The use of remote desktop services on virtualized machines is a general trend to reduce the cost of desktop seats. Instead of assigning a physical machine with its operating system and software to each user, it is considerably easier to manage a light client machine that connects to a server where the instance of the user’s desktop machine actually executes. Citrix and VMware have been major suppliers of these systems in private clouds. Desktop-as-a-Service solutions such as Amazon WorkSpaces offer a similar functionality, yet in a public cloud environment. In this paper, we review the main offerings of remote desktop protocols for a cloud deployment. We evaluate the necessary network resources using a traffic model based on self-similar processes. We also evaluate the quality of experience perceived by the user, in terms of image quality and interactivity, providing values of Mean Opinion Score (MOS). The results confirm that the type of application running on the remote servers and the mix of users must be considered to determine the bandwidth requirements. Applications such as web browsing result in unexpectedly high traffic rates and long bursts, more than the case of desktop video playing, because the on-page animations are rendered on the server. Traditional desktop computers executing local productivity applications are evolving into light local computers used as remote displays for centralized machines. This is the scenario of Remote Desktop (RD) systems , where aThe centralized computing resources are typically virtualized. A single host can offer independent desktops to dozens of users in what is called a Virtual Desktop Infrastructure (VDI) . These rRD systems provide reduced computer management costs owing to centralized backups, updates, security control, and other system functions. The capital expenditure in desktop hardware is also reduced as it functions with inexpensive thin clients. New features can be easily offered, such as access to the user’s desktop from any device, even mobile devices (from smartphones or tablets). The main disadvantages to these RD systems are the requirement for a centralized infrastructure that offers the real desktop and the requirement of high speed and low latency network access from the users to the infrastructure.The centralized infrastructure in a VDI is typically a private server farm, datacentre, or private cloud, for the exclusive use of the company employees. However, a VDI is complex and hence is difficult to manage and maintain. The consequence of these challenges has been the increase of Virtual Desktop Clouds (VDCs) and Desktop-as-a-Service (DaaS) solutions. They offer the capability to outsource the infrastructure and its management to a public cloud. However, VDC usability imposes more severe requirements on the minimum network speed and maximum latency for the company Internet access links and the path from the local network to the closest DaaS cloud . Some prThere is a significant diversity in RD solutions. Remote FrameBuffer protocol (RFB)  and RemoPCoIP  is a proOwing to the popularity of Microsoft Windows as a desktop operating system, large RD deployments are based on Windows desktops and use RD services optimised for Windows. Citrix is based on the Independent Computing Architecture (ICA) protocol and represents more than 50% market share within the RD space, mainly for Windows desktops . It is fIn this paper, we compare the most important Remote Desktop protocols and software implementations, including the effects of WAN access and public cloud sharing for a DaaS scenario. We deploy the setups in the Amazon cloud environment . We present a novel comparison of the most representative Remote Desktop protocols using virtualized hosts in an Amazon datacentre accessed by clients from a campus LAN. We deploy server software for ICA HDX (used in VDI Citrix systems ), RFB  presents a reasonable quality, however its network bandwidth usage is high. Conversely, TeamViewer sacrifices video quality and interactivity to contain network usage.This paper is organised as follows. The next section compares this work with previous related papers in the literature. The section “Methodology and experimental setup” describes the experimental environment including the hardware and software installations, user profiles, and evaluation metrics. Section “Evaluation of PCoIP” presents the measurements and performance results for the PCoIP protocol (Amazon WorkSpaces DaaS), followed by a section that compares the results from PCoIP to the other RD protocols. The section “Recommendations for remote desktop protocol design” provides suggestions to improve the user experience in these scenarios. Finally, conclusions are presented.There are works in the literature that compare RD protocols using different metrics, however none of these offer traffic models or QoE measurements with virtualized hosts in a public cloud. In  the authet al. ; ho; ho25]; In this paper, we compare the remote desktop protocols RDP, ICA, PCoIP, RFB, and TeamViewer. RDP is used in Microsoft Azure VDI , Amazon In this section, we first highlight the characteristics in each protocol that are relevant for this comparison. We then present the hardware and software tools used for the evaluation, describe the user profiles, define the measurement metrics, and finally introduce the characteristics of self-similar arrival processes that are used for user traffic modelling.ICA (1989) is a proprietary solution by Citrix Systems. It was developed for remote access to Microsoft Windows desktops; today, it also offers access to Linux hosts. It uses mainly TCP as a transport protocol , howeverVNC (1998) implements the RFB protocol, specified today in . There eRDP 1998) is a protocol developed by Microsoft for Windows systems based on the T.120 family of protocols from the International Telecommunication Union, with third party client and server implementations for other platforms . It uses TCP as the transport protocol and supports multiple parallel channels for the transmission of different flows of data  is a pro.TeamViewer (2005) uses its own intermediate servers to provide the service in a manner that facilitates the connection with remote desktop servers that are behind a Network Address Translator (NAT). Authentication is performed from the client and remote desktop server against the TeamViewer servers. When the identity is verified, the intermediate servers control connecting the client to the remote desktop server. The control communication is implemented over TCP  and the The PCoIP (2008) protocol was developed by Teradici Corporation. It is today offered by VMware View installations  and the The characteristics of these RD protocols are summarised in The scenario of remote desktop solutions in a real cloud environment, accessed from users through the public Internet, has not been studied in the literature. To address this deficiency, we deploy different remote desktop servers in the Amazon public cloud (Amazon EC2). We selected the Amazon datacentre in Ireland owing to its close location to our remote desktop clients in Spain.We created two similar virtual servers in the cloud. For the PCoIP evaluation, we selected the “value package” template in Amazon WorkSpaces. This is a Windows 7 installation on one CPU core running at 2.4 GHz and using 2 GB of RAM. The second virtual server was created to evaluate RDP, RFB, and TeamViewer. To achieve a fair comparison, we selected the most similar offering in the same datacentre. It was an EC2 instance using Windows Server 2012 on one CPU core at 2.4 GHz and 2 GB of RAM. On this virtual server, we evaluated RDP8 , RFB , and TeamViewer v11. For ICA HDX (Citrix XenDesktop 7.5), we created a local installation on a server with one CPU core at 2.4 GHz and 4 GB RAM.All the tests were performed using the same desktop PC as the client in our local network at the Public University of Navarre in Spain. For each remote desktop service, the most recent client version was selected. The Amazon WorkSpaces client v.2.1 was used to control the first virtual server (the one in the Amazon cloud). The client for RDP was the one from the Windows 10 installation, using RDP8 as the maximum common version between the client and server. The VNC client was Real VNC Viewer v5.3, and for the ICA protocol, the client from the Citrix XenDesktop 7.5 installation was selected. The local video resolution was 1280×1024 pixels. The setup is displayed in The access link to our Internet Service Provider (ISP) was recently updated to full-duplex 10 Gb/s. During the experiments, we conducted network bandwidth and delay tests between our local network and the Amazon datacentre in Ireland, achieving average rates of approximately 60 Mb/s downstream and 90 Mb/s upstream. The average measured round-trip time (RTT) was 53 ms. This is an average delay well below the maximum recommended value for interactive applications . Based on these measurements, we did not expect any limitation due to the access link.To measure the performance of remote desktop services, we defined three user profiles similar to those in  41]: of: of41]: We recorded user actions (keyboard presses and mouse movement events) using the Macro Recorder tool . The actFor the office user profile, we recorded the actions of a LibreOffice Writer user. He launched the text editor, wrote text, changed text styles, added images, and saved the document. All these steps required text selections, interaction with menus, and pop-up windows.For the “web browsing” profile, a user wrote a URL in a web browser and a rich content webpage was loaded. He scrolled around the page and clicked on hyperlinks. Three different webpages were loaded during the recorded test, including a newspaper landing page, a university homepage, and a web containing online courses. These were considerably different in the amount of multimedia content  included.The “video” user profile was based on a user viewing several video files. The same video files were reproduced at low and high resolution (from 144p to 1080p) using YouTube video qualities, including scenarios using full-screen mode. Some RD systems use a channel to transfer the multimedia file from the server to the client, whereas others send an on-the-fly compressed version of the video screen, extracted directly from the framebuffer. We expected large differences in video quality from one RD solution to another, especially for the 1080p resolution. Previous published works have used lower resolution video files ; today, We selected metrics for the evaluation of the network usage and QoE. These were based on the network traffic and video streams at both the server and client. A network usage profile is required for any link dimensioning to determine the minimum available capacity required in the path between a set of clients and servers in a VDC scenario. As the network path was not congested during the conducted experiments, we provide a measurement of maximum bandwidth required. The RTT was considered approximately constant for all the experiments