The recognition of clinical manifestations in both video images and physiological-signal waveforms is an important aid to improve the safety and effectiveness in medical care. Physicians can rely on video-waveform (VW) observations to recognize difficult-to-spot signs and symptoms. The VW observations can also reduce the number of false positive incidents and expand the recognition coverage to abnormal health conditions. The synchronization between the video images and the physiological-signal waveforms is fundamental for the successful recognition of the clinical manifestations. The use of conventional equipment to synchronously acquire and display the video-waveform information involves complex tasks such as the video capture/compression, the acquisition/compression of each physiological signal, and the video-waveform synchronization based on timestamps. This paper introduces a data hiding technique capable of both enabling embedding channels and synchronously hiding samples of physiological signals into encoded video sequences. Our data hiding technique offers large data capacity and simplifies the complexity of the video-waveform acquisition and reproduction. The experimental results revealed successful embedding and full restoration of signal's samples. Our results also demonstrated a small distortion in the video objective quality, a small increment in bit-rate, and embedded cost savings of −2.6196% for high and medium motion video sequences. Video technology continues to improve the safety and effectiveness in healthcare. Today, physicians and engineers rely on rigorous video-based studies to improve medical practices and procedures. These studies are necessary for the identification of clinical manifestations in patients and for the reduction of errors during medical procedures. Extending the video-based studies to incorporate the analysis of physiological-signal waveforms further enhanced the recognition of clinical manifestations and the reduction of false positive cases. Physicians can rely on simultaneous video-waveform observations to recognize difficult-to-spot signs and symptoms. These observations can also expand the recognition coverage to abnormal health conditions.The synchronization between the video images and the physiological-signal waveforms is fundamental for enhanced recognition of the clinical manifestations. The identification of signs and symptoms invisible during specific diagnosis is possible with synchronized video-waveform observations. Physicians are able to diagnose seizures in neonates after creating and analyzing a permanent record . The perThree commonly used techniques are suitable for video-signal synchronization . The timThe second technique relies on synchronization marks. The technique sends synchronization marks from a transmission node. The disadvantage of this technique is the need of an additional assistant communication channel to transmit the synchronization mark. The third technique is multiplexing. This technique maintains the correlation among the media streams during the transmission process. However, multiplexing-based synchronization usually results in a loss of agility and integrality.Data hiding is another alternative for video-waveform synchronization. Data hiding has the goal of embedding information into encoded video sequences with a minimum amount of perceivable degradation . The embThis paper introduces an improved data hiding technique with a larger data-hiding capacity in the context of medical healthcare. Our data hiding technique synchronously embeds physiological-signal samples into H.264/AVC-encoded video sequences. The implementation of data hiding is simpler than other synchronization techniques. The data-hiding technique requires only one encoder to process video and signal's samples. Data hiding also offers the advantage of a unique communication channel for video-audio transmission and requires no complex tasks related to timestamps, synchronization marks, or multiplexing .  FigureExisting data hiding techniques are able to hide information in the video frames during the video encoding process. In , authorsIn , the datIn , the datIn , the proIn , the autIn , the proIn , 5, the Y , and bit-rate distortion. These schemes offer relatively low values for the three metrics. Therefore, the main limitation resides in the data hiding capacity. In [Y distortion and less than 1.32% of bit-rate distortion. Our proposed technique extends the quarter-pixel motion estimation scheme to satisfy the data hiding capacity needs and to ensure the low PSNRY and bit-rate distortions.The desirable features of a data hiding technique suitable for embedding physiological signals into encoded video sequences are a large data hiding capacity, a low impact in video quality, and a minor effect in bit-rate of the video. city. In , the quaJmode) optimization. In this equation, λmode is the Lagrangian multiplier, SSD is the sum of the squared difference between the original and the reconstructed block, and R is the number of bits of MB parameters such as quantization parameter, header, motion vectors, and residue coefficients: Motion estimation (ME) is an important element in the H.264/AVC interprediction process. For a given frame, the ME goal is to find the best predictions for both levels: macroblock (MB) selection and motion vector (MV) estimation. A MB is an array of 16 × 16 pixels. The MB selection process assumes the partitioning illustrated in The motion estimation process computes motion vectors for each macroblock partition found in each video frame. At a given frame, the ME process searches for the new MB position of each MB located in the reference frame. The ME process calculates motion vectors based on these new MB positions and encodes these vectors in the encoded frames. This section presents additional examples of medical applications related to the simultaneous observation of video and physiological-signal waveforms. 2), endtidal CO2 level, in-video leg movement, and in-video rapid eye movement (REM).In , physiciIn , the purIn , clinicaOur proposed technique hides streams of data samples into encoded video sequences. The implementation of our technique is a set of software routines added to the original H.264/AVC codec. Ji is the Lagrangian rate distortion parameter:Our technique modifies the H.264/AVC interprediction process to hide the samples of the physiological signals. Our technique relies upon the quarter-pixel motion estimation process, part of the H.264/AVC encoder, to hide these samples into encoded video frames. Our technique divides the search points into four groups to improve the data hiding capacity of the original technique . EquatioOur proposed technique, illustrated in Our technique also incorporates an approach to overcome the data-hiding capacity limitation found in low-motion video sequences. PSKIP blocks are the most common block partition found in encoded low-motion video sequences. A large number of PSKIP blocks limit the data hiding capacity of our technique due to few number of motion vectors found in the low-motion video sequence. Therefore, our proposed technique forces the H.264/AVC encoder to replace the PSKIP block by a P16 × 16 block partition. This PSKIP replacement adds a motion vector to the data hiding capacity of our technique. The PSKIP replacement also contributes to maintaining the synchronization between the encoded video and the physiological signals. However, this replacement may also result in an increment in the bit-rate of the sequence. Our data hiding technique extracts the samples of the EEG signal; at the same time, conventional video H.264/AVC decoding process takes place. This is an important feature at the time to do synchronized playback of both video and physiological-signal waveforms. The sample extraction algorithm in our technique catches and gathers sample data. This extraction algorithm, illustrated in x and MVy components into  of every macroblock partition and inputs the MVnts into  to identped into :(4)type∈http://www.physionet.org). The experimental setup included 6 signal electrodes at 256 samples per second and 12-bit sample resolution [The experimental setup, illustrated in The implementation of encoding and decoding processes needed the modification of the JM reference software version 16.1. The video test sequences had a CIF (352 × 288) resolution and a 4:2:0 YUV format. The name of the test sequences are akiyo, bridge-far, carphone, football, foreman, mobile, and neonatal. Neonatal is not considered a standard video test sequence. Neonatal was introduced to match the context of the application example related to EEG seizures on neonates. The selected coding structure of the bit stream is “IPPP…” to have an intraframe encoded in the first frame and interframes encoded in the remaining frames.M and N are the height and the width of the video frame, respectively. Iij and Iij′ represent the original pixel and the processed pixel, respectively [Ydiff metric, illustrated in (Y) samples impact the video quality after embedding the physiological samples. PSNRY′ represents the impact of luma samples generated by embedding the samples, and PSNRY represents the impact of luma samples generated with the original H.264/AVC encoder:Metrics to evaluate the effectiveness of our proposed technique are video objective quality, bit-rate difference, embedding cost, and perceptual quality of the image. The peak signal-to-noise ratio (PSNR), illustrated in , is an oectively . The PSNrated in , indicatR is the original bit-rate and R′ is the embedding samples bit-rate. Equation  less number of encoders and decoders, (2) no timestamps needed, (3) no software needed for synchronization of video and signal streams, and (4) higher data capacity compared to other data hiding techniques, especially for high motion sequences.Y difference, and bit-rate resulted in both no impacts in video-waveform synchronization and minimum distortions in video quality. For storage and transmission purposes, the embedded cost for high and medium motion video sequences represent savings. For low motion video sequences the experimental results ranged from –0.003 dB to −0.002 dB in PSNRY difference, from 33.3143% to 36.575% in the bit-rate difference, and from 15.8725% to 19.3017% in embedded cost. The changes in bit-rate were higher compared to the high and medium video sequences.The experimental results demonstrated minimum degradation in video quality and data savings in terms of storage/transmission. The experimental results for high and medium motion video test sequences ranged from −0.007 dB to 0.011 dB in PSNR luma difference, from 0.4459% to 1.3446% in the bit-rate difference, and from −2.6196% to −0.1273% in embedded cost. The changes in PSNR


Mycobacterium marinum (M. marinum) is a ubiquitous waterborne organism that grows optimally at temperatures around 30°C. It is a nontuberculous Mycobacterium found in nonchlorinated water with worldwide prevalence. It is the most common atypical Mycobacterium that causes opportunistic infection in humans. M. marinum can cause superficial infections and localized invasive infections in humans, with the hands being the sites most frequently affected. It can cause skin lesions, which are either single, papulonodular lesions, confined to an extremity, or may resemble cutaneous sporotrichosis. This infection can also cause deeper infections including tenosynovitis, bursitis, arthritis, and osteomyelitis. Disseminated infections and visceral involvements have been reported in immunocompromised patients. We here report a case of severe deep soft tissue infection with necrotizing fasciitis and osteomyelitis of the left upper extremity (LUE) caused by M. marinum in an immunocompromised patient. Mycobacterium marinum was first isolated in 1926 by Aronson from salt water fish carcasses in the Philadelphia aquarium [ M. platypoecilus [poecilus .M. marinum is a nontuberculous mycobacterium belonging to Runyon group I, a photochromogen [ M. marinum has a worldwide distribution and primarily infects fish that can secondarily contaminate aquaria, swimming pools, rivers, and seawater. When transmitted to animals such as amphibians, fish, mice, and bats, it can be highly prevalent in fish tanks and cause infections and death in various fish species [ M. marinum preferentially grows at cold temp between 30 to 32°C and thus, infections with M. marinum are usually localized primarily to the skin. Less commonly it extends to involve deeper structures such as joints and tendons. Dissemination has been reported, but is distinctly unusual [ M. marinum infection in humans is comparatively rare. The approximate annual incidence in the United States is 0.27 confirmed cases per 100,000 inhabitants [hromogen . M. mari species . The col unusual . In geneabitants . In 90%  M. marinum both in immunocompromised and immunocompetent individuals. We found eleven published cases of adult M. marinum