Fadi Boulos

Wei Chen

Benoît Parrein

Patrick Le Callet

A NEW H.264/AVC ERROR RESILIENCE MODEL BASED ON REGIONS OF INTEREST

Keywords: Eye tracking, region of interest, packet loss, error resilience, perceptual quality

Video transmission over the Internet can sometimes be subject to packet loss which reduces the end-user's Quality of Experience (QoE). Solutions aiming at improving the robustness of a video bitstream can be used to subdue this problem. In this paper, we propose a new Region of Interest-based error resilience model to protect the most important part of the picture from distortions. We conduct eye tracking tests in order to collect the Region of Interest (RoI) data. Then, we apply in the encoder an intra-prediction restriction algorithm to the macroblocks belonging to the RoI. Results show that while no significant overhead is noted, the perceived quality of the video's RoI, measured by means of a perceptual video quality metric, increases in the presence of packet loss compared to the traditional encoding approach.

INTRODUCTION

With Internet becoming the cheapest and preferred medium of communication, video traffic over IP is in constant and sharp increase. On one hand, this is made possible by the increasing broadband speeds and the variety of multimedia services offered by Internet Service Providers, e.g., triple-play offers. On the other hand, recent video coding standards such as H.264/AVC [1] allow compression rates for up to twice those of their predecessors, thus making it possible to stream Standard Definition (SD) or High Definition (HD) video contents over the Internet.

However, packet loss still characterizes the best-effort Internet. To overcome this problem, several solutions have been proposed at both the channel and source levels. Mechanisms like Forward Error Correction (FEC) or Automatic Repeat reQuest (ARQ) can be used to compensate for the packets lost during transmission. At the source level, some error resilience features used during the encoding process can help in attenuating the impact of packet losses. In the H.264/AVC standard, Flexible Macroblock Ordering (FMO), Data Partitioning (DP) and Redundant Slice (RS) are examples of error resilience features.

Due to the very nature of video compression techniques, a packet lost from the encoded bitstream has generally a spatiotemporal propagating effect. This is largely due to spatiotemporal dependency between parts of the bitstream. In an earlier study [START_REF] Boulos | Perceptual Effects of Packet Loss on H.264/AVC Encoded Videos[END_REF], we showed that the following two parameters have a great impact on the perceived quality: (1) the spatial position of the loss in the picture, i.e., if it belongs or not to the Region of Interest (RoI) of the picture; and (2) the temporal position of the loss in the sequence. In this work, we propose to link these two parameters to prevent the error from propagating to the RoIs of the video sequence. To this end, we force the macroblocks that belong to an RoI to be coded in intra-prediction mode, thus removing their temporal dependency on macroblocks in other pictures. We also propose an extension to this algorithm to remove the spatial dependency.

The outline of the paper is as follows: in Section 2, we give an overview of some state-of-the-art RoI-based error resilience models. Then we describe the eye tracking tests we performed in order to determine the saliency maps of SD and HD video sequences in Section 3. We also present the methodology of transforming these maps into RoIs. In Section 4, we propose several variants of our method to take advantage of the RoIs by forcing RoI macroblocks to be coded in intra-prediction mode. We validate our method by simulating packet losses in the RoI and assessing the perceived video quality by means of a perceptual video quality metric, namely VQM [3]. The block diagram of the whole processing chain, from encoding to performance evaluation is depicted in Figure 1.

RELATED WORK

While RoI-based video coding is probably the most used application for deriving RoIs, e.g., in [START_REF] Agrafiotis | Multiple Priority Region of Interest Coding with H.264[END_REF][START_REF] Dhondt | Flexible macroblock ordering as a content adaptation tool in H.264/AVC[END_REF], using the RoI in error resilience models has also been investigated. In all of the following research works, RoI-based models have been coupled with an H.264/AVC error resilience feature, namely 978-1-4244-4652-0/09/$25.00 ©2009 IEEE FMO. FMO allows the ordering of macroblocks in slices according to a predefined map rather than using the raster scan order, e.g., to improve the robustness of the video against transmission errors or to apply an Unequal Error Protection (UEP). When coupled with RoI-based coding, FMO is generally used to assemble the RoI macroblocks into a single slice.

In [START_REF] Van Leuven | An Implementation of Multiple Region-Of-Interest Models in H.264/AVC[END_REF], multiple RoI models are proposed to enhance the quality of video surveillance pictures. The RoIs are defined by the user in an interactive way: the mouse pointer is put over the RoI and the coordinates of the pixel position pointed to are transmitted to the encoder. Then, every model will build its own RoI (e.g., square-shaped, diamond-shaped) coupled with an FMO type. For more information about FMO types, the reader is referred to [START_REF] Lambert | Flexible macroblock ordering in H.264/AVC[END_REF]. Results show that a convenient selection of the RoI shape, the quantization parameter and the FMO type can reduce bandwidth usage while maintaining the same video quality.

In [START_REF] Baccichet | Systematic Lossy Error Protection based on H.264/AVC Redundant Slices and Flexible Macroblock Ordering[END_REF], an error resilience model where RoIs are derived on a per picture basis is proposed. Each picture RoI is determined by simulating slice losses and corresponding error concealment at the encoder to build a distortion map. Macroblocks with the highest distortion values are coded into Redundant Slices (RS), which is another H.264/AVC resilience feature and background macroblocks are signalled using FMO type 2. Simulation results demonstrate the efficiency of this method compared to the traditional FEC approach in the presence of packet loss.

The work reported in [START_REF] Kodikara Arachchi | Unequal Error Protection Technique for ROI Based H.264 Video Coding[END_REF] aims at improving the robustness of the video by applying UEP wherein the RoI benefits from an increased protection rate along with a checkerboard FMO slicing. The authors conclude that their approach outperforms UEP and Equal Error Protection (EEP) for lower Signal-to-Noise-Ratio (SNR) values.

A robustness model for RoI-based scalable video coding is proposed in [START_REF] Chen | Robust Region-of-Interest Scalable Coding with Leaky Prediction in H.264/AVC[END_REF]. The model divides the video into two layers: the RoI layer and the background layer. Dependencies between the two layers are removed to stop the error from propagating from the background layer, which is less protected than the RoI layer, to the latter in case of packet loss. This process decreases coding efficiency in error-free environments but enhances the video robustness in the presence of packet loss.

EYE TRACKING TESTS

The goal of performing eye tracking tests is to record the eye movement of the viewers while they are watching video sequences. These data can then be used to achieve a RoI-based error resilient video coding. In this section, we first describe the test setup and the set of videos used then we explain how the RoIs are generated for each video sequence. We also present the results of the tests and discuss them.

Setup

We use a dual-Purkinje eye tracker (Figure 2) from Cambridge Research Systems. The eye tracker is mounted on a rigid EyeLock headrest that incorporates an infrared camera, an infrared mirror and two infrared illumination sources. The camera records a close-up image of the eye. To obtain an accurate measure of the subject's pupil diameter, a calibration procedure is needed. The calibration requires the subject to stare at a number of screen targets from a known distance. Once the calibration procedure is completed and a stimulus has been displayed, the system is able to track a subject's eye movement. Video is processed in real-time to extract the spatial location of the eye's position. Both Purkinje reflections (from the two infrared sources) are used to calculate the location. The sampling frequency is 50 Hz and the tracking accuracy is in the range 0.25 -0.5 degree.

The video testbed contained 30 SD and 38 HD source sequences, 23 of which were common to both resolutions. These 23 720 × 576 SD sequences were obtained from 1920 × 1080 HD by cropping the central region of the picture (220 pixels from right and left borders) and resampling the obtained video using a Lanczos filter. Several loss patterns were applied to 20 SD and 12 HD source sequences, thus increasing the total number of videos to 100. The sequences had either an 8-second or a 10-second duration. To ensure that the RoI extraction is faithful to the content independently of other parameters, all of the sequences were encoded such as to obtain a good video quality. Bitrates were in the range of 4 -6 Mbs and 12 -16 Mbs for SD and HD sequences, respectively. The video sequences were encoded in High Profile with an IBBPBBP... GOP structure of length 24. The JM 14.0 [START_REF]AVC reference software[END_REF] encoder and decoder were used.

We formed two sub-tests of 50 videos each to avoid having a content viewed twice (in both resolutions) by the same subject during a sub-test, which could skew the RoI deriving process. We also randomized the presentation order within the sub-test.

Eye tracking data of 37 non-expert subjects with normal vision (or corrected-to-normal vision) were collected for every video sequence. The test was conducted according to the International Telecommunication Union (ITU) Recommenda- tion BT.500-11 [START_REF]Methodology for the subjective assessment of the quality of television pictures[END_REF]. Before starting the test, the subject's head was positioned so that their chin rested on the chinrest and their forehead rested against the head-strap. Subjects were seated at a distance of 3H and 6H for HD and SD sequences, respectively. All sequences were viewed on an LCD display. The average sub-test completion time was 25 minutes.

RoI generation

A saliency map describes the spatial locations of the eye gaze over time. To compute a saliency map, the eye tracking data are first analyzed in order to separate the raw data into fixation and saccade periods. Fixation is defined as being the status of a region centered around a pixel position which was stared at for a predefined duration. Saccade corresponds to the eye movement from one fixation to another. The saliency map can be computed for each observer and each picture using two methods. The first method is based on the number of fixations (NF) for each spatial location; hence, the saliency map SM (k) N F for viewer k is given by:

SM (k) N F (x, y) = N F P j=1 δ(x -x j , y -y j )
where N F P is the number of fixation periods and δ is the Kronecker function. Each fixation has the same weight.

The second method is based on the fixation duration (FD) for each spatial location. The saliency map SM (k) F D for viewer k is given by:

SM (k) F D (x, y) = N F P j=1 δ(x -x j , y -y j ).d(x j , y j )
where d(x j , y j ) is the fixation duration at pixel (x j , y j ).

To determine the most visually important regions, all saliency maps are merged yielding an average saliency map SM . The average saliency map is given by:

SM (x, y) = 1 K K k=1 SM (k) (x, y)
where K is the total number of viewers. Finally, the average saliency map is smoothed with a 2D Gaussian filter which gives the density saliency map:

DM (x, y) = SM (x, y) * g σ (x, y)
where the standard deviation σ depends on the accuracy of the eye tracking device.

