance components analysis. Sets of tomograms took ∼2 s to acquire. Biometric data could be obtained for 98% of the 130 eyes scanned. The 95% limits of repeatability ranged from ±6 to ±16 μm for the axial ocular component dimensions. The heritability of the axial ocular components was 0.6–0.8, except for corneal thickness, which had a heritability not significantly different from zero. In conclusion, axial ocular component dimensions are highly heritable in mice, as they are in humans. OCT with extended depth range scanning can be used to rapidly phenotype individual mice with sufficient accuracy and precision to permit gene mapping studies.

An amylosucrase gene was subjected to high-rate segmental random mutagenesis, which was directed toward a segment encoding amino acids that influence the interaction with substrate molecules in subsites −1 to +3. A screen was used to identify enzyme variants with compromised glucan chain elongation. With an average mutation rate of about one mutation per targeted codon, a considerable fraction (82%) of the clones that retained catalytic activity were deficient in this trait. A detailed characterization of selected variants revealed that elongation terminated when chains reached lengths of only two or three glucose moieties. Sequencing showed that the amylosucrase derivatives had an average of no more than two amino acid substitutions and suggested that predominantly exchanges of Asp394 or Gly396 were crucial for the novel properties. Structural models of the variants indicated that steric interference between the amino acids introduced at these sites and the growing oligosaccharide chain are mainly responsible for the limitation of glucosyl transfers. The variants generated may serve as biocatalysts for limited addition of glucose moieties to acceptor molecules, using sucrose as a readily available donor substrate.

PURPOSE To demonstrate high-speed, high axial resolution optical coherence tomography (OCT) at 1060 nm with penetration to the sclera. The clinical feasibility of dense, high-speed sampling for higher levels of detail at the macula and optic nerve head is explored with respect to motion artifacts. METHODS A three-dimensional (3D) OCT system making use of a high-speed camera operating at 47,000 depth scans/s was developed. The 1010- to 1080-nm wavelength band leads to 6.7 microm effective axial resolution and enables the acquisition of retinal and choroidal 130 Megavoxel volumes of human subjects within 7 seconds. Motion artifacts were reduced by numeric postprocessing techniques. RESULTS Drift motion artifacts could be suppressed within fields up to 38 degrees x 38 degrees (approximately 1 cm(2)) using acquisition speeds of up to 74 frames/s at 512 x 512 pixel/frame. This isotropic OCT sampling of the human retina in vivo allowed reconstruction of the retinal microvasculature solely on vessel reflectivity, without the use of contrast agents, and revealed three interconnected capillary meshworks. Simultaneously in the choroid, the structure of the choriocapillaris, Sattler's layer, and Haller's layer were differentiated, and the choroidal-scleral interface was clearly delineated in densely sampled narrow- and wide-angle scans (>38 degrees). At the optic nerve head, the 3D fine structure of the lamina cribrosa and the circle of Zinn-Haller were visualized. CONCLUSIONS OCT almost centered within the 1060-nm water transmission window significantly profits from lower scattering and allows investigation of the retina and choroid at an unprecedented combination of penetration and high speed at high resolution and may provide superior clinical feasibility to commercial 800-nm devices.

The glycosylation of bioactive compounds, such as flavonoids, is of particular relevance, as it modulates many of their pharmacokinetic parameters. This article reviews the literature between 2010 and the end of 2015 that deals with the enzymatic O-glycosylation of this class of compounds. Enzymes of glycosyltransferase family 1 remain the biocatalysts of choice for glycodiversification of flavonoids, in spite of relatively low yields. Transfers of 14 different sugars, in addition to glucose, were reported. Several Escherichia coli strains were metabolically engineered to enable a (more efficient) synthesis of the required donor during in vivo glycosylations. For the transfer of glucose, enzymes of glycoside hydrolase families 13 and 70 were successfully assayed with several flavonoids. The number of acceptor substrates and of regiospecificities characterized so far is smaller than for glycosyltransferases. However, their glycosyl donors are much cheaper and yields are considerably higher. A few success stories of enzyme engineering were reported. These improved the catalytic efficiency as well as donor, acceptor, or product ranges. Currently, the development of appropriate high-throughput screening systems appears to be the major bottleneck for this powerful technology.