complicated with osteomyelitis. Four of them had compromised immune systems. This case review aims to focus on severe deep soft tissue infection and osteomyelitis caused by M. marinum.We searched the published English literature for cases of severe deep soft tissue infection and osteomyelitis caused by Staphylococcus aureus. Patient was discharged home on trimethoprim/sulfamethoxazole oral tablets but readmitted seven days later with pain and pustules now mainly on all of the fingers of the left forearm  which has later gotten incision and drainage. At that time, only bacterial cultures were sent, which grew methicillin-sensitive forearm . M. marinum was identified phenotypically in our lab when the culture grew at 30 degrees Celsius. The sample was also sent out for biochemical sequence and further identified as M. marinum . He was empirically treated with intravenous imipenem, linezolid, and azithromycin. During this second admission, magnetic resonance imaging (MRI) of the left arm was done which showed multiple ulcerations extending from the skin surface into the subcutaneous fat with the largest visualized at the palmar aspect of the wrist, extending deep to the level of the carpal tunnel/hook of the hamate, at the dorsal and ulnar aspects of the mid- and distal forearm at two sites and at the ulnar aspect of the distal forearm suggesting tenosynovitis with necrotizing fasciitis. He recovered well from the surgery and was discharged on doxycycline and azithromycin while waiting for susceptibility. Despite being adherent to this regimen for four weeks, he was ultimately readmitted a third time with new abscess formation on medial aspect of his proximal LUE and repeated MRI showing osteomyelitis of the left distal ulna  superficial cutaneous infections . Type II includes numerous (>3) lesions in a sporotrichoid distribution pattern or with inflammatory nodules, abscesses, and granulomas. Type III includes deep infections with or without skin involvement, including tenosynovitis, arthritis, bursitis, and/or osteomyelitis and Type IV refers to disseminated infection, lung involvement, and other systemic manifestations. Bacteremia is usually seen in immunocompromised patients but is considered very rare [There are many different clinical presentations ofthe rule . Inflammthe rule . This “sthe rule . Infectiery rare , 20. Our M. marinum presented with deep tissue infection and osteomyelitis. The first case was published in 1972 involving a 36-year-old healthy Vietnamese female who was exposed to salt water and fishing presented with deep infection and osteomyelitis of the hand leading to amputation. Among the eleven cases, there were seven cases of immunocompetent patients and four cases with underlined immunocompromised status as listed in In the literature, there are eleven cases where M. marinum infection is usually delayed, suggesting that most physicians are not familiar with the disease [ M. marinum infections are a high index of suspicion raised by negative bacterial tissue cultures, poor response to conventional antibiotic treatments, and a history of aquatic exposure [ M. marinum and the majority of the cases had history of exposure to aquatic environments.Diagnosis of disease . This isexposure , 18. A dHowever, in practice, the diagnosis remains largely presumptive, based on clinicohistological features and the response to appropriate antimicrobial treatment, regardless of culture results . In addi M. marinum has been reported in the literature. The main purpose of therapy aims for a rapid recovery from the infection and the prevention of progression to deeper structures [Occasional spontaneous resolution of soft tissue infection byructures . Monothe M. marinum. A literature review that was published in 2007 suggests that topical therapy as a sole treatment is completely ineffective and unnecessary [There have been no comparative trials of different treatment regimens for soft tissue infection byecessary . In limiBased on the available literature review, a reasonable approach is to treat patient with two active antimicrobials for at least one to two months after resolution of symptoms –7. This M. marinum is usually susceptible to rifampin, rifabutin, ethambutol, clarithromycin, sulfonamides or trimethoprim/sulfamethoxazole, doxycycline, and minocycline [ M. marinum was resistant to rifampin and unfortunately could not be used as one of the agents in the combination regimen , surgical treatment may be required if the infection has not been controlled by chemotherapy. Deeper infections (Type III) may require prolonged systemic treatment and repeated surgical debridement. However, the selection of cases and the time of surgical intervention require good judgment. In disseminated infection or bacteremia (Type IV), combined  intravenous therapy of three drugs may be required , 9, 10.The use of isoniazid, streptomycin, and pyrazinamide as empirical treatment options should be avoided, as the resistance of the organism to these agents is well documented , 21. Cry M. marinum are uncommon, but not rare. M. marinum infection should always be included in the differential diagnosis of all cases with poorly healing wounds in upper extremities and in persons with a history of exposure to aquariums. The diagnosis requires both a detailed history and sophisticated microbiological and PCR-based investigations. No large systemic studies have been performed to determine the optimal treatment regimen. In most cases a combination of antibacterial drugs should be given as well as long-term therapy depending on the duration and severity of infection.In conclusion, infections due to


Hand hygiene is an important component in infection control to protect patient safety and reduce health care-associated infection.Our aim was to evaluate the efficacy of different social media on the promotion of a hand hygiene (HH) program.The observational study was conducted from May 5 to December 31, 2014, at a 2600-bed tertiary care hospital. A 3-minute video of an HH campaign in 8 languages was posted to YouTube. The Chinese version was promoted through three platforms: the hospital website, the hospital group email, and the Facebook site of a well-known Internet illustrator. The video traffic was analyzed via Google Analytics. HH compliance was measured in November 2013 and 2014.P<.001). HH compliance sustained from 83.7% (473/565) in 2013 to 86.7% (589/679) in 2014 (P=.13) among all HCWs.There were 5252 views of the video, mainly of the Chinese-language version . The NTUH website had 24,000 subscribers, and 151 of them viewed the video . There were 9967 users of the hospital email group and the connection rate was 0.91% (91/9967). The connection rate was 6.17%  from Facebook, significantly higher than the other 2 venues (both Facebook had the highest connection rate in the HH video campaign. The use of novel social media such as Facebook should be considered for future programs that promote hand hygiene and other healthy behaviors. Since the World Health Organization (WHO) launched the first global hand hygiene (HH) campaign to protect patient safety in 2005, more than 130 WHO member states and 17,000 health care facilities have committed to improving HH [We have therefore questioned whether novel methods can be used to engage our health care workers (HCWs) and accurately measure the efficacy of an HH promotion. “Net generation” has been used to describe the generation who grew up in the era of electronic devices and social media . A 2012 In Taiwan, the WHO’s HH program was centrally sponsored by the Centers for Disease Control (CDC) of Taiwan from 2009-2011 in three demonstration centers. After that, the program was distributed throughout the nation via HH certification programs . In 2014This study was approved by the ethics committee of the NTUH. Informed consent was waived under the agreement of the committee (201501083W).The NTUH  is a university-affiliated medical center with 2600 beds. Alcohol-based hand hygiene equipment has been provided hospital-wide since 2004 . A centrThe video script was written by one of the authors (PSC) and produced by the FTIG Company . The main actors were HCWs in the NTUH. Eight versions of the videos were produced in different languages, including the most common languages in Taiwan  and languages commonly used by international patients .The 3-minute video covered three main topics: an introduction to the importance of HH and the commitment of the NTUH, a short drama to demonstrate five different moments for HH, and a dancing section that demonstrated the technique of HH.th year medical student at the NTU [The videos were uploaded to YouTube on the official site of the Center for Infection Control, NTUH. The videos can be found by searching for keywords such as “National Taiwan University Hospital (NTUH)” and “hand hygiene.” The videos were launched on May 5, 2014. The Chinese version was promoted through three sites: (1) the website of NTUH via an article introducing World HH day, (2) NTUH group email, and (3) the well-known Facebook site of a 6 the NTU . This stThe method used to determine HH compliance was in accordance with WHO . Covert P value ˂.05 was defined as statistically significant. The statistical analyses were performed using STATA 11.0.The number of website views was determined by Google Analytics. The connection rate was calculated by the number of video views divided by the users subscribed to the specific platforms and compared by chi square test. A From May 5 to December 31, 2014, there were 5252 views of the video, mostly of the Chinese version  see . The proP<.001 for both comparisons). On Facebook, 525 visitors  clicked on “Like” for the HH campaign and 21 visitors  shared the video  was female and 46.99% (1649/3509) male. The main age group was among 25-34 years old, which accounted for 34.0% of the viewers. We used three different Internet-based platforms to promote the Chinese version of the video. At the hospital level, the official website of NTUH had 24,000 subscribers, and 151 of them opened the link to the video . A total of 9967 people received the hospital group email, and 91 of them opened the link to the video . There was no feedback from the email or website promotions. On Facebook, there were 13,080 clicked to the site, and 807 opened the link to the video. The connection rate for Facebook  was significantly higher than from the other two platforms , followed by NTUH website (n=19) and hospital group email (n=19), and e-newspaper (n=5). One traditional newspaper reported the news on the following day.During the following year after the video launched , 69.68% (2445/3509) of the views of the Chinese version of the video were during the first month. After excluding 1259 direct links to the video (which cannot be readily tracked), 38.09% (857/2250) of the views were from Facebook, 7.60% (171/2250) from the NTUH website, 4.31% (97/2250) from the NTUH group email, 29.60% (666/2250) from other sources , and 20.40% (459/2250) from search engines, including YouTube  and Google  see . The vieOverall, the video was distributed in 49 countries, and on average, viewers spent 1.57 minutes on the 3-minute video. The Chinese version was the most popular , followed by the Indonesian version  see . There wP=.36)  than computer , although not statistically significant (.36) see .P=.13) (P=.02).The hospital-wide HH compliance improved from 83.7% (473/565) in 2013 to 86.8% (589/679) in 2014, although this change was not significant (P=.13) . Among pDuring the hand hygiene video campaign in 2014, the promotion on Facebook was significantly more successful than promotion on two traditional Internet-based communication tools, that is, the hospital website and group email.Different marketing strategies have been used in the promotion of HH ,10; howeThrough quantitative analysis, we revealed that connecting to the HH video is more efficient through Facebook than the traditional hospital website and group email. The difference in connection rate may be due to the fact that shared information among peers is an important influence on behavior . The FacIn addition to the ability to connect to the right community and create interaction, social media also allows consumption and control, per the social media “4 Cs”  . The worOf note, the proportion of the video viewers via Facebook decreased after the first month that the video launched. The potential pros of Facebook as rapid information sharing can also be a cons as the message may disappear rapidly. But while we stored the video campaign on the Infection Control Center’s YouTube site in December 2014, 34% of the traffic to the video was via search engine . Thus, since search-engine optimization has been an important marketing tool , simple Also from the point of view of marketing, the efficacy of a social media campaign would be considered as the return on investment (ROI). However, health care is different from commercial operations and online merchandise, so we cannot provide a traditional ROI as “cost per acquisition.” However, we calculated the equivalent cost per click as US $1.2, and this is within the average cost of online advertising .There may have been some regional differences among our viewers. According to our analysis of the eight different versions of video, we found that those who viewed the Indonesian version had greater utilization of smartphones. This is consistent with the findings of a 2013 national survey in Taiwan, which found that new immigrants were more likely to own a smartphone than a computer (82.5% vs 61.4%) . Thus, fA limitation of our study would be that HH behavior may be influenced by other interventions at the same time. In Taiwan, the national HH program was promoted by CDC, Taiwan, in 2012. There were no other concurrent interventions for HH after that within the institution. The HH compliance was also measured by professional ICNs, and objective compliance rate was used for evaluation.+, and Vimeo. Future research could focus on regional differences in the use of different social media websites.Another limitation of this study is that we did not examine the use of multiple social media platforms. In particular, different geographical regions tend to prefer different social media sites, as is the case with Facebook and Myspace . SurprisBernoff et al stated that social media “is a phenomenon of people connecting with each other and drawing strength from each other” . This ec


