Individuals were more willing to view  and share  COVID-19 videos with high emotional consistency with virus variant topics. (4) Conclusions: The results of empirical analysis showed that content relevance and emotional consistency between videos and virus variant topics significantly positively impacted video views and shares. The trained SVM classifier can support public health departments in monitoring and assessing the COVID-19 pandemic. Our study provides management advice while helping individuals reduce harm and inform next-step decisions.(1) Background: The coronavirus variants have posed serious challenges for the prevention and control of the COVID-19 pandemic. Individuals selectively watch and forward videos that help them reduce the damage caused by the virus. Therefore, the factors influencing video viewing and sharing in the context of the COVID-19 pandemic caused by virus variation must be explored. (2) Method: Based on a combination of uncertainty reduction theory and functional emotion theory, this paper designed hypotheses regarding how content relevance and emotional consistency affect video views and shares. We used the support vector machine (SVM) classification algorithm to measure the content relevance between videos and virus variant topics. We performed sentiment analysis of video text to evaluate the emotional consistency between videos and virus variant topics. Then, we used empirical analysis to build the model. (3) Results: The trained SVM classifier was effective in judging whether the video text was related to virus variant topics (F = 88.95%). The content relevance between COVID-19 videos and virus variant topics was generally low. The results showed that the higher the content relevance, the more views (IRR = 1.005,  Since the COVID-19 pandemic spread around the world, the damage and changes to the global economy and trade, as well as people’s lifestyles and health, have been immeasurable ,3. The CCOVID-19 belongs to the coronavirus family and has a single plus-stranded RNA genome. The unstable single-stranded structure means COVID-19 is prone to variation . VariantCrisis refers to unexpected, sudden, and uncommon  events tInternet platforms provide the easiest access to information, and more than 70% of adults use them when their health is at risk . InformaAny form of interaction between individuals on Internet platforms during a crisis is meaningful  as interIn the context of the COVID-19 pandemic situation caused by the emergence of virus variants, in this study, we aimed to explore the impact of content relevance and emotional consistency between videos and virus variant topics on the number of views and shares of videos. We used the support vector machine (SVM) classification algorithm to calculate the content correlation between novel coronavirus videos and virus variant topics and then used sentiment analysis to calculate the emotional consistency between COVID-19 videos and virus variant topics. In this study, we included the calculated content relevance and emotional consistency scores in the independent variable index, and we conducted empirical to test the study hypothesis.Many researchers have focused on the COVID-19 pandemic. The information factors that have been mined include information content , informaThe studies on emotion during the COVID-19 pandemic have mainly been conducted within the field of public opinion and have rarely considered the number of views and shares of videos. Researchers have focused on understanding public attitudes by analyzing public responses and interactive texts with the public health sector and news media. Huyen (2021) explored the public’s concern about the COVID-19 pandemic in Vietnam on different Internet platforms and found that the public’s attitude toward COVID-19 pandemic information changed from negative to positive as the COVID-19 pandemic progressed . In addiSocial psychology scholars think that only when people feel sure about their environment can they feel confident in their following actions and have reasonable expectations for the future . UncertaUncertainty is a primary state experienced by humans; as such, uncertainty management is a fundamental activity . URT, deCrises create a high degree of uncertainty. Because crises are unpredictable and irregular events, uncertainty exists at all stages of the life cycle of a crisis . URT staDue to aversion to uncertainty, also known as ambiguity aversion, individuals reduce uncertainty in the environment by collecting accurate details and sharing them with others . AlthougEmotion is an individual’s internal psychological state as different responses to events, agents, or objects . FET staBased on the above theories, scholars have described the process of FET as follows: an individual perceives the environment or an event related to them, the individual produces a unique emotional experience, and specific emotions manage the individual to produce a specific state of readiness for action or action-oriented state; in the end, the specific emotions affect individual cognitive or action, with the purpose