as all the servers were co-located in the same datacentre and there was a common client. We attempted to correlate the user perceived quality with network usage, as these were expected to be tightly coupled. The downstream throughput was the principal component, as the upstream flow contained mainly user input (keyboard and mouse), protocol “keepalives”, and acknowledgements. We used tcpdump  at the cThe service quality experienced by the user of an RD system depends on the video quality and interactivity. A system can apply high compression techniques and produce a low bandwidth stream. The result is a lower video quality due to the high compression rate and a reduced interactivity due to the increased compression delay. Conversely, a system that applies low compression techniques produces higher video quality and lower delays; however, this is realised at the expense of higher traffic bitrates.The QoE is measured based on the difference between the video stream directly at the video output in the source desktop server and at the destination RD client. We use the PSNR  as an obThe video streams at the RD server and client are recorded simultaneously using Badicam software . Both viA reduced PSNR between both video streams could be the consequence of a loss of video quality due to a high compression rate at the server. The majority of remote desktop services use TCP at the transport layer and therefore network losses do not introduce video quality degradation. However, network losses (due to their recovery time), network one-way delay variations, and slower compression result in greater delays between the video at the RD server and the client. The result is a stream desynchronization that also produces reduced PSNR values. The remote desktop systems recover quickly from the desynchronization; however, the PSNR has already been locally impacted. EvalVid offers a relation between the measured values of the PSNR and an estimated MOS . The MOSFor every combination of RD system and user profile, the above-mentioned metrics were recorded. For each experiment, the procedure was: launch traffic capture at the client, launch desktop video capture at the server and the client, and finally, play the recorded user events (using a macro) for the selected user profile. After an experiment completed, both video streams were collected at the same machine, re-synchronized, and EvalVid was executed, obtaining the PSNR and MOS results. Based on the capture of network traffic, a network traffic profile was obtained.For scenarios with cloud deployment of remote desktops, the traffic from this service uses the company’s Internet access link. Sizing the required capacity for the access link and its packet buffers is vital for an adequate QoE. This dimensioning requires characterising the statistical behaviour of the remote desktop traffic.It has been reported for two decades that, contrary to traditional teletraffic theory, Internet traffic cannot be adequately modelled by processes with independent or short-range dependent random variables. High-resolution traffic measurements in LAN and WAN scenarios  52] [53 [5352]  and VariZ(t), t ∈ R be the continuous process of the number of bytes arriving in time interval . Fo. Fon → ∞l models  59] [60 [60n → ∞FH(t) is a Gaussian process that satisfies:A FBM Z(t) where the FGN models the arrival process X of bytes per interval.An Fractional Gaussian Noise (FGN) is defined as the increments of an FBM. An FBM is used as a model for the cumulative arrival process σ2, the aggregated process with level δ presents a variance δσ2. However, for an FBM the variance in the aggregated process For a process with independent increments whose marginal distribution has variance s σδ2 is :σδ2=δ2HH > 0.5 the result is larger variability in the arrival process of traffic to a network link, longer delays waiting in queue and larger packet loss probabilities [For bilities .In this paper, we study the long-range dependence in remote desktop traffic based on estimations of the Hurst parameter. We evaluate its value for different protocols and user profiles and its influence for large user aggregation levels.In the following sections, we first evaluate Amazon WorkSpaces in terms of transfer rate and QoE. Then, this is compared with the other remote desktop protocols.This section presents the evaluation results for the PCoIP protocol as deployed in Amazon WorkSpaces DaaS. It is a novel scenario, offering a massive deployment for the provision of virtualized desktops in the cloud. We identify the network and server requirements for each user profile as defined in a previous section and evaluate the resulting QoE. We model the user traffic as a self-similar arrival process, with different parameters for each user profile, which influence network link dimensioning. In a later section, we compare the results from PCoIP (Amazon WorkSpaces) to RDP, VNC, ICA, and TeamViewer.The access-link available bandwidth and link usage are fundamental characteristics as they limit the number of users for which remote desktop services can be deployed. Peak bitrate and its average are strongly dependent on user behaviour.the network connection should provide at least 300 Kb/s, with capability to provide over 1 Mb/s when viewing video or using graphic-intensive applications”. We must note that text editing is typically not a graphic-intensive application; however, it presents spikes in network usage consistent with this recommendation. It is demonstrated later that for video playback, the network requirements are considerably greater than those recommended.Traffic behaviour was consistent with Amazon recommendations for Amazon WorkSpaces , where iThe second visited web site was a university landing page. The user browsed through the news and information regarding the academic degrees offered. There were moving banners and automatic slideshows in the web page that resulted in continuous screen changes and therefore sustained traffic rates greater than 6 Mb/s. This is a clear indication of how rich content in a web page based on JavaScript, harmless in a local desktop environment, can result in high bandwidth requirements in a remote desktop deployment. The changing images on the screen were not large files, yet because of the animations, they became a video stream.The third visited web page was the landing page of a news site. The user scrolled the news headers and visited some of these. The page did not contain moving banners and hence did not result in sustained high bitrates. However, the multimedia linked files (images) were large, surpassing more than 9 Mb/s for some of the screen updates. The main insight from If the user presents the video in full screen mode, the transfer rate is similar for every video file resolution. The video playback program uses interpolation techniques to produce a higher resolution video stream that fills the screen, instead of presenting a simple scaled version of the video. Therefore, changes occur everywhere in the screen and, as indicated in A parameter related to the transfer rates is the packet size. In conclusion, the transfer rates identified for Amazon WorkSpaces differ substantially from the rates that Amazon itself recommends for the deployment of its services , with a We compared the desktop video stream recorded at the server (sent) and the client (received). Highly lossy compression and delay variations result in changes between both video streams. We obtained a PSNR time series of these changes using EvalVid. From this PSNR, a corresponding MOS value was obtained from However, in this scenario without bandwidth limitations, image quality is, in general, “Good” or “Excellent”. Even though there are differences between the source and destination streams, there are no noticeable compression artefacts. The differences could be noticeable through a heatmap, however not directly by the eye of a user.We summarise the PSNR time series for each user profile using the first, second (the median), and third quartiles. We display these values in Surprisingly, the PSNR values were less in the web browsing profile compared to the video profile. Moreover, the web browsing profile resulted in a higher data rate than the video profile. The web browsing profile using images, animations, and advertisements was more demanding in PCoIP DaaS than video streaming.Applications such as the web or variable bit rate video generate self-similar traffic. Therefore, it was expected that remote desktop traffic would exhibit this property. We evaluated the presence of this property by estimating the Hurst parameter for the traffic arrival process. Many of the proposed algorithms for the Hurst parameter estimation are based on the variance aggregation plot, R/S  statistic , periodoα for each data set. The resulting Hurst parameter is computed as H = (1 + α/2). H and the coefficient of determination in the regression (r2), measuring the quality of the fit.H > 0.5). For the video user, the scaling changes and is not as well fit by a strictly self-similar process (FBM). It continues to provide an estimation of H greater than 0.5 for the scales of interest and indicates that the model is sufficiently accurate.The linear fit is acceptable for the office and web users, which indicate clear long-range dependence , and Citrix ICA protocols. We follow the same procedure used in the previous section and present the results for network bandwidth usage, self-similarity, and QoE for each of the three user profiles.The results are consistent among the five remote desktop systems. The user profiles with larger and more frequent screen changes require more link capacity (web and video profiles); however, the rates vary substantially among the different systems. Attention must be addressed to the logarithmic scale employed for the downstream rate in Regarding packet sizes, TeamViewer consistently provided lower values of MOS than the other RD systems. This is because of the lossy compression it applies. For the office user profile, the other RD systems analysed offered excellent quality (MOS “5”) for at least 75% of the frames, whereas TeamViewer never achieved this quality and its median value was in MOS “3” (“Fair” or “Slightly annoying”).The web browsing user profile demonstrated the highest variability in quality for those protocols that send bitmaps from the server to the client . TeamViewer and VNC provided median MOS values of “2” and “3” for this profile, whereas PCoIP, RDP, and ICA remained above MOS “5” at least 50% of the time.For the video user profile, fast screen changes have an important influence on QoE because of the additional delay they introduce. VNC and TeamViewer offered the lowest qualities whereas PCoIP maintained an MOS greater than “4” more than 75% of the time, even though they all employ bitmap transfers.TeamViewer demonstrated a reduced MOS because it increases the compression degree when there are rapid changes in the image. It prioritises a fast screen update at the client, at the cost of a lower image quality. The comparison of the