Flavonoids possess a wide range of biological activities. Their glycosylation is of considerable interest, as it often positively influences their pharmacokinetic and other molecular properties. We recently showed that two non-Leloir glycosyltransferases that use sucrose as carbohydrate donor, an amylosucrase from Neisseria polysaccharea (Ams-Np) and a glucansucrase from Streptococcus oralis (GtfR-So), were hardly able to glucosylate flavones, but accepted flavanes as substrates. We now examined compounds from two other flavonoid classes, flavanones and isoflavones for glucose transfer by these enzymes. Taxifolin was investigated as a flavanone analogue of both, the accepted pentahydroxyflavane catechin and the non-accepted pentahydroxyflavone quercetin. It was glucosylated by both enzymes, but much better by GtfR-So than by Ams-Np due to apparent strong inhibition of the latter. The acceptance of a collection of isoflavones strongly depended on the substitution pattern of the core. Only two of the 10 compounds examined yielded glucosides in satisfactory amounts. With these substrates, both enzymes catalyzed formation of a range of products, differing in the number of saccharide units. The structures of mono- and diglycosylated compounds obtained in higher amounts were elucidated. While GtfR-So attached glucose to taxifolin in the B ring at O4', both enzymes glucosylated the isoflavones in the A ring at O7. All products were α-glucosides. Interglycosidic linkages formed by Ams-Np were α1-4. To our knowledge, this is the first report of glucosylation of flavanone and isoflavone aglycones by an amylosucrase. All characterized compounds have not previously been described.

It has repeatedly been shown that aryl-hydroxylating dioxygenases do not possess a very high substrate specificity. To gain more insight into this phenomenon, we examined two powerful biphenyl dioxygenases, the well-known wild-type enzyme from Burkholderia xenovorans LB400 (BphA-LB400) and a hybrid enzyme, based on a dioxygenase from Pseudomonas sp. B4-Magdeburg (BphA-B4h), for their abilities to dioxygenate a selection of eight biphenyl analogues in which the second aromatic ring was replaced by aliphatic as well as aliphatic/aromatic moieties, reflecting a variety of steric requirements. Interestingly, both enzymes were able to catalyse transformation of almost all of these compounds. While the products formed were identical, major differences were observed in transformation rates. In most cases, BphA-B4h proved to be a significantly more powerful catalyst than BphA-LB400. NMR characterization of the reaction products showed that the metabolite obtained from biphenylene underwent angular dioxygenation, whereas all other compounds were subject to lateral dioxygenation at ortho and meta carbons. Subsequent growth studies revealed that both dioxygenase source strains were able to utilize several of the biphenyl analogues as sole sources of carbon and energy. Therefore, prototype BphBCD enzymes of the biphenyl degradative pathway were examined for their ability to further catabolize the lateral dioxygenation products. All of the ortho- and meta-hydroxylated compounds were converted to acids, showing that this pathway is quite permissive, enabling catalysis of the turnover of a fairly wide variety of metabolites.

Glycosylation is one of the most important tailoring reactions for natural products. It typically exerts profound direct or indirect effects on their biological activity. The dihydrochalcone phloretin and its known sugar derivatives, particularly phlori(d)zin, have been shown to influence various cellular processes. We found that a non-Leloir glycosyltransferase, amylosucrase from Neisseria polysaccharea, is an excellent catalyst for the stereospecific glucosylation of phloretin at the 4' position. Three novel phloretin derivatives were obtained, the first ones in which the sugar-aglycone bond possesses the configuration. A first biological characterization in a cell viability assay showed that each sugar attachment reduced the compound toxicity approximately two-fold.