To generate RoI maps from saliency maps, some parameters need to be set. The parameters are: fixation duration threshold, fixation velocity threshold, σ and the RoI threshold. The fixation duration threshold (in milliseconds) is the minimal time a region must be viewed for it to be considered as a fixation region. The fixation velocity threshold (in degrees per second) is the eye movement velocity threshold below which the velocity must remain for fixation duration threshold ms. RoI threshold is the minimal number of viewers who must view a region to be considered as a fixation region. The values of fixation duration threshold, fixation velocity threshold and σ were 200 ms, 25 °/s and 1.5, respectively, for both resolutions while the RoI threshold values were 4 and 2 for SD and HD sequences, respectively. Examples of saliency and RoI maps obtained with these parameter values are illustrated in Figures 3 and4

Results and discussion

Depending on its saliency map, a video sequence can have or not an RoI. In Figure 3, it is clear that the harpist in the video attracts the visual attention of the viewers. By contrast, the sparse saliency regions in Figure 4 do not result in any RoI for this particular video sequence.

We draw two main conclusions from the eye tracking test data: • The RoI of a video content is identical for both SD and HD resolutions.

• In the presence of a packet loss, the spatial position of the RoI can change depending on several parameters stated below.

While the first conclusion is somewhat expected, the second is worth discussing. The same packet loss pattern was applied to all the sequences to be impaired. The loss pattern consisted of losing five slices from the 6th I-picture of the sequence, two of them being the first two slices of the picture and the three others its last three. Thus, the Packet Loss Rate (PLR) in the I-picture was in the ranges 5% -20% and 2% -5% for SD and HD sequences, respectively. The losses having occurred in the top and bottom regions of the picture, they were not generally in its RoI. In a video sequence having a clear RoI (e.g., the ball in a football game, the face in a head-and-shoulder scene), a loss in an unimportant region of the picture might not be perceived by the user, whose attention is focused on the action in the RoI. However, when there is no clear RoI, any small loss may attract the user's attention. The nature of the scene content also influences the perception of a loss outside the RoI. While this topic deserves to be investigated more deeply, it is not covered by the scope of this paper.

RESILIENCE MODEL

We implement an RoI-based error resilience model in the H.264/AVC encoder. The model reduces the dependencies between important and unimportant regions of the picture. To test its efficiency, we perform a controlled packet loss simulation on the encoded bitstream. We then decode the distorted bitstream and evaluate the quality of the decoded video using a perceptual quality metric.

Forced intra-prediction

In order to prevent error propagation from reaching the RoI in B and P-pictures, we propose to force the macroblocks in an RoI in these pictures to be predicted in intra-prediction mode. This makes the RoI independent from past or future pictures. To this end, we implement in the JM encoder an algorithm that operates as follows: for each macroblock of a B or Ppicture, it checks if the macroblock belongs to the picture's RoI by comparing its coordinates to the coordinates given to the encoder in the form of an RoI text file. When a macroblock is flagged as being an RoI macroblock, its prediction type is forced to be intra. The selection of a macroblock's prediction type in H.264/AVC being based on the minimization of a distortion measure between the original and the predicted pixels, we choose to force the encoding algorithm to change the prediction type of an RoI macroblock (from inter to intra) by increasing the distortion measure computed for this choice. The process is illustrated in Figure 5(a) and the pseudocode of the algorithm is given below.

Algorithm 1 Forced RoI intra-prediction. while reading(eye tracking data)

for all B and P-pictures for all MBs in a picture if MB ∈ RoI then while (predT ype == anyInterpredT ype) do increase cost function end while else proceed normally end if end for end for end while By forcing some macroblocks in B and P-pictures to be coded in intra mode, the quality of the video may decrease. The explanation is the following: the encoding is done at a constant bitrate, thus the number of bits to be consumed is the same for all coding schemes. The intra-coded macroblocks consume more bits than they would have if they were coded in inter-prediction mode. This results in higher quantization parameters for some macroblocks in the video.

Forcing the RoI macroblocks to be coded in intra does halt completely the temporal error propagation but not the spatial propagation. In fact, the macroblocks to the top and left of an RoI macroblock in a B or P-picture might not belong to the RoI. Thus, these macroblocks can be coded in inter-prediction mode. If the reference macroblocks of these inter-predicted macroblocks are lost and/or concealed, the error can propagate to these macroblocks. The intra-prediction being spatial, using these macroblocks to perform the intra-prediction propagates the error to the RoI.

We propose to extend the algorithm in order to cope with this situation. We establish a "security neighborhood" around each RoI macroblock to attenuate the impact of using as a reference, a macroblock using itself a lost and/or concealed reference. This security neighborhood consists of coding the top left, top center, top right and left macroblocks surrounding an RoI macroblock in intra-prediction mode. If one of those four macroblocks is in the RoI, it is not considered as it will definitely be intra-coded. This is best illustrated in Figure 5(b) where we can see that some of the dependencies in Figure 5(a) have disappeared.

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Loss simulation

We use a modified version of the loss simulator in [START_REF]Joint Video Team (JVT) of ISO/IEC MPEG and ITU-T VCEG. SVC/AVC Loss Simulator[END_REF] to generate the transmission-distorted bitstreams. This simulator provides the possibility of finely choosing the exact slice to lose in the bitstream. At the encoding side, we take some practical considerations into account, namely we set the maximum slice size to 1450 bytes which is less than the Maximum Transmission Unit (MTU) for Ethernet (1500 bytes). The unused bytes (i.e., 50) are left for the RTP/UDP/IP headers (40 bytes) and the possible additional bytes that could be used beyond the predefined threshold. In this case, every Network Abstraction Layer Unit (NALU), which contains one slice of coded data can fit in exactly one IP packet. This makes our simulation more realistic because we can map the Packet Loss Rate (PLR) at the NAL level to the PLR at the application layer (e.g., RTP).

In our simulation, we never lose an entire picture; rather, we lose M slices of a picture where M < N , N being the total number of slices in the picture. When parts of a picture are lost due to packet loss, the error concealment algorithm implemented in the JM decoder is executed. This non-normative algorithm performs a weighted sample averaging to replace each lost macroblock in an I-picture and a temporal error concealment (based on the motion vectors) for lost macroblocks in a B or P-picture. The algorithm is described in detail in [14].

Because the macroblocks of an RoI are not confined in one slice, the RoI generally spans over three or more slices. To lose part or all of the RoI, we simulate the loss of three and five slices in the RoI of the 5th I-picture to evaluate the error propagation impact on quality. We use two loss patterns for quality evaluation: three contiguous slices and five noncontiguous slices, all containing RoI macroblocks. The goal of the loss simulation is to test the efficiency of our approach w.r.t. error propagation. Thus, we only target I-slices and look at how the algorithm copes with the spatio-temporal error propagation in subsequent B and P-pictures.

Quality assessment

To assess the quality of a video sequence one could either perform subjective quality tests or use an objective quality metric. During a subjective test, a group of viewers is asked to rate the quality of a series of video sequences. The quality score is chosen from a categorical (e.g., bad, excellent) or numerical (e.g., 1 -5, 1 -100) scale. An objective video quality metric evaluates the quality of a processed video sequence by performing some computations on the processed video and often on the original video too. While subjective tests are the most reliable way of assessing the quality of video sequences, they are time-consuming and require a large number of participants. Hence, a number of objective metrics providing a reliable quality assessment have been proposed to replace the subjective tests. The most widely used objective quality metric is the Peak Signal-to-Noise Ratio (PSNR). However, the performance of the PSNR metric is controversial [START_REF] Wang | Objective Video Quality Assessment[END_REF]. Therefore, we propose to use in this work a perceptual video quality metric: VQM.

Video Quality Metric

Video Quality Metric is a standardized objective video quality metric developed by the Institute for Telecommunication Sciences (ITS), the engineering branch of the National Telecommunications and Information Administration at the U.S. Department of Commerce [16]. VQM divides original and processed videos into spatio-temporal regions and extracts quality features such as spatial gradient, chrominance, contrast and temporal information. Then, the features extracted from both videos are compared, and the parameters obtained are combined yielding an impairment perception score. The impairment score is in the range 0 -1 and can be mapped to the Mean Opinion Score (MOS) given by a panel of human observers during subjective quality tests. For example, 0.1 and 0.7 are mapped to MOS values of 4.6 and 2.2 on a 5-grade scale, respectively. Note that VQM can be applied over a selected spatio-temporal region of the video to assess exactly its quality. In our experiments, we used the VQM Television model which is optimized for measuring the perceptual effects of television transmission impairments such as blur and error blocks.

Results

We compare the quality of the same sequence (Harp) encoded with the unmodified JM encoder and with three variants of our algorithm: the first one is the classical approach, denoted hereafter by RoI coding 1. RoI coding 2 denotes our approach taking into account the security neighborhood. We also implemented a variant RoI coding 3 of our approach that considers P-pictures only. The slices are lost from frame 97 which is the 5th I-picture of the sequence.

In Figure 6, the VQM impairment perception scores are plotted for each coding scheme. These scores are computed over the full spatial and temporal resolutions of the video. For the no loss case, the encoding quality of all schemes is practically the same. For the two loss patterns, the impairment generally seems to be more annoying when using the variants RoI coding 2 and RoI coding 3 of our algorithm. This trend is inverted in Figure 7, where the impairment perception scores are computed only over the RoI of the picture. Only the scores of the two loss patterns are plotted in this figure . The results show that all three variants of the algorithm outperform the Normal coding approach, although sometimes slightly. We also note that the impairment perception score for the 3-slice loss is greater than the 5-slice loss' for RoI coding 2 and RoI coding 3. This probably results from the contentdependency of the error concealment algorithm because the two loss patterns hit different slices.

Figure 8 depicts the impairment scores calculated over a smaller temporal region, namely 100 consecutive frames. The spatial dimensions of the evaluated region are delimited by the RoI of the picture as in the previous case. Reducing the temporal length of the region to be evaluated makes the distortion impact measure more accurate. The length is chosen such as to cover the GOP that contains the I-picture where the slices are hit and following B and P-pictures. We select 100 frames because VQM requires a temporal region of at least four seconds. The scores in Figure 8 demonstrate the efficiency of the proposed algorithm on the local region where it performs. For instance, the impairment perception score of the 3-slice loss case is 0.34 for Normal coding scheme while it decreases to 0.14 for RoI coding 1.

