Jiazhou Chen

Yujun Chen

Xavier Granier

Jingling Wang

Qunsheng Peng

Importance-Driven Composition of Multiple Rendering Styles

Keywords: stylization, nonphotorealistic rendering, importance map, composition

We introduce a non-uniform composition that integrates multiple rendering styles in a picture driven by an importance map. This map, either issued from saliency estimation or designed by a user, is introduced both in the creation of the multiple styles and in the final composition. Our approach accommodates a variety of stylization techniques, such as color desaturation, line drawing, blurring, edge-preserving smoothing and enhancement. We illustrate the versatility of the proposed approach and the variety of rendering styles on different applications such as images, videos, 3D scenes and even mixed reality. We also demonstrate that such an approach may help in directing user attention.

I. INTRODUCTION

The use of multiple styles has been adopted in many artworks, mostly modern ones, to highlight the important meanings. However, it can only be conducted by skillful artists. Some examples are shown in Figure 1. DeCarlo de al. [START_REF] Decarlo | Stylization and abstraction of photographs[END_REF] cites Toulouse-Lautrec's poster as an example to show that different regions can be abstracted in different levels. We also cite this poster in the left of Figure 1 to show that artists also employ significantly different styles for different regions. In the other two pieces of artwork in the right, different styles, such as watercolor painting, line drawing, luminance, are combined in different but reasonable manners to exhibit the creators' special design intents for different contents, which also presents the maximal of visual information.

In Computer Graphics, many rendering papers focusing on a single style have been published, while few works consider multiple styles. Image abstraction techniques [START_REF] Decarlo | Stylization and abstraction of photographs[END_REF], [START_REF] Santella | Abstracted painterly renderings using eye-tracking data[END_REF], [START_REF] Winnemöller | Real-time video abstraction[END_REF], [START_REF] Kang | Flow-based image abstraction[END_REF], [START_REF] Zhao | Realtime saliency-aware video abstraction[END_REF], [START_REF] Kyprianidis | Image and video abstraction by anisotropic kuwahara filtering[END_REF] adopt edge-preserving smoothing that gives the freedom to omit or remove visual information under the guide of user interaction, eye tracking data or automatic saliency estimation. Level-of-Detail rendering of 3D scenes [START_REF] Luebke | Level of Detail for 3D Graphics[END_REF], [START_REF] Ni | Multi-scale line drawings from 3D meshes[END_REF] is achieved by either pre-building the hierarchical structure of the mesh or adjusting the size of strokes adaptively. Importance-driven volume rendering [START_REF] Viola | Importancedriven focus of attention[END_REF], [START_REF] Krüger | Clearview: An interactive context preserving hotspot visualization technique[END_REF] shares the same idea with X-Ray vision [START_REF] Kalkofen | Interactive focus and context visualization for augmented reality[END_REF], [START_REF] Chen | On-line visualization of underground structures using context features[END_REF], [START_REF] Sandor | An Augmented Reality X-Ray system based on visual saliency[END_REF] in Augmented Reality that focus and context are rendered differently to display the interior or hidden structures. Unfortunately, they severely restrict the choices of styles. A pioneer work to extend this compatibility is called stylized focus [START_REF] Cole | Directing gaze in 3d models with stylized focus[END_REF], which directs the attention of viewers to areas of c Luis Ruiz c Henri de Toulouse-Lautrec c Vic Kulihim Illustration Figure 1: Three examples of artistic illustration with multiple styles. The poster in the left, "Mouline Rouge-La Goulue", adopts detailed painting to exhibit the pose of the woman dancer and the material of her dress, uniform color plus line drawing for her partner, silhouette filled with black color for all the spectators in the background. In the righttop painting, the cathedral painted with watercolor stands out from surrounding building drawn by lines, and shows a clear overall arrangement of this city. In the right-bottom illustration, the line drawing in the middle of the car exhibits the interior structures while the luminance in the head and the tail shows the global appearance of the car.

interests in architectural rendering of 3D models. User can select several elaborate focus models and rendering effects, but gaze-based importance definition can not satisfy complex compositing tasks.

In this paper, we introduce a non-uniform compositing approach that combines multiple rendering styles. The compositing is driven by an importance map. It independently renders the images, videos or 3D scenes with different styles selected by users conforming to this importance map. Different images are then composited in screen space. Our approach shares a lot of similarities with the classical compositing tools, but brings three key advantages:

• It composites multiple rendering styles non-uniformly guided by an importance map instead of one or two styles;

• It can process different input data, such as images, videos, 3D scenes, or even mixed reality data;

• It is compatible with different importance maps, even user-created or user-edited ones.

The goal of this paper is not to introduce a new rendering style, but an importance-driven approach using multiple existed styles. We introduce our general approach on an image example in Section III, after reviewing the previous work in Section II. We then describe all the details of each step in our compositing pipeline in Section IV and V. We also demonstrate the compatibility for various input data, styles, and applications in Section VI. The final Section VII presents a conclusion and potential future extensions.

II. PREVIOUS WORK

Most of the previous work only combines two styles: on one side the lines and strokes and on the other side, color abstractions. DeCarlo and Santella [START_REF] Decarlo | Stylization and abstraction of photographs[END_REF], [START_REF] Santella | Abstracted painterly renderings using eye-tracking data[END_REF] first proposed the idea of "Image Abstraction" that offers such a style with a spatially variant abstraction omits or removes redundant visual information, as mentioned in Section I. Orzan et al. [START_REF] Orzan | Structurepreserving manipulation of photographs[END_REF] uses the Gaussian scale-space theory to deal with the discontinuity problem for computing a perceptually meaningful hierarchy representation.

Inspired by these pioneer works, many extensions on image or video abstraction have been published [START_REF] Winnemöller | Real-time video abstraction[END_REF], [START_REF] Kang | Flow-based image abstraction[END_REF], [START_REF] Zhao | Realtime saliency-aware video abstraction[END_REF], [START_REF] Kyprianidis | Image and video abstraction by anisotropic kuwahara filtering[END_REF], [START_REF] Kang | Shape-simplifying Image Abstraction[END_REF]. In general, they reduce the contrast in lowcontrast regions while increase the contrast in high-contrast regions. Different from the hierarchical structure analysis used in [START_REF] Decarlo | Stylization and abstraction of photographs[END_REF], [START_REF] Santella | Abstracted painterly renderings using eye-tracking data[END_REF], they employ various image filters, such as edge-preserving smoothing filter, Flow-based Differenceof-Gaussian (FDoG) filter, shock filter and Kuwahara filter, to achieve the goal of reducing or increasing the contrast. Recent techniques take the advantages of faster performance with parallel implementations and improved temporal coherence, but still severally restrict in a particular rendering effect.

All these techniques are based on an underlying importance map either extracted from Eye-tracker data [START_REF] Santella | Abstracted painterly renderings using eye-tracking data[END_REF], or from information extracted from images. One more and more popular way to extract visual importance information is to compute a saliency map [START_REF] Itti | A model of saliency-based visual attention for rapid scene analysis[END_REF], [START_REF] Liu | Learning to detect a salient object[END_REF], [START_REF] Zhao | Realtime saliency-aware video abstraction[END_REF], [START_REF] Bhat | GradientShop: A gradient-domain optimization framework for image and video filtering[END_REF]. These techniques detect low level features (such as intensity variance, orientation and color contrast, texture difference) and higher level features (such as horizon lines, human faces, cars) using bottom-up computation, but they don't match actual eye movements [START_REF] Judd | Learning to predict where humans look[END_REF]. Thus, Judd et al. [START_REF] Judd | Learning to predict where humans look[END_REF] introduces the eye tracking data to learn a saliency model to predict where humans look. Zeng et al. [START_REF] Zeng | From image parsing to painterly rendering[END_REF] analyzes top-down semantics of the image by image parsing, but requires much more user interactions to segment and label the image. Kang et al. [START_REF] Kang | A unified scheme for adaptive stroke-based rendering[END_REF] defines an importance map based on outline, and controls the abstraction level by adaptively adjusting the stroke attributes.

Using saliency or any other perception-based importance map only allows controlling the level of stylization according to what a common user will perceive in an image. A wider range of applications are possible if we want to use such a map to control the gaze. Focus+Context rendering is a well-known expressive rendering mechanism in Augmented Reality (AR) and Volume Rendering domain. Kalkofen et al. [START_REF] Kalkofen | Interactive focus and context visualization for augmented reality[END_REF], Chen et al. [START_REF] Chen | On-line visualization of underground structures using context features[END_REF] and Sandor et al. [START_REF] Sandor | An Augmented Reality X-Ray system based on visual saliency[END_REF] render the focus (the hidden scenes in the X-Ray vision of AR) and the context (other visible scenes) with different methods, and composite them in the screen to reveal the correct occluding order. Viola et al. [START_REF] Viola | Importancedriven focus of attention[END_REF] and Krüger et al. [START_REF] Krüger | Clearview: An interactive context preserving hotspot visualization technique[END_REF] composite interior structures and exterior surfaces by dynamically adjusting the transparency in volume rendering.

The gaze simulation in [START_REF] Cole | Directing gaze in 3d models with stylized focus[END_REF] is the closest related work of our approach. The authors introduce stylized focus to draw viewer's gaze to the emphasized area through local variations in shading effects and line qualities. Though four focus models are proposed, some easy-use interaction tools are expected but missed unfortunately. And the stylized focus supports single-point based emphasized area only, which loses the compatibility with either eye tracking data or saliency map, and also the ability to fulfill complex stylization tasks.

In this paper, we present an improved approach that allows the combination of a larger set of styles while allowing direct user controls of the spatial variation of both styles and combination.

III. GENERAL APPROACH

Our approach is a generalization of image abstraction systems employed by many previous works [START_REF] Winnemöller | Real-time video abstraction[END_REF], [START_REF] Zhao | Realtime saliency-aware video abstraction[END_REF], [START_REF] Judd | Learning to predict where humans look[END_REF] and first described in DeCarlo and Santella's work [START_REF] Decarlo | Stylization and abstraction of photographs[END_REF]. This generalization is in two-folds: 1) it permits more than two rendering styles to be integrated into an image; 2) it allows users to modify easily the important map. The former brings richer visual information and more stylization effects and the latter provides a controllable interface to alter the visual importance.

To demonstrate the proposed approach, we adopt three rendering styles, including original color (original color is considered as a stylization for simplification), color desaturation and line drawing (see the overview of our approach in Figure 2). The importance for each style is illustrated by different colors, such as red, green and blue color in Figure 2. We may employ an image saliency estimation technique [START_REF] Judd | Learning to predict where humans look[END_REF] to initialize the importance, to produce results similar to [START_REF] Decarlo | Stylization and abstraction of photographs[END_REF]. Or, we can let the user define it with interaction tools.