Flavonoids are a large group of plant secondary metabolites that exert various biological and pharmacological effects. In this context, the generation of derivatives is of considerable interest. The introduction of hydroxy groups is of particular relevance, as they are known to be involved in many of the biological interactions and furthermore enable additional modifications, such as glycosylations. Bacterial aryl-hydroxylating dioxygenases (ARHDOs) have proven to be very useful for the conversion of aromatic structures into versatile building blocks for different kinds of derivatizations. Such enzymes have been used with varying success for the oxidation of flavonoids. In order to find better ARHDOs for the hydroxylation of such substrates, we carried out biotransformation trials with a collection of hybrid ARHDOs of different origin, using resting cells of recombinant strains. This identified enzymes able to transform all of the flavonoids examined, typically in yields above 50%. It also showed that moderately reactive substituents of flavonoids, such as hydroxy or amino groups, can lead to spontaneous follow-up reactions with the dienediol structures generated by dioxygenation. A report of flavanone epoxidation, a reaction never before observed to be catalyzed by an ARHDO, is challenged by our results. All ARHDOs examined converted this substrate into a dehydrogenase-transformable dihydrodiol. All dihydrodiols obtained by dioxygenation of the examined flavonoids were successfully re-aromatized into catechols by a bacterial dehydrogenase. These metabolites were usually stable. However, the catechols formed from flavanone and 2'-hydroxy-chalcone, respectively, were interconvertible under mild conditions. Altogether, we isolated and characterized 13 compounds that have not previously been described. The biotransformations reported here give access to novel flavonoid derivatives that may be applied for biological screens as well as for further modification, such as glycodiversification.

Burkholderia sp. strain LB400 is one of the most potent aerobic polychlorobiphenyl (PCB)-degrading microorganisms that have been characterized. Its PCB-dioxygenating activity originates predominantly or exclusively from the biphenyl dioxygenase encoded by its bph gene cluster. Analysis of the dioxygenation products of several di- to pentachlorinated biphenyls formed by this enzyme revealed a complex dependence of the regiospecificity and the yield of dioxygenation on the substitution patterns of both the oxidized and the nonoxidized rings. No dioxygenolytic attack involving chlorinated meta or para carbons was observed. Therefore, the ability of the enzyme to hydroxylate chlorinated carbons appears to be limited to the ortho position. However, it is not limited to monochlorinated rings, as evidenced by dioxygenation of the 2,4-disubstituted ring at carbons 2 and 3. This site of attack is strikingly different from that of the 2,5-dichlorinated ring, which has been shown to be dihydroxylated at positions 3 and 4 (J. D. Haddock, J. R. Horton, and D. T. Gibson, J. Bacteriol. 177:20–26, 1995). These results demonstrate that a second substituent of ortho -chlorinated rings crucially influences the site of dioxygenation at this ring and thereby determines whether or not the initial chlorobiphenyl oxidation product is further metabolized through the bph -encoded pathway. The 2,4-dichlorinated ring can alternatively be attacked at carbons 5 and 6. The preferred site crucially depends on the substitution pattern of the other ring. The formation of more than a single dioxygenation product was found predominantly with congeners that contain two chlorinated rings, both of which are similarly prone to dioxygenation or one is substituted only at carbon 3.

cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of polychlorinated biphenyls (PCBs). The crystal structure of the NAD+-enzyme complex was determined by molecular replacement and refined to an R-value of 17.9% at 2.0 A. As a member of the short-chain alcohol dehydrogenase/reductase (SDR) family, the overall protein fold and positioning of the catalytic triad in BphB are very similar to those observed in other SDR enzymes, although small differences occur in the cofactor binding site. Modeling studies indicate that the substrate is bound in a deep hydrophobic cleft close to the nicotinamide moiety of the NAD+ cofactor. These studies further suggest that Asn143 is a key determinant of substrate specificity. A two-step reaction mechanism is proposed for cis-dihydrodiol dehydrogenases.

Optical coherence tomography (OCT) has revolutionises the diagnosis of retinal disease based on the detection of microscopic rather than subcellular changes in retinal anatomy. However, currently the technique is limited to the detection of microscopic rather than subcellular changes in retinal anatomy. However, coherence based imaging is extremely sensitive to both changes in optical contrast and cellular events at the micrometer scale, and can generate subtle changes in the spectral content of the OCT image. Here we test the hypothesis that OCT image speckle (image texture) contains information regarding otherwise unresolvable features such as organelle changes arising in the early stages of neuronal degeneration. Using ultrahigh resolution (UHR) OCT imaging at 800 nm (spectral width 140 nm) we developed a robust method of OCT image analyses, based on spatial wavelet and texture-based parameterisation of the image speckle pattern. For the first time we show that this approach allows the non-invasive detection and quantification of early apoptotic changes in neurons within 30 min of neuronal trauma sufficient to result in apoptosis. We show a positive correlation between immunofluorescent labelling of mitochondria (a potential source of changes in cellular optical contrast) with changes in the texture of the OCT images of cultured neurons. Moreover, similar changes in optical contrast were also seen in the retinal ganglion cell- inner plexiform layer in retinal explants following optic nerve transection. The optical clarity of the explants was maintained throughout in the absence of histologically detectable change. Our data suggest that UHR OCT can be used for the non-invasive quantitative assessment of neuronal health, with a particular application to the assessment of early retinal disease.

