Structural and functional insights into the unique CBS-CP12 fusion protein family in cyanobacteria.

Cyanobacteria are important photosynthetic organisms inhabiting a range of dynamic environments. This phylum is distinctive among photosynthetic organisms in containing genes encoding uncharacterized cystathionine ß-synthase (CBS)-chloroplast protein (CP12) fusion proteins. These consist of two domains, each recognized as stand-alone photosynthetic regulators with different functions described in cyanobacteria (CP12) and plants (CP12 and CBSX). Here we show that CBS-CP12 fusion proteins are encoded in distinct gene neighborhoods, several unrelated to photosynthesis. Most frequently, CBS-CP12 genes are in a gene cluster with thioredoxin A (TrxA), which is prevalent in bloom-forming, marine symbiotic, and benthic mat cyanobacteria. Focusing on a CBS-CP12 from Microcystis aeruginosa PCC 7806 encoded in a gene cluster with TrxA, we reveal that the domain fusion led to the formation of a hexameric protein. We show that the CP12 domain is essential for hexamerization and contains an ordered, previously structurally uncharacterized N-terminal region. We provide evidence that CBS-CP12, while combining properties of both regulatory domains, behaves different from CP12 and plant CBSX. It does not form a ternary complex with phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase. Instead, CBS-CP12 decreases the activity of PRK in an AMP-dependent manner. We propose that the novel domain architecture and oligomeric state of CBS-CP12 expand its regulatory function beyond those of CP12 in cyanobacteria.

Non-canonical localization of RubisCO under high-light conditions in the toxic cyanobacterium Microcystis aeruginosa PCC7806.

The frequent production of the hepatotoxin microcystin (MC) and its impact on the lifestyle of bloom-forming cyanobacteria are poorly understood. Here, we report that MC interferes with the assembly and the subcellular localization of RubisCO, in Microcystis aeruginosa PCC7806. Immunofluorescence, electron microscopic and cellular fractionation studies revealed a pronounced heterogeneity in the subcellular localization of RubisCO. At high cell density, RubisCO particles are largely separate from carboxysomes in M. aeruginosa and relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of MC to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO2 fixation product 3-phosphoglycerate are significantly higher in the MC-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction of M. aeruginosa with its heterotrophic microbial community.

Metabolomic analysis indicates a pivotal role of the hepatotoxin microcystin in high light adaptation of Microcystis.

Microcystis is a freshwater cyanobacterium frequently forming nuisance blooms in the summer months. The genus belongs to the predominant producers of the potent hepatotoxin microcystin. The success of Microcystis and its remarkable resistance to high light conditions are not well understood. Here, we have compared the metabolic response of Microcystis aeruginosa PCC7806, its microcystin-deficient ΔmcyB mutant (Mut) and the cyanobacterial model organism Synechocystis PCC6803 to high light exposure of 250 µmol photons m(-2) s(-1) using GC/MS-based metabolomics. Microcystis wild type and Mut show pronounced differences in their metabolic reprogramming upon high light. Seventeen per cent of the detected metabolites showed significant differences between the two genotypes after high light exposure. Whereas the microcystin-producing wild type shows a faster accumulation of glycolate upon high light illumination, loss of microcystin leads to an accumulation of general stress markers such as trehalose and sucrose. The study further uncovers differences in the high light adaptation of the bloom-forming cyanobacterium Microcystis and the model cyanobacterium Synechocystis. Most notably, Microcystis invests more into carbon reserves such as glycogen after high light exposure. Our data shed new light on the lifestyle of bloom-forming cyanobacteria, the role of the widespread toxin microcystin and the metabolic diversity of cyanobacteria.

Microcystin production revisited: conjugate formation makes a major contribution.