of behavior being consistent with the emotional target . In a stIn a crisis, information can help individuals to reduce ambiguity , distingCrises are constantly changing, and unexpected events are uncontrollable . When acHypothesis 1 (H1):When the COVID-19 coronavirus variation causes a crisis, the more relevant the video content to the virus variant topics, the more likely the video to be viewed by more people.Hypothesis 2 (H2):When the COVID-19 coronavirus variation causes a crisis, the more relevant the video content to the virus variant topics, the more people were likely to forward the video.Emotions are considered distinctive, transient, and targeted, triggering different adaptive behaviors in response to the event causing the emotion . In the Hypothesis 3 (H3):When the COVID-19 coronavirus variation causes a crisis, the higher the emotional consistency between videos and the virus variant topics, the more likely the video will be viewed by more people.Hypothesis 4 (H4):When the COVID-19 coronavirus variation causes a crisis, the higher the emotional consistency between videos and virus variant topics, the more likely the video will be forwarded.On video platforms, to motivate creators, platforms will hold various events from time to time, providing creators with video exposure and creation incentives. Videos posted by creators participating in activities will gain additional exposure, and individuals may choose to watch and forward the video to engage in the activity. To push high-quality content to users, video platforms have established some leaderboards, which attract traffic to the videos on the leaderboard in the top positions. The content accuracy and video information quality released by public health departments and news media are higher than those of ordinary video producers. Hence, individuals trust them more. Individuals may view and forward their videos without trust in public health departments and news media. Therefore, we hypothesized that whether the video is an activity, whether it is included on a leaderboard, and whether the source is public health departments and news media would interfere with our study. We considered these variables as control variables in the model.The independent variables in this study included content relevance and emotional consistency. Content relevance referred to the proportion of sentences referring to virus variant topics in the effective sentences in the video. We considered emotional consistency as the difference between the video’s emotional score and virus variant topics. The dependent variables included the numbers of video views and shares, and the control variables included activity videos, being listed on a leaderboard, and video source. The definitions of the variables are provided in During data preprocessing, we used the SVM algorithm to evaluate the content relevance between videos and virus variant topics, and we used sentiment analysis to evaluate the emotional consistency between videos and virus variant topics. For hypothesis testing, we used empirical analysis to test the impact of content relevance and emotional consistency between videos and virus variant topics on the number of video views and shares. The research workflow is shown in Step 1: Audio to text: We used mature audio-to-text technology to convert video audio into text.Step 2: Sentence segmentation: We segmented the video audio that was processed into text, and we segmented the video audio text into several sentences.Step 3: Data cleaning: We eliminated valid data should when the audio was converted to text and sentences were segmented.Step 4: Conversion to text and sentence segmentation: With stratified random sampling and labeling, we randomly selected 4 sentences from each video as the labeling set. We invite researchers to label the sentences as related to virus variant topics or not, which formed the labeled data set.Step 5: SVM classification: We used the annotated data set as the training set to train the SVM classifier with the linear kernel function, and all valid sentences were classified. The output of two data sets included a sentence set related to and a sentence set unrelated to virus variant topics. Then, according to the content relevance calculation formula, we calculated the content relevance between video and virus variant topics.Step 6: Emotion analysis: We used the mixed emotion dictionary to analyze the emotion score of the two data sets, and we obtained the emotion score of each sentence and the emotion consistency score of each video.Step 7: Negative binomial regression: We used the extracted independent variables (content relevance and emotion consistency) and dependent variables to perform negative binomial regression and hypothesis testing.Step 8: Robustness test: We used the replacement variable measurement method to test the robustness of the model.The research data in this paper comes from a well-known Chinese video platform, which is characterized by attention to originality and strong social functions. Originality ensures that videos on the platform are produced and