Polychlorinated biphenyls (PCBs) are one of the most widely distributed chlorinated pollutants in the environment. Bioremedation seems to be a promising approach for the clean-up of PCB-contaminated soils and aquatic environments. Both anaerobic and aerobic bacteria have the potential to attack PCBs. However, degradation is inefficient and/or incomplete. To develop rational strategies for improvement of PCB-degrading pathways it is necessary to acquire detailed information on the genetics, the enzymes involved and the metabolites formed. We set out to tackle this problem by isolation and characterization of the locus bph involved in PCB degradation by Pseudomomas sp. LB400, a strain particularly noteworthy for its ability to oxidize a broad spectrum of PCBs. This locus encodes seven polypeptides responsible for the four catabolic steps of the ‘upper’ pathway which transforms biphenyl to benzoate and 2-hydroxypenta-2,4-dienoate, three enzymes required for conversion of 2-hydroxypenta-2,4-dienoate to Krebs cycle intermediates, and a glutathione S-tranferase. Based on genetic analysis, recombinant E. coli strains encoding subsets of bph genes were constructed for a detailed analysis of pathway metabolites. Biochemical analysis showed that with many ortho-chlorinated congeners attack by biphenyl-2,3 dioxygenase directly led to dihydroxybiphenyls by dehalogenation. The ‘upper’ pathway converted the majority of mono- and dichlorinated biphenyls into chlorobenzoates. However, more highly chlorinated PCBs were incompletely degraded in most instances. Identification of dead-end metabolites indicated that, depending on the congener, different reactions of this metabolic route limit the degradation of PCBs.

Total extracted DNA from two heavily polychlorobiphenyl-contaminated soils was analyzed with respect to biphenyl dioxygenase sequences and activities. This was done by PCR amplification and cloning of a DNA segment encoding the active site of the enzyme. The translated sequences obtained fell into three similarity clusters (I to III). Sequence identities were high within but moderate or low between the clusters. Members of clusters I and II showed high sequence similarities with well-known biphenyl dioxygenases. Cluster III showed low (43%) sequence identity with a biphenyl dioxygenase from Rhodococcus jostii RHA1. Amplicons from the three clusters were used to reconstitute and express complete biphenyl dioxygenase operons. In most cases, the resulting hybrid dioxygenases were detected in cell extracts of the recombinant hosts. At least 83% of these enzymes were catalytically active. Several amino acid exchanges were identified that critically affected activity. Chlorobiphenyl turnover by the enzymes containing the prototype sequences of clusters I and II was characterized with 10 congeners that were major, minor, or not constituents of the contaminated soils. No direct correlations were observed between on-site concentrations and rates of productive dioxygenations of these chlorobiphenyls. The prototype enzymes displayed markedly different substrate and product ranges. The cluster II dioxygenase possessed a broader substrate spectrum toward the assayed congeners, whereas the cluster I enzyme was superior in the attack of ortho -chlorinated aromatic rings. These results demonstrate the feasibility of the applied approach to functionally characterize dioxygenase activities of soil metagenomes via amplification of incomplete genes.

Detection of acoustic waves is the cornerstone of photoacoustic tomography (PAT). Detection has conventionally been performed mechanically using ultrasonic transducers, or optically by interferometric techniques. We propose an interferometric detection scheme using low coherence interferometry (LCI) and discuss the challenges, advantages and limitations of applying this technique to photoacoustics.

Standard FD-OCT systems suffer from a limited useful depth range due to the inherent complex conjugate artifacts and continuous fall-off in sensitivity with distance from the zero delay. The techniques of dispersion encoded full range (DEFR) frequency-domain optical coherence tomography (FD-OCT) and its enhanced version fast DEFR use the dispersion mismatch between sample and reference arm to double the imaging depth range by iteratively suppressing complex conjugate artifacts. Previously the computational complexity of DEFR prevented its application to fields where real-time visualization or large volumetric datasets are needed. A graphics processing unit (GPU) with hundreds of processing cores provides highly parallel computation capability to FD-OCT in which processing for each A-line is identical and independent. In this paper, we adopted GPUs to accelerate DEFR, thereby significantly improving reconstruction speed by a factor of >90 in respect to CPU based processing. A maximum display line rate of ~21 k-lines/ s for 2048 points/A-line using 10 iterations of the fast DEFR algorithm has been successively achieved, thereby enabling the application of DEFR in fields where real time visualization is required. By comparison in the conjugate artifact suppressed cross-sectional image of a mouse eye, there is no significant qualitative difference between the corresponding CPU- and GPU-processed images.