Based on the importance map I, we are now ready to compose the stylized result. For each position x in the image, a non-uniform composition of N stylized images S i (x, I), i ∈ [1, ..., N ], using weights W i (x, I) is: where

C(x) = 1 K N i=1 W i (x, I)S i (x, I), (1)
K = N i=1 W i (x, I
) is a normalizing term. We take the importance map as the weighting functions to control the effect of stylization.

IV. IMPORTANCE MAP

Our importance map is presented as a gray-scale map with values between 0 and 1. In order to composite more than 2 styles, we subdivide the entire gray-scale interval into several sub-intervals at E i , with

1 ≥ E 0 > E 1 > ... > E N -1 ≥ 0.
Each partitioning point corresponds to a rendering style. Then the importance value of pixel x must fall into a specific sub-interval and determines W i (x, I) in equation 1 according to some rules.

There are many ways to create the importance map. They can be roughly divided into two categories: saliency estimation or feature extraction, and user interaction. Our approach does not try to improve any of these techniques, but provides the compatibility to utilize any of them.

A. Construction from original image

We construct the saliency map I according to Judd et al [START_REF] Judd | Learning to predict where humans look[END_REF]. This map can be further improved using different image-processing tools. In our approach, we have experimented the following two. One tool involves cross bilateral filtering (CBF) the importance map taking the color difference in neighborhood pixels of the original image into account:

I c (x) = 1 K c (x) y∈S G σs (|x -y|)G σr (|C(x) -C(y)|)I(y) (2) where K c (x) = y∈S G σs (|x -y|)G σr (|C(x) -C(y)|) is a normalizing term, G σ (x) = exp(-x 2 /(2σ 2 ))/ √ 2πσ 2 is
Gaussian smoothing, S is a set of neighboring pixels and C(x) presents the color of pixel x in original image. A CBF-based improved importance map I c and its relative result are shown in the third row of Figure 3. Compared with the original result (zoomed-in image in blue frame), I c (zoomed-in image in red frame) removes small spots while preserves the apparent edges.

The second one concerns averaging importance values in the same segmented region, which is produced by Mean-Shift image segmentation [START_REF] Comaniciu | Mean shift: A robust approach toward feature space analysis[END_REF]:

I s (x) = 1 K s (x) L(y)=L(x) I(y) (3)
where L is the segmentation label for each pixel, and K s (x) is the amount of pixels whose label is the same as L(x).

A segmentation-based importance map I s and its relative result are shown in the forth row of Figure 3. Compared with the original result and CBF-based improved one, I s (zoomed-in image in green frame) ensures the consistency in each segmented regions.

B. User interaction

Users may decide themselves the expected importance value E i for each rendering style. To facilitate the users to assign a selected style to a region on the image with specified importance value distribution, we provide them with a classical gradient slider to adjust the location of E i at the gray-scale interval (see the top image in Figure 3). Initially, the interval is partitioned uniformly. Then users adjust the sub-intervals by dragging the bars.

Thanks to this gradient slider, a full gradient-based interaction tool is naturally developed. Users can directly We take three styles for example, and use red, green and blue color to present these styles, whose importance is 1, 0.5 and 0 respectively. Different user inputs are shown in the left column. In the middle column, we visualize the importance maps using one color for each style and composite them together using the weights described in equation 8, the composition results are shown in the right column. draw a line segment on the image to modify or create a new importance map. Similar to many painting softwares, we provide three gradient modes: radial gradient, linear gradient and symmetrical gradient, using the following three equations:

I r (x) = 1 -c(|x -x B |/|x E -x B |) (4)
I l (x) = 1 -c((x -x B ) • (x E -x B )/|x E -x B | 2 ) (5) I sy (x) = |1 -2c((x -x B ) • (x E -x B )/|x E -x B | 2 )| (6)
where c(•) is a function clamping value to [0, 1] and x B , x E are the beginning and end points of the line segment. A radial gradient example is the sixth row in Figure 3.

To provide even more freedom to satisfy complex tasks, we also design a stroke-based interaction tool. Users can directly draw strokes on the image, composed by a series of circles with different radii, to indicate different styles in different regions. We propose the following interpolation scheme to generate a whole importance map: 1) it ensures a smooth importance value transition between neighboring pixels, 2) it favors pure selected style for user drawn regions. Although, radial basis function is a well-known smooth interpolation function, its computation costs a lot, since it requires to solve a linear equation whose dimension is the amount of all the drawn pixels.

We propose a fast approximate solution based on the distance function:

I st (x) = 1 K N i=0 E i N j=1,j =i D(x, j) (7)
where

K = N i=0 N j=1,j =i D(x, j
) is a normalizing term and D(x, j) is the distance between the current pixel x and the nearest circle related to the jth style. This distance can be easily computed with GPU, by the distance to the center of circle subtracted by its radius, thus the proposed solution achieves fast performance. Our GPU implementation includes two steps: 1) recording and storing the centers and radii of every circle of each stroke in a 1D texture, 2) computing the minimal distance from each pixel on the screen to these circles in parallel. The acceleration is owing to the fact that the amount of centers is much less than the pixels covered by these circles. This fast computation meets all the required properties, see the bottom row in Figure 3.

V. NON-UNIFORM COMPOSITION

A. Multiple weights

During the multi-style composition, each style is associated with a specific weight map, thus N weight maps are needed in total. But, our importance map initialized by saliency map is only a scalar image whose values are within [0, 1]. Therefore, we need to find a mapping to set up N weight maps based on the importance map and E i regarding the N rendering styles. We create the weight maps using a piecewise linear interpolation: where |I(x) -E i | is a distance function between the importance value of current pixel and the expected importance level of the ith style. To simplify the formula, we mark E -1 as 1 and E N as 0. In the gradient tool, the user can see the interpolation result illustrated by different colors, as long as all E i are given. The importance map is illustrated in the same way before the stylization, as the middle column of Figure 3. Note that highest importance is presented by red, while least importance is presented by blue in this paper, but the user can select any color for them.

W i (x, I) =        |I(x)-Ei-1| |Ei-Ei-1| , I(x) ∈ [E i , E i-1 ] |I(x)-Ei+1| |Ei-Ei+1| if I(x) ∈ [E i+1 , E i ) 0, others (8)

B. Spatial varying stylization

Besides the composition, the importance map is also used to guide the stylization step. A uniform stylization without importance will introduces ghosting artifacts in the final compositing results, see Figure 4 for example. The goal of the composition in this example is to draw lines in near-by scene (the top part of the image), blur the distant scene (the bottom part) and enhance the color in the middle as focus. The original image and an gradient importance map is given in the left top image. A simple composition can be obtained by a composition of uniform stylized images. But, the color ghosting artifacts occurs in the regions where more than two styles exist, see the blue zoomed-in image.

Our method adapts the kernel size of filter according to the weights, which are computed by equation 8. In the regions with zero weight, the kernel size become zero, thus filter is invalid in these regions. Compared with simple color composition using uniform stylization, our method achieves higher quality. In Figure 4, the lines extracted by our method become thinner when the weights are getting smaller, while the lines in simple composition have uniform thickness which leads to ghosting artifacts. The same phenomenon can be observed for image enhancement and blurring.

We have implemented various stylization techniques to demonstrate the effectiveness of our approach:

• Color desaturation is an easy method to reduce the color cues. We reduce the two chromatic channels in the CIE Lab color space [START_REF] Wyszecki | Color Science: Concepts and Methods, Quantitative Data and Formulae[END_REF]. Because it's a pixel-wise conversion, its conversion does not need to be adapted according to the importance as the previous ones.

• Enhancement technique increases the sharpness in the region with high contrast. We employ Unsharpmasking [START_REF] Ritschel | 3D Unsharp Masking for Scene Coherent Enhancement[END_REF] to enhance both images and 3D objects in high important regions.

• Gaussian smoothing is an ideal effect for distant scenes.

• Edge-preserving smoothing has the ability to smooth the low contrast regions while preserve the sharp edges.

Bilateral filter [START_REF] Tomasi | Bilateral filtering for gray and color images[END_REF] is a spatial variable technique which meets the requirement.

• Line drawing is the very common but effective method to outline the shape of the objects. We use Differenceof-Gaussian operator [START_REF] Winnemöller | Real-time video abstraction[END_REF] to detect lines for images and 3D objects.

All listed rendering techniques, except the color desaturation, are based on local filters. We thus adapt the kernel size of their filters, which use smaller filter kernel where the importance is low, to ensure the composition quality. The original color is sometimes needed to be preserved in some case, such as the important region in Figure 2, we consider it as a special style. We apply these styles directly on the images or each frame of videos. For 3D scenes, we use them either on luminance of their shading or normal map of the 3D meshes. Combination with three styles are employed in Figure 2, 3, 4, more results combined with more styles will be shown in next section.

VI. RESULTS

The parameters of our approach are E i (expected importance value for each style) for the composition and the ones from each stylization, such as the maximal enhancement strength, maximal Gaussian smoothing kernel, spatial kernel and range kernel for bilateral filter, maximal spatial scale, sensitivity and sharpness of DoG edge extraction.

Figure 6 contains various stylized results generated by our system. In the first row, the left image show an example of architectural 3D models, which are rendered by photorealistic manner and then stylized with lines, desaturated colors and enhancement from left to right. The image in the middle mimics depth-of-field phenomenon approximately, by applying a linear gradient Gaussian blurring. For 3D scenes, we first render them using classic photorealistic manner, and then stylize the rendered image. A X-ray vision in AR example is given on the right, the virtual underground pipelines are rendered using X-toon shading [START_REF] Barla | X-toon: An extended toon shader[END_REF], the moving car is segmented out [START_REF] Zhong | Confidence-Based Color Modeling for Online Video Segmentation[END_REF] and presented in the front of the pipelines as the original color while lines extracted to represent shape features are preserved in both background and composited region. The second row displays three frames from a video experiment, the dolphin is highlighted by increasing the luminance, the color on the boat behind the dolphin is desaturated and smoothed with edgepreservation while lines are used for sea in the background. A example on large scale image, part of the Chinese ancient painting "Qingming Festival by the Riverside" painted by Song painter Zhang Zeduan, the bottom row of Figure 6, shows the ability of our approach for complex stylization tasks.

One interesting potential of our approach is to direct viewer's attention by combining multiple rendering styles. The saliency in regions where a high-preserving style is applied will be increased, while the one in regions stylized by a low-preserving style will be reduced, as demonstrated in Figure 5. According to the saliency map of the original image, full color information (as a high-preserving style) is preserved in high-saliency regions, while desaturated (as a low-preserving style) in low-saliency regions. We then estimate a new saliency map of the processed result. Compare with the original saliency map, the new one gathers the saliency to the high-saliency regions, which shows the potential of our method to direct viewer's attention.