The impact of environmental stimuli on the production of the widespread cyanobacterial hepatotoxin microcystin (MC) is under debate. Whereas transcriptional studies of the biosynthetic genes suggest a clear influence of light conditions on toxin production the data for the metabolite itself are inconsistent and highly strain-specific. Here, we have reassessed the MC content by using two immunological detection techniques that allow a parallel quantification of MC in the methanolic extracts and the residual pellet fraction that contains high molecular weight proteins. Our results show a significant proportion of MC in the protein bound fraction in strains of Microcystis and Planktothrix and of the related toxin nodularin (NOD) in Nodularia. Moreover, we could show a very strong increase of MC after high light illumination in the protein fraction contributing to a significant overall increase in MC production under these conditions that is not seen in extracts analysed by LC-MS and ELISA. The fact that a considerable portion of MC is neglected with current analysis techniques was also confirmed for selected field samples. Immunofluorescence studies suggest strain-specific differences in the amount of MC conjugate formation.

Total liquid ventilation: a new approach to improve 3D OCT image quality of alveolar structures in lung tissue.

Little is known about mechanical processes of alveolar tissue during mechanical ventilation. Optical coherence tomography (OCT) as a three-dimensional and high-resolution imaging modality can be used to visualize subpleural alveoli during artificial ventilation. The quality of OCT images can be increased by matching the refractive index inside the alveoli to the one of tissue via liquid-filling. Thereby, scattering loss can be decreased and higher penetration depth and tissue contrast can be achieved. We show the liquid-filling of alveolar structures verified by optical coherence tomography and intravital microscopy (IVM) and the advantages of index matching for OCT imaging of subpleural alveoli in a mouse model using a custom-made liquid ventilator.

Optical coherence tomography in biomedical research.

Optical coherence tomography (OCT) is a noninvasive, high-resolution, interferometric imaging modality using near-infrared light to acquire cross-sections and three-dimensional images of the subsurface microstructure of biological specimens. Because of rapid improvement of the acquisition speed and axial resolution of OCT over recent years, OCT is becoming increasingly attractive for applications in biomedical research. Therefore, OCT is no longer used solely for structural investigations of biological samples but also for functional examination, making it potentially useful in bioanalytical science. The combination of in vivo structural and functional findings makes it possible to obtain thorough knowledge on basic physiological and pathological processes. Advanced applications, for example, optical biopsy in visceral cavities, have been enabled by combining OCT with established imaging modalities. This report gives an outline of the state of the art and novel trends of innovative OCT approaches in biomedical research in which the main focus is on applications in fundamental research and pre-clinical utilization.

Alveolar dynamics in acute lung injury: heterogeneous distension rather than cyclic opening and collapse.

OBJECTIVES: : To analyze alveolar dynamics in healthy and acid-injured lungs of ventilated mice. Protective ventilation is potentially lifesaving in patients with acute lung injury. However, optimization of ventilation strategies is hampered by an incomplete understanding of the effects of mechanical ventilation at the alveolar level. DESIGN: : In anesthetized and ventilated Balb/c mice, subpleural alveoli were visualized by darkfield intravital microscopy and optical coherence tomography. SETTING: : Animal research laboratory. SUBJECTS: : Male Balb/c mice. INTERVENTIONS: : Lung injury was induced by intratracheal instillation of hydrochloric acid. In control animals and mice with lung injury, ventilation pressures were varied between 0 and 24 cm H2O at baseline, 60 mins, and 120 mins, and alveolar distension and cyclic opening and collapse of alveolar clusters were analyzed. MEASUREMENTS AND MAIN RESULTS: : In normal lungs, alveolar clusters distend with increasing ventilation pressure in a sigmoid relationship. Although an increase in ventilation pressure from 0 to 24 cm H2O increases alveolar size by 41.5 +/- 2.3% in normal lungs, alveolar distension is reduced to 20.6 +/- 2.2% 120 mins after induction of lung injury by acid aspiration. Cyclic opening and collapse of alveolar clusters are neither observed in normal nor acid-injured lungs. Alveolar compliance is highest in small and distensible alveolar clusters, which are also most prone to acid-induced injury. CONCLUSIONS: : Over the applied pressure range, volume changes in control and acid-injured mouse lungs result predominantly from alveolar distension rather than cyclic opening and collapse of alveolar clusters. Preferential loss of compliance in small alveolar clusters redistributes tidal volume to larger alveoli, which increases spatial heterogeneity in alveolar inflation and may promote alveolar overdistension.