video feed at the server and the client in these situations results in a reduced PSNR and hence, a lower MOS value.VNC not only suffers delays due to a greater amount of data to transfer on fast screen changes but also renders the screen as it receives the data for different sections. The result is that a part of the screen could be displaying a previous video frame and the remainder displaying the new frame. The resulting PSNR of comparing the video feed at the server and the client is seriously hampered in these situations, providing a reduced MOS value.H = 0.75). Conversely, the web user profile creates the traffic with the greatest value of H or the strongest long-range dependence. This consistent behaviour implies that the reason for the LRD is not related as much to the characteristics of the remote desktop protocol as it is to the user actions. For any of these protocols, the web users are those who create the traffic with the strongest LRD and therefore, the poorest behaviour in router queues. Although the video users present the highest average bit rates . In the second scenario, we consider a mixture of users from the three different profiles.We computed the average traffic, variance, and Hurst parameter for every combination of protocol and user profile. From these parameters, we can generate synthetic FBM traffic traces using one of the existing FBM generation techniques. For this paper, we used the Random Midpoint Displacement (RMD) method, a fast and efficient generation method adequate for qualitative studies . For eveIn the case of a mixture of processes with different values of H (different user profiles), it has been demonstrated that the resulting process is dominated by the largest value of H in the mix . HoweverTo compare to the previous homogeneous case, we again multiplexed 90 users for each protocol; 30 users from each of the users profiles. This means that, for example, we created 30 FBM traffic traces using the parameters from ICA office users and multiplexed them to 30 FBM traffic traces from ICA video users and 30 FBM traces from ICA web users. The values of H in the multiplex are not always near the largest H in the mixed set; however, they are always in the range of values in the mixture see . For exaThe final evaluation considers the opposing metrics of bandwidth usage and QoE. Typically, a higher quality requires greater bitrates; hence, the tradeoff of achieving the best quality with the lowest bitrate is important.In the lower left corner, TeamViewer presents the lowest PSNR; however, it also consumes the least amount of bandwidth. Other protocols requiring less than 1 Mb/s do not sustain this rate for all user profiles. For example, RDP requires less than 1 Mb/s for the office and web browsing user profiles, yet requires an average of 3 Mb/s for the video user.TeamViewer simplifies link bandwidth dimensioning when measuring only average bit rates. However, different user profiles present significant differences in the traffic long-range dependence, which influence packet buffer dimensioning. TeamViewer is an extreme case of this situation as it indicates a Hurst parameter as low as 0.693 for an office user and as high as 0.946 for a web user . It doesVNC requires a large link capacity for any dynamic content (the web browsing and video profiles), obtaining low QoE owing to the delays in rendering. It is a reasonable solution only for an office user with infrequent changes of large parts of their screen.PCoIP maintains an acceptable quality (MOS at least “3”) for every user profile, with a reasonable link capacity requirement for the office and web browsing profiles. The video case requires several megabits per second, however it offers an increased quality compared to every other desktop system.For the office user profile, the best quality at the least cost is obtained by the protocols that transfer system graphics commands (ICA and RDP). This is true both on bit rates and on values of H. They do not require sending screen bitmap updates; rather, they send the instructions to recreate the GUI status at the client . This typically requires smaller downstream updates and shorter bursts. For video playback and some video content in web browsers, these systems transfer the video file for local playback using an independent communications channel, obtaining acceptable quality with a reasonable link capacity, as the original compressed file they transfer is typically smaller than the result of the on-the-fly compression of the screen updates.Based on the MOS scale, certain combinations of user profile and RD system should be excluded. VNC is not suitable for a video user and TeamViewer does not provide sufficient quality for video and web browsing with highly dynamic content. For an office user, TeamViewer does not provide sufficient quality. Other solutions with a similar bitrate provide a superior experience.For a web browsing user, RDP and ICA likely offer the best trade-off between bitrate and quality. PCoIP must compress the animations in the web page as a video stream and therefore obtains lower quality, even with higher bitrates.For a video user, RDP, ICA, and PCoIP present acceptable quality, with the lowest bitrate achieved by the RDP solution. Between ICA and PCoIP, the latter offers improved quality at the same cost.Optimum link capacity cannot be determined based only on the average expected traffic. The self-similar nature of remote desktop traffic is clear and it is not alleviated with reasonable degrees of traffic multiplexing. For a mixture of users, the worst profile (the web profile) dominates in the resulting traffic. Depending on the number of users and the number from each profile in the traffic mix, the result will be closer to the behaviour of the strongest long-range dependent traffic.The most important suggestions that can be extracted to improve user experience in DaaS solutions are:Protocols that transfer system graphics commands (ICA and RDP) are better suited to office user profiles because functions such as the frequent opening and closing of system windows, menu scrolling, and text inputs are not transferred as screen image updates through the network. They avoid streaming the user screen as video, as they transmit system graphics commands. This means lower traffic bit rates with high image quality, achieving low response times, and therefore the best QoE.Protocols that transfer system graphics commands (ICA and RDP) also achieve acceptable results in web browsing and video profiles because they use specific channels to transfer the content . Each content is coded according to its nature and, if possible, is transmitted without further compression, using the original source data that is already compressed and adapted to be streamed over the network . RDP and ICA offer the best trade-off between bitrate and quality. However, the client PCs must be more powerful  because they must process content from the specific channels, sometimes using complex codecs.Multiplexing hundreds of users with an office profile provides less long-range dependence (lower Hurst parameter) for ICA and RDP, as they use system graphics commands instead of streaming a video from the full screen as in other protocols. Even with the web and video profiles, the resulting H value for multiplexed users is better than for the other protocols. This means that the required bandwidth in the Internet link will increase smoothly with the number of simultaneous users.In some protocols , all content is streamed as screen bitmap updates. Therefore, the differences among office, web, and video profiles are related to the size and speed of the changes in the screen images. In this case, the web profile has, surprisingly, the highest H value and larger link data rate requirements than the video profile for the same MOS. This is because of the full screen updates required when scrolling a web page or the embedded advertisements.Protocols can offer low bit rates using complex codecs with lossy compression (TeamViewer is an example). However, they accomplish this at the expense of a reduced MOS and in some situations, they result in a greater degree of self-similarity in the traffic. This makes link capacity dimensioning more complex and packet buffers less effective to reduce losses, as the traffic contains larger bursts.We compared five of the most popular remote desktop protocols and offered models for their traffic arrival based on self-similar processes. They were deployed in a public virtual cloud as a DaaS solution. The protocols were: PCoIP as used in the Amazon WorkSpaces, Microsoft RDP, TeamViewer, VNC (RFB), and Citrix ICA. We evaluated the network transfer rate and its relation to the quality experienced by the DaaS user. We compared three different user behaviours based on productivity: an office software suite, web browsing to modern and dynamic websites, and a video user accessing low and high quality video streams.The QoE measurement was accomplished by comparing the desktop video stream at the source (the server in the cloud) and the destination (the user client). An objective PSNR time series was obtained from the comparison and from this, we produced subjective MOS values. This evaluation considered not only image degradation due to lossy compression but also loss of interactivity from an increased delay, as resulted in video stream desynchronization.The results demonstrate that the Amazon WorkSpaces solution (based on PCoIP) presents a reasonable quality for the three user profiles, although its network bandwidth usage for a video user is considerably greater than the recommended values suggested by Amazon. We confirmed that the recommended traffic rates of 300–1000 Kb/s are reasonable for the office profile. However, for the web browsing and video profiles, we determined that sustained rates up to 10 Mb/s are common. Moreover, the degree of self-similarity in network traffic is greater for web users than for the other user profiles, including video consumers. A network administrator must consider this when dimensioning an access link for a population of Amazon WorkSpaces users.Protocols based on the transfer of graphics primitives (such as RDP and ICA) offer high quality with a low traffic bit rate for a normal productivity desktop user. For multimedia playback, they include parallel channels for the transfer of video files instead of streaming an on-the-fly compressed video extracted from the screen.Solutions such as VNC and TeamViewer are less suited for a DaaS deployment and a better fit for remote control of physical desktops during short tasks. TeamViewer primes a low network bandwidth usage at the expense of the quality, hence it is an acceptable solution in remote assistance scenarios where the interaction is short and high quality is not required. VNC is the simplest system; hence, it offers minimal optimisation compared to the other analysed solutions. The result is high traffic bitrates and less than proportional quality as the compression task introduces delays that degrade the interactivity.