published by the video owner. Videos on the platform rarely have different people publishing precisely the same content. Intense sociability indicates that users not only use the platform to obtain information but also are willing to socialize on the platform, such as by forwarding information. The platform and data characteristics met our study needs.This paper used Chinese words related to virus variants and virus variant names such as “omicron” as search keywords to obtain video data. Our results of data analysis showed that May to August 2021 was the platform’s hottest time for virus variant topics. The number of videos published was above 500 per month, higher than the average of 238 in other months. In addition, the monthly growth rate in May 2021 was 25.37%, decreasing in August to 34.81%. Therefore, this article selected video data from May to August 2021, for a total of 4049 videos. The video data included the video screen and audio, video duration, views, shares, uploader certification type, activity video identification, leaderboard ranking, and other tags.Platform’s search mechanism finds keywords that appear in the video’s title, tag, description, or the publisher’s nickname, and then displays the search results. Therefore, our search results were not necessarily related to the virus variant crisis. Most videos were 2–16 min in length, and videos less than 2 min contained limited information. In a crisis, users’ negative emotions make needs for information acquisition or sharing urgent, so videos longer than 16 min do not conform to user habit of fragmentary information acquisition. Therefore, we eliminated videos that were entirely irrelevant to the COVID-19 topic through manual reading, and we removed videos that were shorter than 2 min and longer than 16 min, for a total of 2501 remaining videos.This study used mature speech-to-text technology to convert the data of the 2501 videos from audio to text. The conversion accuracy was more than 99%, which met the study requirements. Through text processing, we found that some videos did not use human voice dubbing, so the audio-to-text result was empty. We removed the videos for which the result of audio-to-text was empty, the language was not Chinese, or the language was utterly illogical and rough. We finally obtained 2037 videos that met the study requirements.A video 2–16 min in length contains multiple sentences. This article uses the Chinese ending words as the segmentation basis and divided the 2037 videos into several sentences. The video text contained the uploader’s opening and closing remarks, transitions between topics, and other content unrelated to the video content, which needed to be eliminated. The final total was 39,703 sentences.SVM is a machine learning algorithm based on statistics. It has substantial advantages in binary classification problems, is widely used in natural language processing, so was appropriate for our study . This paThis paper used the “Jieba” library for word segmentation, processing the words after eliminating useless sentences. We used the disabled dictionary to remove meaningless words, imported the custom dictionary to increase the accuracy of the segmentation result, and replaced synonyms for words with the same meaning. Due to the limited information expressed by a single character, we retained only phrases with a character length greater than 2 in word segmentation. Then, we used TF-IDF to process the word vectors of the segmented data. The SVM using a linear kernel learned whether sentences were classifiers for virus variant topics. The accuracy of the SVM classifier was 89.37%, and the F1 value was 88.95%, indicating that the training was effective.We obtained the data used in training the classifier from 39,703 sentences after segmentation, and each video had data included in the classifier. Therefore, our classifier could be appropriately applied in our experiments. Next, we used the trained classifier to analyze all 39,703 data items to determine whether they were related to virus variant topics.The training classifier determined whether each sentence was related to virus variant topics, so that we could judge the correlation between the video and virus variant topics. The number of sentences related to virus variants indicated how many sentences related to virus variant topics were in a video. The total number of sentences indicatedSentiment analysis is a text analysis method used to explain emotion intensity . We adopWe performed the empirical analysis in this study with Stata 16.0. The descriptive statistical analysis results of the 2037 video data are shown in This paper used canonical correlation analysis to test whether the independent variables were strongly correlated, leading to multicollinearity. The results of the canonical correlation analysis  showed tIn addition, this paper evaluated the variance inflation factor of the independent variables. The results showed  that theIn this study, we incorporated the independent variables  and control variables  into the regression model. The dependent variables were the numbers