Inositol phosphates were extracted from leaves with diluted HCl and concentrated on small anion exchange columns. Reversed-phase solid phase extraction cartridges were used to remove compounds that give peaks that sometimes interfere during HPLC. The method permitted the determination of InsP6 and InsP5 concentrations in leaves as low as 10 µM and 2 µM, respectively. Most plants analyzed contained a high ratio of InsP6 to InsP5. In contrast, certain members of the Malvaceae family, such as cotton (Gossypium) and some hibiscus (Hibiscus) species, had a preponderance of InsP5. Radiolabeling of cotton seedlings also showed increased amounts of InsP5 relative to InsP6. Why some Malvaceae species exhibit a reversal of the typical ratios of these inositol phosphates is an intriguing question for future research.Methods used to quantify inositol phosphates in seeds lack the sensitivity and specificity necessary to accurately detect the lower concentrations of these compounds contained in the leaves of many plants. In order to measure inositol hexakisphosphate (InsP The Pasteur pipet with the unfurling cotyledon emerging from the top was placed inside a small room-temperature incubator with supplemental lighting, for four days, then harvested and frozen at −80 °C. Arabidopsis seedlings and leaf punches from a pre-flowering G. hirsutum plant were labeled as described with 100 µCi myo-[2-3H(N)]-inositol in 150 microliters and 1 mL 0.5× MS, respectively [Individual soil-grown, newly-emerged whole cotton (ectively . InsPs wectively .6 to InsP5. In contrast, certain members of the Malvaceae family, such as cotton and some hibiscus species, contained mostly InsP5. Additional work is needed to explain the reason for this difference.Vegetative tissues from most plants analyzed contained a high ratio of InsP


Throughout the western United States, native perennial grasses are being supplanted by aggressive non-native annuals. We show that giving native grasses just a two-week germination 'head start' over exotic invasive grasses shifts the competitive edge strongly in their favour. We also show that the strength of this advantage differs strikingly depending on the site where the experiment is carried out, and the weather in the initial weeks of the experiment. These results a) give insight into the reasons for the competitive advantage that annuals usually demonstrate, and b) are an example of the likelihood that ecological experiments often produce results that are limited to a particular time and place, and less general than we might wish to believe. Ecological communities are increasingly being recognized as the products of contemporary drivers and historical legacies that are both biotic and abiotic. In an attempt to unravel multiple layers of ecological contingency, we manipulated (i) competition with exotic annual grasses, (ii) the timing of this competition  and (iii) watering  in a restoration-style planting of native perennial grasses. In addition, we replicated this experiment simultaneously at three sites in north-central California. Native perennial grasses had 73–99 % less cover when planted with exotic annuals than when planted alone, but this reduction was greatly ameliorated by planting the natives 2 weeks prior to the exotics. In a drought year, irrigation significantly reduced benefits of early planting so that these benefits resembled those observed in a non-drought year. There were significant differences across the three sites (site effects and interactions) in (i) overall native cover, (ii) the response of natives to competition, (iii) the strength of the temporal priority effect and (iv) the degree to which supplemental watering reduced priority effects. These results reveal the strong multi-layered contingency that underlies even relatively simple communities. Ecological interactions are increasingly recognized as being highly contingent on their context, shaped by forces that are both historical and contemporary as well as biotic and abiotic . If we wUnderstanding such contingencies is also crucial for successfully restoring ecosystems. One emerging theme is the phenomenon of priority—how differences in arrival times by different species may have profound effects on the long-term trajectories of communities e.g. . Such prA number of experimental studies on perennial herbaceous plants have shown that a 1- to 3-week priority can significantly affect initial community structure .Priority effects may be particularly relevant for testing the mechanisms underlying the competitive advantage of invasive annual plants over native perennials. In many western US ecosystems, these invasives have become community dominants . SeveralThe structure of communities may also be dependent on conditions in the year in which they were established e.g. , and resCommunity structure may be also contingent on site conditions, and the relative abundances of different species may change over relatively small environmental gradients  competition with exotic annual grasses, (ii) the relative timing of seed arrival , (iii) rainfall addition and (iv) geographical location (site effect). We also tested the interactions among priority, rainfall addition and site effects.At three different sites, we manipulated the timing of competition between native perennial grasses and exotic annual grasses (priority effect) with four different planting treatments, and crossed these with two watering treatments to simulate rainfall differences between years.In November 2011, we established a set of experimental plots as part of a long-term study of priority effects, site effects and rainfall in the context of grassland/prairie restoration. The entire experiment is replicated over three sites in north-central California which have similar relatively fertile clay loam soils, but differ moderately in elevation and climate , and weed challenge , we had collected seeds of local provenance at each of the three sites (where possible) from four native perennial grasses and four exotic annual grasses  natives sown alone (N), (ii) natives sown together with exotics (NE), (iii) natives sown and exotic sown 2 weeks after the next germinating rain (NtE) and (iv) exotics sown alone 2 weeks after the next germinating rain (tE). Planting treatments were implemented in an additive design . Each plot was lightly raked, sown and then raked again to increase seed–soil contact. There was a second germinating rain on 24 November. Two weeks later, the plots designated to receive a second sowing (of exotics) where sown. Unusually, there had been little rain in the intervening 2 weeks, and there was no rain in the 5 weeks that followed the second sowing. Therefore, to simulate an early-season rain that was more similar to a normal year, the four treatments designated for rainfall manipulation (‘Watering’ treatment) in each block were watered with the equivalent of 1.25 cm of rain immediately after the second sowing pass (5–7 December).P < 0.001).Over the following weeks, plots were weeded of volunteer forbs. Because grasses are difficult to reliably identify at the seedling stage and because there were volunteer seedlings of sown species at two of the three sites  vs. natives seeded together with exotics (NE). Only unwatered plots were included in this analysis.To determine the effects of priority, watering and site on native and exotic cover, we compared two planting treatments—natives and exotics seeded together (NE) and exotics seeded after natives (NtE). Planting treatment, watering treatment, site, and all two-and three-way interactions were included in models of native cover and exotic cover (separately). Priority treatment was nested in watering treatment to account for the split-plot design. Due to large and significant site effects and priority treatment interactions with site, we also analysed the same dataset for each site separately.P < 0.002). At the coolest site (McLaughlin) the sown natives (and exotics) achieved the lowest total cover. Exotic annual grasses greatly suppressed the native perennials in all the three sites. When the native perennial grasses were sown together with exotic annual grasses, they achieved 73–99 % less cover than when seeded alone . These differences among sites were themselves significant, with the site that had lower cover overall showing less suppression of natives by exotics . Across all treatments and sites, cover by seeded exotic annual grasses was strongly negatively correlated with cover by seeded native perennial grasses at the plot level . However, this priority effect varied significantly across sites, ranging from 60 to 90 % increases in cover (Table P < 0.0001). The significant interaction between site and priority was because sites with high cover in general (both natives and exotics) had greater reductions in native cover with competition.The analysis of priority effects (natives and exotics seeded together (NE) vs. exotics seeded after the natives (NtE)), and how these priority effects differed between watering treatments and across sites revealed a variety of significant main effects and interactions. First, giving the native perennial grasses a short-term advantage over exotic annual grasses (seeded 2 weeks after the first germinating rains following native seeding) greatly increased their success , where total cover of all plants was highest. For exotic cover, this correlation was associated with significant site, priority and site × priority effects , where native grasses achieved little cover in the first year even when planted alone Fig. , they weAlthough in practice weed control often seeks to greatly reduce the challenge of exotic annuals for at least the first year of native planting, our results show that even a much briefer respite can have a profound effect. When exotic annual grasses were seeded just 2 weeks after germinating rains for the natives, their ability to suppress these natives was greatly reduced Fig. . This prThis experiment was initiated in a year 2011/12) when there was a 4-week drought following a few weeks of germinating rains in November  partly offsets the difference between the long-term mean and the rainfall lost during the fall drought of 2011/12, and the result was a priority effect much more similar to 2008 than unwatered 2011/12 Fig. . These rThe three-way interaction  only partly mitigates for their more troubling implications. Our results suggest that not only is the success of restored native perennial grasses significantly affected by site effects and year effects, but that the basic conclusions from experimental manipulations can differ dramatically across relatively similar sites and conservatively simulated year differences.This also raises the uncomfortable possibility that results of many ecological field experiments initiated in a single site and/or a single year run the risk of being idiosyncratic rather than general see also . This maElvinia Slosson Endowment and NSF DEB 10-50543.This study was supported by grants from the All authors have made a substantial contribution to the manuscript and/or the research presented. T.P.Y. conceived the project and did data collection. T.P.Y., S.F. and K.J.V. implemented the experiment. S.F. and E.P.Z. oversaw seed collection and preparation, and weed control. E.P.Z. and T.P.Y. did statistical analyses. T.P.Y. wrote the first and final drafts. All authors contributed to editing of the manuscript.None declared.