Specific loci that form the genetic framework underlying high Mg2+ homeostasis, however, are not well understood. By using GWA mapping on 388 accessions of Arabidopsis thaliana selected from a worldwide collection and genotyped at approximately 250,00 SNPs, we successfully identified 109 and 74 putative genetic regions associated in nutrient traits under normal  and high Mg2+ , respectively. Above 90% SNPs associated with nutrients including Mg2+ and only two SNPs shared between normal and high Mg2+. A single strong peak of SNPs associated with Ca concentration corresponding to candidate gene At1g60420 ARABIDOPSIS NUCLEOREDOXIN (AtNRX1) under high Mg2+ was further determined. Compared with wildtype, mutants of Atnrx1-1 and Atnrx1-2 supplied with high Mg2+ had higher Ca concentrations in the plant, and higher cytosolic Ca2+ concentrations during root elongation, as well as higher fresh weight and lateral-root number. This suggests that AtNRX1 was a critical gene negatively regulating Ca uptake under high Mg2+ conditions. The discovery could help to breed/select crops that can adapt to high-Mg2+ soils such as serpentine soils (high ratio of Mg2+: Ca2+) or Mars soil with high levels of magnesium sulfate.High magnesium (Mg Magnesium in soils originates from source rock material containing various types of silicates and carbonates. In extreme serpentine  soils2 and in semi-arid regions, where water stress conditions3 can lead to Mg accumulation. In addition, Mars regolith  is a potential medium for plant growth in bioregenerative life support systems. However, analyses by the Mars Exploration Rover Landers at Meridiani Planum and Gusev Crater have also suggested there are particularly high levels of magnesium sulfate minerals in outcrops and soils6.Magnesium (Mg) is the 82+ for normal growth and development, excessive Mg2+ accumulation often causes toxicity to plant cells. Plants grown in high-Mg2+ soils like serpentine or Mars soils may alleviate Mg toxicity by limiting internal Mg accumulation, Mg excretion from leaves and/or increasing plant tolerance to high Mg2+ concentrations via altering leaf size, sclerophylls, stature and root systems8. Previous reciprocal transplant experiments between serpentine populations (or species) and their spatially adjacent populations (or sister species) growing on serpentine soils demonstrated unequivocally that adaptation to serpentine soils had a genetic basis12. Several quantitative trait loci that control contrasting adaptive traits in serpentine-tolerant Microseris douglasii and serpentine-intolerant M. bigelovii have also been identified13. A high degree of polymorphism has been reported among plant species in terms of growth tolerance to serpentine soils16.Although plants rely on a sufficient supply of Mg17, to identify candidate genes likely to be involved in adaptation to high Mg2+, based on 388 accessions of Arabidopsis thaliana selected from a worldwide collection. Arabidopsis thaliana has a broad geographical distribution and consequently is subject to varying nutritional environments, which makes it a useful model species for studying nutrient homeostasis19 in a wide variety of environments20. Importantly, more than 1300 distinct accessions have been genotyped21 and to date more than 1000 inbred lines have been ‘fully sequenced’. Meanwhile, automated phenotyping platforms have been developed24, allowing for precise and high-throughput measurements of plant growth. These developments enhance our ability to map causal genetic polymorphisms through GWAS. Moreover, high Mg2+ decreased the growth of Arabidopsis including biomass production, root hair development and the absorption of other nutrients28. To further identify the likely candidate genes that are involved in adaptation to high-Mg2+ soils, we used a chemically-defined nutrient solution that contained 10,000 μM MgSO4 of maximum authentic high Mg2+ soils to perform a viability screen on a large collection of wild-type accessions of Arabidopsis thaliana. This study aimed to (1) investigate natural variations in growth response to high Mg2+, and (2) identify novel genes tolerant to Mg2+ excess.This present study employed genome-wide association studies (GWAS), an approach that has been applied successfully to identify many different traits ranging from genetically simple traits to more complex featuresArabidopsis Biological Resources Center stock center (http://nasc.nott.ac.uk). The accessions were selected without any a priori consideration of their natural habitat, including geographical distribution or precipitation levels, and were collected from the wild and available in the stock center or soon-to-be-released collections. Most of these accessions were originated from Europe including Sweden, Germany, France, Czech Republic and United Kingdom, and from North America and Middle Asia, representing the sampling strategy of the 1,307 worldwide accessions21. This was consistent with the distribution of the 1,307 Arabidopsis lines collected in RegPanel.Seeds of 388 accessions with accessions CS76636, CS76427, CS78885 and CS22660 and two T-DNA insertion lines SALK_100357C and SALK_113401C were derived from the Arabidopsis thaliana were grown under 2 Mg2+ treatments, which were achieved by altering the concentration of MgSO4 in the basal medium. To minimise maternal effects prior to phenotyping, natural accessions were grown for one generation during 2015 under controlled greenhouse conditions at Zhejiang University . For surface sterilization, seeds of various accessions were placed for 1 h in open 200-μL PCR tubes in a sealed box producing chlorine gas generated from 13 mL of 10% sodium hypochlorite and 350 μL of 37% hydrochloric acid. Sterile seeds were then put on the surface of 30 mL agar media, containing 1.2% (w/v) agar and 0.6% (w/v) sucrose  in 10 × 10-cm2 plates with grid schematic engraved below the plate. Plates were positioned in racks and oriented in a vertical position, and were kept at 4 °C for 48 h in the dark for seed stratification. Thereafter, the racks containing the plates were transferred to a growth chamber under a 10-h light/14-h dark photoperiod at constant temperature of 22 °C, 60% relative humidity and light intensity of 120 µmol photons m−2 s−1. Twelve seedlings were grown in each plate and each treatment received at least four independent replicates. The racks were removed to the image acquisition room once per day and then immediately returned to the growth chamber. Throughout the experiments, the plate position within the box and box position in the growth chamber were re-randomized daily. For assays on agar plates, studies were performed on 8-d-old plants; that is, at an early stage of their stability and homogeneity growth phase. Moreover, many previous publications adopted 6- to 8-d-old Arabidopsis seedlings under stresses for determining Arabidopsis morphology, physiology, and development as well as responses to nutrient stresses31.The 388 accessions of 2+, N), which was used as control, contained (µM) 1500 KNO3, 500 NaH2PO4, 1000 CaCl2, 250 (NH4)2SO4, 1000 MgSO4, 25 Fe-EDTA, 10 H3BO3, 0.5 MnSO4, 0.5 ZnSO4, 0.1 CuSO4 and 0.1 (NH4)6Mo7O24. The normal and high Mg2+ treatments contained 1,000, and 10,000 µM MgSO4, respectively, which was in accordance with Mg2+ concentrations in soil solutions27. 10,000 μM Mg2+was selected in the present study for the quantitative analysis of the natural variation of Arabidopsis in response to high Mg2+ stress because this concentration had an adverse effect on growth without toxic symptoms of most natural accessions32. Besides, the concentrations of Mg2+ in the control were 1000 µM, that has been adopted for Arabidopsis growth by many plant biologists35. Though the concentration of SO42− increased 10 times comparing with that in the normal medium, SO42− had hardly affected morphogenesis of Arabidopsis36. The pH of the growing media was adjusted to pH 5.8 with MES (N-morpholino) ethane-sulphonic acid)-KOH buffer before autoclaving.The basal medium 27. The length of primary roots, rosette diameter and epicotyl were determined across the median seedling using Image J. Lateral-root number was determined by counting the number of true roots  per primary root. For each condition, an independent sample accession consisted of four plants at the same growth stage, and the time of sampling was the end of the light period of day 8.Each line had four biological replicates for each treatment. Seeds that had not germinated at day 6 were discarded from further analysis, resulting in approximately 3–4 seedlings analyzed per genotype per treatment. The 21 traits under each treatment were classified into 2 categories: 7 morphological traits and 14 nutrient traits. Seedlings were photographed with a high-resolution digital camera  daily for determination of root and shoot growth. Root or shoot biomass, and the number of days from seeding to emergence (>50% seedlings having the first radicle or cotyledon), were recorded. Meanwhile, photographs after 8 days of treatment were analyzed and quantified for phenotype using the public domain image analysis program Image J version 1.43 , calcium (Ca), Magnesium (Mg), sulfur (S), iron (Fe), manganese (Mn) and sodium (Na) in the digests were analyzed by ICP-MS , the contents per plant were calculated. Mineral analysis was done in the whole-plant.After 8-d growth at various Mg2+-sensitive fluorescent dye 1-[2-amino-5-phenoxy] -2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid, pentaacetoxymethyl ester (Fluo-4/AM) ester was used to estimate cytosolic Ca2+ concentration in the roots. The Fluo-4/AM ester was loaded into the root at 37 °C in the dark at a final concentration of 5 μM Fluo 4-AM dye and Pluronic F-127 (0.05%) in the Hank’s Balanced Salt Solution (HBSS) solution. After 1-h incubation, the roots were washed with the standard HBSS medium three times and left at 25 °C for 30 min. The samples were mounted and photographed with a Zeiss LSM 780 . Fluorescence was detected using 488 nm excitation and 505–545 nm band pass filter. Roots including root hairs were placed on the stage of a Zeiss Axiovert inverted microscope attached to a LSM780 laser scanning confocal microscope, and imaged using a Zeiss 20×, N.A., wet objective. Each frame represents a 7-s scan of the laser.The Ca2+ (Supporting Information Table http://gwas.gmi.oeaw.ac.at/) interface using the mixed model algorithm method37. A kinship matrix was generated using the identity in state of SNPs between each pair of accessions38. The P-value above bias due to population stratification was evaluated with Q–Q plots. Candidate loci were selected based on the minor allele frequency, LOD score and