Discussion

The test results show that the video quality for all loss patterns was generally in the higher values of the quality scale (lower impairment perception scores). Visually, this was not always true. Some distorted frames of the Harp sequence given in Figure 9 illustrate this claim. This is probably due to the VQM not being perfectly adapted for RoI-based losses, i.e., no special weights are attributed to the losses in the RoI during error pooling.

Figures 7 and8 clearly demonstrate that RoI coding 1 is the most adapted coding scheme in the presence of bursty losses while RoI coding 3 works best for single losses. The almost equal impairment scores given by VQM for the no loss case in Figure 6 show that the algorithm used does not incur a significant extra encoding cost, namely a quality decrease. Results for the 3-slice and 5-slice losses in this same figure might indicate that the algorithm fails to cope with the loss patterns. However, the error propagation for Normal coding and RoI coding 1 schemes, illustrated in Figure 9, shows that our algorithm performs well in the presence of losses. Further, while the error propagation is progressively attenuated in Normal coding scheme, it is drastically in RoI coding 1 starting from frame 99 which is 2 frames away from the I-picture hit.

In Figure 10, two differently encoded versions of frame 103 of Harp sequence are depicted. The green box indicates the RoI of the picture. While the shape of the face is generally preserved when using the RoI intra-coding scheme (Figure 10(b)), we can see clearly that this is not the case with Normal coding (Figure 10(a)). The block effect appearing in the RoI of the picture for RoI coding 1 scheme is due to the spatial dependency between the macroblocks adjacent to the RoI and the RoI macroblocks. When coding a macroblock in intraprediction mode, the encoder checks if any of the upper and left macroblocks are available (i.e., existing or coded in intra mode). If no macroblock is available, it uses the DC intraprediction mode (intra-coding mode 2) which computes the average of the upper and left samples. The upper and left macroblocks being inter-predicted from a lost and/or concealed reference, a block distortion appears in the RoI. The high impairment perception scores obtained in Figure 6 could be due to the fact that VQM penalizes the block effects much more than other distortions. Note that using a security neighbor-hood did not show a significant improvement over RoI coding 1.

To overcome the spatial error propagation limitation, and in an RoI-based video coding perspective, we propose to create a new RoI-based prediction type that will be applied to all RoI macroblocks which do not have an upper or left available RoI macroblock. In this case, the RoI macroblock would be intra-coded as if it were the top left macroblock of the picture. However, doing so will render the bitstream non-standard compliant. To counter this problem, we suggest to use a specific signalling for this prediction type. In the worst case, this scheme would create an overhead of one bit per intra-coded macroblock to signal this new intra-prediction type. Intracoded macroblocks in a sequence comprise all macroblocks of I-pictures, the occasional intra-coded macroblocks and RoI macroblocks of B and P-pictures. We believe that this slight modification in the H.264/AVC can greatly improve the robustness of the bitstream against packet loss while not incurring a significant overhead. This new coding scheme can be thought of as a "cheap" FMO (in terms of overhead) because it creates totally independent regions in the picture.

On the other hand, to improve the security neighborhood variant which did not significantly increase the resilience model performance, we propose to force any RoI macroblock with at least one available RoI macroblock for intra-coding to use it as a reference to avoid that it uses a non-RoI macroblock.

CONCLUSION AND FUTURE WORK

We presented in this paper a new H.264/AVC error resilience model implemented in the encoder. The model, which is based on RoI data collected through an eye tracking experiment, aims at removing the dependencies between the RoIs in B and P-pictures and the reference picture(s). We described the test procedure and the post-processing that is applied to the saliency maps in order to obtain the RoI maps. We tested the efficiency of the proposed model by simulating packet loss on RoI-encoded video sequences and then evaluating their perceived quality. Results show that the RoI intra-coding algorithm outperforms the normal encoding locally and preserves the shape of the RoI.

This work can be further improved by (1) completely removing the spatial dependency between RoI macroblocks and adjacent non-RoI macroblocks; (2) performing subjective tests for video quality assessment; and (3) incorporating an objective saliency computational model (e.g., [START_REF] Itti | A Model of Saliency-Based Visual Attention for Rapid Scene Analysis[END_REF]) in the encoder which would steer the intra-prediction restriction algorithm. If the model chosen is reliable, it could be used as an alternative to eye tracking tests which are expensive in terms of both time and human resources. We are also working towards the development of an RoI-based UEP scheme.

Fig. 1 .

 1 Fig. 1. Block diagram of our work.

Fig. 2 .

 2 Fig. 2. The eye tracker.

  .

Fig. 3 .

 3 Fig. 3. Saliency map (left) and the resulting macroblockbased RoI (right) of Harp sequence.

Fig. 4 .

 4 Fig. 4. Saliency map of Marathon sequence. This saliency map does not yield any RoI.

Fig. 5 .

 5 Fig. 5. RoI intra-coding. Gray and marked macroblocks are RoI and lost macroblocks, respectively. Arrows indicate interpredictions. (a) Raw algorithm. (b) Algorithm with security neighborhood ("1" macroblocks).

Fig. 6 .

 6 Fig.6. VQM impairment perception scores for all coding schemes. VQM is applied over the full temporal and spatial resolutions of the video.

Fig. 7 .

 7 Fig. 7. VQM impairment perception scores for all coding schemes. VQM is applied over the full temporal resolution and the RoI of the video.

Fig. 8 .

 8 Fig.8. VQM impairment perception scores for all coding schemes. VQM is applied over 100 frames and the RoI of the video.

Fig. 9 .

 9 Fig. 9. From left to right: frames 97, 99 and 116 of Harp sequence. Top: Normal coding. Bottom: RoI coding 1. The green box indicates the RoI.

Fig. 10 .

 10 Fig. 10. Frame 103 of Harp sequence. The shape of the RoI is better preserved in (b) than in (a).

Acknowledgment

The authors would like to thank Romuald Pépion for setting up the eye tracking tests and helping in processing their results.


Denise Biagini
email: denise.biagini@dcci.unipi.it

Silvia Ghimenti

Alessio Lenzi

Andrea Bonini

Federico Vivaldi

Camille Oger

Jean-Marie Galano

Laurence Balas

Thierry Durand

Pietro Salvo

Fabio Di Francesco

Tommaso Lomonaco

Salivary lipid mediators: Key indexes of inflammation regulation in heart failure disease

Keywords: Lipid mediator, mass spectrometry, inflammation, saliva, cardiovascular disease, heart failure

Introduction

Cardiovascular diseases (CVDs) represent a global burden whose incidence continues to increase [START_REF] Capó | Resolvins as proresolving inflammatory mediators in cardiovascular disease[END_REF]. CVDs refer to all the disorders involving the heart and blood vessels, such as coronary heart disease, peripheral vascular disease, and heart failure (HF) [START_REF] Capó | Resolvins as proresolving inflammatory mediators in cardiovascular disease[END_REF]. A non-healthy lifestyle, e.g. smoking, bad dietary habits, physical inactivity or chronic stress, are associated with the principal CVDs risk factors, including hypertension, hypercholesterolemia, obesity and diabetes [START_REF] Ruiz-Núñez | Lifestyle and nutritional imbalances associated with Western diseases: causes and consequences of chronic systemic lowgrade inflammation in an evolutionary context[END_REF]. Oxidative stress and inflammation have gained increasing attention as key pathophysiological factors in cardiovascular events [START_REF] Cammisotto | The role of antioxidants supplementation in clinical practice: focus on cardiovascular risk factors[END_REF], such as HF syndrome. HF is a leading cause of hospitalization and mortality in the West [START_REF] Bui | Epidemiology and risk profile of heart failure[END_REF], as well as a key comorbidity in cognitive impairment in the elderly [START_REF] Bennett | Cognitive Deficits in Patients With Heart Failure: A Review of the Literature[END_REF]. HF disease has been associated with poor quality of life, recurring hospitalizations, and a reduced survival rate [START_REF] Bennett | Cognitive Deficits in Patients With Heart Failure: A Review of the Literature[END_REF][START_REF] Almeida | Two-year course of cognitive function and mood in adults with congestive heart failure and coronary artery disease: The Heart-Mind Study[END_REF]. The diagnosis of HF is established only after a clear appearance of the clinical symptoms, thus highlighting the importance of understanding and modulating its pathophysiological mechanisms for a successful patient management [START_REF] Polidori | Increased F2 isoprostane plasma levels in patients with congestive heart failure are correlated with antioxidant status and disease severity[END_REF]. Dysfunction of cardiac mitochondria is a hallmark of HF and a leading cause of oxidative stress, which in turn causes myocardial tissue damage and inflammation thus contributing to heart failure progression [START_REF] Aimo | Oxidative stress and inflammation in the evolution of heart failure: From pathophysiology to therapeutic strategies[END_REF]. Oxidative stress and inflammation are strictly related to each other, both in the acute phase of the disease, e.g., after myocardial infarction, and during chronic cardiac remodelling [START_REF] Aimo | Oxidative stress and inflammation in the evolution of heart failure: From pathophysiology to therapeutic strategies[END_REF]. Antioxidants or anti-inflammatory drugs have thus been proposed as supporting therapies for subjects either at high risk or who suffer from established heart failure [START_REF] Aimo | Oxidative stress and inflammation in the evolution of heart failure: From pathophysiology to therapeutic strategies[END_REF]. The immune-inflammatory response is regulated among cells by macrophages through the expression and secretion of various receptors and soluble molecules, such as cytokines, chemokines and lipid mediators [START_REF] Jessup | Lipid oxidation in atherogenesis: an overview[END_REF]. Lipid mediators, also termed oxylipins, are generated both by omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) through enzymatic (e.g., prostanoids, epoxy, and hydroxy fatty acids) and non-enzymatic (e.g., isoprostanoids) oxidation reactions [11,[START_REF] Galano | Isoprostanes, neuroprostanes and phytoprostanes: an overview of 25 years of research in chemistry and biology[END_REF]. Oxylipins are implicated not only in the initial phases of inflammation, but also in the precise regulation of its course and resolution. At the very beginning of the inflammatory insult, M1-type macrophages induce the release of pro-inflammatory mediators, such as prostaglandins, thromboxanes, and leukotrienes, leading to the classic signs of inflammation [START_REF] Serhan | Pro-resolving lipid mediators are leads for resolution physiology[END_REF]. The production of oxylipins then undergoes a lipid mediator class switching because of the shift from M1-to anti-inflammatory M2-type macrophages. M2-macrophages play a key role in producing pro-resolving mediators (SPMs), including lipoxins, resolvins, protectins and maresins [START_REF] Levy | Lipid mediator class switching during acute inflammation: signals in resolution[END_REF], roughly 12h after the beginning of the inflammatory response. SPMs lower the inflammatory response and even promote its resolution by decreasing the influx of neutrophils to tissues, enhancing non-phlogistic recruitment of monocytes into tissues, as well as decreasing the production of pro-inflammatory mediators [START_REF] Sugimoto | Resolution of inflammation: what controls its onset?[END_REF]. The lack of control of the process and SPM deregulation can lead to a perpetuating inflammatory cycle [START_REF] De Renaud | Wine, alcohol, platelets, and the French paradox for coronary heart disease[END_REF], typical of chronic inflammation. This form of low-grade chronic inflammation often accompanies metabolic disorders, such as obesity and hypertension, and represents a fundamental player in the onset of CVDs [START_REF] Van Linthout | Inflammation -Cause or Consequence of Heart Failure or Both?[END_REF]. During chronic inflammation, both pro-inflammatory and pro-resolving mediators are present, with specific molecules being over-or under-expressed, according to the different inflammatory diseases and tissues.