Two biphenyl dioxygenases (BphAs) were shown to catalyze dioxygenation of biphenyldienediol in the nonoxidized ring to form the respective symmetrical biphenyl-bis-dienediol. This novel metabolite served as a growth substrate for both BphA source strains. Its catabolism through the upper bph pathway of Burkholderia xenovorans LB400 was analyzed.

To map choroidal (ChT) and retinal thickness (RT) in healthy subjects and patients with diabetes with and without maculopathy using three dimensional 1060-nm optical coherence tomography (3D-1060nm-OCT).Sixty-three eyes from 42 diabetic subjects (41-82 years of age; 11 females) grouped according to a custom scheme using Early Treatment Diabetic Retinopathy Study definitions for pathology within 1 disc-diameter of fovea (without pathology [NDR], microaneurysms [M1], exudates [M2], clinically significant macular edema [CSME]) and 16 eyes from 16 healthy age matched subjects (38-79 years of age; 11 females) were imaged by 3D-1060nm-OCT performed over a 36° × 36° field of view. Axial length, 45° fundus photographs, body mass index, plasma glucose, and blood pressure measurements were recorded. The ChT at the subfoveal location and ChT maps between RPE and the choroidal-scleral interface were generated and statistically analyzed.RT maps show thinning in the NDR group but an increase in thickness with increasing maculopathy in the temporal and central regions (unpaired t-test; P0.05). ChT mapping of all diabetic patients revealed central and inferior thinning compared to healthy eyes (unpaired t-test; P0.001). Subfoveal ChT (mean ± SD) for healthy eyes was 327 ± 74 μm, which was significantly thicker than all diabetic groups (214 ± 55 μm for NDR, 208 ± 49 μm for M1, 205 ± 54 μm for M2, and 211 ± 76 μm for CSME (ANOVA P0.001; Tukey P0.001).3D-1060nm-OCT has shown that the central choroid is thinner in all type 2 diabetic eyes regardless of disease stage. The choroidal thinning may exceed the magnitude of possible choriocapillaris atrophy. In contrast to the conventional assessment of pathologic thickness change in several locations, thickness maps allow investigation of the choroid over the extent of affected areas.

A noninvasive, multimodal photoacoustic and optical coherence tomography (PAT/OCT) scanner for three-dimensional in vivo (3D) skin imaging is described. The system employs an integrated, all optical detection scheme for both modalities in backward mode utilizing a shared 2D optical scanner with a field-of-view of ~13 × 13 mm(2). The photoacoustic waves were detected using a Fabry Perot polymer film ultrasound sensor placed on the surface of the skin. The sensor is transparent in the spectral range 590-1200 nm. This permits the photoacoustic excitation beam (670-680 nm) and the OCT probe beam (1050 nm) to be transmitted through the sensor head and into the underlying tissue thus providing a backward mode imaging configuration. The respective OCT and PAT axial resolutions were 8 and 20 µm and the lateral resolutions were 18 and 50-100 µm. The system provides greater penetration depth than previous combined PA/OCT devices due to the longer wavelength of the OCT beam (1050 nm rather than 829-870 nm) and by operating in the tomographic rather than the optical resolution mode of photoacoustic imaging. Three-dimensional in vivo images of the vasculature and the surrounding tissue micro-morphology in murine and human skin were acquired. These studies demonstrated the complementary contrast and tissue information provided by each modality for high-resolution 3D imaging of vascular structures to depths of up to 5 mm. Potential applications include characterizing skin conditions such as tumors, vascular lesions, soft tissue damage such as burns and wounds, inflammatory conditions such as dermatitis and other superficial tissue abnormalities.