of views and shares. The hypothesis test results of negative binomial regression are shown in p = 0.017). So, H1 was supported. The results of the test of H2 proved that video shares  increased by 8% when the content relevance increased by one unit, and the effect was significant, supporting H2.H1 stated that the more relevant the video content to virus variant topics, the more views the video would receive. Model 1 in the table shows that when the content relevance score increased by one unit, the number of views increased by 0.5%, which was statistically significant (p < 0.001), so H3 was supported as well.Model 1 also showed that when the video emotional consistency score increased by one unit, the number of views significantly increased by 52.5% , which supported H4.We found from the test results of Model 2 regarding emotional consistency that with an increase in emotional consistency, video shares significantly increased (IRR = 1.761, This paper used punctuation marks as the standard when segmenting the sentences in the videos, where each segmented sentence expressed complete semantics. Therefore, we chose the number of sentences as the standard for calculating content relevance. Although sentences with shorter character lengths were eliminated, the character lengths of the sentences widely differed. In terms of video length, we found large differences in the proportions of each sentence. Therefore, this paper changed the method of calculating content relevance and used the character length of the sentence as the calculation standard to test the regression results and the robustness of the original model. We also proved that the model that replaced the method of calculating content relevance had no multicollinearity. The experimental results of negative binomial regression are shown in The model results after changing the method of calculating content relevance showed that the emotional consistency and content relevance between videos and virus variant topics significantly impacted video views and shares. The robustness test results supported the original model’s regression results.From the theoretical combination URT and FET, this paper used emotion analysis, the SVM algorithm, and empirical analysis to explore the factors influencing the number of video views and shares when COVID-19 variation caused tension among people.First, functional emotion theory well explained the impact of emotional consistency on the number of views and shares in this study. This paper used the combination of URT and FET to explain the complexity of individual acquisition of information and sharing behaviors. FET could explain the impact of emotion on the number of video views and shares during the COVID-19 crisis. URT is a theory commonly used in the study of public health emergencies, but uses of FET to explain individual information collection and sharing behaviors in crises have been relatively rare. FET has often been used to understand the motivational effect of emotional perception on behavior. Previous studies focused on the influence of negative emotions on adopting protective behaviors. Nabi proposed discrete emotions as a psychological framework. In drunk driving scenarios, emotions stimulate relevant behavioral tendencies, which promote selective allocation of attention and information-seeking behaviors . HoweverSecond, this paper trained an SVM classifier to automatically divide the audio text into a set of sentences related to virus variant topics and a set of unrelated sentences. The trained SVM was effective, with an accuracy of 89.37% and an F1 of 88.95%. Previous researchers mostly used content analysis to classify video content , which rThird, the results of empirical analysis showed that the content relevance between videos and virus variant topics positively correlated with the number of video views and shares. Compared with historical pandemic video information, viewers are more likely to watch and forward videos related to COVID-19 . BecauseFourth, our results showed that the higher the emotional consistency, the more a COVID-19 video is viewed and shared. Emotion, as the external manifestation of an individual’s feelings, is reflected in individual language and writing, which reflect the individual’s attitude toward events. In studies on emotion in crisis, researchers determined individuals’ emotional expression from individual language and writing to measure individual feedback on public crisis management . HoweverCOVID-19 prevention and control have always had to face the challenges produced by new crises, such as the emergence of new virus variants. From our findings, we provide several recommendations for public health departments, news media, and individuals concerned with the COVID-19 crisis.First, the SVM algorithm classifier could identify whether the text in COVID-19 videos was related to virus variant topics. The coronavirus variations have led to crises during the COVID-19 pandemic, which have posed constant challenges to the prevention and control of the pandemic. The classifier could monitor the relevance between