All this evidence suggests that several families of bioactive lipid mediators coexist which are differently expressed during the various stages of inflammation, and that these bioactive compounds are needed to work together to regulate the inflammatory response. To date, a fully detailed temporal pag 4 of 20 characterization of these mediators during the different stages of inflammation is still lacking and represents a future challenge in the study of CVDs. In the present work we thus propose a powerful MS-based platform for the quantitation of sixty salivary oxylipins in CVD samples. Saliva, which represents a non-invasive and painless alternative to blood, was collected from patients suffering from acute and chronic heart failure, obesity and hypertension for a comprehensive lipid mediator profiling of one of the most studied CVDs and its main risk factors.

Materials and methods

Chemicals and materials

Acetonitrile and methanol hypergrade for LC-MS LiChrosolv ® (purity ≥ 99.9%), and water for chromatography (LC-MS grade) LiChrosolv ® (purity ≥ 99.9%) used for sample treatment and UHPLC-ESI-MS/MS analysis were from EDM Millipore (Milan, Italy). Commercially available oxylipins (purity ≥99%), 15-F 2t -isoprostane, 15-F 2t -isoprostane-d4, 15-E 2t -isoprostane, 15-E 2t -isoprostane-d4, prostaglandin E 2 , prostaglandin E 2 -d4, prostaglandin D 2 , 15-deoxy-Δ

(R)-Resolvin-D 1 , 17(R)-Resolvin- D 1 -d5, 17(R)-Resolvin-D 3 , 17(R)-Resolvin-D 4 , neuroprotectin D 1 ,
protectin DX, maresin-1, maresin-1-d5, 7-epi-maresin-1, maresin-2, 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, 20-HETE, 20-HETE-d6, 8,9-DiHETE, 11,12-DiHETE, 14,15-DiHETE, 13-hydroxy-9Z,11Eoctadecadienoic acid (HODE), 8,9-epoxyeicosatrienoic acid (EET), 11,12-EET, 14,15-EET, 14,15-EET-d11, adrenic acid (AdA), eicosapentaenoic acid (EPA), alpha-linolenic acid (ALA) docosahexanoic acid (DHA), arachidonic acid (AA), docosapentaenoic acid (DPA), linoleic acid (LA) and LA-d4 were from Cayman Chemical (Michigan, USA). Non-commercially available oxylipins, i.e. 5-F 2t -isoprostane, 5-epi-5-F 2t -isoprostane, 8-F 3t -isoprostane, 8-epi-8-F 3t -isoprostane, 18-F 3t -isoprostane, 20-F 4t -neuroprostane, 20-epi-20-F 4t -neuroprostane, 10-F 4t -neuroprostane-d4, 10-epi-10-F 4tneuroprostane-d4, 14(R,S)-14-F 4t -neuroprostane, 14(R,S)-14-F 3t -neuroprostane, 4(R,S)-4-F 4tneuroprostane, C21-15-F 2t -isoprostane, tetranor-NPD 1 , dinor-NPD 1 , 7(R,S)-ST-Δ 8 -11-dihomo-isofuran, ent-7(R,S)-7-F 2t -dihomo-isoprostane, 17-F 2t -dihomo-isoprostane, linotrin, diH n-3 -DPA, diH n-6 -DPA and diH-AdA were synthesized at the Institut des Biomolecules Max Mousseron (IBMM) (Montpellier, France), according to procedures reported elsewhere [START_REF] Oger | Stereocontrolled access to isoprostanes via a bicyclo [3.3. 0] octene framework[END_REF][START_REF] Balas | An overview of their synthesis and biological significance[END_REF][START_REF] Balas | Rapid metabolization of protectin D1 by β-oxidation of its polar head chain[END_REF].

Sterile polypropylene containers were from Eppendorf (Milan, Italy), whereas Phenex™-RC syringe filters (0.2 μm regenerate cellulose, 4 mm diameter) were from Phenomenex (California, USA). A VELP Scientifica ZX4 Advanced Vortex Mixer (Usmate, Italy) and a Hermle Z-326 K Centrifuge, (Wehingen, Germany) were used for sample vortex-mixing and centrifugation, respectively. The removable needle micro-extraction by packed sorbent (MEPS) 250 μL syringe for HTA 300APlus (Thermo Scientific & Varian 8400 systems) and MEPS silica-C18 Barrel Insert and Needles (BINs) were purchased from SGE Analytical Science (Melbourne, Australia). The automated HT4000 Series Sample Prep workstation was purchased from HTA S.R.L. (Brescia, Italy). pag 5 of 20

Preparation of standard solutions and quality control samples

Stock solutions of commercially available oxylipins (100-1000 µg/mL in organic solvent [see product insert from Cayman Chemicals]), as well as those synthesized at IBMM in Montpellier, France (1000 µg/mL in methanol), were stored at -80 °C and used for the preparation of intermediate standard mixtures containing all the 60 oxylipins in methanol:acetonitrile (1:1, v/v) mixed solvent.

Working standard solutions of the 60 oxylipins were prepared by diluting intermediate stock solutions with LC-MS water (stored at 4 °C for up to one month). A mixture of deuterated internal standards (ISs, 15-F 2t -isoprostane-d4, 15-E 2t -isoprostane-d4, 10-F 4t -neuroprostane-d4, 10-epi-10-F 4tneuroprostane-d4, C21-15-F 2t -isoprostane, prostaglandin E 2 -d4, lipoxin A 4 -d5, resolvin D 1 -d5, 17(R)-Resolvin-D 1 -d5, maresin-1-d5, 20-HETE-d6, 14,15-EET-d11 and LA-d4,) was prepared at 20 ng/mL through sequential dilutions of the stock solutions (100 µg/mL in organic solvent [see product insert from Cayman Chemicals]) with methanol:acetonitrile (1:1, v/v). Working standard solutions were used to obtain five-point calibration curves (see concentration levels in Table 1). These solutions, to which 20 µL of the ISs mixture (20 ng/mL) were added, were then subjected to the MEPS procedure and analysed by UHPLC-ESI-MS/MS. Saline solutions of copper sulphate pentahydrate (10% w/v) and sodium tungstate dihydrate (12% w/v) were prepared by dissolving the required amount of each salt in LC-MS water.

Pooled saliva samples were obtained by mixing known aliquots of stimulated saliva samples collected from healthy volunteers by Salivette ® polyester swab (Sarstedt, Nümbrecht, Germany). This pooled sample was spiked with a known amount of the intermediate stock solutions to obtain quality control samples (QCs) at low, medium, high levels (i.e. levels 1, 3, and 5 of the calibration curve), which were used for method validation. The volume of the spiked standard solution never exceeded 10% of the total saliva sample volume.

Saliva sample extraction and analysis by MEPS-UHPLC-MS/MS

Sample treatment and analysis were performed by extending our analytical protocol previously developed in plasma [START_REF] Biagini | MS-based targeted profiling of oxylipins in COVID-19: A new insight into inflammation regulation[END_REF] to the less conventional saliva matrix. Our protocol successfully couples the new micro-extraction by packed sorbent (MEPS) technique to liquid chromatography tandem mass spectrometry for the straightforward analysis of oxylipins in various biofluids [START_REF] Biagini | MS-based targeted profiling of oxylipins in COVID-19: A new insight into inflammation regulation[END_REF][START_REF] Ghimenti | Salivary lactate and 8isoprostaglandin F2α as potential non-invasive biomarkers for monitoring heart failure: a pilot study[END_REF][START_REF] Biagini | Saliva as a non-invasive tool for monitoring oxidative stress in swimmers athletes performing a VO2max cycle ergometer test[END_REF].