In-vivo Fourier domain optical coherence tomography as a new tool for investigation of vasodynamics in the mouse model.

In-vivo imaging of the vascular system can provide novel insight into the dynamics of vasoconstriction and vasodilation. Fourier domain optical coherence tomography (FD-OCT) is an optical, noncontact imaging technique based on interferometry of short-coherent near-infrared light with axial resolution of less than 10 microm. In this study, we apply FD-OCT as an in-vivo imaging technique to investigate blood vessels in their anatomical context using temporally resolved image stacks. Our chosen model system is the murine saphenous artery and vein, due to their small inner vessel diameters, sensitive response to vasoactive stimuli, and advantageous anatomical position. The vascular function of male wild-type mice (C57BL/6) is determined at the ages of 6 and 20 weeks. Vasoconstriction is analyzed in response to dermal application of potassium (K(+)), and vasodilation in response to sodium nitroprusside (SNP). Vasodynamics are quantified from time series (75 sec, 4 frames per sec, 330 x 512 pixels per frame) of cross sectional images that are analyzed by semiautomated image processing software. The morphology of the saphenous artery and vein is determined by 3-D image stacks of 512 x 512 x 512 pixels. Using the FD-OCT technique, we are able to demonstrate age-dependent differences in vascular function and vasodynamics.

Comparison of two in vivo microscopy techniques to visualize alveolar mechanics.

OBJECTIVE: In conventional in vivo microscopy, a three dimensional illustration of tissue is lacking. Concerning the microscopic analysis of the pulmonary alveolar network, surgical preparation of the thorax and fixation of the lung is required to place the microscope's objective. These effects may have influence on the mechanical behaviour of alveoli. Relatively new methods exist for in vivo microscopy being less invasive and enabling an observation without fixation of the lung. The aim of this study was to compare a fibered confocal laser scanning microscopy (FCLSM) with optical coherence tomography (OCT) in a mouse and a rabbit model. Moreover, FCLSM was also used endoscopically in the rabbit model. METHODS: Smallest possible thoracic windows were excised at the lower margin of the upper right lung lobe and an interpleural catheter inserted before re-coverage with a transparent membrane foil. The OCT-scanner was positioned by a motor driven translation stage. The imaging was gated to endinspiratory plateau. For CLSM, Fluorescein 0.1% was given into the central venous streak line. The confocal probe with a diameter of 650 microm was carefully positioned at the very same lung region. Images were directly recorded real-time and the observed region qualitatively compared with FD-OCT images. Additionally, in the rabbit model, CLSM was used endoscopically under bronchoscopic sight control. In a postprocessing analysis, images taken were analyzed and compared by using an "air index" (AI). RESULTS: In the mouse model, the very same region could be re-identified with both techniques. Concerning alveolar shape and size, qualitatively comparable images could be gained. The AI was 40.5% for the OCT and 40.1% for the CLSM images. In the rabbit, even an endoscopic view on alveoli was possible. Likewise AI was 43.2% for CLSM through the thoracic window and 43.6% from endoscopically. For the OCT an AI of 44.6% was analysed in the rabbit model. CONCLUSIONS: Both FD-OCT and CLSM provide high-resolution images of alveolar structure giving depth information that is beneficial to conventional microscopy. CLSM also facilitates endoscopic view on alveoli being well comparable to images gained through a thoracic window.

In-ovo sexing of 14-day-old chicken embryos by pattern analysis in hyperspectral images (VIS/NIR spectra): A non-destructive method for layer lines with gender-specific down feather color.