The dispersion mismatch between sample and reference arm in frequency-domain OCT can be used to iteratively suppress complex conjugate artifacts and thereby increase the imaging range. We propose a fast dispersion encoded full range (DEFR) algorithm that detects multiple signal components per iteration. The influence of different dispersion levels on the reconstruction quality is analyzed for in vivo retinal tomograms at 800 nm. Best results have been achieved with about 30 mm SF11, with neglectable resolution decrease due to finite resolution of the spectrometer. Our fast DEFR algorithm achieves an average suppression ratio of 55 dB and converges within 5 to 10 iterations. The processing time on non-dedicated hardware was 5 to 10 seconds for tomograms with 512 depth scans and 4096 sampling points per depth scan. Application of DEFR to the more challenging 1060 nm wavelength region is demonstrated by introducing an additional optical fibre in the sample arm.

Optical coherence tomography (OCT) is a non-invasive in vivo biomedical imaging modality capable of three-dimensional visualization of tissue morphology permitting imaging at high speed and sensitivity. Coherent Anti- Stokes Raman Scattering (CARS) is a nonlinear spectroscopic technique which provides molecular information due to a four wave mixing process. In order to extend the performance of OCT towards detecting the molecular fingerprint of biological samples a combined CARS/OCT setup has been developed that employs only a single ultrashort pulse Ti:Sapphire laser which enables high axial resolution OCT and simultaneously combined with a spectral shaper a CARS setup. During first measurements the same area of a sample was imaged twice, applying OCT and CARS consecutively. OCT was used to perform three-dimensional morphological screening. Due to CARS additional chemical information could be gained for two dimensions. The spectrum was modified computer controlled to match the requirements for the generation of a CARS signal whereas for OCT the unmodified spectrum was applied. Fluids such as dimethylsulfoxide (DMSO) and PBS were compared in a cuvette to demonstrate the functionality of the multimodal setup. As a biological sample a 100 m thickcr oss section through a human optic nerve surrounded by sclera was investigated.

A novel non-invasive in vivo multimodal optical coherence tomography (OCT)/photoacoustic tomography (PAT) imaging system capable of obtaining structural and functional information simultaneously has been demonstrated in skin. A 1060 nm OCT system acquiring 47k depth-scans/s with ~ 7 μm axial and ~ 20 μm transverse resolutions has been incorporated into a backward-mode PA system based on a planar, optically-transparent Fabry-Perot interferometer (FPI) sensor. In this study, the excitation wavelength was set to 670 nm and a focused laser beam at 1550 nm was used as the sensor interrogation beam. OCT and PAT images were obtained sequentially and the coregistered images were combined to form the final 3D image. OCT/PAT images obtained in vivo from the skin of a hairless mouse and human palmar skin demonstrated the ability of this multimodal imaging system to provide complementary structural and functional information from deeper depths with increased contrast.

Visualization of cell migration during chemotaxis using spectral domain optical coherence tomography (OCT) requires non-standard processing techniques. Stripe artefacts and camera noise floor present in OCT data prevent detailed computer-assisted reconstruction and quantification of cell locomotion. Furthermore, imaging artefacts lead to unreliable results in automated texture based cell analysis. Here we characterize three pronounced artefacts that become visible when imaging sample structures with high dynamic range, e.g. cultured cells: (i) time-varying fixed-pattern noise; (ii) stripe artefacts generated by background estimation using tomogram averaging; (iii) image modulations due to spectral shaping. We evaluate techniques to minimize the above mentioned artefacts using an 800 nm optical coherence microscope. Effect of artefact reduction is shown exemplarily on two cell cultures, i.e. Dictyostelium on nitrocellulose substrate, and retinal ganglion cells (RGC-5) cultured on a glass coverslip. Retinal imaging also profits from the proposed processing techniques. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Purpose. To evaluate the performance and potential clinical role of three-dimensional (3D) 1060-nm OCT by generating choroidal thickness (ChT) maps in patients of different ages with different degrees of ametropia and axial lengths and to investigate the effect of cataract grade on OCT retinal imaging quality. \ud \ud Methods. Axial lengths (ALs) and 45° fundus photographs were acquired from 64 eyes (34 healthy subjects, 19 to 80 years, ametropia +3 to −10 D). 3D 1060-nm OCT was performed over a 36° × 36° field of view with ∼7-μm axial resolution and up to 70 frames/s (512 A-scans/frame). ChT maps between retinal pigment epithelium and the choroidal–scleral interface, were generated and statistically analyzed. A further 30 eyes (19 subjects), with cataracts assessed with the LOCS III scale, were imaged with 3D 1060-nm OCT and 800-nm OCT, and visualization of the posterior segment was compared qualitatively. \ud \ud Results. In 64 eyes, ChT maps displayed a thickness decrease with increasing AL. Subfoveal ChT was 315 ± 106 μm (mean ± SD), negatively correlated with AL (R2 = −0.47, P < 0.001). Averaged ChT maps of eyes with AL < 23.39 mm showed an increased ChT in an area ∼1500 μm inferior, compared with subfoveal ChT. Eyes with AL > 24.5 mm showed a larger variation and a thicker ChT superiorly than inferiorly. Reduced signal strength in cataractous eyes was found in 65% of the 800-nm OCT images, but in only 10% of the 1060-nm OCT images. \ud \ud Conclusions. The imaging performance of 3D 1060-nm OCT is unique, producing maps that show the variation in ChT over the entire field of view, in relation to axial length. This imaging system has the potential of visualizing a novel clinical diagnostic biomarker. Compared with 800-nm OCT, it provides superior visualization of the posterior pole in cataractous eyes.