trait-associated SNPs and their position in the genome to pinpoint candidate genes within 5 kb up- and down-stream of the identified SNP (Supporting Information Table Arabidopsis.org).The GWA mapping was performed on the values of epicotyl length, rosette width, primary root length, and lateral root number of the median seedling of each accession. For traits of and nutrients, means values of 4 biological replicates were used. We linked 21 traits under normal or high MgArabidopsis after grown at normal and high Mg2+ treatments. For each condition, four samples were taken on independent seedling materials from eight different plants at the same growth stage and the time of sampling was the end of the light period of day 8. Total DNA was extracted by Plant DNA Isolation Reagent (Takara) from about 80 mg of fresh seedling tissues of four independent biological replicates. Two homozygous T-DNA insertion lines  and  was conducted by PCR with gene-specific primers SALK_100357C LP: 5′-AACCCAACCATCTTTGGACTC-3′; SALK_100357C RP: 5′-TCAAGACTTCAAGACCAAGCC-3′ and T-DNA border primer LBb1.3:5′-ATTTTGCCGATTTCGGAAC-3′; SALK_113401C, LP: 5′-ATCCACTTTTGTCGTTGAACG-3′; SALK_113401C RP: 5′-CGATCGCAACTTCTTCTGATC-3′ and T-DNA border primer LBb1.3:5′-ATTTTGCCGATTTCGGAAC-3′; Total RNA was extracted by RNAisoPlus  from about 70 mg of fresh seedling tissues of four independent biological replicates. All RNA samples were checked for DNA contamination before cDNA synthesis. cDNA was synthesized, and possible residual genomic DNA contamination was verified as described in our previous study39. The mRNA levels of AT1G60420 in two lines of SALK_100357C and SALK_113401C were detected by the SYBR Green RT-PCR kit (Takara) with following pairs of gene-specific primers: AT1G60420, fw: 5′- TTCTCGGCCCTGATGGAAAAACC-3′, rv: 5′- GCTTCATTCCGCTCCTTTATCTGCT-3′ A pair of housekeeping gene of UBQ10  was used for a control PCR. Melting curve analysis and gel electrophoresis of the PCR products were used to confirm the absence of non-specific amplification products. Relative expression levels were calculated by subtracting the threshold cycle (Ct) values for UBQ10 from those of the target gene (to give ∆Ct) and then calculating 2−∆Ct, where Ct refers to the cycle number at which the fluorescence rises above the set threshold qPCR. UBQ10 was chosen as the housekeeping reference40.Samples were collected from 8-day-old www.1001genomes.org). All statistical analyses were conducted with DPS software . Means were compared by the t test or the Fisher’s least significant difference test at P = 0.05.DNA sequences were obtained from the 1001 Genomes project  had an adverse effect on the growth of most natural accessions. This high Mg2+ concentration induced a large degree of phenotypic variation and thus was selected for the quantitative analysis of the natural variation of Arabidopsis in response to high Mg2+ stress .Distribution of 388 ess Fig. . A subse+ supply 0,000 μM 2+ absorption under high Mg2+) and Mg-C-H (Mg2+ concentration under high Mg2+) showed a great right-skewed probability density with the right-skewed being greater for Mg-C-H than that for Mg-A-H, suggesting that high Mg2+ supply increased Mg concentration of most Arabidopsis accessions at least in the early phase of development. However, only slight skewness and kurtosis was observed for S absorption and concentration  were used to examine the natural variation within the population of quantified traits between normal and high Mg2+ conditions (Table 2+ treatments (r2 = 0.53) (Table 2+ treatments (r2 < 0.2), suggesting different physiological functions of nutrients under different Mg2+ supplies.Pearson’s correlation coefficients r were use2+ would compete for uptake with Ca2+, Fe2+ and Na2+ in most Arabidopsis accessions when supplied with high Mg2+. Increased Mg2+ absorption would decrease the uptake of these ions and hence plant growth. However, Mg-A, Mg-C, S-A, S-C, K-A and K-C were positively correlated only under high Mg2+  showed a negative correlation between Ca-C-H, Fe-C-H and Na-C-H and Mg-C-H Fig. , suggestg2+ Fig. , indicatg2+ Fig. . In contply Fig. .2+ conditions (Supporting Information Table 10(p) > 6.15 as the nominal genome-wide significance threshold, the GWASs had 109 hits in total, 32, 10, 25, 18 and 24 located on chromosomes (Chr) 1, 2, 3, 4, and 5, respectively, under normal Mg2+, and 79 hits in total, 14, 13, 18, 10 and 19 located on chromosomes 1, 2, 3, 4, and 5, respectively, under high Mg2+  and concentration (Mn-C-N), and accounted for 7.47% and 2.67% of the total phenotypic variance, respectively. 17 and 21 loci were associated with Fe absorption (Fe-A-N) and concentration (Fe-C-N), and accounted for 3.60% and 5.04% of total phenotypic variance individually  and concentration (Mn-C-H), and accounted for 2.93% and 12.04% of total phenotypic variance individually. Thirty three and 11 loci were associated with Fe absorption (Fe-A-H) and Fe concentration (Fe-C-H), which accounted for only 5.07% and 2.60% of total phenotypic variance individually  1_3884526  was found to associate with SG-H, RG-H, RG-N and Na-C-N. It had a Minor allele frequency (MAF) of 0.0633, and is within the 2+ (Mg-A) and Ca absorption (Ca-A) under normal Mg2+, but 2 loci were identified to associate with these traits under high Mg2+. Furthermore, no significant associations were found for Mg2+ (Mg-C) and S concentration (S-C) under high Mg2+, whereas 7 and 6 loci were identified to associate with these traits under normal Mg2+  > 6.9) corresponding to only a candidate gene At1g60420 (AtNRX1) g2+ Fig. . This SNX1) Fig. , and wasAtnrx1-1 and Atnrx1-2) and physiological and morphological comparisons in mutants and wild-types. Before this, we further verified that the mutant stocks were homozygous T-DNA insertion lines, by PCR with gene-specific primers LP, RP and LB. As expected, the results confirmed that both Atnrx1-1 and Atnrx1-2 were only mutation in At1g60420 . These results indicate that AtNRX1 is a special and critical negative gene regulating Ca uptake and probably the plant growth under high Mg2+ stress.Compared with the wildtype, both g2+ Fig. . Under h2+ in the environment, plants have evolved mechanisms of reduced ion uptake and/or sequestering excessive ions in the vacuole or developing higher Mg2+ requirement for the maximum growth, which finally improves the tolerance to the stress of certain elements. By linking the mapping with reverse genetic experiments, we identified the causal gene and established that polymorphisms at AtNRX1 were the major genetic determinant for the variation in Ca concentration in this global A. thaliana population  > 6.15 reflects the high complexity of the underlying genetic architecture or that these traits were not influenced by any of the SNPs alone. This notion is supported by the presence of obvious sub-threshold peaks associated with shoot and root morphology under both normal and high Mg2+ supply. Meanwhile, the low heritability of investigated traits in the present study suggested a weak genetic component determining the observed phenotypic variability. Hopefully, it is known that many traits of Arabidopsis are polygenic with small effect size, and that increasing the sample size would improve the power to recover meaningful associations42. Therefore, increasing sampling size was likely to underestimate the range natural variation in photomorphogenic responses or growth-related pathways and processes that strongly interact with the high Mg2+ stress.By using the phenotypic data of 388 2+, the effective number of SNPs (−log10(p) > 6.15) was also different 43. Likewise, SNP 3_19424421 of p-value 12.55, associated with Mn-C under the normal Mg2+ condition, had a MAF of 0.058 and was within gene AT3G52380 CHLOROPLAST RNA-BINDING PROTEIN 33 (CP33) that is co-expressed with SEN1. SEN1 gene was reported encoding an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar Fe/Mn transporter of Saccharomyces cerevisiae of Arabidopsis41. Besides, SNP 1_210424, associated with Fe-C, had a MAF of 0.055 and was within gene FRO2 which encodes the low-Fe-inducible ferric chelate reductase. It is suggested to be the major Fe(III) chelate reductase in Arabidopsis44. The most SNP hits under high Mg2+ were identified associations with Fe-A and Fe-C and then Mn-A and Mn-N. SNP 1_26930918 had a MAF of 0.074 and was located within AT1G71480 and AT2G33410 genes which are involved in nuclear transport factor 2 family gene, and are co-expressed with Fe transporter gene FERRIC REDUCTION OXIDASE 6 (FRO6) and 7 (FRO7)45 and 1 (FRO1)46. Furthermore, SNP 2890675, associated with Fe-C under the high Mg2+ on Chromosome 5, had a MAF of 0.074 and was located within AT5G09310 gene and co-expressed with Fe transporter gene FRO146. Likewise, SNP 3_6977829 of P-value 8.40, associated with Fe-A, within gene AT3G20015 (ASPG2), and was co-expressed with H( + )-ATPASE 2 (AHA2), a main actor in Fe uptake and signaling47.Though not all cases, the chromosomal regions harbouring the significantly associated SNPs are coincided with the location of the gene(s) co-expressed with known-function genes that control these traits. Of the 105 loci under normal Mg2+, had a MAF of 0.079 and was located within AT4G01800 ALBINO OR GLASSY YELLOW 1 (AGY1) gene which was co-expressed with MAGNESIUM CHELATASE I2 (CHLI2) while CHLI2 regulates the function of Mg2+ chelatase48. It should be mentioned that SNP 1_21762851 of P-value 9.05, associated with S-C under the normal Mg2+, had a MAF of 0.090 and contained only one gene, AT1G51980, which encodes an insulinase and is co-expressed with SULFATE TRANSPORTER 1;2  that is involved in sulfur metabolism49. As discussed above, changing the Mg2+ availability in the soil would alter absorption and utilization of other elements, and transporters, channels, or/and the genes that encode and regulate them in Arabidopsis. However, a few of the genes in proximity of the most significant associations were among the major known regulators of ion uptake or transport, highlighting the potential of GWAS to identify previously unknown regulators50.In addition, SNP 4_772594 associated with Mg + C under the normal MgA. thaliana, but the effect may vary among populations or genotypes53. Decreases in nutrient availability result in phenotypic responses  that vary across A. thaliana genotypes.Changes in nutrient availability influence plant vegetative growth and phenotypic traits of 2+) act as an osmoticum within vacuoles, a stabilizing element in the membranes, a strengthening agent in cell walls, and a secondary messenger for a multitude of signals56. By using GWA mapping on the 388 A. thaliana accessions, we successfully identified