videos and virus variant topics and evaluate the change in viewer attention to virus variant topics. It can also provide technical support for popularizing highly relevant videos when a new COVID-19 variant emerges.Second, this paper revealed that when the COVID-19 virus variant, the number of video views and shares is related to content relevance and the emotional consistency between the videos and virus variant topics. Specifically, the public is more willing to watch and forward videos highly related to virus variant topics and that have similar emotional expressions. Public health departments and news media use the Internet to provide information to the public and support for the public to use the Internet to collect and share information. This finding suggests that public health departments and news media should consider both the relevance of videos and virus variant topics, as well as whether the video’s emotion is close to the crisis emotion when providing video information to people in areas where the COVID-19 pandemic is serious.Finally, for individuals in crisis, our findings explain what kind of video can help them understand the crisis and is suitable for sharing with others. In the case of unpredictable danger and the timing of crises, our findings can help individuals quickly find the information they need when a crisis occurs. Individuals can also reduce their uncertainty by watching and forwarding videos and feel sure about the next steps in a crisis.This study has many limitations. First, the sectional data we used could not reflect the changes over time in the number of video views and shares. The situations caused by the pandemic are variable, resulting in sudden tension, followed by relaxation as waves caused by the variants pass. The given situation considerably and quickly changes individual information-seeking and -sharing. Therefore, the time when a video is uploaded during a crisis is critical. In future studies, panel data of the number of video views and shares on specified days can be considered. The classification of time can also be more detailed. For example, when the information demand is high, we can use the classification of the video release time based on the search volume on Internet engines as the classification standard. Second, due to the limitation of the study methods and data, we did not consider the entertainment experience when individuals watched the videos. In addition to information and emotional needs, watching videos is a form of entertainment. Factors affecting individual viewing and immersion perception, such as the camera position of the video producer when recording the video, will also impact the number of video views and shares .From the perspective of video content relevance and emotional consistency between videos and virus variant topics, in this study, we explored the factors that affected the number of video views and shares. The study leveraged the SVM classification algorithm to assess video content relevance and used sentiment analysis to provide support for scoring the sentiment coherence of the videos. The results showed that when the emergence of virus variants caused sudden tension during the COVID-19 pandemic, individuals tended to watch and forward videos whose content and emotional expression were similar to virus variant topics. Our findings can help public health departments better monitor the development of the COVID-19 pandemic and cooperate with news media to push information that more in line with the needs of the public. Our study can also help with providing timely, rapid, and accurate advice for individuals to respond to a new crisis, helping them confidently make decisions. The limitations in data and variable extraction in this study need to be addressed. In future studies, we will use panel data to improve consideration of time and other experimental methods to measure individual entertainment needs.


A key feature of compulsive alcohol drinking is continuing to drink despite negative consequences. To examine the changes in neural activity that underlie this behavior, compulsive alcohol drinking was assessed in a validated rodent model of heritable risk for excessive drinking  rats). Neural activity was measured in dorsal medial prefrontal cortex  and assessed via change point analyses and novel principal component analyses. Neural population representations of specific decision-making variables were measured to determine how they were altered in animals that drink alcohol compulsively. Compulsive animals showed weakened representations of behavioral control signals, but strengthened representations of alcohol seeking-related signals. Finally, chemogenetic-based excitation of dmPFC prevented escalation of compulsive alcohol drinking. Collectively, these data indicate that compulsive alcohol drinking in rats is associated with alterations in dmPFC neural activity that underlie diminished behavioral control and enhanced seeking. Compulsive alcohol drinking is a core feature of alcohol use disorder. Here the authors find that in rodents, neural signals in a