Briefly, an aliquot of saliva sample (500 µL) to which 20 µL of the ISs mixture (20 ng/mL) were added, was then treated by the sequential addition of salts (i.e., 250 µL of CuSO 4 ·5 H 2 O 10% w/v and 250 µL of Na 2 WO 4 ·2 H 2 O 12% w/v) and acetonitrile (500 µL) for protein precipitation. After vortexmixing (2000 rpm for 1 min), centrifugation (7000 rpm for 5 min), and filtration at 0.2 µm, the supernatant was diluted (1:5 v/v) with water and subjected to a clean-up procedure by the MEPS technique. The MEPS C18 cartridge was activated by drawing and discharging 100 µL of methanol (3 x 100 µL) three times, which was then conditioned with 3 x 100 µL of water at 0.6 mL/min. The diluted supernatant (3000 µL) was loaded up and down twelve times at 0.3 mL/min by discarding it. The cartridge was then washed with 100 µL of a water:methanol (95:5 v/v) mixture at 0.6 mL/min to remove potential interferences. Analytes were eluted with 50 µL of methanol at 0.3 mL/min and then injected into the UHPLC-MS/MS instrument. pag 6 of 20

The instrument consisted of an Agilent 1290 Infinity II LC system coupled to a 6495 Triple Quadrupole mass spectrometer, which was equipped with a Jet Stream electrospray (ESI) ionization source (Agilent Technologies, USA). The chromatographic separation was achieved at 0.7 mL/min using a Polaris 3 C18-A column (50 × 4.6 mm, 3 μm, Agilent Technologies, USA) and a gradient elution with a mobile phase consisting of 0.1% aqueous formic acid (A) and 50:50 v/v methanol:acetonitrile (B). The multisampler and the column compartment were set at 4 and 25 °C, respectively. The injection volume was 20 μL. The Agilent 6495 Triple Quadrupole mass spectrometer detector operated in ESI negative ionization mode and performed multiple reaction monitoring with unit mass resolution. Mass spectrometer control, data acquisition and data analysis were performed with MassHunter Workstation software (B.07.00). Each analyte was detected using two specific MRM transitions. The most abundant one was used for the quantification (Q) of the target compound whereas the other was used for its identification (q). A deviation ≤ ± 0.10 min of the expected retention time compared to working standard solutions and a qualifier/quantifier (q/Q) ratio within 20% of the ratio measured in working standard mixtures were required for the identification of the analyte in the saliva. Detailed chromatographic parameters, and ESI and MRM operating conditions are shown elsewhere [START_REF] Biagini | MS-based targeted profiling of oxylipins in COVID-19: A new insight into inflammation regulation[END_REF].

MEPS-UHPLC-MS/MS method validation

The analytical method was validated according to the IUPAC guidelines [START_REF] Thompson | Resulting from the Symposium on Harmonization of Quality Assurance Systems for Analytical Laboratories[END_REF]. This included the evaluation of the limits of detection (LODs), calibration curves, matrix effect, recovery, and intra-day and inter-day precision. LODs were calculated as three times the standard deviation (s.d.) of the "low level spiked blank". Five-point external and internal calibration curves were obtained by analyzing in triplicate working standard solutions and pooled saliva samples spiked with known amounts of standards, respectively. As well as the spiked saliva samples, 20 µL of the ISs solution (20 ng/mL) were added to these solutions, which were then subjected to the MEPS procedure and analysed by UHPLC-ESI-MS/MS.

The analyte (a) to internal standard (IS) peak area ratios (Y, A a /A IS ) were plotted versus the corresponding concentration ratios (X, C a /C IS ). Calibration curves (Y=mX) for all the analytes were fitted by the Deming regression, which considers measurement errors for both dependent and independent variables. The matrix effect was evaluated by comparing, at a confidence level of 95%, the slopes of the calibration curves obtained with working solutions (external) and spiked saliva samples (internal). In case of a matrix effect, internal calibration curves were used for analyte quantitation.

Recovery was assessed by analysing a pooled saliva sample spiked at three concentration levels (low, mid, and high) in triplicate (n =3 at each concentration level) within the same day and on three consecutive days. The analyte recovery was calculated as the percentage ratio of the difference between the average analyte concentration measured in the spiked and non-spiked samples to the expected concentration. Intra-and inter-day precision were expressed as the relative standard deviation (RSD) of measurements performed on the spiked samples on a single day and on three consecutive days, respectively. pag 7 of 20

Study design

The study was carried out within the framework of the KARDIATOOL project ('An integrated PoC solution for diagnosis and therapy monitoring of heart failure patients', Grant agreement N°: 768686) and was approved (protocol number: 54764) by the local ethics committee (Area Vasta Nord-Ovest, CEAVNO). The study was conducted in accordance with the 1964 Declaration of Helsinki and all subjects gave written informed consent after receiving appropriate information on the protocol. The study population consisted of patients admitted to hospital with acute heart failure (AHF, n =17), patients with symptoms of HF and known HF, i.e. chronic stable HF (CHF, n=7), patients with symptoms without HF, but with obesity (n =10) and patients with symptoms without HF, but with hypertension (n = 7). Each AHF subject was monitored at hospital admission (due to a re-cutisation episode), at hospital discharge and one month later during the follow-up visit. Patients with hypertension and obesity were monitored twice a month.

The inclusion criteria for the present study were: patients with a diagnosis of AHF requiring hospital admission or outpatient intravenous diuretics; patients with known stable heart failure; patients with a diagnosis of heart failure (with reduced or preserved ejection fraction) and any underlying aetiology, according to the European Society of Cardiology guidelines 2016 [START_REF] Ponikowski | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC) Developed with the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF]; patients with obesity (with a body mass index greater than 30 kg/m 2 ) and HF symptoms but no evidence of ventricular dysfunction on echocardiography or raised natriuretic peptides; and patients with hypertension defined as systolic blood pressure ≥ 140 mmHg ± diastolic blood pressure ≥ 90 mmHg or >1 month use of antihypertensive medications, and HF symptoms but no evidence of ventricular dysfunction on echocardiography or raised natriuretic peptides.

The exclusion criteria included patients aged under 18 years of age, patients unable or unwilling to give informed consent, patients with cognitive disorders, mental disorders or psychiatric disorders, with alcohol abuse or drug abuse, pregnant or breast-feeding female patients, patients on vegan and/or vegetarian diets, patients with a diagnosis (other than HF) compromising survival over the study period, patients with severe co-morbidities such as: severe COPD (e.g., patient is on oxygen therapy or on nebulizers), uncontrolled dysthyroidism, i.e. TSH < 0.03 or > 7, decompensated diabetes, chronic inflammatory intestinal diseases, severe/active rheumatological diseases, active oncological disease on current or previous <1 year therapy of any kind, liver failure, severe kidney disease, i.e. eGFR < 30 ml/min/1.73m 2 .

For each patient, information on demographics, clinical history, co-morbidities, previous therapies and physical status were collected. In the case of a HF re-acutisation episode and the subsequent hospitalization, patients were treated in accordance with the recommendations for attending physicians from the ESC guidelines [START_REF] Ponikowski | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC) Developed with the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF].

Saliva sampling was performed in accordance with the procedures already developed in our laboratory and reported elsewhere [START_REF] Lomonaco | Influence of sampling on the determination of warfarin and warfarin alcohols in oral fluid[END_REF][START_REF] Lomonaco | The effect of sampling procedures on the urate and lactate concentration in oral fluid[END_REF]. Briefly, stimulated saliva samples were collected by asking the patients to roll a Salivette polyester swab in the mouth for two minutes. Circadian cycle effects and interference from fasting were minimized by always collecting samples in the morning (10.00 to 12.00 AM). After sample collection, salivary pH was measured using a narrow range pH paper. The salivary flow rate (mL/min) was calculated from the ratio between the weight difference (g) of the sampling device before and after sampling and the collection time (min), considering the density of sample equal pag 8 of 20 to 1 g/mL [START_REF] Chicharro | Saliva composition and exercise[END_REF]. The sample was recovered by centrifuging the swab at 7000 rpm and 4 °C for 5 min and was stored in a polypropylene tube at -80 °C until use.

Statistical analysis

GraphPad Prism (v. 9.0) and MetaboAnalyst v. 5.0 (Wishart Research group, The Metabolomics Innovation Centre (TMIC), University of Alberta, Canada (http://www.metaboanalyst.ca)) were used for the statistical data analysis. The statistical relationship between variables was examined by Pearson's correlation. Univariate (non-parametric Wilcoxon and Kruskal-Wallis test) and multivariate analysis ( (PCA and cluster analysis [START_REF] Oliveri | Data Analysis and Chemometrics[END_REF]) were performed on the dataset. The original data were preprocessed (data integrity and missing value check) and normalized (i.e., data transformation and data scaling) before the statistical analysis. Continuous variables with a normal distribution were reported as mean ± standard deviation, whereas variables with skewed distribution were described by the median with lower (25 th percentile) and upper (75 th percentile) quartiles.

Results and discussion

Optimization of saliva sample extraction

Sample pre-treatment is a critical step in the analysis of biofluids [START_REF] Clark | Sample preparation for bioanalytical and pharmaceutical analysis[END_REF]. The mucins and other proteins in saliva prevent its direct loading onto the MEPS cartridge, thus a preliminary sample treatment step prior to MEPS extraction is required to minimize the saliva viscosity and extend the lifetime of the cartridge. Here, we compared the analytical performances of a combined addition of salts and acetonitrile (method A [START_REF] Biagini | MS-based targeted profiling of oxylipins in COVID-19: A new insight into inflammation regulation[END_REF]) and a simple and straightforward 6-fold saliva dilution with water (method B [START_REF] Biagini | Saliva as a non-invasive tool for monitoring oxidative stress in swimmers athletes performing a VO2max cycle ergometer test[END_REF]). Method A entailed the consecutive addition of 250 µL of CuSO 4 ·5 H 2 O 10% w/v and 250 µL of Na 2 WO 4 ·2 H 2 O 12% w/v and 500 µL of acetonitrile to 500 µL of spiked saliva samples (medium concentration level). After vortex-mixing (2000 rpm for 1 min) and filtration at 0.2 µm, the resulting supernatant was then diluted (1:5 v/v) with water. In method B, the spiked saliva sample was directly diluted with water (1:5 v/v) and filtered at 0.2 µm. Each procedure was performed in quintuplicate. For both methods the diluted supernatant/sample (3000 µL) was loaded up and discharged 12 times by MEPS and the target analytes were eluted with 50 µL of methanol, maintaining the same sample-toelution solvent volume ratio across all the experiments.

Both procedures showed a comparable analytical performance in terms of analyte peak area and extraction efficiency. The analyte recovery was almost quantitative for both the combined addition of salts and acetonitrile (87-111%, RSD <15%) and the 6-fold water dilution (75-119%, RSD <15%).