The method is illustrated through its application to a video photoplethysmogram acquired during a porcine model of acute desaturation. The modular continuous wavelet transform-based approach is advocated by the author as a powerful methodology to deal with noisy, non-stationary biosignals in general.A novel method of extracting heart rate and oxygen saturation from a video-based biosignal is described. The method comprises a novel modular continuous wavelet transform approach which includes: performing the transform, undertaking running wavelet archetyping to enhance the pulse information, extraction of the pulse ridge time–frequency information [and thus a heart rate (HR HR has been tackled by many groups using a variety of signal processing approaches [et al. [et al. [vid and SvidO2) is described which comprises a number of distinct algorithm modules based on the continuous wavelet transform (CWT) [Remote acquisition of physiological signals from video image streams is attracting much attention in the bioengineering space. In particular, the video photoplethysmogram may be used to derive the two main vital signs displayed on a pulse oximeter device: heart rate (HR) and oxygen saturation  , 12.2R and G video signals (from RGB). This was found to be the best performing of the three possible two-signal combinations. , of each signal is first computedψ*(t) is the complex conjugate of the wavelet function ψ(t), a is the dilation or scale parameter of the wavelet, b is the location parameter of the wavelet and x(t) is the signal under investigation [TRWA, is generated using a weighted averaging scheme as followsw is a predefined weight, TRWA) is the previous archetype value separated from the current value by a period P(a) and T is the currently computed wavelet transform. In the method, each time a value of T is computed, it is used with the previous archetype transform value to form a new value of the archetype transform. As the wavelet transform already separates out the signal information into natural temporal scales according to the periodicity of the wavelet function, the characteristic period of the wavelet may be used at each scale for P(a). Thus, it can be seen that there is no requirement for the determination of fiducial points in the method, as the wavelet information is naturally ‘rolled up’ at each scale using P(a) based on the wavelet function itself. The RWA method, fully described in [The flow diagram in Fig. channels , 10). A tigation . A tunabtigation . Runningtigation . The arcribed in , producevid. In addition, a wavelet ratio surface is computed from the RWAs by dividing the modulus of the two RWA transforms. .Once the RWA is computed from the CWT, a ridge extraction algorithm is employed to extract the instantaneous frequency of the pulse ridge from the RWA transform. The ridge algorithm detects local maxima of the pulse band with respect to wavelet characteristic frequency. The ridge locus provides the instantaneous video-based HR signal, HRribed in , but usi3R and G signals were extracted from the video image, which was zoomed in on visible skin around the face region. The AC components from each signal were extracted by high-pass filtering at 0.25 Hz to remove respiratory and other lower frequency signal components, and then normalised by their DC component. The normalised AC signals were then wavelet transformed using a Morlet wavelet with characteristic frequency ω0 = 15 [R signal is shown in Fig. 2a with the corresponding wavelet modulus plot in Fig. 2b. A distinct pulse band can be seen across the transform modulus plot at around 85 beats per minute. The corresponding processed RWA scalogram is shown in Fig. 2c, where the smoothing of the band relative to the original scalogram in Fig. 2b is evident.The method described above was applied to a standard video sequence from a commercially available standard RGB video camera (Panasonic HDC-TM10 High Definition Video Camera). Video footage was acquired during a porcine model undergoing a rapid desaturation event where the arterial oxygen saturation dropped from 100% to around 40% for 2 min before being brought back to 100%. A Nellcor pulse oximeter  was also attached and provided a reference HR and oxygen saturation. The  ω0 = 15 . The R sR RWA transform modulus by the G RWA transform modulus is shown in Fig. 4a. Apart from localised edge effects, the video and pulse oximeter HRs stay within 2 BPS of each other during the period of investigation . The projection of the pulse rate onto the ratio surface is shown in Fig. 4b and the subsequent extracted ratio is shown in Fig. 4c. This ratio is then calibrated using the existing oximeter reading. The calibrated video saturation signal is presented in Fig. 5d along with the original oximeter-based saturation (SpO2) signal. The original R and G signals and the extracted HRs are also shown in Figs. 5a–c, respectively. Note that the SvidO2 appears to fall more rapidly than the SpO2. This is a manifestation of the difference in filtering characteristics of the two signals: one is wavelet based, applied to a video signal, and the other uses traditional pulse oximeter algorithmic components and is applied to a photoplethysmogram acquired at the finger.The wavelet ratio surface derived by dividing the 4A method has been detailed for the computation of HR and oxygen saturation from video signals comprising a series of code modules built around manipulations of the CWT. The method allows for the main vital signs currently measured by a pulse oximeter to be measured using a video system. However, the use of such CWT-based modular approach is advocated by the author as a powerful methodology to deal with noisy, non-stationary biosignals in general. In addition, other modules could be added to further enhance the method. These include reassignment or synchrosqueezing to enhance the definition of the pulse band –19, crosvidO2 approximation of SpO2 may be calibrated during spot checks and calculated continuously between these manual checks using the method described here.The signals used in this study came from an opportunistic capture of video during a porcine model of desaturation involving an anesthetised animal where ambient light and motion noise was not an issue. However, significant noise on the signal before and after the hypoxic episode was observed when movement of the investigators in the room caused major fluctuations in ambient light levels. This caused poor-quality signal prior to the desaturation phase and also subsequent to resaturation. In addition, the method as currently set up requires the input of calibration information from an existing oximeter. In practice, this system may be used as a trend and/or event monitor. Alternatively, the absolute saturation value may be calculated in a system that employs intermittent calibration during bedside observation by the caregiver using a pulse oximeter device. In the latter mode, the S2 to track oximeter-derived values is that both ‘devices’ may produce a different time sequence of output values due to internal filtering characteristics and/or internal reporting delays. Thus, matching the two signals may be difficult during rapid changes in the parameter . The study described is, in effect, a tougher challenge than that for a ‘standard’ oximeter hypoxia study [2 is altered in discrete steps , and where at each step or ‘plateau’ the values are measured only after they become stable. These are then compared with either another device or a reference comprising several blood draws for arterial oxygen saturation values. In contrast, in the study described here, the values were compared dynamically, which is much more challenging and does not lend itself to rigorous comparison. However, this is a consequence of the nature of the study where the data was collected opportunistically and there was no fine control over the SpO2 changes.In the preliminary investigation described in this Letter, close agreement between the video-derived parameters and pulse oximeter references was observed. A fundamental problem in attempting to compare the ability of video-derived HR and SpOia study  where thFuture work will include opportunistically adding video capture to other ongoing in-house human hypoxia studies. However, in addition, more specific trials are planned to consider motion and lighting noise in a rigorously controlled environment with reference to the ISO 8060-2-61:2011 (2011)  pulse ox


Shareable online video offers the potential for spreading a health message across online and real world social networks. Seeding a message in a clinical setting may be advantageous.To investigate the potential of an online video to spread a health message about juvenile idiopathic arthritis (JIA) when delivered or seeded in a clinical setting and investigate factors that influence sharing behavior.Multimethod proof of concept study. Concepts for two different styles of video were developed using focus groups and interviews and reviewed by an online market research panel. We compared dissemination of the two videos from two specialist pediatric rheumatology clinics in NHS Hospitals. Participants were 15 patients, family members, and clinical staff with knowledge of JIA at concept stage; 300 market research panel members in development stage; and 38 patients and their parents or guardians in the seeding stage. Newly diagnosed patients with JIA and/or parents or guardians were invited to view and share an online video with a health message about JIA across real-life and electronic social networks. Main outcome measures were viewing statistics, sharing behavior and patterns, and participant feedback.Of 38 patients and/or their parents or guardians given links, 26 visited the video webpage and shared the link, 2 visited and did not share, and 10 did not visit. Most links were viewed and shared within a few days. A total of 3314 pageviews were recorded with a mean of 89.6 pageviews per link (range 0-1245). Links were accessed from 26 countries, with most viewers in the United Kingdom (82.5%). Mothers were the most active group of sharers.Distribution of a video link in a clinical setting may be an effective way to spread a health message. Parents or guardians of children with JIA are more likely to share a link than young people. Dissemination depends on a small number of active sharers, the content of the video, and the willingness of participants to share health information about themselves.UK Clinical Research Network Study Portfolio ID (UKCRN): 13747; http://public.ukcrn.org.uk/Search/StudyDetail.aspx?StudyID=13747 (Archived by WebCite at http://www.webcitation.org/6eeXlMmM6). Health promotion and early diagnosis are core components of the National Health Service Improving Quality program . TraditiViral campaigns can reach large numbers of people through active sharing, but success depends upon the willingness of individuals to share messages. Shared online health messages are not simply passed from producer to consumer but are mediated before dissemination across social and other networks . The potential advantages of online sharing of health messages include cost-effective dissemination of bottom-up advice, greater reach than read-only information, the ability to trigger debate and generate support within networks, and encouragement for patients to “come out” as living with a chronic condition. Open discussion within social media forums can decrease stigma attached to health conditions such as mental illness .We were therefore interested in finding out whether