We report a high-speed, dispersion-encoded, full-range (DEFR) swept-source optical coherence tomography system for in vivo ocular imaging and biometry of small animals. The fast DEFR algorithm removes the depth ambiguity, gives access to objects located at the zero delay position, and doubles the sampling depth to 2x5.0 mm (at -101 to -71 dB sensitivity) in a single scan using 2048 samples/depth scan 0.43 nm line width of a light source operating at 1056 nm with 70 nm tuning range. The acquisition speed (frames of 512 depth scans in 18.3 ms) permits precise on-line monitoring during positioning and provides cross-sectional views of the mouse eye. Preliminary studies demonstrate high-throughput, reproducible assessment of multiple biometric features (e.g., day-to-day reproducibility of axial length measurement +/-5.3 microm) that is insensitive to eye motion sufficient for long-term monitoring.

Two high-level heterologous expression systems for amylosucrase genes have been constructed. One depends on sigma-70 bacterial RNA polymerase, the other on phage T7 RNA polymerase. Translational fusions were formed between slightly truncated versions of the gene from Neisseria polysaccharea and sequences of expression vectors pQE-81L or pET33b(+), respectively. These constructs were introduced into different Escherichia coli strains. The resulting recombinants yielded up to 170 mg of dissolved enzyme per litre of culture at a moderate cell density of five OD(600). To our knowledge, this is the highest yield per cell described so far for amylosucrases. The recombinant enzymes could rapidly be purified through the use of histidine tags in the N-terminally attached sequences. These segments did not alter catalytic properties and therefore need not be removed for most applications. Investigations with glucose and malto-oligosaccharides of different lengths identified rate-limiting steps in the elongation (acceptor reaction) and truncation (donor reaction) of these substrates. The elongation of maltotriose and its reversal, the truncation of maltotetraose, were found to be particularly slow reactions. Potential reasons are discussed, based on the crystal structure of the enzyme. It is furthermore shown that amylosucrase is able to synthesise mixed disaccharides. All of the glucose epimers mannose, allose, and galactose served as acceptors, yielding between one and three main products. We also demonstrate that, as an alternative to the use of purified amylosucrase, cells of the constructed recombinant strains can be used to carry out glucosylations of acceptors.

A novel combined photoacoustic tomography (PAT), optical resolution photoacoustic microscopy (ORPAM) and optical coherence tomography (OCT) instrument has been developed for imaging biological tissues. The system is based on the use of a Fabry-Perot (FP) polymer film ultrasound sensor. This is designed to be transparent to wavelengths between 590nm and 1200nm so that photoacoustic excitation laser pulses in this spectral range can be transmitted through the sensor into the underlying tissue to allow backward mode operation. The dual PAT-ORPAM capability of the system was demonstrated by imaging a tissue phantom composed of 7μm diameter carbon fibres immersed in an optically scattering liquid. The lateral and vertical spatial resolutions in ORPAM mode are approximately 7μm and 10μm respectively for sub-mm depths. In PAT mode, the lateral spatial resolution is less than 50μm for depths up to 5mm and the vertical resolution is approximately 10μm. The transparent nature of the FP polymer film ultrasound sensor offers a convenient platform for combining other optical imaging modalities with PAT and ORPAM. To illustrate this, a frequency-domain OCT system operating at 1060nm was integrated into the system and combined PAT/OCT images of the skin of a mouse were obtained in vivo.