The performance of our approach mainly depends on the stylization approaches we employed. Fortunately, our system prefers spatial variable stylization techniques, which are naturally parallel. Therefore, we have implemented our system in GPU architecture, achieve a real time performance.

VII. CONCLUSION

In this paper, we have introduced a non-uniform compositing approach that combines multiple rendering styles driven by an importance map. This map is employed to guide both stylization and composition, which improves the quality and compatibilities with different input data and user interactions.

We have shown that various styles can be adopted in our system, but they are still limited in spatially varying stylization techniques. More sophisticated use of importance map may solve this limitation. Moreover, temporal coherence has not been discussed yet, though it is not a big issue in our video experiments.

Another interesting future extension is to exploit the potential ability of our approach in controlling the attention. We have discussed this possibility in Section VI, but some further development has to be done. To achieve this goal, there are at least two main issues to deal with: 1) the importance map depends mostly on saliency map, while saliency estimation techniques in the state-of-art are far from detecting accurate visual attention; 2) our system requires the user to select styles and adjust the expected importance values for each style. Though a default mode and userfriendly gradient tools are provided, arbitrarily selection by fresh users may produce unexpected results or even reverse the visual attention. A reasonable strategy to help the users to do a good selection will probably improve our system for attention control. We believe this direction of research deserves future endeavor.

Figure 3 :

 3 Figure 3: Five importance map examples computed by different user inputs.We take three styles for example, and use red, green and blue color to present these styles, whose importance is 1, 0.5 and 0 respectively. Different user inputs are shown in the left column. In the middle column, we visualize the importance maps using one color for each style and composite them together using the weights described in equation 8, the composition results are shown in the right column.

Figure 4 :

 4 Figure 4: Compare with uniform stylization. In this experiment, we apply line extraction, image enhancement and blurring driven by an importance map. The first row contains the original image (with an importance map), a simple composition result and a composition produced by our method. A zoomed-in comparison is shown below. To compare each component of these two compositions, we illustrate all the stylized images one-by-one in the last two rows.

Figure 5 :

 5 Figure 5: Overview of our evaluation on estimating the visual attention before and after our stylization by the way of saliency Estimation.

  Overview of our system. Three example styles (original color, desaturated color, line drawing) are presented by red, green and blue color respectively in the importance map. One of the main contributions of our paper is that we use the importance map to guide not only stylization, but also the composition, illustrated by thick red arrows. The user, presented by a green box, has to select the styles, and modify the importance map optionally.

	Input	Selection of Styles	Stylization 1 Stylization 2 Stylization N	Output
			User
	Saliency Map		Importance Map
	Figure 2:

ACKNOWLEDGEMENTS

This research is supported by National Basic Research Program of China (973 Program, Grant No.2009CB320802), National Natural Science Foundation of China (Grant No.60970020), and the Animare Project of French (ANR-08-JCJC-0078-01).

Architectural 3D models

Appr. depth-of-field X-Ray vision in AR Three frames from our video example Complex stroke-based interaction Figure 6: Various results. Each result is shown with its scaled-down original image and importance map. In the top row, an example of architectural 3D models, which includes a photorealistic rendered image, gradual and final result from top to bottom, is given on the left, a depth-of-field effect is approximated using symmetrical gradient importance in the middle, and the right image exhibits an example for X-Ray vision in AR. The middle row shows three result frames in our video experiments, their importance maps produced by video segmentation are also given. We also experiment complex strokebased user interaction on large scale image in the bottom: trees are all outlined with thick lines, and buildings are silhouetted by thin lines, and the edge-preserving smoothing is applied on the pedestrians.


Oleg Gluz

Peter Wild

Cornelia Liedtke

Ronald Kates

Heiko Mendrik

Elisabeth Ehm

Verena Artinger

Raihanatou Diallo-Danebrock

Evelyn Ting

Svjetlana Mohrmann

Christopher Poremba

Nadia Harbeck

Ulrike Nitz

Arndt Hartmann

MD Andreas Gaumann
email: andreas.gaumann@klinik.uni-regensburg.de

Tumor angiogenesis as prognostic and predictive marker for chemotherapy dose-intensification efficacy in high-risk breast cancer patients within the WSG AM-01 trial

Keywords: high-risk breast cancer, angiogenesis, high-dose chemotherapy, immunohistochemistry, molecular subtypes

Purpose: The goal of this analysis was to characterize the survival impact of angiogenesis in patients with high-risk breast cancer (HRBC), particularly the predictive impact on benefit from dose intensification of adjuvant chemotherapy.

Methods: Formalin-fixed tissue sample of 152 patients treated as part of the WSG AM-01 trial by either high-dose (HD) or conventional dose-dense (DD) chemotherapy were analyzed. Angiogenic activity was measured using microvessel count (MVC) and vascular surface area (VSA) determined by the expression of vascular markers CD31 (n=128) and CD105/endoglin (n=130). Protein molecular breast cancer subclasses were analyzed by k-means clustering(k=5). The univariate impact of factors on event-free (EFS) and overall survival (OS) was tested by log-rank statistics and quantified by univariate Cox analysis. Multivariate survival analysis included including factors significant in univariate analysis, as well as interactions was performed for EFS.

Results: Both VSA/CD31 (p=0.004) and VSA/CD105 (p=0.003) were significantly higher among cases with increased Ki-67. A significant association with molecular subtypes was also found for VSA/CD105: In patients with basal-like/Her-2 subtypes, mean was 1.72 vs. 1.24 in patients with other subtypes (p<0.001). Elevated VSA/CD105 was associated with both significantly decreased EFS (p=0.01) and OS (p=0.02). Increased tumor size and positive Her-2 status were also prognostic for poorer EFS. The benefit of dose intensification for EFS was seen in those low-VSA/CD105 patients. The result was evident both in univariate and in multivariate survival analysis including all factors that were significant at the univariate level.

Discussion: Expression of angiogenesis markers may mirror or confer resistance to chemotherapy in patients with breast cancer, particularly within the context of dose intensified chemotherapy. Highly angiogenic tumors may not derive sufficient benefit from dose intensification of chemotherapy alone. Our findings may serve as a rationale for further exploring anti -angiogenic treatment options in patients with such highly angiogenic tumor subtypes.

Introduction

High-risk primary breast cancer (HRBC) is characterized by extensive axillary lymph node involvement. Ten-year mortality of patients with very high lymph node involvement (i.e., more than 10 positive nodes) is around four times as high as in node-negative patients [START_REF] Fisher | Relation of number of positive axillary nodes to the prognosis of patients with primary breast cancer. An NSABP update[END_REF]. Their unfavorable outcome persists despite improvement in drug selection and scheduling in adjuvant chemotherapy of breast cancer (BC). To date, there is only limited data regarding the survival impact of factors within this high-risk population. A small number of studies have reported poor outcome among this subgroup of patients in association with increased tumor size, poor grade, estrogen/progesterone receptor negativity or overexpression of Her-2 and p53 [START_REF] Gluz | Triple-negative high-risk breast cancer derives particular benefit from dose intensification of adjuvant chemotherapy: results of WSG AM-01 trial[END_REF][START_REF] Kroger | Prognostic and Predictive Effects of Immunohistochemical Factors in High-Risk Primary Breast Cancer Patients[END_REF][START_REF] Faneyte | Predicting early failure after adjuvant chemotherapy in high-risk breast cancer patients with extensive lymph node involvement[END_REF] Dose intensification and densification of adjuvant chemotherapy remains one of the most controversial issues in breast cancer systemic therapy. The WSG AM-01 trial, using rapidly cycled tandem high-dose (HD) compared with dose-dense (DD) conventional design, is the only study resulting in a significant improvement of event free survival (EFS) and overall survival (OS) in patients with more than nine positive lymph nodes [START_REF] Nitz | Comparison of rapidly cycled tandem high-dose chemotherapy plus peripheral-blood stem-cell support versus dosedense conventional chemotherapy for adjuvant treatment of high-risk breast cancer: results of a multicentre phase III trial[END_REF].

Recently, molecular breast cancer subtypes based on microarray analysis have been shown to carry a significant prognostic impact [START_REF] Perou | Molecular portraits of human breast tumours[END_REF][START_REF] Sorlie | Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF]. We previously stratified patients with HRBC into molecular subgroups based on a set of immunohistochemical markers and identified poor outcome of patients with basal-like and Her-2 subtypes [START_REF] Diallo-Danebrock | Protein Expression Profiling in High-Risk Breast Cancer Patients Treated with High-Dose or Conventional Dose-Dense Chemotherapy[END_REF][START_REF] Hannemann | Molecular subtypes of breast cancer and amplification of topoisomerase II alpha: predictive role in dose intensive adjuvant chemotherapy[END_REF]. The most pronounced benefit of HD chemotherapy was seen in patients with both highly-proliferating triple-negative and/or high-grade tumors (assessed by conventional immunhistochemistry) and with basal-like and/or Her-2 subtypes (defined by clustering analysis). Moreover, in an independent analysis based on the same study population, an increased efficacy of HD was found among patients with YB-1 positive, i.e. potentially drug-resistant tumors [START_REF] Gluz | Y-Box-Binding Protein YB-1 Identifies High-Risk Patients With Primary Breast Cancer Benefiting From Rapidly Cycled Tandem High-Dose Adjuvant Chemotherapy[END_REF] Several studies as well as comprehensive meta-analyses have shown that the angiogenetic potential of BC as assessed by tumor microvessel density (MVD) correlates with progression and metastasis; it thus predicts for clinical outcome [START_REF] Gasparini | Tumor microvessel density, p53 expression, tumor size, and peritumoral lymphatic vessel invasion are relevant prognostic markers in node-negative breast carcinoma[END_REF][START_REF] Heimann | Angiogenesis as a Predictor of Long-term Survival for Patients With Node-Negative Breast Cancer[END_REF][START_REF] Uzzan | Microvessel Density as a Prognostic Factor in Women with Breast Cancer[END_REF]. MVD or vascular surface area (VSA) can be evaluated by applying immunohistochemistry to tissue sections using different antibodies. In the past, Factor VIII as well as CD31 have been extensively used to analyze tumor vascularisation. CD 31 is a cellular adhesion molecule (PECAM-1), which has been shown to reliably detect neoangiogenesis in both malignant and benign tissue, despite the fact that it also labels other hematopoetic cells such as plasma cells.