seeding a viral campaign in a clinical setting could be effective in spreading a health message. We thought this could be successful because the message came from a trusted source, and members of patients’ social networks might be interested in the patients and their new diagnoses. However, we recognized this approach could fail if there was reluctance to disclose personal information   or if thThe diagnosis of musculoskeletal conditions such as juvenile idiopathic arthritis (JIA), muscular dystrophies, and bone cancer in children and young people is often delayed, which has a negative impact on clinical outcomes and experience of care -11. We tWe aimed to explore in this proof of concept study whether a health message with a shareable online video would be disseminated after initial distribution in a clinical setting by newly diagnosed children and young people with JIA and their parents or guardians. We also aimed to evaluate the feasibility of this approach and factors that might influence the distribution of such a video.The specific objectives wereTo investigate whether an online video distributed in a clinical setting is sharedTo determine whether video style and content influence sharingTo obtain user feedback in order to develop this approachThis was a multimethod study in 4 stages see . EthicalIn this first stage we explored ideas about online sharing and developed key concepts for video development. Focus groups were held with newly diagnosed and established children and young people with JIA (2-16 years of age) and their parents or guardians. Interviews were held with other interested parties including health care professionals caring for patients with JIA. Participants were recruited from one center (Newcastle).A researcher (MF) led each focus group using a topic guide including the following themes: (1) awareness of JIA before diagnosis, (2) journeys to diagnosis and information seeking, (3) views about information that others should have and how to communicate that information, (4) how children and young people and parents or guardians share experiences, and (5) the reactions of others to a diagnosis of JIA. In interviews with other interested parties, themes emerging from the focus groups were further explored. Focus groups and interviews were audio recorded, and key themes were identified using grounded theory .In the second stage, a digital communications agency  developeTo ensure videos were appropriate, informative, and likely to be shared, animatics were reviewed by an independent market research testing panel comprising 300 people without previous first-hand experience with JIA. Panel members were grouped for analysis as follows: 13- to 16-year-olds , 17- to 25-year-olds , and mothers of children up to 16 years of age. Panel feedback was incorporated into the final scripts. Two videos, each approximately 1 minute in duration, were cast, filmed, and edited to agreed final versions.Participants were recruited using a criterion sampling method in which children and young people within 6 months of diagnosis of JIA were identified in 2 centers (Newcastle upon Tyne and Liverpool). The parent or guardian was sent an information sheet before the clinic appointment, and participants (patients or their parents/guardians) were recruited in the clinic by the researcher or a clinical staff member.Participants were handed a postcard with a unique web link (bit.ly) and asked to access the link, view the video, and share it across their real-life and electronic social networks. Each link was associated with one of the 2 randomly assigned videos. Researchers were blinded to the allocation. Within 4 weeks, participants were interviewed by telephone for feedback about the study.Videos were hosted on a private, purpose-built website comprising multiple pages with unique 3-digit identifiers, each of which could be tracked. Each page comprised a video and share buttons . At the end, viewers were asked to complete an online questionnaire (SurveyMonkey) including demographic details, opinion of the video, reasons for sharing or not sharing, and social network use.Standard web analytic tools  tracked link activity. Google Analytic algorithms count a pageview when a user loads a page. Technology platform and some other metrics are reported by session: a session starts when a user accesses a website and ends after 30 minutes of inactivity or when the user moves to another website. Data collection was terminated when activity fell to very low levels, approximately 6 weeks after recruitment of the final patient. Analysis included geographic location of viewers, viewing platform , referring site, number of pageviews and sessions, and time spent on the website. Results for the 2 videos were compared. Participants were telephoned 2 weeks after recruitment. Results from the online questionnaire were compiled using standard metrics.Two focus groups were held, each comprising children and young people with JIA and their parents or guardians (n=9). Participants felt there was little awareness among the public and health care professionals that arthritis can affect children and treatment is often successful. Because effective treatments are available, appropriately treated JIA is an invisible albeit chronic condition. Although this means children and young people may not have to disclose their diagnosis, low visibility of physical changes may perpetuate lack of awareness and therefore delayed diagnosis. Interestingly, participants were concerned the public may have little interest in JIA because it is a manageable condition.Participants felt that children and young people and parents or guardians are often better informed than health professionals and indeed may have suggested the possibility of JIA to their general practitioner before diagnosis. They also reported low levels of awareness in schools that arthritis affects children and young people. Therefore, support is often lacking, affected families are left to inform schools, and children and young people have to negotiate with peers. Adolescents may be reluctant to tell others about their condition to avoid being perceived as “boring” or “crippled” if unable to participate in certain activities, and this may encourage teasing or bullying.Although fathers frequently attend hospital appointments with their child, participants felt mothers would be more engaged in information gathering, disease management, and exchange with others . Participants identified 2 key target audiences: mothers of children and young people with arthritis and mothers of adolescents and young adults with arthritis. Videos provoking an emotional reaction were thought likely to be shared more widely than those resembling a fact sheet.Other interested parties (n=6) were interviewed: a general practitioner with a special interest in pediatric rheumatology, a clinical nurse specialist in pediatric rheumatology, 2 consultant pediatric rheumatologists, the founder of a large parent or guardian support and awareness-raising network, and the communications officer of a relevant medical charity. Additional issues that emerged were around video style and content. Participants felt that to be widely shared, videos should be cute or humorous with key messages that children and young people can have arthritis and with timely treatment many maintain good quality of life. In addition, videos should not be heavily educational or overly alarmist while discussing symptoms . Participants suggested the most motivated sharers would be mothers of younger children, parents or guardians of children with more severe disease, and those who had suboptimal diagnostic experiences.Concepts and animatics for 2 contrasting videos were developed by the creative team. The 2 target audiences in Stage 1 were selected: parents or guardians (mothers in particular) of younger children and adolescents and young adults. Key messages wereAnyone can get arthritis, at any ageEarly treatment is importantThe video concepts were (1) “How old do you need to be…?”  and (2) A series of children approximately 4-10 years of age are shown answering “How old do you need to be...?” questions: “How old do you need to be to make a cup of tea/drive a bus/buy a house/get married/bake a cake/go on an airplane by yourself?” The answers, delivered to camera, are diverse, lighthearted, humorous, and real. Eventually we arrive at the last question—“How old do you need to be to get arthritis?”—to which children reply with high numbers . The last child we see tells us you can be any age to get arthritis. It ends with the caption “You don't have to be old to get arthritis” and a call to action to share the link.An older woman is seen in a teenager's bedroom playing drum and bass on a turntable and moving to the music. Her voice is dubbed by a teenage female talking about the joy of listening to music and dancing. As her tone changes from happy and animated to sad, she talks about restricted movement, swelling, and pain, which at first she could not make sense of. The video culminates in a “big reveal” where the viewer discovers that the old woman is in fact a teenage female. The viewer sees the old woman put down her headphones, and the camera moves up to meet the face of the teenager whose voice we have been hearing. The message is that what started out as diffuse and inexplicable pains was, in fact, JIA. The video ends with the caption “You don’t have to be old to get arthritis” and a call to action to share the link.Market research panel feedback about the animatics suggested the concepts successfully communicated key messages and would appeal to the target audiences. Concept 1 was considered more humorous and shareable and likely to appeal to mothers of young children. Concept 2 was considered more shocking and likely of greater appeal to 13- to 16-year-olds. Although the panel suggested 13- to 16-year-olds shared online video content most frequently, mothers were felt most likely to share a health message about their children online. Suggested barriers to sharing included lack of relevance, reluctance to share content about health, and self-consciousness about sharing content.A total of 38 participants were recruited between January and May 2014 in 2 centers (23 from Newcastle upon Tyne and 15 from Liverpool). Links were distributed to the participants and/or their parents or guardians: 21 were to Video 1 (“How old do you need to be…?”) and 17 to Video 2 (“Old to young”). One participant mislaid the paper link which was replaced. Only one potential participant, a 16-year-old male, declined. In general, even when offered to children and young people considered old enough to use them, the links were accepted by a parent, guardian, or other family member instead.P=.47 and P=.14, respectively).The mean age of the children and young people was 7.6 years (range 2-15); 28 were female and 8 male (2 unknown). Age, gender, and video allocation did not vary significantly by center. Children and young people allocated Video 1 were not significantly different from those allocated Video 2 in terms of age or gender .new in 82.10% (2657/3236) of pageviews and returning in the remainder. The average session duration was 1:14 minutes.Of 20 links to Video 1, 1 was not accessed. The remaining 19 links achieved a total of 2868 pageviews . One 12-year-old shared the link with her teacher and classmates but did not share online. Of 18 links to Video 2, 9 were not viewed. The remaining 9 links achieved a total of 368 pageviews  . VisitorMost links were viewed and shared within