Substantial increase in detection efficiency for filter array-based spectral sensors.

A method that significantly increases the detection efficiency of filter array-based spectral sensors is proposed. The basic concept involves a wavelength-dependent redistribution of incident light before it reaches the filter elements located in front of the detector. Due to this redistribution, each filter element of the array receives a spatially concentrated amount of a pre-selected and adjusted spectral partition of the entire incident light. This approach can be employed to significantly reduce the reflection and absorption losses of each filter element. The proof-of-concept is demonstrated by a setup that combines a series of consecutively arranged dichroic filters with Fabry-Perot filter arrays. Experimentally, an efficiency increase by a factor larger than 4 compared to a reference system is demonstrated. The optical system is a non-imaging spectrometer, which combines the efficiency enhancement module with the filter arrays, is compact (17.5mm×17.5mm×7.8mm), and integrated completely inside the CCD camera mount.

An approach to "escape from flatland": chemo-enzymatic synthesis and biological profiling of a library of bridged bicyclic compounds.

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.

Recent developments in the enzymatic O-glycosylation of flavonoids.

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.

An aryl dioxygenase shows remarkable double dioxygenation capacity for diverse bis-aryl compounds, provided they are carbocyclic.

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.

Permissivity of the biphenyl-specific aerobic bacterial metabolic pathway towards analogues with various steric requirements.

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.

Flavonoid glucosylation by non-Leloir glycosyltransferases: formation of multiple derivatives of 3,5,7,3',4'-pentahydroxyflavane stereoisomers.

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.

Characterization of biphenyl dioxygenase sequences and activities encoded by the metagenomes of highly polychlorobiphenyl-contaminated soils.

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.

Dioxygenation of the biphenyl dioxygenation product.

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.

Heritability of ocular component dimensions in mice phenotyped using depth-enhanced swept source optical coherence tomography.

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.

In vivo, in situ imaging of microneedle insertion into the skin of human volunteers using optical coherence tomography.

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 <3 µm effective axial resolution was used to visualise the microneedle-treated area during insertion and/or following removal of the device, without any tissue processing. RESULTS: Transverse images of a microneedle device, in situ, were captured by the OCT system and suggest that the stratified skin tissue is compressed during microneedle application. Following removal of the device, the created microchannels collapse within the in vivo environment and, therefore, for all studied devices, microconduit dimensions are markedly smaller than the microneedle dimensions. CONCLUSIONS: Microchannels created in the upper skin layers by microneedles are less invasive than previous histology predicts. OCT has the potential to play a highly influential role in the future development of microneedle devices and other transdermal delivery systems.

High level expression of a recombinant amylosucrase gene and selected properties of the enzyme.

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.

Robust segmentation of intraretinal layers in the normal human fovea using a novel statistical model based on texture and shape analysis.

A novel statistical model based on texture and shape for fully automatic intraretinal layer segmentation of normal retinal tomograms obtained by a commercial 800nm optical coherence tomography (OCT) system is developed. While existing algorithms often fail dramatically due to strong speckle noise, non-optimal imaging conditions, shadows and other artefacts, the novel algorithm's accuracy only slowly deteriorates when progressively increasing segmentation task difficulty. Evaluation against a large set of manual segmentations shows unprecedented robustness, even in the presence of additional strong speckle noise, with dynamic range tested down to 12dB, enabling segmentation of almost all intraretinal layers in cases previously inaccessible to the existing algorithms. For the first time, an error measure is computed from a large, representative manually segmented data set (466 B-scans from 17 eyes, segmented twice by different operators) and compared to the automatic segmentation with a difference of only 2.6% against the inter-observer variability.

Fast dispersion encoded full range optical coherence tomography for retinal imaging at 800 nm and 1060 nm.

The dispersion mismatch between sample and reference arm in frequency-domain optical coherence tomography (OCT) can be used to iteratively suppress complex conjugate artifacts and thereby increase the imaging range. In this paper, 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 experimentally using a multilayered scattering phantom and in vivo retinal tomograms at 800 nm. Best results have been achieved with 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 typically 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 also demonstrated by introducing an additional optical fibre in the sample arm.

Topsoil removal reduced in-situ methane emissions in a temperate rewetted bog grassland by a hundredfold.

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.

Generation of amylosucrase variants that terminate catalysis of acceptor elongation at the di- or trisaccharide stage.

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.

Dispersion encoded full range frequency domain optical coherence tomography.

We propose an iterative algorithm that exploits the dispersion mismatch between reference and sample arm in frequency-domain optical coherence tomography (FD-OCT) to effectively cancel complex conjugate mirror terms in individual A-scans and thereby generate full range tomograms. The resulting scheme, termed dispersion encoded full range (DEFR) OCT, allows distinguishing real structures from complex conjugate mirror artifacts. Even though DEFR-OCT has higher post-processing complexity than conventional FD-OCT, acquisition speed is not compromised since no additional A-scans need to be measured, thereby rendering this technique robust against phase fluctuations. The algorithm uses numerical dispersion compensation and exhibits similar resolution as standard processing. The residual leakage of mirror terms is further reduced by incorporating additional 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 complex FD-OCT techniques which use multiple A-scans.