key decision-making brain region (dmPFC) shift from behavioral control to alcohol seeking during compulsive alcohol drinking behaviour. Developing novel ways to treat addiction at this stage requires the identification of the brain mechanisms that underlie the decision to compulsively use alcohol. Isolating these mechanisms is difficult as decision-making deficits can exist both as a risk factor for and consequence of addiction4. Towards this goal, the current study assessed the decision to drink alcohol in a rodent model of compulsive drinking.Continued use of alcohol despite negative consequences 7. Furthermore, mPFC plays a critical role in many forms of decision-making including drug seeking and extinction10, thus highlighting the importance of this region for expression of pathological drinking. It is not clear how the computational properties of mPFC are altered during addiction, but alcohol (and other appetitive) cues increase neuronal activity in human medial PFC areas12, where activity is linked to perceived reward value14. In addition, mPFC plays a key role in guiding decision-making during conflict16, and compulsive drinking has been conceptualized as impaired processing of conflict between desire to avoid punishment and drive to consume alcohol17.Impairments in decision-making are observed following excessive alcohol exposure2. Rodents with heritable risk for excessive drinking rapidly progress to ARD19, suggesting a pathophysiology in circuits required to control compulsive drinking. In support of this view, previous work in rats bred for excessive drinking, alcohol preferring rats (P rats), identified alterations in mPFC neural activity that reflected a weakened representation of the “intention” to drink alcohol20, indicating that the role of this brain region in decision-making was compromised in non-compulsive alcohol drinking.In animal models, compulsive drinking is frequently operationally defined as aversion-resistant drinking (ARD)2, which is mediated by connections from mPFC to ventral striatum21. Also, decreased drive from mPFC to periaqueductal gray contributes to compulsive drinking in mice22. In heavy-drinking humans, compulsive responding for future alcohol at the risk of immediate punishment corresponded with increased activity in mPFC during alcohol-associated cues23. These mPFC findings synergize with behavioral evidence that compulsive drinking involves a “head down and push” strategy17 where animals endure the aversive stimulus to obtain alcohol. Therefore, it is critical to determine how altered computational properties of mPFC neural ensembles contribute to expression of compulsive drinking.Outbred rats exhibit compulsive drinking following prolonged alcohol exposure25) neurons in rodent models of compulsive drinking and non-compulsive drinking during a simple cued-access protocol task. To assess compulsive (ARD) intake, alcohol was adulterated with the bitter tastant quinine in selected drinking sessions 2. Previous work from our group established that following a short (2–4 week) drinking induction, compulsive drinking patterns were consistently observed in alcohol preferring P rats whereas non-compulsive consumption was consistently observed in Wistars18. This strong relationship between genotype and phenotype is not surprising given the well-established existence of heritable risk for AUD27 and previous research demonstrating that P rats are a valid pre-clinical rodent model of genetic risk for AUD28.To directly examine the importance of mPFC networks for compulsive drinking, we recorded single unit activity from large ensembles of dorsal mPFC  across neural populations of varying size. By analyzing populations of neurons at high temporal resolution35 it was possible to examine how large-scale patterns of neural activity evolve during the compulsive decision to drink. We deconstructed the decision-making process into multiple stages by comparing different trials and different time points36 including pre-trial seeking state, alcohol-availability cues, sipper approach initiation, and drinking. Critically, we also utilized change point analyses37 to identify changes in animal seeking state and sipper approach likelihood. With these methods, we tested the hypothesis that representations of behavioral control signals are weakened in dmPFC during compulsive drinking.We evaluated neuronal representations of decision-making relevant signals by dmPFC populations during alcohol and alcohol+quinine sessions using principal component analysis (PCA)40 could reduce craving and intake in patients diagnosed with a substance use disorder42. Thus, we used a chemogenetic approach to stimulate neurons in dmPFC to test the hypothesis that increasing dmPFC activity would reduce compulsive drinking.Finally, emerging data in humans suggests that targeting the mPFC44. Following initial alcohol exposure in home cage (Intermittent Access Protocol (IAP), Fig. The 2-Way Conditioned Access Protocol (2CAP) task was previously developed by our group to assess cued drinking with or without aversive consequences45) for these test