Another marker to detect angiogenetic activity is CD34. However, this molecule also labels fibroblasts as well as hematopoetic stem cells and thus may not be useful under all circumstances for angiogenesis detection. CD105, known as endoglin, is a member of the TGF-receptor family [START_REF] Kumar | Vascular Density Determined Using CD105 Antibody Correlates with Tumor Prognosis[END_REF]. CD105 is expressed by vascular endothelial cells and plays an important role in angiogenesis of breast cancer [START_REF] Bodey | Over-expression of endoglin (CD105): a marker of breast carcinoma-induced neo-vascularization[END_REF] and other malignancies [START_REF] Dallas | Endoglin (CD105): A Marker of Tumor Vasculature and Potential Target for Therapy[END_REF] In contrast to other angiogenic markers, CD 105 / endoglin seems to be more specific for malignant angiogenesis [START_REF] Kumar | Vascular Density Determined Using CD105 Antibody Correlates with Tumor Prognosis[END_REF][START_REF] Dallas | Endoglin (CD105): A Marker of Tumor Vasculature and Potential Target for Therapy[END_REF][START_REF] Li | Both high intratumoral microvessel density determined using CD105 antibody and elevated plasma levels of CD105 in colorectal cancer patients correlate with poor prognosis[END_REF]. Several studies have shown that increased vascular density as assessed by a CD 105 antibody as well as elevated serum CD 105 were associated with poorer overall und disease free survival in different types of cancer [START_REF] Kumar | Vascular Density Determined Using CD105 Antibody Correlates with Tumor Prognosis[END_REF][START_REF] Dallas | Endoglin (CD105): A Marker of Tumor Vasculature and Potential Target for Therapy[END_REF][START_REF] Fujita | Endoglin (CD105) as a urinary and serum marker of prostate cancer[END_REF][START_REF] Yao | Prognostic significance of microvessel density determined by an anti-CD105/endoglin monoclonal antibody in astrocytic tumors: Comparison with an anti-CD31 monoclonal antibody[END_REF]. as well poorer survival in patients with higher vascular counts (measured by CD 31 antibody) treated by HD chemotherapy [START_REF] Nieto | Prognostic analysis of tumour angiogenesis, determined by microvessel density and expression of vascular endothelial growth factor, in high-risk primary breast cancer patients treated with high-dose chemotherapy[END_REF] Although the impact of these markers is still a matter of debate, consensus conferences on tumor angiogenesis have supported the notion that all of these molecules are useful for evaluating angiogenesis [START_REF] Vermeulen | Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation[END_REF][START_REF] Vermeulen | Second international consensus on the methodology and criteria of evaluation of angiogenesis quantification in solid human tumours[END_REF].

The objective of the present investigation was to identify a hypothetical clinically relevant prognostic and/or predictive signature of angiogenesis factors among HRBC patients treated by different adjuvant chemotherapy dose regimens. To this end, the impact of angiogenesis parameters (CD 105 and CD 31, measured retrospectively) on event-free survival in different therapy arms of a randomized clinical trial was studied using univariate and multivariate interaction analysis.

Material and Methods

The West German Study Group (WSG) AM-01 trial and tumor samples

In the present study, paraffin-embedded breast cancer specimens from 181 randomized HRBC patients with more than nine affected axillary LN were available. These patients were previously enrolled in the prospective multi-center WSG AM-01 trial comparing tandem HD with DD conventional chemotherapy. Details of this trial were described previously [START_REF] Nitz | Comparison of rapidly cycled tandem high-dose chemotherapy plus peripheral-blood stem-cell support versus dosedense conventional chemotherapy for adjuvant treatment of high-risk breast cancer: results of a multicentre phase III trial[END_REF]. The present study is based on archived tissue samples from the clinical trial, as described elsewhere [START_REF] Gluz | Triple-negative high-risk breast cancer derives particular benefit from dose intensification of adjuvant chemotherapy: results of WSG AM-01 trial[END_REF]. The final study population consisted of 152 cases (84%) after exclusion of 29 cases without sufficient remaining material for accurate morphometric analysis.

After immunostaining, 128 (70%) and 132 (73%) cases were available for analysis of CD31 and CD105, respectively (See consort diagram, figure 1).

Immunohistochemistry (IHC):

Immunostaining for vascular markers CD31, CD105 (Endoglin).

3 µm sections were cut and stained for the vascular markers. Briefly, sections were dewaxed and nonspecific binding was blocked by incubation with 3% H 2 O 2 and subsequent incubation with 20% goat serum in phosphate buffered saline.

Microwave antigen retrieval (citrate buffer pH 6.0 microwave; 320 watt for 30 min in 0.1 mol/l citrate buffer, pH 6.0) or pressure cooker (120°C, 5min) was utilized.

Staining for CD31 and CD105 were performed manually. CD105 (DAKO Cytomation, Germany) was detected using the CSA amplification kit (DAKO Cytomation, Germany) following the manufacturers' protocol without antigen retrieval. CD31 (DAKO Cytomation, Germany) staining was performed by using an antigen retrieval solution (DAKO Cytomation, Germany) for 20 min at 120°C (pressure cooker). After intermediate washing steps, the primary antibody was incubated overnight at 4°C. Subsequently, sections were incubated with a biotinylated secondary antibody and connected with Avidin-Biotin coupled with Alkaline Phosphatase or Peroxidase.

Finally, staining was developed with Fast Red reagent or DAB; the reaction was stopped under microscopic control. Paraffin sections from colon carcinomas served as positive controls. As negative controls, primary antibodies were omitted or replaced by non -specific immunoglobulins.

Evaluation of vascular markers by morphometry

Immunohistochemical staining was independently assessed by at least two different observers (A.G.; and H. M. or E. E. or V. A. for either CD105 or CD31). For each case with excellent staining quality (at least three invasive tumor containing areas), up to 6 areas of invasive carcinoma with the highest vascular density were identified by low power magnification (100 X; Zeiss axiophot microscope, (Zeiss AG, Germany)) in the tumor center as well as the tumor periphery based on the criteria of Weidner [START_REF] Weidner | Tumor angiogenesis and metastasis--correlation in invasive breast carcinoma[END_REF]. [START_REF] Weidner | Tumor angiogenesis and metastasis--correlation in invasive breast carcinoma[END_REF]. The selected areas were digitally photographed at high magnification (200x) with the AxioVision software (rel 4.5) (Zeiss AG, Germany). Subsequently, every picture was printed and the microvessel density (MVD) and vascular surface area (VSA) were assessed. The latter parameter was evaluated by using a transparent reticule, which was overlayed over every individual print; the vessel area was counted and expressed as percent of the whole area.

Microvessels and vascular surface were assessed in a 0.141 mm 2 area.

Mean and median values of vascular markers were calculated for each individual tumor; the median was used in further analysis. In order to analyze inter-observer variability, 10% randomly selected cases were re-evaluated without knowledge of the primary results by an experienced surgical pathologist (A.G.) for all three markers.

Statistical Methods

Bivariate correlations of continuous variables were assessed by Spearman's correlation; associations among discrete variables were assessed by Fisher's exact test. Associations between continuous and discrete variables were analyzed by the t-test or by one-way ANOVA. The primary endpoint for survival analysis was eventfree survival (defined as time from the randomization to first relapse, secondary malignancy or death); overall survival was considered to be a secondary endpoint.

In survival analysis, variables were classified as follows: age (<50 vs. >50 years old), tumor size (>3 cm vs. <3 cm), tumor grade (3 vs.1 and 2), therapy arm (HD vs. DD), centrally measured expression of ER, PR, Her-2, ki-67/MIB-1 (positive (>10% strong nuclear staining) vs. negative), and markers for angiogenesis such as VSA/CD 105 (< sample median vs. > sample median). Molecular subtypes definition based on expression of 24 proteins was described previously [START_REF] Diallo-Danebrock | Protein Expression Profiling in High-Risk Breast Cancer Patients Treated with High-Dose or Conventional Dose-Dense Chemotherapy[END_REF][START_REF] Hannemann | Molecular subtypes of breast cancer and amplification of topoisomerase II alpha: predictive role in dose intensive adjuvant chemotherapy[END_REF]. The Kaplan-Meier method was used to estimate cumulative survival time probabilities. The log-rank test (p<0.05) was applied to test for survival differences by treatment arm (possibly stratified by factors). The study was performed in accordance to the REMARK criteria [START_REF] Mcshane | Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK)[END_REF] A preliminary univariate Cox analysis for EFS was performed on each individual marker; those markers with significant univariate impact, as well as treatment arm, were entered into multivariate forward stepwise Cox analysis for EFS, with main effects in the first block and (factor times therapy) interactions in the second block. To test the biological hypothesis that low levels of angiogenesis markers are predictive for response to HD, the appropriate coding in Cox analysis was zero if > sample median and one if < sample median. Confidence intervals are reported at the 95% level. All statistical calculations were performed using the statistical software package SPSS 17.0 for Windows.

Results

Patients, follow up and treatment arms

The treatment groups of the 152 patients were well balanced in terms of baseline

CD31 and CD105 Scoring

Evaluation of MVD revealed a median of 13 microvessels (mean 13.7 ± 5.26) by CD31 and of 11.25 microvessels (mean 12.9 ± 6.66) by CD105. Assessment of VSA showed a median of 2.52 % (mean 2.67 ± 1.14) for CD31 and 1.30 (mean 1.43 ± 0.72) for CD105. For both parameters, the sample median levels (i.e. in CD31: 13 (MVD) and 2.52 % (VSA)) were selected as cut-offs to define two distinct groups (low vs. high vessel density). For both CD31 and CD105, a significant positive correlation was found between increased VSA and MVD (p<0.001 in each case). Conversely, for both VSA and MVD, a significant positive correlation was found between CD31 and CD105 (p<0.001 in each case).

Correlations and associations

In bivariate correlation analysis between clinical-pathological variables and angiogenic markers, no correlation was found among age, tumor size, number of involved lymph nodes, tumor grade, hormone receptors (ER/PR) and MVD or VSA (for both CD31 and CD105). However, increased proliferation as determined by MIB-1/Ki-67 expression was significantly associated with increased VSA for CD105 (mean 1.58 vs. 1.19; p=0.003) and CD31 (mean 2.92 vs. 2.32; p=0.004), but not for MVD of both markers. A significant association with molecular subtypes was also found for VSA/CD105: In patients with "basal-like" or "Her-2" subtypes, the mean was 1.72, compared to 1.24 in patients with luminal A, luminal B, or multiple marker negative subtypes (p<0.001).

Univariate influence of markers on survival

According to Kaplan-Meier estimates on the entire collective, after median follow up of 65.5 months, VSA CD105 was the only significant angiogenesis factor for EFS (median EFS for VSA CD105 high vs. low: 81 months vs. 46 months, p=0.01) and OS (median OS high vs. low: 104 months vs. 87 months, p=0.02) (Figures 3,4). In univariate Cox analysis for EFS, the significant markers for poorer EFS were VSA CD105, as well as the established markers tumor size (>3 cm) and positive Her-2 status (Table 1). Table 1. Univariate Cox analysis for EFS (markers and therapy arm).

HD therapy, positive ER and/or PR status and negative Her-2 and low proliferation index as measured by MIB-1/Ki-67 status were favorable predictors for OS (data not shown).