a single strong peak of SNP 1_22263976/22264331/22262848  associated with Ca concentration under high Mg2+  during root elongation, meanwhile produced higher biomass and more lateral roots as compared with wildtype 58. Study of Tang et al.59 showed that tonoplast CBL–CIPK calcium signaling network regulated ion homeostasis and vacuolar sequestration of Mg2+, thereby protecting plants from Mg2+ toxicity60. Besides, loss-of-function cax1 (gene encoding vacuolar H+/Ca2+) mutations could produce phenotypes characteristic of plants adapted to serpentine8.It has been known that AT1G60420 (AtNRX1), a gene involved in catalytic activity of NADP-NADPH and disulfide-reductase activity61. In special cases, toxic ROS molecules are largely accumulated under excess stresses and diverse enzymes played as ROS scavengers. Arabidopsis contains 3 NADPH-dependent thioredoxin reductases (NTRs) for scavenging ROS and Nucleoredoxins (NRX) are potential nuclear TRX found in most eukaryotic organisms61. Recently, excellent research presents evidence that activity of the plant stresses-inducible TRX superfamily member, NRX1 is necessary for the integrity of antioxidant systems62. Meanwhile, it is the fact that many publications indicated ROS produced by NADPH oxidase activates Ca2+ channels at the plasma membrane, leading to an increase in Ca2+ concentration and a tip-focused Ca2+ concentration gradient and subsequent signaling inherent to plant growth66. Withal, our previous study also showed that high Mg2+ could inactivate NADPH and the production of ROS27 probably through regulating ABA–DELLA signaling67.This locus has a MAF of 0.0712 and is within the 2+ concentration; While in the nrx1 mutants, ROS could not be effectively removed without protection of NRX1 due to NRX1 mutation, which leads to ROS accumulation. Then ROS activate Ca2+-channels at the plasma membrane and promote cytosolic Ca2+ concentration. When both wild-type and nrx1 mutants under normal Mg conditions, the cell is normally controlled with H2O2 and redox balance, thus the protective effect of NRX1 gene is not prominent, or maybe even little dedication.To sum up, it is likely that under high Mg, the wild-type plant is associated with cellular accumulation of ROS and redox imbalance, which triggers class of antioxidant enzymes activation and subsequently results in a decrease of intracellular ROS. ROS restrains ion channels and Ca2+ concentrations in Atnrx1-1 and Atnrx1-2 mutants was increased compared to that in the wildtype plants under high Mg2+ supply. This implied that nrx1 mutants required for a high concentration of Mg2+for the maximum growth associating with tolerance to high Mg2+ soils. The AtNRX1 gene is co-expressed with MAGNESIUM TRANSPORTER 4 (MRS2-3), a transport gene related to Mg2+ nutrition68. These indicate that Mg2+ transporter genes have some interactions with AtNRX1. However, there is no significant difference in K, Fe, Mn and S concentration in plants between the two mutants and wildtype, but the mutant had a higher Na content (Data not shown). These was consistent with other study showed that the cbl2 cbl3 double mutant was hypersensitive to a handful cations but not to Na+ 59. Further work will be directed to identify other key components in this novel of NRX-signaling pathway in regulation of Ca uptake and how the positive effects of AtNRX1 can be maximized through plant adaptive strategies to improve the tolerance to high Mg2+.Notably, MgAtNRX1 was a critical gene negatively regulating Ca uptake and probably improving the plant tolerance to high Mg2+ soils. The discovery of the functions of AtNRX1 gene helps to breed/select crops that can adapt to high-Mg2+ soils such as serpentine soils (high Mg2+: Ca) and those in semi-arid regions or even serpentine or Mars soils with high levels of magnesium sulfate. Also, new genes associated with natural variation in A. thaliana can be used for future bioengineering.To sum up, our study suggests that Supplemental infomationTable S1Table S2Table S3


Freezing behavior is commonly used as a measure of associative fear memory. It can be measured by a trained observer, but this task is time-consuming and subject to variation. Commercially available software packages can also be used to quantify freezing; however, they can be expensive and usually require various parameters to be adjusted by the researcher, leading to additional work and variability in results. With this in mind, we developed Phobos, a freely available, self-calibrating software that measures freezing in a set of videos using a brief manual quantification performed by the user to automatically adjust parameters. To optimize the software, we used four different video sets with different features in order to determine the most relevant parameters, the amount of videos needed for calibration and the minimum criteria to consider it reliable. The results of four different users were compared in order to test intra- and interobserver variability in manual and automated freezing scores. Our results suggest that Phobos can be an inexpensive, simple and reliable tool for measurement of fear-related behavior, with intra- and interuser variability similar to that obtained with manual scoring. The pairing of a conditioned stimulus  with an aversive unconditioned stimulus  produces an association between stimuli that leads to fear conditioning, a phenomenon that is widely used to study memory in laboratory animals  that render them incapable of making precise measurements . For contact regarding the software, an e-mail address has been setup at phobos.freezing@gmail.com.Code for the software was written in MATLAB 2017 (MathWorks) and is provided as supplementary material along with the user manual. Code can either be run on MATLAB or as a standalone program, both available under a BSD-3 license at The video analysis pipeline performed by the software is described in For calibration, a reference video is chosen to be scored manually by the user using the software interface. The user is asked to press a button each time the animal freezes to start quantification of freezing time, and to press it again to stop it. For each video, an output file is created containing the timestamp for every frame in which the observer judged the rodent to be freezing. A warning message appears if freezing scores for manual quantification represent less than 10% or more than 90% of the total video time, as both situations can compromise calibration.The data is analyzed in blocks of 20 s, and freezing time is calculated for each of these bins. The same video is then analyzed automatically by the software using various combinations of two parameters (freezing threshold and minimum freezing time), and the results for each 20-s block are systematically compared with the experimenter’s manual freezing score for the corresponding epoch. Methods for determining parameters, calibration duration and validation criteria will be detailed in the software validation section.r) are initially chosen, and a linear fit for each one is generated. Among these, the software then selects the five combinations of parameters with the slopes closest to 1, and after that, the one with the intercept closest to 0, in order to avoid choosing a combination of parameters with good correlation and poor linear fit for absolute freezing values. For each reference video, a MAT file containing the best parameters is created and can be used as a calibration reference for use in other videos recorded under similar conditions.The 10 parameter combinations leading to the highest correlation between manual and automatic scoring for 20-s epoch freezing times  the freezing threshold, defined as the quantity of non-overlapping pixels between adjacent frames below which the animal was considered to be freezing; (b) the use of separate thresholds to begin and end the recording of freezing epochs; and (c) the minimum freezing time for an epoch, defined as the minimum amount of frames in which the score needed to remain below threshold to be counted. Thresholds was varied between 100 and 6,000 pixels with steps of 100, while minimum freezing time varied between 0 and 2 s with steps of 0.25. For these validation steps, we used all videos from each set to calibrate the parameters.r. Correlation between automated and manual scores was performed with optimization of each parameter  and without it  in order to assess their importance  curves to predict whether calibration using a specific video would yield an r-value of at least 0.6 between automated and manual scoring in the whole video set, using either the r-value or the slope (β) of the calibration video as a predictor. Values for r and slope leading to optimal sensitivity and sensitivity were selected for further analysis. We then investigated the sensitivity and specificity of these parameters to predict correlation values other than 0.6 in the whole set  manual scores by different users; (b) automated scores based on calibrations by different users; (c) manual and automated scores using the user’s own calibration; and (d) manual and automated scores using another user’s calibration. We also investigated whether the video used for calibration influenced interuser variability by performing linear regressions between the automatic scores of two observers using the same video or randomly chosen videos passing minimum criteria , Matlab 2017 (Fisher Z transformation and Z-test) and Graphpad Prism 7  were used for the analysis.Significance was set at To test whether the use of a variable threshold results in better automated freezing scores than a fixed threshold, we correlated manual freezing scores for the four video sets with those obtained using: (a) a fixed threshold of 500 pixels varying between frames for freezing detection; or (b) a variable threshold ranging from 100 to 6,000 pixels with steps of 100, in which the best parameter was selected based on correlations between manual and automatic freezing detection (see “Methods” section).r-values with both approaches in set 1 , but not in sets 2 (p = 0.86) or 3 (p = 1); in the later, the chosen threshold was the same as the fixed one . There were significant differences between slopes in set 1  and between intercepts in two sets , leading to an improvement in the similarity of absolute values with manual freezing scores in sets 1 and 2. In set 4, in which the quality of video recording was poor, neither approach was able to provide meaningful correlations between both scores.We found a difference between the ixed one . Set 4 hp = 0.003) and different intercepts for sets 2  and 3 . Nevertheless, there was no clear improvement in accuracy . Moreover, the