In the case of water dilution (method B), a pronounced increase in the background noise in the chromatographic profile was observed after five consecutive UHPLC-MS/MS injections. This occurred particularly within the timeframe starting from minute ten up to the end of the chromatographic run and corresponding to the acquisition of pro-resolving mediators, epoxy and hydroxy-fatty acids, and PUFAs signals. A laborious cleaning of both column and ESI source would thus be needed to restore the chromatographic baseline after a typical batch sequence (10-15 samples). The combined addition of salts and acetonitrile was thus preferred to the dilution of the saliva sample prior to MEPS-UHPLC-MS/MS analysis. Compared to the time consuming and multistage procedures reported elsewhere [START_REF] Peña-Bautista | Validated analytical method to pag 23 of 20 determine new salivary lipid peroxidation compounds as potential neurodegenerative biomarkers[END_REF], pag 9 of 20 our approach consists of a single-step saliva clean-up and analyte enrichment (10-fold) in about 30 min using a reduced amount of organic solvents (i.e. 50-100 μL), as well as avoiding any drying step before injection.

Analytical figures of merit

LOD values ranged between 5-800 pg/mL for oxylipins and 30-1000 ng/mL for PUFAs, which in most of cases were much lower than those reported in the literature [START_REF] Cháfer-Pericás | Ultra high performance liquid chromatography coupled to tandem mass spectrometry determination of lipid peroxidation biomarkers in newborn serum samples[END_REF][START_REF] Sánchez-Illana | Novel free-radical mediated lipid peroxidation biomarkers in newborn plasma[END_REF][START_REF] Rund | Development of an LC-ESI (-)-MS/MS method for the simultaneous quantification of 35 isoprostanes and isofurans derived from the major n3-and n6-PUFAs[END_REF], and which were suitable for a reliable determination of oxylipins in saliva [START_REF] Peña-Bautista | Validated analytical method to pag 23 of 20 determine new salivary lipid peroxidation compounds as potential neurodegenerative biomarkers[END_REF]. All the analytes showed adequate linearity with R 2 between 0.96 and 1.00 within the tested concentration range. Recoveries were not significantly different (p> 0.05) across the concentration levels tested and were satisfactory for most of the analytes when performing both intra-(overall range: 88-110%) and inter-day (overall range: 85-119%) experiments. Intra-and inter-day precisions were always lower than or equal to 20%. The slopes of the calibration curves obtained with working solutions (external curve) and spiked samples (internal curve) were not significantly different at a confidence level of 95% for half of the analytes (see Table 1), suggesting the absence of any measurable matrix effect. The difference was slightly significant (p<0.05) for thirty out of sixty oxylipins (see Table 1), for which internal calibration curves were used for analyte quantitation.

Table 1 reports the mean slopes and standard deviations (n=3) of the five-point calibration curves, LOD values and intra-and inter-day recovery, with the corresponding RSD. pag 11 of 20

Preliminary data on heart failure, hypertensive, and obese patients

Within the framework of the clinical pilot study, AHF (n=17) and CHF (n=7), hypertensive (n=7) and obese (n =10) patients were enrolled. AHF subjects were monitored at hospital admission (due to a recutisation episode), at hospital discharge and one month later during the follow-up visit. Hypertensive and obese subjects were monitored twice a month, for a total of 91 samples collected and analysed. For these patients, clinical parameters and oxylipin levels did not change significantly (p>0.05) within that month thus samples collected at those collection points were considered as biological replicates of the same population. Demographic and clinical data of both AHF and CHF, hypertensive and obese patients are reported in Table 2. pag 13 of 20

The demographics were generally similar between the different groups, except in terms of the prevalence of females in obese patients. Pharmacological therapy was almost comparable among the different pathologies except for the high prevalence of beta blockers (80-100%), diuretics (80-100%) and anticoagulants (50-60%) in HF patients, and calcium channel blockers (57%) in patients with hypertension. Most HF patients showed a high incidence of coronary artery disease (76%) as well as diabetes mellitus (47%) in the case of acute decompensation. Oxylipin levels were not significantly different (p>0.05) when patients were grouped according to those comorbidities. AHF patients were characterized by significantly higher levels of urea (p<0.001) and creatinine (p=0.002), and lower levels of potassium (p=0.006), lower full blood count (p<0.001) and iron (p=0.001) content compared with CHF, hypertensive and obese patients. As expected, AHF patients showed very high levels of NT-proBNP which differed significantly (p<0.001) hypertensive and obese patients. When patients were grouped in one of four categories in the NYHA functional class, lipoxin-B 4 , DHA and omega-3 DPA content differed significantly among the four classes (p=0.02).

Oxylipins were quantified in the saliva samples collected from all the participants. Thirty-four out of sixty oxylipins were excluded from the dataset as the concentrations were below the limit of quantification for more than 50% of the samples. This may be expected since only a fraction of the free oxylipins circulating in blood may be transferred in oral fluid depending on the physical and chemical properties of the compound (e.g. molecular size, pKa and lipid solubility [START_REF] Lomonaco | The effect of sampling procedures on the urate and lactate concentration in oral fluid[END_REF]). This aspect will be investigated further in the near future.

The data matrix used for the subsequent statistical analysis therefore consisted of 87 samples and 26 features (oxylipins). Figure S1 shows the chromatographic profiles for the 26 oxylipins, whereas their concentration levels [median (min-max)] are reported in Table S1. Data were analysed by PCA in order to summarize and visualize the information content of our data matrix (Figure 1). In the PCA, variables contributing similar information were grouped together, that is, they were correlated. When the variables correlated negatively, they were positioned on opposite sides of the plot origin. Observations characterized by a high value of a specific variable were located close to this value in the 3D-space. In our case, the three lowest-order principal components accounted for a total explained variance of approximately 36%. Samples were spread throughout the space along both PC1 and PC3 directions. Conversely, PC2 scores contributed to the slight separation between i) AHF patients, and ii) CHF as well as obese patients. An up-to-down switch of the oxylipin profile was observed when moving from an acute to chronic inflammation status, i.e. from AHF to both CHF and obesity [START_REF] Wang | Inflammation, a link between obesity and cardiovascular disease[END_REF]. Hypertensive samples were spread halfway the samples of the two sub-groups. Samples from AHF patients (admission discharge, follow-up) were located in the upper part (dotted red circle, Figure 2A), thus highlighting an over-expression of different families of isoprostanes (e.g. F 2isoprostanes, F 2 -dihomo-isoprostanes (Figure 2B)). These mediators are indexes of oxidative damage and are typically released from the very beginning of an oxidant/inflammatory injury. Otherwise, samples from both CHF and obese subjects were located at the bottom of PC2 (dotted blue circle, Figure 2A) in the direction of omega-3 PUFAs and pro-resolving mediators (Figure 2B), thus suggesting low-grade and late-stage persistent inflammation.

Figure 2 shows the median omega-3 PUFA values (i.e., EPA (20:5), DPA (22:5) and DHA (22:6)) as well as AA-derived pro-resolving lipoxin-B 4 which varied significantly (p<0.05) among groups. The obese population showed significantly higher levels [median (lower and upper quartiles)] of EPA [30(20-55) vs 14(10-18) ng/mL, p < 0.02], DHA [40(32-70) vs 21(14-25) ng/mL, p < 0.02] and lipoxin-B 4 [75(32-123) vs 16(10-44) pg/mL, p < 0.005], compared to AHF patients monitored during hospitalization. AHF patients at admission presented higher levels of omega-3 DPA [12(8-21) vs 3(3-4) ng/mL, p = 0.001] and lower levels of lipoxin-B 4 [16(10-44) vs 39(30-65) pg/mL, p = 0.044], as opposed to CHF subjects. Interestingly, the lipoxin-B 4 concentration in the AHF population showed a notable increase (6-fold) over time, one month later (follow-up measurement) reaching comparable levels [50(24-85) pg/mL] to those of the CHF [39(30-65) pg/mL] and hypertensive [47(29-66) pg/mL] group (Figure 2). pag 15 of 20 None of the investigated oxylipin levels varied significantly (p > 0.05) between admission and discharge of AHF subjects. The longitudinal monitoring of each subject was hypothesized to be more informative since patients would act as their own controls [START_REF] Lomonaco | Determination of carbonyl compounds in exhaled breath by on-sorbent derivatization coupled with thermal desorption and gas chromatography-tandem mass spectrometry[END_REF][START_REF] Lomonaco | Monitoring of warfarin therapy: Preliminary results from a longitudinal pilot study[END_REF]. Figure 3 shows the changes in oxylipin concentrations for each AHF patient from admission to discharge, normalized with respect to the admission level. HF patients had a notable decrease (40%) of 15-E 2t -IsoP concentration over time (Figure 3), thus showing an incremental ability to differentiate between the admission (acute phase) and discharge point. pag 16 of 20 Bioactive lipid mediators belong to a very complex network of chemicals whose regulation is still not completely understood. A comprehensive study of their temporal production and the bioactive role exerted within the different phases of the inflammatory cascade is challenging. In the present study, we observed the change in the oxylipin pattern that was strictly related to the inflammatory phase expressed in the different populations. The oxylipin profile in AHF patients was rich in isoprostanes, such as 15-F 2t -IsoP, 5-F 2t -IsoP, and 15-E 2t -IsoP, which represent well-known markers of oxidative stress. In a failing heart, excess mitochondrial ROS, as well as the inability to supply energy, are the main causes of mitochondrial dysfunction and contractile failure found in both humans and animal models [START_REF] Dai | Mitochondrial oxidative stress mediates angiotensin IIinduced cardiac hypertrophy and Gαq overexpression-induced heart failure[END_REF]. Mitochondrial damage itself causes further increase in ROS production and more severe injury, revealing the key role of oxidative stress in the onset and progression of CVDs [START_REF] Tsutsui | Oxidative stress and heart failure[END_REF]. Isoprostanes are reliable markers in assessing the oxidative stress status [START_REF] Roberts | Measurement of F2-isoprostanes as an index of oxidative stress in vivo[END_REF]. Apart from being indexes of oxidative damage, these mediators exert various bioactive roles in vivo, such as vasoactivity, stimulation of both mitogenesis and atherogenesis [START_REF] Cracowski | Isoprostanes: An emerging role in vascular physiology and disease?[END_REF][START_REF] Geng | Neuroprotective effects of DHA-derived peroxidation product 4 (RS)-4-F4t-neuroprostane on microglia[END_REF] F 2 -isoprostanes have been found to play a significant role in the progression of HF from asymptomatic to symptomatic and in the deterioration of the functional capacity [START_REF] Cracowski | The putative role of isoprostanes in human cardiovascular physiology and disease: Following the fingerprints[END_REF]. The levels of these lipid mediators were found to correlate consistently to the severity of HF and can thus be tested as long-term prognostic markers [START_REF] Cracowski | Increased formation of F2-isoprostanes in patients with severe heart failure[END_REF]. Interestingly, the urinary 15-F 2t -isoprostane concentration has been found to be significantly correlated with the concentration of plasma BNP and was found to decrease gradually in proportion to the severity of chronic HF during hospitalisation [START_REF] Polidori | Increased F2 isoprostane plasma levels in patients with congestive heart failure are correlated with antioxidant status and disease severity[END_REF][START_REF] Nonaka-Sarukawa | Increased urinary 15-F2t-isoprostane concentrations in patients with non-ischaemic congestive heart failure: a marker of oxidative stress[END_REF][START_REF] Reina-Couto | Impaired resolution of inflammation in human chronic heart failure[END_REF]. In the present work, none of the investigated oxylipins changed significantly during hospitalization, i.e. from admission to discharge, except for 15-E 2tpag 17 of 20 isoprostane. Compared with the F 2 -isoprostanes, E 2 -isoprostanes are more potent vasoactive mediators [START_REF] Cracowski | Isoprostanes: An emerging role in vascular physiology and disease?[END_REF]. 15-E 2t -IsoP is more potent than 15-F 2t -IsoP in systemic and pulmonary vessels, showing vasoconstrictor as well as vasodilator activity through thromboxane (TP)-and prostaglandin (EP)receptors, respectively [START_REF] Catalli | Receptors and signaling pathway underlying relaxations to isoprostanes in canine and porcine airway smooth muscle[END_REF]. The high levels of 15-E 2t -IsoP at hospital admission in AHF subjects suggest a pronounced oxidative insult occurring at the very beginning of the decompensation episode, which faded over the course of the hospitalization. However, the lack of sufficient data available on the production of E 2 -isoprostanes in cardiovascular disorders, makes it almost impossible to draw further conclusions regarding the potential bioactive role exerted in the failing heart. As with AHF patients, hypertensive subjects showed an over-expression of isoprostanes and, thus, their samples were superimposed in the score plot of the PCA (Figure 1). Recent studies have indicated oxidative stress, and in particular 15-F 2t -isoprostane levels, as a risk factor for blood pressure elevation in both severe and essential hypertensive patients [START_REF] Rodrigo | Relationship between Oxidative Stress and Essential Hypertension[END_REF].