Up to now there is no economically maintainable modality for chicken sexing in early embryonic stages (first 3 d) that is suitable for large-scale application in the commercial hatcheries. Hence, the culling of male day-old chicks of layer lines is still the normal procedure.In this paper we present a non-destructive optical technique for gender determination in layer lines with gender-specific down feather color. This particular chicken strain presents a sexual dimorphism in feather color, where the female day-old chicks have brown down feathers and the males have yellow down feathers.The eggs are candled with halogen lamps and a hyperspectral camera collects the transmitted light within the spectral range from 400 nm to 1,000 nm. For data analysis and classification, common methods like principal component analysis and linear discriminant analysis are used. The accuracy of gender determination was determined for 11- to 14-day-old embryos. At 14 d of incubation (7 d before hatch) the sex can be determined with an overall accuracy of approximately 97%.

In vivo imaging of murine vasodynamics analyzing different mouse strains by optical coherence tomography.

BACKGROUND AND AIMS: We tried to circumvent the limitations of standard organ chamber experiments using in vivo optical coherence tomography (OCT) to analyze the vascular function of small arteries in different mouse strains. METHODS: OCT images were acquired with a two-axis galvanometer scanner head. Time series (3 frames per second, 300 × 512 pixel per frame) of cross-sectional images were analyzed with image processing software measuring the time course of vessel lumen dynamics. Vascular function of murine saphenous artery of male C57BL/6 (wild-type) and hypercholesterolemic LDLR knockout (LDLR-/-) mice was analyzed at 6 weeks and after 14 weeks feeding a control or high-fat diet containing 21.2% butter fat and 2.1 mg/kg cholesterol. Vasoconstriction and vasodilation was analyzed by OCT in response to 80 mM K+ and 1 mM SNP. RESULTS: The OCT technique allowed determination of inner diameter, flow resistance, maximal velocity of diameter change and time to half-maximal diameter change in murine saphenous arteries of wild-type and LDLR-/- mice. LDLR-/- had impaired vasodilation and changes in vasodynamics after 14 weeks on control or high-fat diet, compared to wild-type mice. The diameter of the saphenous artery of LDLR-/- mice was reduced after vasoconstriction (38 ± 7 µm vs 12 ± 6 µm) and vasodilation (245 ± 8 µm vs 220 ± 10 µm) (P < 0.05 vs C57BL/6). CONCLUSION: OCT was used as an innovative method to image vascular function of small arteries of wild-type and hypercholesterolemic LDLR-/- mice after high-fat diet. The method offers the ability to display differences in the vasodynamics at early stages of endothelial dysfunction in vivo.

Toward a comprehensive interpretation of intravital microscopy images in studies of lung tissue dynamics.

Intravital microscopy (IVM) is a well-established imaging technique for real-time monitoring of microscale lung tissue dynamics. Although accepted as a gold standard in respiratory research, its characteristic image features are scarcely understood, especially when trying to determine the actual position of alveolar walls. To allow correct interpretation of these images with respect to the true geometry of the lung parenchyma, we analyzed IVM data of alveoli in a mouse model in comparison with simultaneously acquired optical coherence tomography images. Several IVM characteristics, such as double ring structures or disappearing alveoli in regions of liquid filling, could be identified and related to the position of alveoli relative to each other. Utilizing a ray tracing approach based on an idealized geometry of the mouse lung parenchyma, two major reflection processes could be attributed to the IVM image formation: partial reflection and total internal reflection between adjacent alveoli. Considering the origin of the reflexes, a model was developed to determine the true position of alveolar walls within IVM images. These results allow thorough understanding of IVM data and may serve as a basis for the correction of alveolar sizes for more accurate quantitative analysis within future studies of lung tissue dynamics.

Four-dimensional imaging of murine subpleural alveoli using high-speed optical coherence tomography.