a few days. The lifespan of links was variable. For example, the most shared link was first shared 2 days after recruitment and generated 837 pageviews in the first week, decreasing in the subsequent 6 weeks . The secLinks were accessed from 26 countries . Most viOf 3236 pageviews, 2175 (67.21%) were from social networks, 624 (19.28%) from the original bit.ly links, and 437 (13.50%) from direct links. The social networks driving viewers were Facebook , Twitter , Mums in the Know , and Google+ . Of the 3236 pageviews, 2417 (74.69%) were on mobile or tablet devices.There were responses to 78 online questionnaires. All respondents either had arthritis or had a child with arthritis. The majority were female  and aged 25 to 44 years . Most  had been through or were planning to complete higher education.Of 67 respondents to this question, 16 (24%) received the link directly from the research team, 30 (45%) via social networking sites, 5 (7%) from friends and 3 (4%) from a family member; 23 (34%) received the link from someone with JIA or whose child had JIA.Of 72 respondents reporting their social media use, 70 (97%) used Facebook, 28 (39%) Twitter, 17 (24%) Instagram, 14 (19%) Pinterest, 6 (8%) Google+, 3 (4%) Tumblr, 2 (3%) Reddit, and 1 respondent kept a personal blog; 34 (47%) reported being logged on most of the time and 33 (46%) at least daily.Most respondents  shared the video . The mosWhen asked what they liked about the video, responses were “It reminded me of myself/my child/someone I know who has got JIA” , “The way it talks about JIA” , “It was informative” , “It was cute” , “It made me feel quite emotional” , and “It was funny” . Comments included the following:It plays on stereotypes and shows people their preconceptions are wrong.It’s brief and to the point without being overly emotional. I don't share much about my daughter's JIA as it is personal to her but nor do I want her to feel ashamed or different—knowledge empowers and reduces prejudice. I was happy to share this.not sharing the video  reported viewing but he video . The mosOf 78 respondents, 18 specified what they disliked about the videos. Responses were “It reminded me of myself/my child/someone I know who has got JIA” , “It made me feel emotional” , “The way it talks about JIA” , “It was cute” , and “It was funny” . One respondent expressed concern about self-protection, vulnerability, and inviting negative attention or bullying after sharing. Two respondents specified groups they wouldn't share the video with (school friends and work colleagues) stating it would make them feel vulnerable.In terms of content, Video 1 was considered more appealing and shareable. One respondent commented that the tone of Video 2 was “gray and depressing;” another commented that the actor did not come across as “cool” and therefore the video might not be helpful in raising awareness and engagement with the condition and that teenagers might not want to be associated with an older person’s disease.We were able to contact 15 participants for telephone feedback. Those contacted expressed support for the study and were positive and enthused about the video they had watched. Most felt that it was a good way to “get awareness out there.” One mother described the video (Video 2) as “quite catching” and said she wished she had seen it “2-3 years ago” when she first suspected that her daughter might have a medical condition. Participants tended to share the video with friends and family . They reported receiving positive feedback about the videos as well as consistent comments like “I didn’t realize that kids could get arthritis.” Telephone interviews further suggested that in most cases it was the parents or guardians who shared the link via their networks, mostly with family and friends but also through JIA-specific networks such as support groups and general parenting forums. Only 1 participant mentioned explicitly that her 13-year-old daughter shared the link with family and friends on Facebook and Instagram.Two participants had not viewed the video when contacted and were prompted to do so. Reasons for not viewing included loss of the recruitment card, having to attend to family or health-related matters, and stress following their child’s recent diagnosis.The video was also shared offline. One 13-year-old female shared the video with her teacher who then integrated it in a school lesson, although it was not widely shared online. The mother of a 9-year-old newly diagnosed female was very enthusiastic about the project but had recently stopped using social media. She suggested sharing the link through real-life networks and platforms including school notice boards and at extracurricular events.This study has investigated the feasibility of seeding an online awareness campaign for JIA in a clinical setting and has demonstrated that this approach can work in what we believe is a unique study. We have shown that this approach is acceptable to most children and young people and their parents or guardians and that it can reach an audience which far exceeds the geographical and sociodemographic spread of other  information. Dissemination is influenced by the content and its shareability, participant engagement in social networks, and willingness to share personal or family-related health information.The clinical motivation behind this study was to improve the diagnostic experience of patients with JIA. Our impression that awareness of JIA is low was supported by the initial group work in which children and young people and parents or guardians agreed there was little awareness of JIA in the community, schools, and primary care. Studies of delayed cancer diagnosis describe complex and varied pathways to diagnosis: delays can be patient-, family-, or doctor-related and occur between primary and secondary care or within secondary care . RaisingAlthough this was a relatively small study, it demonstrated proof of concept for this technique; each link received a mean of 89.6 pageviews, and links were viewed from a wide range of geographic locations. Concentrations of views in the North East and London in the United Kingdom and within the United States likely reflect the connections of sharers locally as well as the population density and Internet usage in London and the United States.Despite initial apparent enthusiasm from participants, links were not accessed by everyone recruited for the study. Unfortunately, we could not contact all of those concerned to understand why. The success of the project relied on a small number of enthusiastic sharers with appropriate viral dynamics or digital capital and access to and engagement with social media, factors which are crucial for successful seeding .Despite the apparent social media engagement of younger digitally native patients, the most effective sharers were the parents or guardians (particularly mothers) of younger children, as predicted in the first phase. Although we had limited feedback from this population, likely factors for disengagement of JIA patients include unappealing tone and content of videos, reservations about sharing personal health-related information online, and lack of interest in the research. Other approaches like encouraging participants to make their own videos or sharing offline might be more successful.A personal or emotional attachment to the message is important for sharing; the majority shared the video because their child or someone they knew had JIA. The engagement of patients and parents or guardians in spreading a health message represents a reconfiguration of the roles of patients and health professionals in a process which may require monitoring by public health professionals . When spIn terms of absolute numbers of pageviews, Video 1 was more successful than Video 2 (2868 vs 368 views). However, this appeared to be driven by a couple of very successful sharers of Video 1 and not because of a significant difference in the number of pageviews. This emphasizes the impact of small numbers of highly connected and motivated individuals in online sharing. Comments about the videos appeared to favor the lighter and more humorous tone of Video 1. The online content which is shared to some extent reflects on the sharer, and it may be that sharers were happier to be associated with this than the darker tone of Video 2. Facebook was the dominant social network through which links were shared, predominantly to friends or family members. Most views were on mobile or tablet platforms.Key to the transferability of this approach is the condition in question, and this approach may be more applicable to some conditions than others. Given the reservations about what sharers thought the messages said about themselves and their condition, messages about different conditions  are likely to be shared differently. Therefore, while there are clear elements of transferability, relevant condition-specific enablers and barriers should be taken into account when developing Web-based health messages.A major drawback to our study is that although we had viewing statistics, we were unable to measure whether our method had raised awareness of JIA in the population or among those who watched the videos. Testing this concept within a formal study to which patients were required to consent led to some logistical difficulties including the uneven randomization of patients across the 2 videos. Our method of sharing written links to the videos was not ideal; future studies should consider direct electronic sharing of links  to eliminate the need to manually enter the address into a browser and facilitate the sending of reminders if appropriate. We believed that up to 6 months after diagnosis patients would be more motivated to share videos but were unable to test this. Our study was heavily dependent on 2 professionally produced videos, which were relatively expensive to produce and may limit the scalability and transferability of this approach. Furthermore, the dependence on the video content itself means it is possible that different videos would have generated entirely different results. The feedback we received was predominantly from women aged 25 to 44 years, which may not reflect the majority of the viewing audience.In developing an effective awareness campaign, it might be appropriate to target other groups . We could not tell to what extent the links had been shared with health professionals, and some research into sharing behavior might be appropriate. While awareness campaigns can improve outcomes for patients, the impact on primary and secondary care of increased numbers of referrals as a result of a campaign should be considered .The findings of this exploratory study suggest that distributing a link to a shareable online video in a clinical setting is a feasible and potentially effective way of spreading a health message. The tone and content of the message are important factors in the success of this approach, as is an understanding of the population (patients and parents or guardians) most likely to share the resource. Other factors include the condition itself, the willingness of sharers to be identified with the condition, and preexisting awareness of the condition. The parents or guardians of affected children may be the most effective group for spreading health messages about childhood-onset conditions, and future campaigns should consider this. Further work should focus on refining this approach, delivering it at lower cost, and improving its generalizability across age groups and medical conditions.