Adaptive optics optical coherence tomography at 120,000 depth scans/s for non-invasive cellular phenotyping of the living human retina.

This paper presents a successful combination of ultra-high speed (120,000 depth scans/s), ultra-high resolution optical coherence tomography with adaptive optics and an achromatizing lens for compensation of monochromatic and longitudinal chromatic ocular aberrations, respectively, allowing for non-invasive volumetric imaging in normal and pathologic human retinas at cellular resolution. The capability of this imaging system is demonstrated here through preliminary studies by probing cellular intraretinal structures that have not been accessible so far with in vivo, non-invasive, label-free imaging techniques, including pigment epithelial cells, micro-vasculature of the choriocapillaris, single nerve fibre bundles and collagenous plates of the lamina cribrosa in the optic nerve head. In addition, the volumetric extent of cone loss in two colour-blinds could be quantified for the first time. This novel technique provides opportunities to enhance the understanding of retinal pathogenesis and early diagnosis of retinal diseases.

Impact of enhanced resolution, speed and penetration on three-dimensional retinal optical coherence tomography.

: Recent substantial developments in light source and detector technology have initiated a paradigm shift in retinal optical coherence tomography (OCT) performance. Broad bandwidth light sources in the 800 nm and 1060 nm wavelength region enable axial OCT resolutions of 2-3 mum and 5-7 mum, respectively. Novel high speed silicon based CMOS cameras at 800 nm and InGaAs based CCD cameras in combination with frequency domain OCT technology enable data acquisition speeds of up to 47,000 A-scans/s at 1060 nm and up to 312,500 A-scans/s at 800 nm. Combining ultrahigh axial resolution, ultrahigh speed OCT at 800 nm with pancorrected adaptive optics allows volumetric in vivo cellular resolution retinal imaging. Commercially available three-dimensional (3D) retinal OCT at 800 nm (20,000 A-scans/s, 6 mum axial resolution) is compared to ultrahigh speed 3D retinal imaging at 800 nm (160,000 A-scans/s, 2-3 mum axial resolution), high speed 3D choroidal imaging at 1060 nm (47,000 Ascan/ second, 6-7 mum axial resolution) and cellular resolution retinal imaging at 800 nm using adaptive optics OCT at 160,000 A-scans/second with isotropic resolution of ~2 mum. Analysis of the performance of these four imaging modalities applied in normal and pathologic eyes focusing on motion artifact free volumetric retinal imaging and revealing novel, complementary morphological information due to enhanced resolution, speed and penetration is presented.

Engineering the glucansucrase GTFR enzyme reaction and glycosidic bond specificity: toward tailor-made polymer and oligosaccharide products.

Two long-standing questions about glucansucrases (EC 2.4.1.5) are how they control oligosaccharide versus polysaccharide synthesis and how they direct their glycosidic linkage specificity. This information is required for the production of tailor-made saccharides. Mutagenesis promises to be an effective tool for enzyme engineering approaches for altering the regioselectivity and acceptor substrate specificity. Therefore, we chose the most conserved motif around the transition state stabilizer in glucansucrases for a random mutagenesis of the glucansucrase GTFR of Streptococcus oralis, yielding different variants with altered reaction specificity. Modifications at position S628 achieved by saturation mutagenesis guided the reaction toward the synthesis of short chain oligosaccharides with a drastically increased yield of isomaltose (47%) or leucrose (64%). Alternatively, GTFR variant R624G/V630I/D717A exhibited a drastic switch in regioselectivity from a dextran type with mainly alpha-1,6-glucosidic linkages to a mutan type polymer with predominantly alpha-1,3-glucosidic linkages. Targeted modifications demonstrated that both mutations near the transition state stabilizer, R624G and V630I, are contributing to this alteration. It is thus shown that mutagenesis can guide the transglycosylation reaction of glucansucrase enzymes toward the synthesis of (a) various short chain oligosaccharides or (b) novel polymers with completely altered linkages, without compromising their high transglycosylation activity and efficiency.

Re- or displacement of invariant residues in the C-terminal half of the catalytic domain strongly affects catalysis by glucosyltransferase R.

It is shown that exchanges of single invariant amino acids in two C-terminal catalytic domain segments of the glucosyltransferase R (GtfR) strongly affect its catalytic properties. Drastic decreases of activity through re- or displacements of Tyr965 demonstrate a crucial role of this residue. Similarly, exchanges of amino acids Asp1004, Val1006, and Tyr1011 profoundly influenced catalytic parameters. These results are interpreted on the basis of a homology model of the catalytic domain. They are consistent with the view that Tyr965 is a constituent of the substrate-binding pocket and directly contacts the sucrose molecule, whereas the other critical residues contribute to the required positioning of Tyr965 and other active site residues.