The investigation of lung dynamics on alveolar scale is crucial for the understanding and treatment of lung diseases, such as acute lung injury and ventilator induced lung injury, and to promote the development of protective ventilation strategies. One approach to this is the establishment of numerical simulations of lung tissue mechanics where detailed knowledge about three-dimensional alveolar structure changes during the ventilation cycle is required. We suggest four-dimensional optical coherence tomography (OCT) imaging as a promising modality for visualizing the structural dynamics of single alveoli in subpleural lung tissue with high temporal resolution using a mouse model. A high-speed OCT setup based on Fourier domain mode locked laser technology facilitated the acquisition of alveolar structures without noticeable motion artifacts at a rate of 17 three-dimensional stacks per ventilation cycle. The four-dimensional information, acquired in one single ventilation cycle, allowed calculating the volume-pressure curve and the alveolar compliance for single alveoli.

Three-dimensional simultaneous optical coherence tomography and confocal fluorescence microscopy for investigation of lung tissue.

Although several strategies exist for a minimal-invasive treatment of patients with lung failure, the mortality rate of acute respiratory distress syndrome still reaches 30% at minimum. This striking number indicates the necessity of understanding lung dynamics on an alveolar level. To investigate the dynamical behavior on a microscale, we used three-dimensional geometrical and functional imaging to observe tissue parameters including alveolar size and length of embedded elastic fibers during ventilation. We established a combined optical coherence tomography (OCT) and confocal fluorescence microscopy system that is able to monitor the distension of alveolar tissue and elastin fibers simultaneously within three dimensions. The OCT system can laterally resolve a 4.9 µm line pair feature and has an approximately 11 µm full-width-half-maximum axial resolution in air. confocal fluorescence microscopy visualizes molecular properties of the tissue with a resolution of 0.75 µm (laterally), and 5.9 µm (axially) via fluorescence detection of the dye sulforhodamine B specifically binding to elastin. For system evaluation, we used a mouse model in situ to perform lung distension by application of different constant pressure values within the physiological regime. Our method enables the investigation of alveolar dynamics by helping to reveal basic processes emerging during artificial ventilation and breathing.

Intravital microscopy of subpleural alveoli via transthoracic endoscopy.

Transfer of too high mechanical energy from the ventilator to the lung's alveolar tissue is the main cause for ventilator-induced lung injury (VILI). To investigate the effects of cyclic energy transfer to the alveoli, we introduce a new method of transthoracic endoscopy that provides morphological as well as functional information about alveolar geometry and mechanics. We evaluate the new endoscopic method to continuously record images of focused subpleural alveoli. The method is evaluated by using finite element modeling techniques and by direct observation of subpleural alveoli both in isolated rat lungs as well as in intact animals (rats). The results confirm the overall low invasiveness of the endoscopic method insofar as the mechanical influences on the recorded alveoli are only marginal. It is, hence, a suited method for intravital microscopy in the rat model as well as in larger animals.

The cyanobacterial hepatotoxin microcystin binds to proteins and increases the fitness of microcystis under oxidative stress conditions.

Microcystins are cyanobacterial toxins that represent a serious threat to drinking water and recreational lakes worldwide. Here, we show that microcystin fulfils an important function within cells of its natural producer Microcystis. The microcystin deficient mutant ΔmcyB showed significant changes in the accumulation of proteins, including several enzymes of the Calvin cycle, phycobiliproteins and two NADPH-dependent reductases. We have discovered that microcystin binds to a number of these proteins in vivo and that the binding is strongly enhanced under high light and oxidative stress conditions. The nature of this binding was studied using extracts of a microcystin-deficient mutant in vitro. The data obtained provided clear evidence for a covalent interaction of the toxin with cysteine residues of proteins. A detailed investigation of one of the binding partners, the large subunit of RubisCO showed a lower susceptibility to proteases in the presence of microcystin in the wild type. Finally, the mutant defective in microcystin production exhibited a clearly increased sensitivity under high light conditions and after hydrogen peroxide treatment. Taken together, our data suggest a protein-modulating role for microcystin within the producing cell, which represents a new addition to the catalogue of functions that have been discussed for microbial secondary metabolites.

Virtual four-dimensional imaging of lung parenchyma by optical coherence tomography in mice.