Our findings indicate that variants of EGFR and SYNE2 play an important role in p21 regulation and are associated with the clinical outcome of HBV-related HCC in a TP53-indenpdent manner.This study was to explore the association between gene variants and p21 expression and investigate the TP53-independent p21 regulation in hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) patients from Guangxi by genome-wide association study. 426 HBV-related HCC patients were enrolled. Results showed that, after quality control, a total of 21,643 SNPs were identified in 107 p21 positive and 298 p21 negative patients. The variants of epidermal growth factor receptor  and spectrin repeat containing, nuclear envelope 2  were associated with p21 expression. Moreover the haplotype block (rs2227983 and rs6950826, r Investigations have demonstrated that the dys-regulation of cell cycle plays an important role in the occurrence and progression of HCC68108131517Although p21 is a tumor suppressor, it may also promote oncogenesis in certain circumstances. p21-deficient mice may spontaneously develop tumors, supporting the tumor suppressor activity of p21202123112526et al.et al.TP53 249Ser, which has been considered as a molecular fingerprint of HCC pathogenesisKao In the present study, a genome-wide association study (GWAS) using Illumina HumanExome BeadChip-12-1_A was performed in Chinese HBV-related HCC patients from Guangxi, aiming to detect the gene variants associated with p21 expression and reveal the TP53-independent p21 regulation.TP53 249Ser mutation between p21 negative group and p21 positive group (P > 0.05). TP53 expression status and hepatic cirrhosis status were significantly different between p21 negative group and p21 positive group . A significantly positive relationship was observed between p21 expression status and TP53 expression status .The clinicopathological characteristics are shown in After quality control (QC), a total of 21,643 SNPs were identified in 107 p21 positive patients and 298 p21 negative patients. The total genotyping rate in remaining individuals was 98.34%. The principal component analysis (PCA) plot  showed tP = 3.61 × 10−5) was located in the exon 13 of EGFR gene and rs6950826  in the intron 7 of EGFR gene. rs8010699 , rs3829767 , rs4027402 , rs9944035 , rs4902264 , rs4027405  and rs1890908  were found in the SYNE2 gene.Due to the limitation of sample size, we focused on the loci with minor allele frequencies (MAF) > 0.05. Manhattan plot is shown in P = 1.26 × 10–3, OR = 3.23, 95%CI = 1.58–6.58; GG vs. AA, P = 6.44 × 10–6, OR = 5.65, 95%CI = 2.66–12.00), the G allele was strongly associated with positive p21 expression. The genetic model of rs6950826 was over dominant model . The genetic model of rs8010699 was an additive model . The genetic model of rs4027405 was a dominant model . The genetic model of rs1890908 was also a dominant model .2 = 0.378) in EGFR and the haplotype block in SYNE2 ) were identified, and the haplotype A-G of EGFR and haplotype G-A-A of SYNE2 were significantly associated with p21 expression .After above analysis, GeneMania SoftwareThe clinical and pathologic characteristics of patients are shown in P = 0.001, HR = 1.86, 95%CI = 1.28–2.69). Similar findings were also found in rs1890908 . In addition, rs2227983, rs6950826 and rs8010699 were not related to the OS.Cox proportional hazard regression analysis showed rs4027405 and rs1890908 were significantly associated with OS after adjustment for age, body mass index (BMI), smoking status, drinking status, TACE status, serum AFP, radical resection, pathological grade, cirrhosis, intrahepatic metastasis, vascular invasion, antiviral therapies, child-pugh class, BCLC stage and PVTT . rs40274P = 0.001, HR = 2.82, 95%CI = 1.51–5.27) or child-pugh class B . Similar results were also observed in rs1890908.A stratified analysis was performed to evaluate the associations of rs4027405 and rs1890908 genotypes with HBV-related HCC survival by age, smoking status, drinking status, serum AFP level, radical resection, TP53 expression status, intrahepatic metastasis, vascular invasion, antiviral therapies, child-pugh class and BCLC stage . PatientP = 0.003, HR = 2.25, 95%CI = 1.31–3.87). Similar results were observed when the rs1890908 and serum AFP level were considered simultaneously .In this study, GWAS was performed to investigate the p21 regulation in Chinese HBV-related HCC patients in Guangxi. Results showed several SNPs in EGFR gene  and SYNE2 gene  were associated with p21 expression, and the mRNA expression of p21, EGFR and SYNE2 had positive correlations between each other. Furthermore, the p21 expression was significantly associated with the cirrhosis status and TP53 expression, which was consistent with previous results1132According to the NCBI database (NG_007726), EGFR gene is mapped to chromosome 7p12 spanning 188.3 kb and contains 30 exons. EGFR is also known as ErbB1 or HER-1 and is a transmembrane receptor belonging to the family of receptor tyrosine kinases (RTK) including ErbB2/HER2, ErbB3/HER3 and ErbB4/HER436373940et al.In our study, results showed that SNPs  of SYNE2 gene were associated with p21 expression, and these three loci had strong linkage disequilibrium. Haplotype analysis for the association of SNPs  with p21 expression indicated that patients with haplotype A-G-G had a lower p21 expression in HBV-related HCC. SYNE2, also known as EDMD5, NUANCE and Nesprin-2, is mapped to chromosome 14p23.2 and consists of 116 exons. SYNE2 belongs to the family of giant spectrin-repeat (nesprins), which plays an important role in linking the nucleus to the cytoskeleton and is a key component of the linker of the nucleoskeleton and cytoskeleton (LINC) complex4750515053et al.P = 0.443, et al.Kao in vitro studies are required to clarify the underlying mechanisms.However, our study has several limitations. This was a single center study, and a small fraction of clinical data was missing. The SNPs were not functionally characterized to reveal their effects on the p21 expression and clinical outcome of HBV-related HCC. Therefore, additional studies with larger sample size are needed to further validate the association of these SNPs with p21 expression in HBV-related HCC and with the clinical outcome of HBV-related HCC patients, and In conclusion, our results demonstrate that SNPs of EGFR (rs6950826 and rs2227983) and SYNE2  are associated with p21 expression in HBV-related HCC. rs4027405 and rs1890908 are potential biomarkers for clinical outcome of HBV-related HCC Chinese patients after hepatectomy in Guangxi. In GWAS, TP53 expression as a covariate, and hence, our findings indicate that variants of EGFR and SYNE2 genes may play important roles in the regulation of p21 expression and affect the outcome of HBV-related HCC via TP53-indenpdent manner.All experimental protocols were approved by the Ethical Review Committee of the First Affiliated Hospital of Guangxi Medical University . The methods were carried out in accordance with the approved guidelines. Written informed consent was obtained from all subjects before study.A total of 426 patients with pathologically proven HBV-related HCC were enrolled from the First Affiliated Hospital of Guangxi Medical University between 2005 and 2014. HCC tissues were collected during surgery and immediately stored at −80 °C until the DNA extraction. Total DNA was extracted using the TIANamp Genomic DNA Kit . DNA concentration and purity were measured with the NanoDrop2000 system . Immunohistochemical staining of p21 and TP53 in HCC tissues was done in the Department of Pathology at the First Affiliated Hospital of Guangxi Medical University. Finally, 114 patients were found to be positive for p21 expression and 312 negative for p21 expression. Patients with p21 positive expression were included in positive group and those negative for p21 expression of patients in negative group.TP53 249Ser mutation was evidence of AFB1 exposure and detected by sanger sequencing after amplification with polymerase chain reaction (PCR) . Tumor status was classified according to the Barcelona Clinic Liver Cancer (BCLC) staging systemParaffin embedded HCC tissues were used in immunohistochemistry for p21 and TP53 with general two-step method. Briefly, the antigen retrieval was done with EDTA Tris at a high temperature for 2.5 min. Then, sections were treated with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase. After washing thrice with PBS, the mouse against human p21 (1:150)  and mouse against human TP53 (1:150)  antibodies were independently added, followed by incubation overnight at 4 °C. The sections were incubated with secondary antibody  at 37 °C for 30 min. Finally, these sections were visualized with DAB (Dako Cytomation) and counterstained with hematoxylin. Positive and negative controls were included in each immunohistochemical reaction. All the sections were reviewed and scored by two pathologists independently who were blind to the clinical characteristics of patients. At least 10 fields were randomly selected at a high-power (x400 magnifications) at the regions distant from necrotic areas, and the proportion of positive cells was calculated with the following formula: . Positive cells had brown granules in the nucleus. According to previous criteria, positive p21 expression was defined as the presence of ≥5% positive cancer cells282863All samples were genotyped using the Illumina HumanExome BeadChip-12-1_A, which includes 242,901 markers on the protein-altering variants. These markers include non-synonymous single nucleotide polymorphisms (SNPs), SNPs in splice sites, stop variants, SNPs in promoter regions, SNPs in extended MHC region, GWAS tag markers, HLA tags, etc. Details about SNPs can be found at the exome array design webpage. Samples were processed according to the manufacturer’s instructions. Genotype calling was carried out using the Genotyping Module v1.0 in GenomeStudio version 2011.1, and average call rate was 99.84%. A total of 50 samples (>10%) were randomly selected and sequenced for the candidate loci using the ABI Prism 3100 . The candidate loci and primers used for sequencing are shown in All patients were followed up via telephone or hospital visit until death or the last time follow-up was done in September 2014. The median follow-up period in 426 patients was 41 months (ranging: 3–123 months), and the median survival time (MST) was 47 months.−6; (iii) a Minor allele frequencies (MAF) <0.05. 3. PCA: Guangxi in Southern China is a multi-ethnic region. An analysis of population stratification by PCAA quality control (QC) procedure was used before association analysis as follows: 1. The sample was removed if they (i) had an overall genotyping rate of <95%; (ii) had ambiguous gender; (iii) had genome-wide identity-by-descent (IBD) >0.1875; (iv) were outliers in principal components analysis (PCA) for ancestry and population stratification. 2. The SNP was excluded if they had (i) a call rate of <95%; (ii) a P value in Hardy-Weinberg equilibrium (HWE) <1 × 10The association analysis was evaluated using Single Variant TestP < 0.05 was considered statistically significant. Statistical analysis was performed with SPSS version 18.0 .Overall survival (OS) was defined as the time from hepatetomy to death or the last follow-up. MST was calculated for all variables, univariate and multivariate survival analysis were done using the Cox proportional hazard regression model. Hazard ratios (HR) and their 95% confidence interval (CI) were calculated from the Cox proportional hazards regression model with adjustment for age, gender, race, body mass index (BMI), smoking status, drinking status, TACE status, serum AFP level, BCLC stage, child-pugh class, radical resection, pathological grade, hepatic cirrhosis and PVTT. A value of How to cite this article: Han, C. et al. EGFR and SYNE2 are associated with p21 expression and SYNE2 variants predict post-operative clinical outcomes in HBV-related hepatocellular carcinoma. Sci. Rep.6, 31237; doi: 10.1038/srep31237 (2016).