Therapy response

In this studied collective, a trend for increased EFS (median EFS HD vs. DD: 81 vs. 52 months, p=0.08) and significantly better OS (median OS HD vs. DD: 113 months vs. 77 months, p=0.026) in favor of HD was shown after 98 months of median follow up.

EFS and OS by therapy arm in angiogenesis subgroups

Both MVD/VSA of angiogenesis markers were tested for their interaction with therapy but only VSA CD 105 was found to be a significant predictor for efficacy of different chemotherapies.

HD was significantly superior to DD in CD105 VSA low tumors for both EFS (median EFS HD vs. DD: 99 vs. 57 months, p=0.02) and OS (median OS HD vs. DD: not reached vs. 80 months, p=0.01, fugure 6), but not in high CD105 VSA tumors (median EFS HD vs. DD: 41 vs. 49 months, p=0.69, median OS HD vs. DD: not reached vs. 80 months, p=0.38, figure 5)

Multivariate survival analysis for EFS with interactions

Comparison of treatment effects in high-VSA/CD105 tumors (Fig. 4) versus low-VSA/CD105 tumors (Fig. 5) illustrates the qualitatively different treatment impacts on EFS in these subgroups. To test the biological hypothesis that low levels of the angiogenesis marker VSA/CD105 are predictive for benefit from HD compared to DD, VSA/CD105 was coded as described above (zero if > sample median). Thus, the interaction term is 1 for patients with low-VSA/CD105 receiving HD, zero otherwise (either high low-VSA/CD105 or DD).

Consistent with stratified

Discussion:

Endoglin (CD 105) is an integral protein of the TGF-β receptor family, which plays an essential role in regulating cell differentiation and proliferation in malignant tissues through smad proteins as well in haematopoeisis and cardiogenesis and strongly associated with stem cell phenotype of breast cancer cells correlating with basal-like or Her-2 tumor types [START_REF] Shipitsin | Molecular definition of breast tumor heterogeneity[END_REF]. TGF-β is expressed almost exclusively on endothelial cells of both peri-and intratumoral blood vessels and on tumor stromal components as shown by de Caestecker et al [27] . CD 105 is a co-receptor of TGF-β-1 and -2 and antagonizes inhibitory effects of TGF-β on proliferation and migration, thus promoting growth and migration of tumor cells [START_REF] Li | CD105 antagonizes the inhibitory signaling of transforming growth factor {beta}1 on human vascular endothelial cells[END_REF]. Unlike other TGF-β receptors, endoglin was found to be expressed almost exclusively by vascular endothelial cells. Similarly, CD 31 has been discussed as a potential pan-angiogenesis marker, being expressed also by plasma cells, macrophages and neutrophils [START_REF] Leek | The prognostic role of angiogenesis in breast cancer[END_REF]. Thus, CD31 seems to be less specific than CD105 regarding malignant angiogenesis, rather representing angiogenesis in both benign and malignant tissues.

In this study, we have investigated the predictive role of the well established angiogenesis parameter CD31 and CD105 in primary tumor specimens regarding patient outcome in a collective of 152 HRBC patients treated by either conventional DD or tandem HD chemotherapy within the randomized WSG AM-01 trial as reported previously [START_REF] Nitz | Comparison of rapidly cycled tandem high-dose chemotherapy plus peripheral-blood stem-cell support versus dosedense conventional chemotherapy for adjuvant treatment of high-risk breast cancer: results of a multicentre phase III trial[END_REF]. The results of the trial support the significant benefit of rapidly cycled tandem HD over conventional dose DD EC-CMF with regard to both EFS and OS in HRBC. However, despite these findings, HD and DD remains a controversial issue in adjuvant breast cancer therapy due to higher toxicity, costs, and inhomogeneous patient collectives treated within the respective trial. Despite incorporation of modern highly effective agents (such as taxanes) [START_REF] Moebus | Intense Dose-Dense Sequential Chemotherapy With Epirubicin, Paclitaxel, and Cyclophosphamide Compared With Conventionally Scheduled Chemotherapy in High-Risk Primary Breast Cancer: Mature Results of an AGO Phase III Study[END_REF], the outcome in this patient group remains poor. Moreover, no predictive marker for benefit from dose-density or doseincrease has been established for clinical use so far.

We now show for the first time, that VSA measured by CD 105 is significantly associated with increased proliferation as well as basal-like and Her-2 molecular subtypes, which are known to impact unfavorably on disease outcome [START_REF] Sorlie | Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications[END_REF][START_REF] Diallo-Danebrock | Protein Expression Profiling in High-Risk Breast Cancer Patients Treated with High-Dose or Conventional Dose-Dense Chemotherapy[END_REF]. In our analysis, VSA/CD105 was an independent prognostic marker for decreased survival both by univariate and multivariate analysis.

Moreover, our study revealed a predictive value of VSA/CD 105 with regard to efficacy of dose-intensification. In patients with low angiogenetic activity, a significant benefit of HD was observed: Median EFS in this subgroup was 99 months (compared to 57 months for high angiogenesis tumors) in favor of HD. In contrast, in the VSA CD105 high subgroup, median EFS rates were similarly short in both therapy arms.

The impact of VSA/CD 105 on efficacy of HD for improved EFS persisted in interaction analysis: patients with highly angiogenic tumors derived no significant benefit from dose-intensification of chemotherapy in this collective. Thus, highly dysregulated angiogenesis was associated with apparent resistance to both chemotherapy regimens: In our study, patients with low angiogenic tumors (a prognostically favorable factor) treated by HD had a HR of 0.448 for recurrence as compared to those with high angiogensis treated by any regimen. In contrast, in the collective as a whole [START_REF] Gluz | Triple-negative high-risk breast cancer derives particular benefit from dose intensification of adjuvant chemotherapy: results of WSG AM-01 trial[END_REF], high tumor grade, which is a prognostically unfavorable factor, was predictive for response to HD.

Poor outcome of patients with increased angiogenesis as measured by CD 105 is in line with previously published studies in breast cancer and other malignancies [START_REF] Duff | CD105 is important for angiogenesis: evidence and potential applications[END_REF]. Kumar et al. showed shorter disease-free and overall survival in patients with higher microvessel density in a study with 106 breast cancer patients [START_REF] Kumar | Vascular Density Determined Using CD105 Antibody Correlates with Tumor Prognosis[END_REF]. Dales et al. reported decreased survival rates in patients with increased counts for CD105 and CD 31 positive microvessels. However, a statistically significant effect on DFS was only observed for CD105 in their multivariate analysis [START_REF] Dales | Prognostic significance of angiogenesis evaluated by CD105 expression compared to CD31 in 905 breast carcinomas: correlation with long-term patient outcome[END_REF] mirroring CD 31 as less specific marker for tumor angiogenesis [START_REF] Dallas | Endoglin (CD105): A Marker of Tumor Vasculature and Potential Target for Therapy[END_REF], what is in line with with our study. Despite a significant correlation of CD31 with CD105, , no prognostic impact of CD31 was found in our study in contrast to previously reported significant negative prognostic impact of MVD measured only by CD 31 in similar collective of patients treated by HD [START_REF] Nieto | Prognostic analysis of tumour angiogenesis, determined by microvessel density and expression of vascular endothelial growth factor, in high-risk primary breast cancer patients treated with high-dose chemotherapy[END_REF]. However this study supports our data in term of relative chemotherapy resistance of highly angiogeneic tumors. Elevated endoglin levels in serum have also been associated both with presence of metastatic disease [START_REF] Chenggang | Plasma levels of soluble CD105 correlate with metastasis in patients with breast cancer[END_REF] as well as decreased response to endocrine therapy and poorer survival in metastatic breast cancer [START_REF] Vo | Elevated plasma endoglin (CD105) predicts decreased response and survival in a metastatic breast cancer trial of hormone therapy[END_REF]. The predictive effect of CD 105 with regard to dose-dependent efficacy of chemotherapy correlates also with the data of neoadjuvant CEF chemotherapy as reported by Beresford et al [START_REF] Beresford | The relationship of the neoangiogenic marker, endoglin, with response to neoadjuvant chemotherapy in breast cancer[END_REF]. In this study, breast cancer patients with lower CD 105 tumor counts experienced increased chemotherapy benefit compared to patients with higher levels. Similar to our report, global angiogenesis as measured by CD 34 or CD 31 did not reach statistic significance in this study.

Based on our results, we hypothesize that angiogenesis and hypoxia are key factors in mediating chemotherapy resistance. Hypoxia has been shown to induce endoglin expression in vitro and in vivo [START_REF] Warrington | Functional Role of CD105 in TGF-β1 Signalling in Murine and Human Endothelial Cells[END_REF][START_REF] Zhu | Hypoxic Induction of Endoglin via Mitogen-Activated Protein Kinases in Mouse Brain Microvascular Endothelial Cells[END_REF]. Functionally, endoglin causes anti-apoptotic signals in hypoxic cells. But, as shown both in our investigation and in other studies, altered (mostly up-regulated) angiogenesis reflects distinct, more aggressive tumor types (e.g. basal-like or Her-2 subtypes [START_REF] Lopes | Vessel density assessed by endoglin expression in breast carcinomas with different expression profiles[END_REF]) and may be associated with or even cause chemoresistance. The reason that only tumors with low VSA better respond to therapy may be explained by the fact that slower growing tumors induce a less chaotic angiogenic network compared to high angiogenic tumors and thus facilitate a better response to intense chemotherapy regimens. In fact it is already known that histologic high vessel count does not per se indicate a pathophysiological functional dense vascular network susceptible for therapy and disrupting of neovasculature could improve chemotherapy delivery to tumor cells [START_REF] Escorcia | Selective Killing of Tumor Neovasculature Paradoxically Improves Chemotherapy Delivery to Tumors[END_REF][START_REF] Drevs | PTK787/ZK 222584, a Specific Vascular Endothelial Growth Factor-Receptor Tyrosine Kinase Inhibitor, Affects the Anatomy of the Tumor Vascular Bed and the Functional Vascular Properties as Detected by Dynamic Enhanced Magnetic Resonance Imaging[END_REF]. However, in addition, methodic aspects may in part explain the different results when comparing CD105 and CD31.