Apart from being susceptible to ROS, mitochondria are emerging key players in the regulation of innate immunity. Studies on mitochondrial-mediated regulation of inflammation in CVDs are increasing [START_REF] Chen | Mitochondria, Oxidative Stress and Innate Immunity[END_REF]. Endogenous lipids are among the most important mediators involved in the regulation of all the phases of inflammation including initiation and resolution. Lipids are not just the major constituents of cell membranes and sources of energy, but are also key pathophysiological mediators of several intercellular and intracellular processes [START_REF] Chiurchiu | Resolution of inflammation is altered in chronic heart failure and entails a dysfunctional responsiveness of T lymphocytes[END_REF]. Among lipids, omega-3 PUFAs (e.g. ALA, EPA, DHA, n-3 DPA) have been associated with antiarrhythmic, antithrombotic, antiatherogenic and antiinflammatory activities [START_REF] Mcclung | Heme-oxygenase and lipid mediators in obesity and associated cardiometabolic diseases: Therapeutic implications[END_REF]. In mammals, the essential fatty acid ALA is enzymatically elongated and desaturated to produce EPA and subsequently DHA, with omega-3 DPA being the biosynthetic intermediate [START_REF] Honn | The Role of Bioactive Lipids in Cancer, Inflammation and Related Diseases[END_REF]. EPA and DHA exert an intense cardioprotective effect in a wide variety of CVDs, such as myocardial infarction and congestive heart failure. They enhance the production of NO and pro-resolving mediators (e.g. lipoxins, resolvins, protectins), inhibit ACE activity and thus, function as anti-inflammatory and anti-hypertensive molecules [START_REF] Matsuo | High plasma docosahexaenoic acid associated to better prognoses of patients with acute decompensated heart failure with preserved ejection fraction[END_REF].

In our study obese patients were characterized by higher EPA levels than AHF. EPA has been found to exert a key role in the obese population by attenuating some symptoms of obesity through its antioxidant properties [START_REF] Mcclung | Heme-oxygenase and lipid mediators in obesity and associated cardiometabolic diseases: Therapeutic implications[END_REF]. The caloric overload in obesity results not only in a notable lipid accumulation in adipose tissue as well as the circulation of free fatty acids, but also in an increased lipoprotein circulation [START_REF] Ramos-Nino | The role of chronic inflammation in obesity-associated cancers[END_REF]. Lipoproteins are associated with an endotoxin-scavenging role, thus preventing the harmful inflammatory consequences of endotoxemia. Traditional cardiovascular risk factors such as obesity and hypercholesterolemia are thus associated with greater survival among chronic HF patients. This is referred to as the "reverse epidemiology" paradox, which could be explained by the presence of the "malnutrition-inflammation complex syndrome" in chronic HF patients [START_REF] Hasper | Systemic inflammation in patients with heart failure[END_REF]. This suggests that nutritional strategies (i.e. omega-3 PUFAs diet supplementation) could be used in HF to maintain dietary adequacy, reduce the risk of hospitalization and improve survival, as clearly suggested by the European Society of Cardiology as well as the American College of Cardiology/American Heart Association [START_REF] Matsuo | High plasma docosahexaenoic acid associated to better prognoses of patients with acute decompensated heart failure with preserved ejection fraction[END_REF]. Among omega-3 PUFAs, omega-3 DPA has gained increasing attention. The literature on both omega-3 DPA and its oxidation products (e.g., diHDPA n-3 ) is limited because of the poor availability of the pure compound, nevertheless data suggests that omega-3 DPA does not only serve as a reservoir of pag 18 of 20 DHA and EPA in humans [START_REF] Miller | A short-term n-3 DPA supplementation study in humans[END_REF], but it shows beneficial health effects [START_REF] Kaur | Docosapentaenoic acid (22:5n-3): A review of its biological effects[END_REF]. In our study, n-3 DPA was found to be highly expressed in AHF subjects compared to CHF subjects. This was proven during hospitalization, i.e. from admission to discharge, but also at the follow-up sample collection point (after one month). Omega-3 DPA showed an opposite trend compared to EPA and DHA, which is consistent with its role as a biosynthetic intermediate product in the formation of DHA from EPA. Dietary habits, e.g. fish consumption, could impact on circulating omega-3 PUFA levels in patients, more than inflammation status or disease severity. To assess the relationship between the increase in omega-3 DPA levels and the worsening of the clinical situation due to the re-acutisation episode in HF subjects, we evaluated the correlation between the omega-3 DPA concentration and NT-proBNP which is the gold standard biomarker in heart failure. We found a medium positive correlation between the two variables (Spearman r, 0.52) as clearly shown in Figure 4. These results demonstrate that the decrease in omega-3 DPA in the acute setting from admission to follow-up was not only diet-related, but may be also a consequence of a lipid rearrangement typical of the failing heart during acute decompensation. If confirmed, this could open up a new scenario in the use of omega-3 DPA as a potential predictive marker of re-acutisation episodes, thus providing opportunities for preventive interventions and reduced hospitalizations. Apart from PUFAs, many molecules play a central role in regulating the inflammatory response by controlling its magnitude and duration, such as the PUFA-derived pro-resolving mediators (SPMs) [START_REF] Serhan | The scent of a phagocyte: advances on leukotriene B4 receptors[END_REF]. Endogenous SPMs appear to control the host response and ease the resolution of inflammation [START_REF] Diamond | Regulation of leukocyte trafficking by lipoxins[END_REF]. Lipoxins are an important class of lipid mediators with inflammation-resolving properties. Lipoxins are endogenous mediators of resolution that are locally produced at sites of inflammation in the early phases by the sequential lipoxygenation of arachidonic acid during cell-cell interactions [START_REF] Reina-Couto | Impaired resolution of inflammation in human chronic heart failure[END_REF]. To date, two types of native lipoxins, lipoxin-A 4 and its positional isomer, lipoxin-B 4 , have been pag 19 of 20 identified [START_REF] Reina-Couto | Impaired resolution of inflammation in human chronic heart failure[END_REF] and recognized as vasoactive molecules as well as mediators in the regulation of leukocyte functions [START_REF] Badr | Lipoxin A4 antagonizes cellular and in vivo actions of leukotriene D4 in rat glomerular mesangial cells: evidence for competition at a common receptor[END_REF].

In the present work the lipoxin-B 4 concentration varied significantly between AHF patients at hospital admission and CHF patients. In the acute setting patients showed lower levels of lipoxin-B 4 , which gradually increased during the hospitalization, at the follow-up visit reaching comparable levels to the CHF population. Plasma levels of lipoxin-A 4 have been found to be downregulated in chronic HF in all the NYHA classes [START_REF] Reina-Couto | Impaired resolution of inflammation in human chronic heart failure[END_REF], showing a more pronounced decrease in line with the increase in disease severity. Reina-Couto et al. observed inverse correlations between plasma lipoxin-A 4 and the established HF biomarkers (e.g. BNP, troponin I, and myoglobin), as well as the markers of proinflammatory/pro-oxidant injury, such as CRP and uric acid [START_REF] Reina-Couto | Resolving inflammation in heart failure: novel protective lipid mediators[END_REF]. In our case, we found a medium negative correlation between lipoxin-B 4 and NT-proBNP (Figure 4, Spearman r = -0.32). The reduction in lipoxin-B 4 concentration that we observed during the acute decompensation episode thus appeared to be reasonable, as well as its return to the levels of chronic subjects once stable clinical conditions were restored. These results suggest the counter-regulatory and protective role of lipoxins in HF pathophysiology, thus highlighting the importance of measuring these SPMs as tools for risk stratification [START_REF] Reina-Couto | Impaired resolution of inflammation in human chronic heart failure[END_REF]. During the healing phase, macrophages switch from the M1-phenotype to primarily anti-inflammatory macrophages, and then to reparative M2 macrophages, which are the main ones responsible for SPM production [START_REF] Honn | The Role of Bioactive Lipids in Cancer, Inflammation and Related Diseases[END_REF]. As critical regulators of the resolution program, targeting the actions of macrophages could help control inflammation and cardiac remodelling in the context of cardiac healing. We believe that the targeted modulation of endogenous mechanisms of inflammation, rather than its simple suppression, will soon become a key approach to treating chronic inflammatory diseases.