Weakly scattering tree shrew retina has been imaged in vitro with full field swept source optical coherence tomography, visualising multiple intraretinal layers. The system utilises a 50nm bandwidth Superlum SLD, to acheive ~8μm of axial resolution and 4μm of transversal resolution. Volumetric images of retinal tissue with dimensions of 1248x936x678μm (horizontal by vertical by axial) were recorded in two second (equivalent of 153,600 A-scans per second) with a measured signal to noise ratio of 75dB. From the 5mW of SLD optical power available, 720μW illuminates the sample, giving a power per pixel of 4.6nW, ten times less power per pixel then standard FDOCT systems. After upgrading the camera and redesigning the optical beam path, 82dB of SNR was realised.

Conventionally, cell chemotaxis is studied on two-dimensional (2D) transparent surfaces due to limitations in optical and image data-collection techniques. However, substrates which more closely mimic the natural environment of cells are often opaque or three-dimensional (3D). The non-invasive label-free imaging technique of frequency domain optical coherence tomography (OCT) has high axial and transverse resolution of >4μm, comparatively high penetration depth and the ability to acquire volumes in a few seconds, therefore offering the potential to visualize moving cells in 3D (2D+time) and 4D (3D+time). Cell migration is demonstrated in 3D on opaque surfaces, and in 4D within an agarose gel. The speed and directionality of wild type (Ax2) cell movement is seen to be comparable on agar and nitrocellulose filter substrates. Differences can be clearly seen in the character of cell movement between Ax2, myosin knockout (mhcA-) and PLC null ~10μm Dictyostelium discoideum cells in 4D using ultrahigh resolution OCT. OCT is therefore shown to be a useful technique for the study of cell migration.

In this study, depth resolved measurements of absorption profiles in the wavelength range of 800 nm with a bandwidth of 140 nm are demonstrated using high speed spectroscopic frequency domain OCT (SOCT). With proper calibration, SOCT is able to extract absolute, depth resolved absorption profiles over the whole wavelength range at once without the need of tuning and performing measurements at single wavelengths sequentially. In addition, high acquisition speed in excess of 20 kHz allows to measure absorption dynamics with 50 µs time resolution, which might be useful for the investigation of pharmacokinetics or pharmacodynamics. SOCT of ∼600 µm thick single- and multilayered, weakly scattering phantoms with varying absorption in the range of 10–60 cm-1, equivalent to blood absorption in capillaries, is presented. SOCT measurements are compared with those using a spectrometer in transmission mode. For Indocyanine Green (ICG), a dynamic absorption measurement is demonstrated.

Conventionally, cell chemotaxis is studied on two-dimensional (2D) transparent surfaces, due to limitations in optical and image data-collection techniques. However, surfaces that more closely mimic the natural environment of cells are often opaque. Optical coherence tomography (OCT) is a noninvasive label-free imaging technique, which offers the potential to visualize moving cells on opaque surfaces and in three dimensions (3D). Here, we demonstrate that OCT is an effective means of time-lapse videomicroscopy of Dictyostelium cells undergoing 3D (2D+time) cell migration on nitrocellulose substrates and 4D (3D+time) chemotaxis within low-density agarose gels. The generated image sequences are compatible with current computer-based image-analysis software for quantification of cell motility. This demonstrates the utility of OCT for cell tracking and analysis of cell chemotaxis in complex environments. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

We propose an algorithm that effectively cancels complex conjugate mirror terms from single OCT A-scans by utilizing the dispersion mismatch between reference and sample arm to generate full range tomograms. This allows distinguishing between complex conjugate mirror terms and real structures and is therefore called dispersion encoded full range (DEFR). Whereas the computational complexity is higher, acquisition speed is not compromised since no additional A-scans need to be measured which makes this technique also robust against phase fluctuations. The iterative algorithm uses numeric dispersion compensation and exhibits no reduction in resolution compared to standard processing. Residual leakage of mirror terms is reduced by incorporating further knowledge such as the power spectrum of the light source. The suppression ratio of mirror signals is more than 50 dB and thus comparable to other complex FD-OCT techniques which use more than one A-scan.