Targeting of TGF-β signaling leads to depression of angiogenesis (among others through endoglin) and can reverse a stem cell phenotype to a more-differentiated luminal phenotype [START_REF] Nam | An Antiâ€"Transforming Growth Factor β Antibody Suppresses Metastasis via Cooperative Effects on Multiple Cell Compartments[END_REF] These effects can be potentially inhibited by dual blockade of TGF-beta and endoglin pathways [START_REF] She | Synergy between anti-endoglin (CD105) monoclonal antibodies and TGF-beta in suppression of growth of human endothelial cells[END_REF]. Moreover, anti-endoglin antibodies as single agents as well as CD 105 as target for oral DNA vaccine have also been reported as effective in suppressing or preventing tumor progression and prolonging survival in in-vitro models [START_REF] Uneda | Anti-endoglin monoclonal antibodies are effective for suppressing metastasis and the primary tumors by targeting tumor vasculature[END_REF][START_REF] Lee | Endoglin (CD105) is a target for an oral DNA vaccine against breast cancer[END_REF].. Anti-angiogenic therapies are emerging agents for advanced BC (e.g. VEGF antibody bevacizumab) treatment and are currently investigated in (neo)-adjuvant breast cancer clinical trials. These substances are reported to prolong progression-free survival with so far marginal effect on OS rates [START_REF] O'shaughnessy | A meta-analysis of overall survival data from three randomized trials of bevacizumab (BV) and first-line chemotherapy as treatment for patients with metastatic breast cancer (MBC)[END_REF]. This illustrates that optimal use of antiangiogenic agents, preferably in pre-selected patient collectives, are urgently needed for substantial improvement of therapy efficacy in HRBC.

Despite the randomized nature of the patient collective analyzied in our study, the present investigation constitutes a retrospective study performed on archived material and is thus subject to potential biases; in particular, n=29 cases needed to be excluded due to poor immunohistochemical staining quality for both markers. This problem is most likely explained by different fixation protocols of the fifty contributing pathology departments.

In summary, our study suggests an improved efficacy of chemotherapy doseintensification in HRBC in the low-angiogenesis group, as determined by VSA CD105. The VSA/CD105 high phenotype is associated with the aggressive basal-like and Her-2 subtypes. It seems to be resistant to adjuvant chemotherapy of its good overall efficacy in these subtypes with mostly poorly differentiated tumors. To the best of our knowledge, this is the first report on the importance of VSA in a randomized HRBC collective. Our data indicates that increased angiogenesis may be involved in chemoresistance in aggressive BC despite of the overall high chemosensitivity of this breast cancer subtype [START_REF] Liedtke | Response to Neoadjuvant Therapy and Long-Term Survival in Patients With Triple-Negative Breast Cancer[END_REF]. These hypothesis-generating findings warrant validation in future prospective trial concepts.

  characteristics. Median age of patients was 48 years. Median tumor size was 3.0 cm; patients had median of 15 involved lymph nodes; 42 % were G3 tumors. There was no significant difference in the distribution of characteristics between the entire study population (median age: 47.5 years, median tumor size: 3.0 cm; median of 15 involved lymph nodes; poor grade: 59 % by decentral and 42% by central assessment) and the subset presented here. Of our study population, 81 patients (53 %) were randomized to the HD arm and 71 patients (47 %) to the DD arm. In this collective, median follow-up was 65.5 months (range: 4 -121 months); (HD: median 72.5 months; DD: median 60 months).

Figure 1

 1 Figure 1 shows representative examples (maginification 200x) of tissue specimens with low (A: CD31, C: CD105) and high microvessel count (B: CD31, D: CD105).

Figures:Figure 3 :

 3 Figures:Figure3: Prognostic impact of VSA/CD105 on event free survival and 4. overall survival.

Figure 5 :

 5 Figure 5: Event free survival in high-VSA/CD105 tumors by chemotherapy arm

Figure 6 :

 6 Figure 6: Event free survival in low-VSA/CD105 tumors by chemotherapy arm

  Kaplan-Meier analysis, the benefit of dose intensification for EFS in low-VSA/CD105 patients was also significant in multivariate interaction analysis. In the resulting model [including age, therapy, Her-2 , tumor size, ER, PR, Ki-67, VSA CD 105 and interaction of factors significant in the univariate analysis (Her-2, tumor size and VSA CD/105) with therapy] only the interaction of low VSA and HD therapy (p=0.014; HR=0.367, 95% CI: 0.166-0.815) and positive ER status (p=0.027; HR=0.553, 95% CI: 0.327-0.934) were significant (favorable) factors for EFS.

Figure 1 .

 1 Figure 1. Consort diagramm

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5.

Figure 6

 6 Figure 6

Table 1 .

 1 lower VSA CD 105 levels are associated with reduced risk for relapse.

		Event Free Survival
			Univariate
				Hazard ratio §
	Factor	Coding	p
				[95%-CI]
	Therapy	HD vs. DD	0.08	0.68
				[0.45-1.05]
	Tumor size	>3cm vs. <3cm	0.037	1.58
				[1.03-2.41]
	VSA/CD105 Negative vs. Positive	0.01	0.55*
				[0.34-0.88]
	ER	Positive vs. Negative	n.s.	0.68
				[0.43-1.07]

*

Acknowledgments

We very much appreciate the support of the contributing institutions by giving us access to WSG AM-01 patients' archival tumor material. We thank the Jürgen-Manchot-Stiftung, Düsseldorf, Germany, for support of this research project. A.

Gaumann was supported by a DFG grant GA1092/2-1. We are grateful to Martina Waeber and Matthias Hornberg and Rudolf Jung for excellent technical assistance. This publication contains data of the dissertation thesis of V. Artinger, E. Ehm, and H. Mendrik.


Ivanka Petrova Boyadzhieva

Miroslava Atanasova

Elka Emanuilova

A novel, thermostable manganese-containing superoxide dismutase from Bacillus licheniformis

Keywords: Bacillus licheniformis, Enzyme thermal stability, Manganese-containing superoxide dismutase, Purification

A new, thermostable superoxide dismutase (SOD) from Bacillus licheniformis M20, isolated from Bulgarian mineral springs, was purified 11-fold with 11% recovery of activity. From native PAGE and SDS-PAGE, the enzyme was composed of two subunits of 21.5 kDa each. The SOD was inhibited only by NaN 3 , which suggested that this SOD is of the manganese superoxide dismutase type. The purified enzyme had maximum activity at pH 8 and 55°C. The half-life of the SOD was 10 min at 95°C.

Introduction

Superoxide dismutases (SODs, EC 1.15.1.1) are metalloenzymes, classified into three types: Mn-, Fe-and Cu/Zn-SOD according to their metal cofactors (Whittaker 2003). They are involved in the detoxification of the superoxide radicals (О 2 -) to H 2 O 2 , which in turn is broken down to H 2 O by catalases and peroxidases. The superoxide radicals are generated by a single electron transfer to O 2 and can damage DNA, proteins and lipids [START_REF] Halliwell | Biochemistry of oxidative stress[END_REF]).

An important requirement for SODs intended for industrial applications is their thermal stability as thermal denaturation is a common cause of enzyme inactivation. In this connection, an increasing interest to the SODs from thermophiles and hyperthermophiles were observed (Wang 2008). Thermostable SODs have also a higher resistance to most chemical denaturants and could be very useful in the industries of pharmaceuticals, cosmetics, food, and biotechnology and in environmental protection. SODs are crucial components in the physiological response to oxygen toxicity and are actively investigated as potential therapeutic agents in pathological conditions related to oxidative stress. Moreover, SODs offer a great potential for the sensitive quantification of О 2 -in electrochemical biosensors [START_REF] Campanella | Superoxide dismutase biosensors working in nonaqueous solvent[END_REF]).

To our knowledge, studies describing SOD from Bacillus licheniformis are not available. Therefore, in the present work, the purification and biochemical properties of a novel, thermostable SOD from Bacillus licheniformis M20 are reported for the first time.

Materials and methods

Microorganism and growth conditions

Bacillus licheniformis M20 isolated from Bulgarian thermal springs (Dolna banja region), identified by 16S rRNA analysis and deposited in the EMBL GenBank DDBJ under accession number FN597717, was used. The strains was cultivated as described previously [START_REF] Boyadzhieva | Characteristics of bacterial strain, superoxide dismutase producer, isolated from Bulgarian thermal spring[END_REF].

SOD assay

SOD activity was determined by the method of [START_REF] Beauchamp | Superoxide dismutase: improved assays and an assay applicable to acrylamide gels[END_REF]. The reaction mixture in 3 ml was composed of 20 mM phosphate buffer (pH 7.8), 13 mM L-methionine, 75 μM NBT (Nitroblue Tetrazolium), 2 μM riboflavin and 20 μl purified enzyme. The test tubes were placed 30 cm below a fluorescent lamp (40 W).

The reaction was initiated by turning the light on and reduction of NBT was determined by following the reading A 560 for 5 min. One unit of SOD activity was defined as the amount of the enzyme causing one half of the maximum inhibition of NBT reduction.

Protein concentration was estimated according to the Bradford method, using bovine serum albumin as standard.

All analyses of each sample were carried out in two repetitions and the data presented were obtained after three independent experiments, i.e. the data are mean values ±SD, n=6.

Results and discussion

Purification of the SOD A summary of the purification steps is given in Table 1. A purification of 11-fold with a yield of 11% was achieved. The SDS-PAGE and PAGE analysis of the purified enzyme revealed a single band (Fig. 1a). The molecular weight of the SOD was 22 kDa by SDS-PAGE. The native PAGE and the zymogram showed a single active protein band with molecular weight 43 kDa (Fig. 1b). These data indicated that the SOD from B.

licheniformis M20 was dimeric. Most of the reported SODs are homodimers or homotetramers with subunits of about 20 kDa (Whittaker 2003). The purified SOD was completely inhibited by 20 mM NaN 3 , but not by KCN and H 2 O 2 , suggesting that the enzyme belonged to Mn-SOD classification (Fig. 1c).

Properties of the SOD

The SOD from Bacillus licheniformis M20 was catalytically active only in the presence of Mn 2+ (Table 2). The purified SOD was optimally active at pH 7.5-9, with maximum activity at pH 8 (Fig. 2a). The enzyme exhibited 60% relative activity at pH 9.5, unlike other Mn-SODs, which rapidly lost its activity at pH over 7.8 [START_REF] Seatovic | Purification and partial characterization of superoxide dismutase from the thermophilic bacteria Thermothrix sp[END_REF]). The optimum enzyme activity was established at 50-55ºC (Fig. 2b). The purified SOD from thermophilic Geobacillus kaustophilus ATCC8005 is optimal active at the same temperature, but with respect to the enzyme activity, SOD from B. licheniformis M20 exhibited 9780 U mg -1 , compared to 1780 U mg -1 for Geobacillus kaustophilus SOD [START_REF] Huang | Thermostable superoxide dismutase[END_REF]. SOD from B. licheniformis M20 was distinguished with a high thermal stability, as shown in Figure 3. The half-life of the enzyme was 10 min at 95ºC. In comparison with the SOD from thermophilic Rhodothermus sp. XMH10, having half-life 30 min at 80ºC (Wang et al. 2008) and the SOD from thermophile Thermomyces lanuginosus with half-life 30 min at 70-75ºC [START_REF] Li | A thermostable manganese-containing superoxide dismutase from the thermophilic fungus Thermomyces lanuginosus[END_REF], the SOD from B. licheniformis M20 had exceptionally better thermal stability.