Conclusions

Our MS-based platform was shown to be suitable for the simultaneous quantitation of sixty salivary lipid mediators in CVDs samples. A lipid rearrangement typical of the failing heart during acute decompensation was clearly evidenced from our preliminary results. The targeted regulation of the inflammatory storm, rather than the simple suppression of the inflammation, could be the most successful approach for the management of CVDs in the near future.

Figure 1 .

 1 Figure 1. 3D principal component analysis (PC1 (19.1%), PC2 (9.9%), PC3 (7.2%)) based on the 26 features detected in more than 50% of the samples: score plot (A) and loading plot (B). Score plot: saliva samples collected from AHF patients at hospital admission (red), discharge (green) and follow-up one month later (blue), hypertensive (sky blue), obese (pink),

Figure 2 .

 2 Figure 2. Box plots of PUFAs A) EPA; B) DHA; C) DPA, and oxylipin D) lipoxin-B 4 , which differed significantly among groups: AHF at admission (AHF(adm)), AHF at discharge (AHF(dis)), AHF at the follow-up visit (AHF(flw-up)), CHF, hypertensive, and obese; (p<0.05, significantly different). The box-plot shows: the minimum, 25th percentile, median, 75th percentile, and maximum value for each variable investigated. The plus symbol inside the box shows the mean value.

Figure 3 .

 3 Figure 3. Heatmap (left side) of the 26 features detected in saliva samples collected from thirteen AHF patients at both hospital admission and discharge (Kn). Data are normalized with respect to the admission point for each subject. Each square represents a collection point. Box plot (right side) of 15-E 2t -IsoP which differed significantly (p<0.05) in AHF patients between admission and discharge. The box-plot shows: the minimum, 25th percentile, median, 75th percentile, and maximum value for each variable investigated. The yellow diamond inside the box shows the mean value.

Figure 4 .

 4 Figure 4. Spearman correlation heatmap. Negative correlation (blank to red, r= 0 to -1.0) and positive correlation (blank to blue, r= 0 to 1.0) between the concentration level of the most significant oxylipins (EPA, DHA, n-3 DPA and lipoxin B 4 ) and the gold standard biomarker in heart failure blood NT-proBNP.

  12,14 -Prostaglandin J 2 , thromboxane B 2 , leukotriene B 4 , lipoxin A 4 , lipoxin A 4 -d5, lipoxin B 4 , resolvin E 1 , resolvin D 1 , resolvin D 1 -d5, resolvin D 2 , resolvin D 3 , resolvin D 4 , resolvin D 5 , 17

Table 1 .

 1 Full list of quantified oxylipins and relevant analytical parameters i.e., calibration curve, limit of detection, intraassay and inter-assay recovery, intra-assay and inter-assay precision.

	Compound	Calibration levels (pg/mL, *ng/mL)	Slope ± s.d (R 2 )	LOD (pg/mL,	Intra-assay recovery %	Inter-assay recovery %

, NeuroPs, dihomo-isoPs), isofurans (IsoFs) and prostanoids (prostaglandins (PGs), leukotrienes (LEU

						),
	thromboxanes (TX))
	5-F2t-IsoP		0.19±0.02 (1.00) ◊	10	101(3)	111(8)
	5-epi-5-F2t-IsoP		0.15±0.01 (1.00) ◊	15	100(7)	110(10)
	15-F2t-IsoP		1.1±0.1 (1.00) ◊	10	102(8)	109(9)
	8-F3t-IsoP		1.08±0.13 (1.00)	10	101(4)	111(8)
	8-epi-8-F3t-IsoP		1.15±0.03 (1.00)	15	101(7)	107(9)
	18-F3t-IsoP		0.17±0.03 (1.00)	5	103(3)	96(11)
	15-E2t-IsoP		14±1 (0.98) ◊	7	101(8)	93(12)
	4(R,S)-4-F4t-NeuroP	0.05; 0.10; 0.25; 0.50; 1	0.51±0.06 (1.00)	70	100(3)	105(7)
	20-F4t-NeuroP		0.095±0.003 (0.99)	20	101(3)	98(4)
	20-epi-20-F4t-NeuroP		0.07±0.01 (0.99) ◊	610	100(11)	90(15)
	14(R,S)-14-F4t-NeuroP		0.076±0.003 (0.99)	250	100(10)	107(9)
	14(R,S)-14-F3t-NeuroP		0.54±0.05 (1.00)	15	88(11)	95(10)
	ent-7(R,S)-7-F2t-dihomo-isoP		0.52±0.02 (0.99) ◊	5	101(9)	117(13)
	17-F2t-dihomo-isoP		0.78±0.09 (0.99)	70	101(7)	106(6)
	7(R,S)-ST-Δ 8 -11-dihomo-isoF		0.15±0.01 (1.00) ◊	20	102(3)	94(12)
	PGE2		4.9±0.7 (1.00) ◊	4	100(4)	100(12)
	PGD2	0.1; 0.5; 1; 2.5; 5	1.07±0.09 (1.00) ◊	20	101(9)	106(18)
	15-deoxy-Δ 12,14 -Prostaglandin J2		1.1±0.1 (0.98)		100(9)	91(6)
	LEU-B4 TX-B2	0.5; 5; 10; 25; 50	4.2±0.3 (0.98) 0.23±0.02 (0.99) ◊	10 70	97(3) 98(6)	94(5) 103(13)
	Hydroxy/dihydroxy-PUFAs
	5-HETE		31±4 (1.00) ◊	17	100(8)	99(9)
	12-HETE 15-HETE	0.5; 5; 10; 25; 50	26±3 (0.99) ◊ 110±10 (0.97)	83 7	97(9) 104(6)	85(15) 91(7)
	20-HETE		3.5±0.4 (0.98)	15	101(12)	106(17)
	13-HODE	2.5; 25; 50; 125; 250	28±5 (0.97)	1 *	103(10)	115(14)
	8,9-DiHETE		9.3±0.9 (0.96) ◊	11	103(6)	92(11)
	11,12-DiHETE	0.1; 0.5; 5; 10; 25	16±1 (0.97)	5	102(11)	114(13)
	14,15-DiHETE		11±1 (1.00) ◊	10	101(6)	115(12)
	Epoxy-PUFAs
	8(9)-EET		0.67±0.03 (0.97) ◊	180	110(10)	102(12)
	11(12)-EET	0.5; 5; 10; 25; 50	4.3±0.6 (0.96) ◊	50	101(4)	115(8)
	14(15)-EET		4.3±0.4 (0.96) ◊	320	111(6)	106(3)
	Pro-resolving (lipoxins, resolvins, maresins, protectins)
	Lipoxin-A4		80±10 (0.99) ◊	830	105(6)	118(15)
	Lipoxin-B4		1.29±0.09 (0.97) ◊	10	109(6)	113(12)
	Resolvin-D1		1.00±0.08 (0.99)	4	101(7)	110(12)
	Resolvin-D2		0.15±0.01 (1.00) ◊	40	100(7)	97(18)
	Resolvin-D3		0.65±0.03 (1.00) ◊	30	100(11)	95(10)
	Resolvin-D4		0.41±0.07 (0.95) ◊	55	97(11)	97(13)
	Resolvin-D5		4.7±0.5 (0.99)	8	102(7)	119(16)
	17(R)-Resolvin-D1		1.5±0.1 (0.98)	20	101(7)	109(10)
	17(R)-Resolvin-D3		1.02±0.07 (1.00) ◊	5	106(10)	108(13)
	17(R)-Resolvin-D4		0.41±0.07 (0.95) ◊	770	97(11)	97(13)
	Resolvin-E1 Maresin-1	0.1; 0.5; 2.5; 5; 20	1.0±0.2 (0.99) 0.72±0.07 (0.98)	80 130	87(13) 103(6)	91(9) 114(9)
	7-epi-maresin-1		0.95±0.05 (1.00) ◊	5	101(2)	116(7)
	Maresin-2		1.95±0.07 (1.00) ◊	10	100(9)	104(13)
	Neuroprotectin D1		3.8±0.2 (0.98)	15	102(5)	107(8)
	dinor-Neuroprotectin D1		14±2 (1.00) ◊	5	101(6)	114(8)
	Tetranor-Neuroprotectin D1		1.36±0.09 (1.00) ◊	40	101(7)	93(13)
	Protectin DX		7.7±0.5 (0.98)	11	101(10)	114(11)
	diH-AdA		1.9±0.1 (0.98)	50	102(7)	116(10)
	diHn-3-DPA		2.4±0.1 (0.97)	50	101(8)	109(13)
	diHn-6-DPA		11.5±0.8 (1.00) ◊	5	101(3)	103(8)
	Linotrin		14±1 (0.99) ◊	70	103(5)	109(8)

Table 2 .

 2 Demographic and clinical characteristics of enrolled patients (n = 41) at the hospital admission (t 0 ), discharge (t d ) and follow-up 1 month later (T f-up ). Data are shown as median [interquartile range, calculated as 75th -25th percentiles]. Bold values denote statistical significance at p < 0.05 level (non-parametric Kruskal-Wallis test). Only 9 out of 17 patients were monitored at the follow-up visit after one month.

		AHF (n=17)	CHF (n=7)	Hypertensive (n=7)	Obese (n=10)	p
	Age, years (mean±SD)	74±9	70±6	70±10	60±10	0.095
	Sex (M:F)	13:4	6:1	3:4	3:7
			pag 12 of 20

a

Acknowledgement

We would like to acknowledge the support of the KARDIATOOL project (Grant Agreement No. 768686), which received funding from the EU's Horizon 2020 research and innovation program. We would also like to thank Dr. Christina Petersen from the Cardiology Division, Fondazione Toscana Gabriele Monasterio (Pisa, Italy) for managing the saliva sample collection.

Institutional funds from the University of Pisa supported the study.

data and material

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. pag