use of a second threshold led to a significant increase in processing time . Once more, there was a difference in the intercepts of sets 2  and 3 , with an improvement in the correspondence of absolute values in set 3 and a worsening in set 2. As varying the minimum freezing time did not add as much processing time as the dual-threshold approach  was used to calibrate the two variable parameters (variable threshold and freezing time) on the basis of the best fit between manual and automatic calibration. The chosen parameters were then used to automatically score freezing behavior in the remaining videos.r-values for each set after calibration with 2, 4 or 6 min of video time using different videos or video combinations. Using more videos to calibrate the software did not appreciably change the frequency distribution of correlation values. An ANOVA used to compare the r-values among the three approaches in each set detected significant differences only in set 3 (p = 0.011). However, Tukey’s post hoc comparisons only showed a difference between the 4- and 6-min groups . We thus judged that the use of additional videos did not lead to a meaningful improvement in calibration and that a single manually scored 2-min video was enough for selecting parameters.Although adding more videos did not improve calibration, the variability within each group in r or slope values obtained in calibration could be used as thresholds to predict an r-value of at least 0.6 in the whole video set to which the calibration video belonged. For this, we built ROC curves to calculate the thresholds for correlation coefficients and slope values that provided optimal sensitivity and specificity values to detect valid videos in the four sets. The optimal thresholds for r-values and slope in the calibration step were 0.963 and 0.84, respectively  is shown in To establish minimum criteria to validate a video as a calibration template, we asked what ectively , which pTo evaluate the impact of using automated scoring on the intra- and interuser variability of freezing measurements, we had four independent observers manually score the four video sets. We then scored the same videos automatically based on calibrations performed by each of the observers, using the first video that provided valid calibration for all users within each video set. A correlation matrix was then built between the four manual scores and the automatic scores obtained with the four calibrations (p < 0.0001), with r-values above 0.93. Interuser agreement was similar when using manual and automatic scores . Agreement between manual and automatic scores was also similar whether automatic scoring was calibrated by the same user that performed manual counting or a different one , although slightly lower than interuser agreements within the same category . This suggests that, for high-quality videos, manual and automatic scoring provide similarly high interuser agreement.All correlations in set 1 were highly significant . Correlations between automatic and manual scoring were high in set 3 , but not as good in set 2 . In set 4 , with some negative r-values in the correlation matrix. Interuser agreement after automated calibration was also poor in some cases, showing that automatic scoring is heavily dependent on video quality, even after optimal calibration.For sets 2 and 3, agreement was also high among observers using either manual or automated scoring, with r-values, with the exception of set 4, in which video quality was markedly lower than in the other ones. An ANCOVA for comparing slopes revealed no significant difference between the regression lines obtained for manual × manual, automated × automated (same video) and automated × automated (different videos) correlations between these observers .r > 0.86 for correlations involving all but one of the calibration videos that passed minimum criteria. For other sets, the number of videos reaching minimum calibration criteria was insufficient to adequately perform this analysis.Choosing different videos for calibration also led to variability within a single observer’s automatic measurements , althougAfter the main software features were established and intra- and interuser variability was determined, the user interface went through two rounds of beta testing. A first round involved four users, who ran the software on the first version of the Matlab code and suggested features to be added to the user interface. Stopwatches for manual quantification, sliders to set the start and finish time for each video and progress bars for processing steps were added at this point. After this, a second round of testing involved two of these users and an additional researcher who had no previous contact with the software was used to detect bugs in the system—including both the MATLAB code and compiled version—using different hardware and operational systems. Further options for the output file were also added at this step.We have developed a freely-available, self-calibrating software to automatically score rodent freezing behavior during fear conditioning protocols using .avi video files. Our system shows good performance using a combination of movement threshold and minimum freezing time duration as variable parameters set by the system on the basis of manual calibration using a single 2-min video.Existing software to assess freezing behavior use parameters similar to those tested in our study, as well as others such as object intensity and frame rate , avoiding diagonal angles to record the experiment and using the same recording system for all videos in an experiment. Strains of rodents with more than one color and/or rodents markings will affect the number of contrasting pixels during binary conversion; nevertheless, the software was robust to detect freezing performance accurately in these cases . The method was also able to accurately assess freezing with frame rates as low as 5 frames/s, and with resolutions as low as 384 × 288 . We would not advise using rates below that due to lack of testing.By measuring software performance against manual observation during the calibration step, our system also works to ensure that video quality is adequate for freezing measurement. This is an important advantage, as even with calibration, there are conditions that lead to poor system performance (as shown for Set 4 in our case), demonstrating that video quality is an important concern for automated freezing measurement. The use of a minimal threshold for validating calibration thus allows the user to detect low-quality video sets that might be inappropriate for automated assessment.A limitation of our automated calibration approach is that the method is not able to accurately set parameters if an animal presents very low or high freezing percentage in the calibration video, as this reduces the amount of freezing and non-freezing epochs available for correlation. Nevertheless, users are warned by the software to choose another video if this is the case. The criteria for valid calibration also helps to detect this issue, as videos leading to poor calibration due to inadequate freezing time are less likely to reach validation criteria.Another limitation of the software is that it is currently not able to detect active forms of fear responses, such as darting (Gruene et al., Automatic assessment of freezing behavior should ideally provide results with good correlation between manual and automatic scores, as well as low interuser variability (Anagnostaras et al., Although the use of manual calibration may seem like a step back from fully automated analysis, it serves to streamline a process of parameter adjustment that inevitably happens—though usually in a more cumbersome, trial-and-error basis—for any freezing detection software. Thus, even though the use of stringent calibration criteria may lead to the need to quantify several videos until proper calibration is achieved, we feel that the process ultimately saves time for researchers, besides helping to ensure that freezing assessment is accurate. Moreover, if recording conditions are kept similar from one experiment to another, the end user can opt to use a previously generated calibration file, thus skipping the manual step for later experiments.Finally, whereas the majority of the systems available are expensive, our program was developed to be freely available as an open-source code. This approach benefits laboratories with low financial budget which cannot afford commercial software or hardware such as photobeams (Valentinuzzi et al., The datasets generated for this study are available on request to the corresponding author.Ethical review and approval was not required, as all data in the article are based on secondary analysis of previously acquired videos. These procedures had been recorded for distinct studies and reviewed and approved by their respective animal ethics commitees at the Federal University of Rio de Janeiro, Federal University of Rio Grande do Sul and University of Birmingham.FA developed the software main code and user interface, performed the statistical analysis, prepared the figures and wrote the manuscript. TM developed the software main code, performed the statistical analysis and prepared the figures. OA contributed with funding acquisition, supervision and manuscript editing. All authors contributed equally with conceptualization, methodology, data interpretation and manuscript revision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In mitogen-activated protein kinase (MAPK) activation, morin hydrate evidently diminished ERK2 or JNK1 activation, except for p38 MAPK. Additionally, morin hydrate markedly reduced the OH· signals in platelet suspensions but not in the cell-free system (Fenton reaction solution). Moreover, morin hydrate substantially increased the occlusion time of thrombotic platelet plug formation but had no effect on bleeding time in mice. In conclusion, morin hydrate crucially inhibits platelet activation through inhibition of the PLCγ2–PKC cascade and subsequent suppression of Akt and MAPK activation, thereby ultimately inhibiting platelet aggregation. Therefore, this paper suggests that morin hydrate constitutes a novel and potential natural therapeutic product for preventing or treating thromboembolic disorders.Morin hydrate, a bioactive flavonoid, has been proven to prevent inflammation and apoptosis of cells. Flavonoids can reduce the risk of cardiovascular diseases, in which platelet activation plays a major role. This study investigated the effect of morin hydrate on platelet activation in vitro and in vivo. Morin hydrate markedly inhibited platelet aggregation stimulated