The studied SOD from B. licheniformis M20, able to catalyze the conversion of superoxide ions or radicals under the moderate to high temperature environments is exceptionally useful in their removing or subtracting and has valuable potentials in different biotechnological processes. An advantage of the proposed SOD in the present work is its superior thermal stability, which could be provided a relatively longer functional action in the applied processes. The new, thermostable purified Mn-SOD from B. licheniformis M20 with its important features to be active at a high alkaline pH of the reaction media (8; 9-9.5) and high temperatures (75-95ºC) could be a candidate for industrial uses.

Wang X, Yang H, Ruan L, Liu X, Li After 5 h cultivation, the cells were collected by centrifugation (4500 x g for 15 min), and washed with 20 mM phosphate buffer (pH 7.8). The pellets were suspended in the same buffer and the cells were ruptured by ultrasonic disintegration (three times in an ice bath for 2 min at 40 V), and then the cell debris were removed by centrifugation (10000 x g for 15 min, 4°C). The resulting supernatant was precipitated with 40-60% saturated (NH 4 ) 2 SO 4 . After centrifugation (10000 x g, 30 min, 4°C), the precipitate was dissolved in 20 mM phosphate buffer and dialyzed against the same buffer overnight at 4°C. The sample was loaded to a Phenyl-Sepharose column (2.5 x 15 cm), pre-equilibrated with 20 mM Tris/HCl buffer (pH 7.8), containing 2.8 M (NH 4 ) 2 SO 4 . The elution was carried out with the same buffer by stepwise decrease in the ionic strength of (NH 4 ) 2 SO 4 ranging from 2.8 to 0.7 M. The fractions with SOD activity were pooled, dialyzed against 20 mM phosphate buffer and loaded to a DEAE-Sepharose column (2.5 x 20 cm), equilibrated with 20 mM phosphate buffer. Elution of SOD was done with a linear gradient from 20 to 100 mM phosphate buffer, pH 7.8. The active fractions were pooled and concentrated by ultrafiltration using PM-10 membrane (10 kDa; Amicon, Millipore, USA). The enzyme concentrate was applied to a Sephadex G-100 column, and then was loaded on Sephadex G-50 column. The initial activity of native SOD 9700 U mg -1 ± 1.7 % is taken as 100%. enzyme was considered as 100% (9810 U mg -1 ). Standard deviations of residual activities were in range of 0.7-2.7%

Fig. 1

 1 Figure captions

Fig. 2 Fig. 3

 23 Fig. 2 pH (a) and temperature profile (b) of the purified SOD from Bacillus licheniformis M20.(a) SOD activity was measured under standard assay conditions at pH range of 5-12. The following buffers were used: 0.2 M Na-acetate-CH 3 COOH (pH 5-6), 0.2 M potassium-sodium phosphate (pH 6.5-7.5) and 0.2 M glycine-NaOH-NaCl (pH 8-12). The enzyme activity corresponding to 100% was 9670 U mg -1 . Standard deviations of relative activities were in range of 0.5-1.4%. (b) SOD activity was measured under standard assay conditions at temperatures over the range 30-90ºC in 20 mM phosphate buffer, pH 7.8. The enzyme activity corresponding to 100% was 9550 U mg -1 . Standard deviations of relative activities were in range of 1.5-3.4%

  Fig. 1

Table 1

 1 F, Xu X (2008) Cloning and characterization of a thermostable superoxide dismutase from the thermophilic bacterium Rhodothermus sp. XMH10. J Ind Microbiol Purification process of SOD from B. licheniformis M20

	Biotechnol 35:133-139
	Whittaker JW (2003) The irony of MnSOD. Biochem Soc Trans 31:1318-1321

Table 2

 2 Specific activity of native, apo-and metal-reconstituted apo-SOD from B. licheniformis M20

	Purification steps	Total protein	Total activity	Specific activity	Enzyme yield	Purification
		(mg)	(kU)	(U mg -1 )	(%)	( fold )
	Crude extract	390	351	900	100	1
	40%-60% (NH 4 ) 2 SO 4	100	200	2000	57 ± 1.14	2.2
	Phenyl-Sepharose	40	168	4200	48 ± 0.96	4.7
	DEAE-Sepharose	10	60	6000	17 ± 0.34	6.7
	Sephadex G -100	5	41.75	8350	11.8 ± 0.35	9.2
	Sephadex G -50	4	39.12	9780	11.1 ± 0.28	10.8

Gel filtration was carried out with 20 mM phosphate buffer, pH 7.8, on a self-packed column (2.5 x 25 cm)


Stanislav Obruca

Ivana Marova

Ondrej Snajdar

Ludmila Mravcova

Zdenek Svoboda

Section: Microbial and Enzyme Technology Production of poly(3-hydroxybutyrate-co-3hydroxyvalerate) by Cupriavidus necator from waste rapeseed oil using propanol as a precursor of 3-hydroxyvalerate

Keywords: Cupriavidus necator, Polyhydroxyalkanoates, Propanol, Stress conditions, Waste edible oils

Waste rapeseed oil is a useful substrate for polyhydroxyalkanoates (PHA) production employing Cupriavidus necator H16. In fed-batch mode, we obtained biomass and PHA yields of 138 and 105 g l -1 , respectively. Yield coefficient and volumetric productivity were 0.83 g PHA per g oil and 1.46 g l -1 h -1 , respectively. Propanol at 1% (v/v) enhanced both PHA and biomass formation significantly and, furthermore, resulted in incorporation of 3-hydroxyvalerate units into PHA structure. Thus, propanol can be used as an effective precursor of 3-hydroxyvalarete for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. During the fed-batch cultivation, propanol concentration was maintained at 1 % which resulted in 8 % content of 3-hydroxyvalerate in copolymer.

Introduction

Polyhydroxyalkanoates (PHA) are polyesters of hydroxyacids, which are accumulated in bacterial cells in form of intracellular granules. Bacteria use PHA as carbon, energy and reducing power storage materials. Due to their mechanical properties, PHA have attracted much attention as biodegradable alternative to traditional petrochemical plastics (Kessler and Witholt 1999). The member of PHA family, copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HBco-HV)], is one of the best characterized PHA copolymers because of its high commercial potential. Incorporation of 3-hydroxyvalerate (3-HV) units into PHA structure reduces the hardness, crystallinity and melting point of material as compared to homopolymer of 3-hydroxybutyrate (3-HB), poly(3hydroxybutyrate) (PHB). Therefore, copolymer P(HB-co-HV) possesses better mechanical properties and processability (Du et al. 2001).

In spite of many satisfactory properties of PHA, their commercial applications have been limited by their high price. One approach to reduce the cost of PHA production is to use inexpensive carbon sources, such as plant oils, for their production. In contrast to other carbon sources, the theoretical yield coefficients of PHA production from plant oils are over 1 g PHA per g plant oil, since they comprise a much higher number of carbon atoms per weight (Kahar et al. 2003).

Waste edible oils from the food industry and food service industry are recovered as industrial wastes. They can be converted into feed for animals, fatty acids, soap or bio-diesel. Nevertheless, waste oils are largely destroyed in incinerators or are abandoned in the environment, which causes problems with waste management or water pollution (Taniguchi et al. 2003). These problematic waste oils could be used as cheap substrates for microbial production of PHAhighly valuable environmentally-friendly materials. This report describes production of PHB and P(HB-co-HV) from waste rapeseed oil employing Cupriavidus necator H16. In our previous work we reported that exposition of C. necator to controlled ethanol or H 2 O 2 stress enhances PHA accumulation in cells (Obruca et al. 2010a, Obruca et al. 2010b).

Therefore, we decided to test also other aliphatic alcohols as stress factors enhancing PHA yields. Finally, we performed fed-batch cultivation of C. necator in laboratory fermentor under controlled stress conditions using waste rapeseed oil as a carbon substrate.

Material and Methods

Microorganism

Cupriavidus necator H16 (CCM 3726) purchased from Czech Collection of Microorganisms, Brno, Czech Republic, was used in all experiments.

Culture media

Nutrient broth (NB) medium was used for inoculum development. NB consisted of 10 g peptone, 10 g beef extract, 5 g NaCl in 1 l distilled water.

Mineral salt (MS) medium was used in all production experiments. MS medium contained 3 g (NH 4 ) 2 SO 4 , 1 g KH 2 PO 4 , 11.1 g Na 2 HPO 4 .12 H 2 O, 0.2 g MgSO 4 , 1 ml microelement solution and 1 l of distilled water. The microelement solution was composed of 9.7 g FeCl 3, 7.8 g CaCl 2 , 0.156 g CuSO 4 .5 H 2 O, 0.119 g CoCl 2 , 0.118 g NiCl 2, 0.062 g CrCl 2 in 1 l 0.1 M HCl. Plant edible oils (20 g in 1 l of medium) were used as carbon sources in production media. Oils, salt solution and microelement solution were sterilised separately at 121°C for 25 min and then aseptically reconstituted at room temperature prior to inoculation. The pH was adjusted to 7.0 using 2 M NaOH /HCl. Pure oils were purchased in supermarkets; waste frying rapeseed and sunflower oils were obtained locally.

Cultivation in flasks

The microorganism was grown in NB medium at 200 rpm, 30°C for 24 h.

For production cultivation, 100 ml MS medium with oil (20 g l -1 ) was held in 300 ml Erlenmeyer flasks and inoculated with 5 ml culture. The flasks were shaken at (200 rpm and at 30°C).

Fed-batch cultivation in fermentor

Fermentor vessel (2 l, BioFlo Celligen 115, New Brunswick) containing 1.2 l MS medium and waste rapeseed oil (20 g l -1 ) was inoculated with 60 ml 24 h culture grown on NB medium. The temperature was set at 30° C, pH was maintained at 7 by 0.5 M NaOH/H 2 SO 4 . The dissolved oxygen (DO) concentration was monitored by an O 2 electrode. DO value was maintained at the level of 50 % to air saturation by varying the agitation speed and/or air flow rate automatically.

Analytical procedures

To measure biomass concentration (expressed as cell dry weight) and PHA content in cells, samples (10 ml) were centrifuged and the cells were washed with 5 % (v/v) Triton X (10 ml) and then distilled water. The biomass concentration was analyzed as reported previously (Obruca et al. 2010b). PHA content in cells and monomer composition of PHA were analyzed as methyl esters of particular 3hydroxyacids by gas chromatography as reported by [START_REF] Brandl | Pseudomonas oleovorans as a source of poly(beta-hydroxyalkanoates) for potential application as a biodegradable polyester[END_REF]. Residual biomass was defined as biomass minus PHA concentration.

The concentration of oil in the medium was estimated according to [START_REF] Kahar | High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain[END_REF]; (NH 4 ) 2 SO 4 was analyzed spectrophotometrically using Nessler's reagent. Propanol was measured as amount of NADH (A 340nm ) formed after 5 min treatment of sample (100 μl of cultivation medium diluted to 1 ml with 100 mM phosphate buffer pH 7.0) with 30 units alcohol dehydrogenase (Sigma).