In this feasibility study, we present a method for virtual 4-D imaging of healthy and injured subpleural lung tissue in the ventilated mouse. We use triggered swept source optical coherence tomography (OCT) with an A-scan frequency of 20 kHz to image murine subpleural alveoli during the inspiratory phase. The data acquisition is gated to the ventilation pressure to take single B-scans in each respiration cycle for different pressure levels. The acquired B-scans are combined off-line into one volume scan for each pressure level. The air fraction in healthy lungs and injured lungs is measured using 2-D OCT en-face images. Upon lung inspiration from 2 to 12 cm H(2)O ventilation pressure, the air fraction increases in healthy lungs by up to 11% and in injured lungs by 8%. This expansion correlates well with results of previous studies, reporting increased alveolar area with increased ventilation pressures. We demonstrate that OCT is a useful tool to investigate alveolar dynamics in spatial dimensions.

Three-dimensional Fourier domain optical coherence tomography in vivo imaging of alveolar tissue in the intact thorax using the parietal pleura as a window.

In vivo determination of 3-D and dynamic geometries of alveolar structures with adequate resolution is essential for developing numerical models of the lung. A thorax window is prepared in anesthetized rabbits by removal of muscle tissue between the third and fourth rib without harming the parietal pleura. The transparent parietal pleura allows contact-free imaging by intravital microscopy (IVM) and 3-D optical coherence tomography (3-D OCT). We demonstrate that dislocation of the lung surface is small enough to observe identical regions in the expiratory and inspiratory plateau phase, and that OCT in this animal model is suitable for generating 3-D geometry of in vivo lung parenchyma. To our knowledge, we present a novel thorax window preparation technique for 3-D imaging of alveolar dynamics for the first time. The 3-D datasets of the fine structure of the lung beneath the pleura could provide a basis for the development of 3-D numerical models of the lung.

Improved three-dimensional Fourier domain optical coherence tomography by index matching in alveolar structures.

Three-dimensional Fourier domain optical coherence tomography (3-D FDOCT) is used to demonstrate that perfusion fixation with a mixture of glutaraldehyde and paraformaldehyde does not alter the geometry of subpleural lung parenchyma in isolated and perfused rabbit lungs. This is confirmed by simultaneous imaging of lung parenchyma with intravital microscopy. To eliminate the diffraction index interfaces between alveolar pockets and walls, we fill the fixed lungs with ethanol by perfusing with gradually increasing concentrations. This bottom-up filling process leaves no remaining air bubbles in the alveolar structures, thus drastically improving the resolution and penetration depth of 3-D FDOCT imaging. We observe an approximately 18% increase in alveolar area after ethanol filling, likely due in large part to elimination of the air/tissue interfaces. 3-D OCT datasets acquired from ethanol-filled lungs allow segmentation of the ethanol-filled structures, which were formerly air-filled, and 3-D reconstruction of larger areas of subpleural alveolar structures. Our innovative process of filling the lungs with ethanol postperfusion fixation thus enables more accurate quantification of alveolar geometries, a critical component of modeling lung function.

Simultaneous three-dimensional optical coherence tomography and intravital microscopy for imaging subpleural pulmonary alveoli in isolated rabbit lungs.

There is a growing interest in analyzing lung mechanics at the level of the alveoli in order to understand stress-related pathogenesis and possibly avoid ventilator associated lung injury. Emerging quantitative models to simulate fluid mechanics and the associated stresses and strains on delicate alveolar walls require realistic quantitative input on alveolar geometry and its dynamics during ventilation. Here, three-dimensional optical coherence tomography (OCT) and conventional intravital microscopy are joined in one setup to investigate the geometric changes of subpleural alveoli during stepwise pressure increase and release in an isolated and perfused rabbit lung model. We describe good qualitative agreement and quantitative correlation between the OCT data and video micrographs. Our main finding is the inflation and deflation of individual alveoli with noticeable hysteresis. Importantly, this three-dimensional geometry data can be extracted and converted into input data for numerical simulations.