Respiratory rate (RR) is an important vital sign which is strongly correlated with in-hospital mortality. At the same time, RR is the most likely vital sign to be omitted when assessing a patient. We believe that one reason for this could be the difficulty in measure the RR, since it is not read off a monitor, but counted manually. Also there is the possibility of assessment bias and the inter-observer reliability becomes important. We therefore set out to investigate how the nursing staff counting the actual number of respirations per minute would agree with the nursing staff using a predefined ordinal scale.For this prospective study, we recorded five videos of a young healthy man breathing approximately 5, 10, 15, 30 and 60 times per minute. The videos were shown in a random order to a suitable sample of the nursing staff. The participants were randomized into two groups; one to count the exact number of breaths per minute, and one to use a predefined ordinal scale.Comparing the exact number of breaths per minute, the Intra Class Coefficient (ICC) was 0.99 (95% CI: 0.97–1.00). Comparing the RR using the predefined scale, the overall Kappa Fleiss Coefficient was 0.75.The inter-observer agreement was high when comparing the use of the actual number of breaths per minute and substantial when comparing the use of the predefined scale. This is the largest inter-observer study on RR to date. However, further studies on the use of scaled comparisons of RR are needed. Respiratory rate (RR) is an important vital sign in the assessment of patients and has proven to be strongly correlated with in-hospital mortality and serious adverse events (e.g. cardiac-pulmonary arrest or ICU admissions) , 2. SomeAlso, several studies have illustrated that RR measurements are omitted when vital signs are collected at a daily basis during admission in acute hospital wards , medicalSome argue that a reason for not measuring the RR is a lack of knowledge of its importance. It has been suggested that nurses and doctors should be educated better to appreciate the RR as an useful marker in assessing patients in risk of serious adverse events . Others It therefore also becomes challenging and time consuming to measure RR, making the measurement more laborious and therefor tempting to omit.One method to increase the reporting of RR might be to introduce an ordinal-scaled measurement of the RR as very slow, slow, normal, fast and very fast.The majority of vital signs are measured electronically, which reduces the risk of assessment bias. However, when a measurement in clinical medicine is not captured electronically, the risk of assessment bias  is suddenly present. It therefore becomes important to assess the reliability of the measurement. Previous studies into the inter-observer reliability of RR measurements are based on few observers assessing the patients –10. SincWith the present study, we aimed at investigating the inter-observer agreement between nursing staff measuring the RR by counting the exact number of breaths per minutes or by using a predefined ordinal scale. We hypothesized that the use of a predefined ordinal scale would result in a higher inter-observer agreement compared to the group counting the exact number of breaths.We examined the inter-observer agreement in measuring the RR. And as most vital signs, including RR, are assessed by the nursing staff, the observers in our study were nurses and nurses assistants at a medical admission unit in Denmark.The study is a questionnaire study based on video recordings of one patient (simulated). We recorded five videos using a Canon IXUS 70 camera . The videos showed a healthy thirty years old male breathing respectively 5, 10, 15, 30 and 60 times per minute. A picture from the videos can be seen in The videos were shown to a convenient sample of participants consisting of nurses and nurses assistants. The participants were randomized into two groups by handing out alternate forms for them to complete. 18 were asked to count the exact number of breaths per minute, and 19 to use the predefined scale of very slow, slow, normal, fast and very fast. When using the ordinal scale, the participants were not given any indication of where a given RR should be allocated on the scale.The data is presented as median (range). The Intra Class Coefficient (ICC) was used to classify the agreement between the participants measuring the exact numbers of breaths per minute. Fleiss Kappa statistics were used to assess the reproducibility (reliability) of the measurements when comparing the RR in the group using the predefined ordinal scale. Kappa values greater than 0.75 represents an excellent agreement. Kappa values between 0.4–0.75 represents a fair to good agreement, and values less than 0.4 a moderate to poor agreement. There are no established criteria for sample size calculations for this type of study, and therefore none were performed.Finally we stratified the participants into two groups according to experience (above and below the median experience). The agreement between the experienced and the less-experienced was tested by using a Wilcoxon-Rank-Sum test for the counted RR. For the scaled RR we used Spearman’s chi-squared test. A p-value > 0.05 indicates no significant difference in the RR noted by the two groups. In other words, the two groups agree on the RR noted, if the p-value is above 0.05.A statistical analysis was performed using Stata version 11 .According to Danish law, an Institutional Review Board (IRB) (Videnskabsetisk komite Region Syddanmark) approval was not required. The IRB was contacted prior to the study and a waiver of approval was given. The participants were not offered any incentive to participate in the study. All participants gave oral informed consent that their responses were recorded for scientific purposes. Due to the waiver of approval from the IRB, no written consent was obtained. All participants were informed that participation was voluntary; all asked participants wanted to participate. We kept no records of participant consent, since a filled out questionnaire would indicate informed consent to participate.Four nurses assistants and 34 nurses participated in the study. The median age of the participants was 42 (25–63) years, and the median experience was 18 (0–36) years.Overall, the results showed high agreement between the observers.For the group of observers counting the exact RR, the Intraclass Coeffiecient was calculated to 0.99 (95% CI: 0.97–1.00). For video 1  the participants counted the RR to be a median of 64 breaths per minute (50–70), for video 2  8 (8–10), for video 3  5 (4–7), for video 4  10 (9–11) and for video 5  31 (30–32). When using Wilcoxon-Rank-Sum there were no significant differences between the experienced and less-experienced groups in the counted RR for each video.In the group using our predefined ordinal-scale, the overall Fleiss Kappa was calculated to be 0.75, which also indicates a high inter-observer agreement. Kappa was calculated for video 1 at 0.94, video 2 at 0.55, video 3 at 0.78, video 4 at 0.61 and video 5 at 0.88. For video 2, 80% of the participants reported the RR to be normal, 15% for it to be slow, and 5% reported the RR to be very slow. For video 4, 75% of the participants reported the RR to be normal, 20% for it to be slow, and the last 5% reported the RR to be very slow. The lowest Kappa was calculated for video 2 and 4, indicating a fair and substantial inter-observer agreement. Using Spearman’s chi-squared test all p-values were above 0.05.We investigated the inter-observer agreement by having one simulated patient and multiple observers. When asking 38 nurses and nurses assistants to assess the RR, we found a high inter-observer agreement in the group counting the exact RR. Furthermore, we found substantial inter-observer agreement in the group using our predefined ordinal scale. Overall this gives the impression of a high inter-observer agreement in assessing the RR.We expected the inter-observer agreement to be the highest in the group using the predefined scale. This was not confirmed by our results, however. It is noteworthy that the lowest Kappa values are calculated in video 2 and 4. Video 2 shows the simulated patient breathing approximately 10 times per minute, and video 4 shows a RR of 15 breaths per minute. The explanation for the lowest kappa values calculated in video 2 and 4, we believe, is due to the small difference in the RR when comparing the scenarios in those to videos. When having to assess the RR without actually counting the exact numbers of breaths per minute, the small difference becomes harder to detect. Since a RR of 10 and 15 is close to what is considered the normal range of the RR, it is even more difficult for the observers to differentiate between the two categories . For video 1, the simulated patient had a RR of 60, and one nurses assistant noted the RR to be fast, not very fast. For video 5, one nurse noted the RR to be normal even though the real RR was 30. For video 4, one nurse noted the RR to be very slow even though the real RR was 15. If the RR is estimated wrongly, too high or too low, it could lead to under- or over- estimating the patients illness, and can then lead to an incorrect triage, monitoring and treatment. The participants were given no guidance to which levels of RR were to be categorized as very-slow to very-fast. This could account for some of the misclassifications of the RR. Experience in patient treatment and observation could also lead to some differences in the classification of the RR. However, we found no difference between the most experienced and least experienced participants regardless of using the counted- or scaled RR.Overall, our results confirm earlier findings. In a study by Lim et al, the RR was calculated in 245 patients in medical wards or attending the lung function department. Each patient was assessed twice with 15 minutes between the assessments. They found a very good agreement between the observers in the RR measurements . In anotOur study has limitations. The simulated patient recorded in the videos was breathing with a fixed RR during each video sequence, and it was therefore not true to reality, making our study a simulation more than a clinical study. In reality, the RR changes quickly according to the clinical condition, and is thus difficult to compare between observers, unless counted simultaneously. Another limitation is the fact that the participants were fully aware of the purpose of the video sequences, and therefore may have been more thorough in the assessment than they would during clinical practice. In addition, there is a risk of incomplete blinding, and the videos were subject to lighting, blurring and colours.The main strength of our study is that the observer is the only variable, as multiple observers assess the same patient simultaneously. At the same time, we eliminate the possible natural variation and uncertainty of few observers performing independent observations consecutively by using video recordings. Removing the bias of changing the conditions between the observations provides a more accurate impression of the actual inter-observer agreement than designs used in previous studies.We found high inter-observer agreement when assessing the exact RR, and substantial agreement when using a predefined ordinal scale. We expected the inter-observer agreement to be higher when using the predefined scale, but this was not confirmed in our study. Our predefined scale, however, needs further analysis before possible implementation in standard clinical use. Whether an ordinal scale will increase the recording of the RR is unknown, but the study shows that nurses and nurses assistants agree on how to record and report the RR.