by collagen in human platelets but not that stimulated by other agonists. In collagen-activated platelets, morin hydrate inhibited adenosine triphosphate (ATP) release; intracellular Ca Flavonoids are naturally occurring compounds that contain a number of phenolic hydroxyl groups attached to ring structures designated as A, B, and C A. The stChlorophora tinctoria), Osage orange (Maclura pomifera), almond, mill (Prunus dulcis), fig (Chlorophora tinctoria), onion, apple, and other Moraceae, which are used as dietary agents and herbal medicines i mobilization and DAG stimulates protein kinase C (PKC), giving an approximately 47 kDa protein that is principally phosphorylated  and causes ATP release [2+]i mobilization (Phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-bisphosphate to produce the secondary messengers inositol 1,4,5-trisphosphate , 260.7 ± 41.9 s , and 271.3 ± 66.7 s   C. Each mOur study was the first to demonstrate that morin hydrate shows in vitro antiplatelet effect and effectively inhibit arterial thrombosis in vivo. In general, dietary intake from the natural sources is likely insufficient for attaining plasma concentrations that can inhibit platelet activation in vivo, however, several nontoxic prophylactic agents such as food products and nutritional supplements is ideal for stopping atherothrombotic events. Thus, morin hydrate may signify a novel antithrombotic agent for use in humans.2β1 and glycoprotein (GP) VI i mobilization and ATP production is critical for assessing the antiplatelet effects of a compound.Platelets adhere to the subendothelial matrix protein collagen, which alters their shape and releases granular contents of ATP, Cagregates . Agonist and ATP , as well2X1-mediated Ca2+ influx and activates ERK2, thereby increasing the phosphorylation of myosin light chain kinase i levels during the initial phase of platelet activation, and PKC is involved in receptor-mediated production of free radicals in platelets i concentration was determined using Fura-2AM, as described previously i and eventually inhibit platelet aggregation. Moreover, morin hydrate does not affect platelet permeability or induce platelet cytolysis. These findings may recommend that morin hydrate establish a novel therapeutic agent for treating thromboembolic disorders.This paper reports that morin hydrate inhibits collagen-stimulated platelet activation by hindering the PLCγ2–PKC cascade and hydroxyl radical generation and consequently by inhibiting Akt and MAPKs activation. Together these alterations reduce the level of [Ca


S, is organised into three regions: an inner region of fixed radius, r < rb, which is fully saturated with injection fluid, S = 1; a frozen finger region, rb < r < rf (t), in which the saturation is independent of time, S(r) = (r/rb)−0.3; and an outer growing finger region, rf(t) < r < 1.44 rf(t), in which the saturation decreases linearly to zero from the value (rf/rb)−0.3 at rf(t). For a given injected volume per unit thickness of the cell, V ≫ πrb2, we find rf = 0.4rb (V/rb2)1/1.7. This apparent universality of the saturation profile of non-linear fingers in terms of the inner region radius, rb, and the injected volume V, demonstrates extraordinary order in such a complex and fractal instability. Furthermore, control strategies designed to suppress viscous fingering through variations in the injection rate, based on linear stability theory, are less effective once the instability becomes fully nonlinear.Complex fingering patterns develop when a low viscosity fluid is injected from a point source into the narrow space between two parallel plates initially saturated with a more viscous, immiscible fluid. We combine historical and new experiments with (a) a constant injection rate; (b) a constant source pressure; and (c) a linearly increasing injection rate, together with numerical simulations based on a model of diffusion limited aggregation (DLA), to show that for viscosity ratios in the range 300–10,000, (i) the finger pattern has a fractal dimension of approximately 1.7 and (ii) the azimuthally-averaged fraction of the area occupied by the fingers,  Linear stability theory suggests that the fingering process may be suppressed if the injection rate is varied with time, with the optimal profile involving a linearly increasing injection rate2. However, it is not known whether there is any benefit to such an approach once the finger pattern has developed into a fully nonlinear regime. The lack of a simple model for the non-linear fingering process makes it challenging to address such questions. Classical experiments3 have shown that the nonlinear finger pattern has a fractal dimension of 1.70 ± 0.01, and simulations with diffusion limited aggregation (DLA) models4 have replicated many of the features of viscous fingering in cases with relatively large flow rates. Guided by this historical work, we now explore the impact of changes in the injection rate with time on the structure of the finger pattern and, in particular, the variation of the saturation with radius.If air is injected from a point source into a second immiscible fluid confined within a narrow planar channel, then, with a constant injection rate, the interface eventually becomes unstable and a series of viscous fingers develop8 and (c) a series of new experiments, some of which have injection rate which varies with time: each experiment analysed is listed in the 3, which involves much more tip-splitting than in Fig. 3 shows that in this case the injection rate evolves approximately as 9, which replicates many features of the nonlinear fingering process.We have analysed the output from (a) a series of new DLA calculations, (b) many of the classical radial viscous fingering experimentsr S dr, in the annular ring bounded by the circles with radii r and r + dr, with dr ≪ r. This reveals the variation of saturation S with radius at a series of times t. Figure 11. The thickness of the film scales with b(μU/σ)2/312, where b is the cell thickness, μ and U the fluid viscosity and speed, and σ the surface tension, and this has value of order 0.01b, suggesting that the residual liquid film is very thin relative to the gap width. This is consistent with estimates of the injected volume of air based on the product of the gap width and the finger area, which agree within measurement error with the actual volume injected.In order to characterize the fingering pattern, for each experiment we have computed the area covered by fingers, 2π fully flooded region, r < rb, in which S = 1, where rb depends on the injection rate but is approximately independent of time, (ii) an inner frozen finger zone, rb < r < rf(t), where the saturation is independent of time and gradually decays with radius, and (iii) an active outer finger zone, rf (t) < r < rt(t), in which the fingers advance in time but also thicken and undergo tip-splitting events. Historical data have shown that the viscous fingering pattern is fractal with dimension D = 1.7. If the saturation in the frozen finger region decays with radius asrf  rb, integration shows that this saturation profile is consistent with the region covered by the frozen finger pattern being fractal  < r < rt(t), the saturation decreases to zero approximately linearly with radius. This suggests a simple model for the saturation in this region given byrt(t), corresponds to the minimum radius at which S = 0, and we define rt = rf.Observations of the saturation in the finger patterns shown in Fig. V(t), the invaded area per unit thickness of the cell at time t, satisfies V(t) ≫ πrb2. In order that this expression represents a fractal shape for all values of rf(t), with a constant value of D, we expect that Integration of the model saturation profile across the frozen finger zone and the advancing front region leads to the approximate relationD = 1.79 are rb = 0.14 ± 0.01, where lengths have been scaled with the radius of the particle. To find the best fit, we minimise the quantity:By comparing the saturation profile for the DLA calculations with eqn  at a serrb we show the contour on which E = 0.1, as a function of rb with D = 1.7  the point at which the saturation first decreases below unity  = Q0tα, μ is the viscosity, b the cell thickness, and σ the surface tension. It is seen that rb/r0 increases with C* corresponding to there being a larger fully saturated region in a more intense flow. It is interesting to note that rb 0, which indicates that the fully saturated zone extends some distance beyond the radius at which the linear instability initially sets in. This may be expected since at the onset of instability, the interface is still migrating outwards, and as the radius increases, the wavenumber of the most unstable mode also changes2.Figure rf, and the growing finger zone, rt, scaled with the best-fit values of rb for each experiment. Data are given for a large number of experiments from both the literature and our new experiments. We have also evaluated rf/rb , where V is defined in terms of the number of particles added as V = nπ.As a further test of the model, Fig. f/rb eqn  using thrf and rt as a function of V/rb2, irrespective of whether the experimental injection rate varies in time or is constant, and also for the DLA simulations. This is consistent with Fig. Figure 13, in which varying the injection rate with time but injecting a fixed volume leads to differences in the amplitude of the most unstable mode2, the apparent universality of the non-linear finger pattern, as characterised by the saturation profile shown herein, does not seem to depend on the injection rate history for a given total volume injected.The universality of the model for the variation of the saturation profile with radius, and the recognition that there is a transition from the frozen non-linear finger region to the growing non-linear finger region in the leading part of the flow provide new insight about and a succinct description of the evolution of such nonlinear viscous fingers. In contrast to predictions of linear stability theoryμ = 50 mPa s; σ = 31 mN/m2). Another experiment, marked C4 in Fig. et al. and used the same setup10 but is previously unpublished. It involved the displacement of silicone oil  by water. In the DLA simulations, we used code supplied by the co-authors of publication9 and used the mean of 7 saturation profiles for each aggregate size.In our experiments, two World Precision Instruments AL1000-220 pumps were used to inject air into a Hele-Shaw cell of radius 30 cm, with plate thickness 1 cm, and gap width of either 1 mm or 1.5 mm set by positioning micrometers. The cell was initially filled with rapeseed oil (Supplementary information