RESULTS AND DISCUSSION

PHB production from various oils

To investigate the ability of Cupriavidus necator H16 to utilize plant oils and produce PHA, we performed cultivation of this bacterial strain on selected oils of various origins (Tab. 1). Growth of the bacterial culture and production of PHB homopolymer were observed with all tested plant oils. The highest yields were reached using waste frying oils. During the frying process, the oil undergoes some changesfatty acid composition is slightly changed, the concentration of free fatty acids is enhanced and also some components of fried food such as proteins, lipids or carbohydrates migrate to the oil (Matthäus 2007). It is likely that these changes were beneficial for bacterial growth and PHB accumulation. We expected that also application of other alcohols may have induced similar response of the culture resulting in enhanced PHA yields. Therefore, we added methanol ethanol and propanol into cultivation media at various times of cultivation (Table 2).

In terms of biomass as well as PHA yields, the most efficient was addition of alcohols after 24h. The alcohols not only enhanced PHA accumulation in comparison with the control culture but, surprisingly, also increased bacterial growth. The more non-polar alcohol was applied, the more pronounced growth was observed. The explanation of such an effect could be that the alcohols supported solubilization of triacylglycerols, which made them more susceptible to extracellular lipases. This is likely to have increased concentrations of products of lipolysis (glycerol and fatty acids) in medium that could support bacterial growth.

Ethanol and propanol enhanced PHA production significantly. In addition, metabolism of propanol resulted in incorporation of 3-hydroxyvalerate into PHA, which could improve mechanical properties of produced material. For instance, incorporation of 10 % 3-hydroxyvalerate increases elongation to break from 3 % (homopolymer PHB) to 20 % (copolymer (Kessler and Withholt 1999).

To our knowledge, the application of propanol as a precursor of 3hydroxyvalerate in P(HB-co-HV) has not been reported so far. In comparison with other commonly used precursors of 3-hydroxyvalerate, such as propionate or valerate, propanol has several advantages. Apart from the enhancement of PHA yields and the support of bacterial growth on oils, propanol is also much cheaper.

In Table 3, propanol is compared with other commonly used precursors of 3-HV.

We assume that the metabolism of propanol in C. neactor is similar to that of ethanol (Obruca et al. 2010b). Thus, propanol is oxidized and transformed into propionyl-CoA. During oxidation, reduced coenzymes are formed, which again supports PHA synthesis in the same way as in ethanol. Further, propionyl-CoA is coupled with acetyl-CoA in reaction catalyzed by -ketothiolase. -ketothiolase encoded by the gene phbA is specific only for acetyl-CoA, therefore, this step must be catalyzed by the enzyme encoded by the gene bktB which is placed out of phaCAB operon of C. necator (Slater et al. 1998). NADPH-dependent stereospecific reduction of 3-ketovaleryl-CoA could be catalyzed by acetoacetyl-CoA reductase. The final step of copolymer synthesis is catalyzed by PHB synthase which is, thanks to its low substrate specificity, able to build 3hydroxyvalerate units into PHA structure (Fig. 1).

The highest growth and PHA production was with 1 % (v/v) propanol but the highest content of 3-HV in PHA was with 0.75 % (v/v) propanol. (Table 4).

Controlled application of propanol as stress factor not only enhanced total PHB yields but also increased PHB content in cells. This would reduce the costs of PHB recovery, since PHB content in cells strongly affects the efficiency and the cost of down-stream processing (Lee and Choi 1999).

PHA production in fed-batch mode

Because production of P(HB-co-HV) from waste rapeseed oil using propanol as a stress agent seemed to be a prosing strategy, we performed fedbatch cultivation in a laboratory fermentor (Fig. 2).

After 72 h the cell dry weight was 138 g l -1 . Cells contained 76 % PHA with 8 % content of 3-HV. Total yield of PHA was 105 g l -1 . Volumetric productivity yield was 1.46 g l -1 h -1 and yield coefficient was 0.83 g PHA per g oil. To our knowledge, these yields achieved are among the highest reported for PHA production from oils. [START_REF] Kahar | High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain[END_REF] performed fed-batch cultivation of C. necator on soybean oil; yield coefficient and volumetric productivity yield were 0.76 g PHA per g oil and 1 g l -1 h -1 , respectively. Thus, we gained better yields and, moreover, we only used cheap waste oil instead of pure oil.

Conclusion

We have shown that utilization of waste edible oils is a promising strategy facilitating an economically feasible process of PHA production. Waste oils are lower in price as compared to other pure substrates. Moreover, problematic waste would be transformed into high value product. In fed-batch mode of cultivation, we gained high yield coefficient and volumetric productivity yield 0.83 g PHA per g oil and 1.46 g l -1 h -1 , respectively. Furthermore, we suggest propanol as a novel precursor of 3-hydroxyvalerate for P(HB-co-HV) copolymer production.

Apart from the fact that propanol is built into PHA structure, which improves mechanical properties of material, it also enhances PHA yields and supports bacterial growth on oils. b Molar content (%) of particular monomer in polymer.

Results are in form: mean ± standard deviation, each cultivation was performed in triplicate and analyzed in triplicate as well. Fig. 2 PHA production form waste rapeseed oil in fed-batch mode. Propanol was added at 18 h and after that its concentration was maintained at 1 % (v/v), (NH 4 ) 2 SO 4 was maintained at 3 g l -1 until 30 h when nitrogen feeding was stopped to induce nitrogen limitation. The concentration of carbon sourcewaste rapeseed oil was kept at 20 g l -1 . a = biomass, PHA and residual biomass developments. b = ammonium sulfate and oil concentrations. c = propanol concentration and 3-HV content in the copolymer. Each sample was analyzed in triplicate.

  Although waste oils seem to be a promising feedstock for PHA production, only a few authors have reported PHA production on these substrates.Taniguchi et al. (2003) reported PHA production from waste sesame oil employing C.necator; the highest achieved PHB yield was 4.6 g l -1 .Chan et al. (2006) studied PHA production from various plant oils including waste oil using Pseudomonas aeruginosa, but PHA content in cells was only about 5 % of dry cell weight.Finally, Vidal-Mas et al. (2001) cultivated Pseudomonas aeruginosa on waste frying oil and reached PHA yields of 3 g l -1 . Nevertheless, in this work we obtained PHB yields which were significantly higher than those reported in literature. The highest PHB yields were achieved on waste rapeseed oil coming from the university canteen and, therefore, this oil was used as a carbon substrate in further cultivations.Introduction of stress conditions by alcoholsIn our previous work we observed that exposition of C. necator to ethanol significantly enhanced PHB accumulation in cells(Obruca et al. 2010b). Briefly, ethanol was metabolized via oxidation while reduced coenzymes NAD(P)H were formed. The final product of ethanol metabolization was acetyl-CoA, the key substrate of PHB biosynthesis. Reduced coenzymes both inhibited Krebs cycle and served as co-substrates of acetoacetyl-CoA reductase -the second enzyme of PHB biosythetic pathway. As the consequence of Krebs cycle inhibition, the flux of acetyl-CoA into PHB biosynthetic pathway was increased. Simultaneously, less free CoA, which inhibited -ketothiolase -the first enzyme of PHB biosynthesis, was formed.

Fig. 1

 1 Fig. 1 Proposed mechanism of metabolization of propanol by C. necator H16 and its incorporation into P(HB-co-HV) copolymer structure.

Table 1

 1 Growth and PHB production of C. necator H16 on various oils a

		Biomass	PHB	PHB
	Oil	[g.l -1 ]	[g.l -1 ]	[% w/w]
	Olive	8.1 ± 0.2	3.3 ± 0.3	40
	Corn	10.6 ± 0.6	4.4 ± 0.4	40
	Soybean	7.5 ± 0.4	2.0 ± 0.1	28
	Sunflower	9.4 ± 0.2	4.6 ± 0.4	49
	Rapeseed	9.5 ± 0.4	4.8 ± 0.4	50
	Waste -Household (sunflower)	10.3 ± 0.6	5.8 ± 0.4	57
	Waste -University canteen (rapeseed)	12.8 ± 0.5	7.7 ± 0.4	60
	Waste -Restaurant (sunflower)	11.4 ± 0.5	6.7 ± 0.1	59
	Waste -Chips manufactory (rapeseed)	12.2 ± 0.6	6.8 ± 0.5	55
	a Cultivation conditions: MS medium, oil 20 g l -1 , Erlenmeyer flasks, 30° C, 200 rpm, samples
	were withdrawn at the 72 nd hour of cultivation.
	Results are in form: mean ± standard deviation, cultivations were performed in triplicate and
	analyzed in triplicate as well.

Table 2

 2 Biomass and PHA production after alcohols applications a .

Table 3

 3 Production of P(HB-co-HV) on waste rapeseed oil using selected precursors of 3-HV a . Cultivation conditions: MS medium, oil 20 g l -1 , Erlenmeyer flasks, 30° C, 200 rpm, samples were withdrawn at 84 h. Propanol was added at 24 h.

	Propanol	Biomass	PHA	PHA	3-HB	3-HV
	[% v/v]	[g.l -1 ]	[g.l -1 ]	[% w/w]	[% ]	[% ]
	0.5	12.3 ± 0.7	7.8 ± 0.1	63	94	6
	0.75	12.9 ± 0.4	9.1 ± 1.0	70	86	14
	1	13.7 ± 0.2	10.8 ± 0.3	79	89	11
	1.25	13.0 ± 0.1	8.7 ± 0.5	67	90	10
	1.5	12.1 ± 0.2	8.3 ± 0.7	69	91	9
	Control	11.2 ± 0.7	6.4 ± 0.6	58	100	0

a

Ackonowledgements This work was supported by projects MSM 0021630501 and CZ.1.05/2.1.00/01.0012 of the Czech Ministry of Education

Propanol (1 % v/v) 14.7 ± 0.3 11.7 ± 0.7 80 91 9

Propionate (5 g l -1 ) 14.1 ± 0.3 7.3 ± 0.8 52 87 13

Valerate (5 g l -1 ) 12.1 ± 0.5 6.5 ± 0.2 53 82 18 Control 11.2 ± 0.6 5.9 ± 0.4 53 100 0

a Cultivation conditions: MS medium, oil 20 g l -1 , Erlenmeyer flasks, 30° C, 200 rpm, samples were withdrawn after 84 h. Precursors were applied at 24 h. Propanol concentration in medium was set at 1 % (v/v), propionate and valerate was 5 g l -1 .

b Molar content (%) of particular monomer in polymer.

Results are in form: mean ± standard deviation; cultivations were performed in triplicate and analyzed in triplicate as well.