sessions. Multiple compulsive drinking tests sequences (baseline and test day) were performed and analyzed. Results for the first compulsive drinking test sequence will be presented first, followed by results for subsequent compulsive drinking tests in later sections. We chose to initially present the first quinine exposure because these sessions were unbiased by possible effects from acclimation to the taste of quinine and to improve clarity in the results by removing compulsive drinking test number as an additional variable. In total, 7 Wistars and 7P rats received electrophysiology probe implants. The initial compulsivity test data for one Wistar was lost, resulting in 6 Wistars and 7P rats for this portion of the analysis .Following training in the 2CAP task with 10% ethanol (v/v) only, selected animals were implanted with multielectrode probes (Cambridge Neurotech) in dmPFC. After recovery and task reacclimation, animals underwent a two-day compulsive drinking test sequence that consisted of a baseline regular 2CAP session with 10% ethanol only and a compulsive 2CAP session with 10% ethanol adulterated with 0.1 g/L quinine Fig. . Electro18, we found that Wistars decreased consumption significantly when alcohol was adulterated with quinine  = 10.05, p = 0.0044, post-hoc Bonferroni corrected t-test, Wistar alcohol vs. alcohol+quinine: t(10) = 5.33, p = 9.9*10−4). Conversely, P rats showed a smaller reduction in consumption of alcohol-quinine, indicating that P rats exhibited compulsive drinking to a greater degree than Wistars . P rats exhibited a preference for unadulterated alcohol during a separate alcohol vs. alcohol+quinine choice test  = 2.68, p = 0.18, P Rat/Alcohol: t(6) = 5.42, p = 0.0064, P Rat/Alcohol+Quinine: t(6) = 3.82, p = 0.035). As there was no punishment for approaching on a CS− trial, animals still frequently approached on CS− trials. The reduced consumption and time at sipper in Wistars were driven behaviorally by a tendency in alcohol-quinine sessions to approach the sipper, but then leave soon after arriving  to examine the representations48 of decision-making variables by populations of neurons in dmPFC  or behaviorally  to assess neural population representations of a given variable that differed across the trial types. Note that we do not attempt to identify which trial type represents the signal, rather we only quantify separation as a measure of representation strength. In this way, we can compare neural representations between groups to determine if the neural population in one group is more selective for a certain trial type or is more distinct between trial types than in another group. Also, we note that separation between PC trajectories is not necessarily a measure of the subjective strength of a given signal to the animal . We developed a method that used principal component analysisWe used four example neurons to demonstrate this analysis methodology Fig. . DistancWhen PCA is performed on neural populations of different sizes, there is a risk that the most variance explaining dimensions are mostly driven by the larger population, thus resulting in the spaces from the smaller population being warped to fit the larger one. To address this, we developed a novel method for selecting PCs that relies on subsampling neurons to identify stable PCs across different groups of animals  = 237, p = 6.3*10−18, interaction: F = 27.5, p = 6.2*10−6, post-hoc Tukey’s test, Wistar/Alcohol vs. Wistar/Alcohol+Quinine: p = 3.8*10−9, Wistar/Alcohol vs. P Rat/Alcohol: p = 3.8*10−9, P Rat/Alcohol vs. P Rat/Alcohol+Quinine: p = 8.7*10−8). Increased seeking representation strength implies neural firing patterns are more distinct in dmPFC, not necessarily that animals have an increased desire for alcohol or alcohol + quinine. Collectively, these data show increased seeking representation during challenged drinking and in compulsive rats during alcohol drinking.Trial by trial change in average firing rate across all neurons exhibited an increase at the session change point above randomized data Fig. . After pp = 6.2*10−11, time main effect: F = 17.2, p = 3.3*10−13, strain/liquid interaction: F = 115, p = 4.8*10−13).The CS+ alerted animals to trial onset and identified the correct sipper where access would be provided Fig. . We compp = 0.0054, strain/liquid interaction: F = 9.93, p = 0.0032, post-hoc Tukey’s test, Wistar/Alcohol vs. Wistar/Alcohol+Quinine: p = 0.99, Wistar/Alcohol vs. P Rat/Alcohol: p = 0.79, P Rat/Alcohol vs. P Rat/Alcohol+Quinine: p = 6.1*10−4). Overall, cue representation strength was similar in compulsive and non-compulsive rats, except for decreased representation strength in compulsive rats during challenged drinking.Next, we compared firing patterns during the CS+ and CS- to examine representations of CS+/CS- discrimination. PC trajectories for CS+ and CS− trials were similar following the initial CS onset, but CS− trajectories returned to the pre-CS position in activity space while CS+ trajectories did not Fig. . All grop = 3.4*10−31, liquid main effect: F = 27.0, p = 7.2*10−6, strain/liquid interaction: F = 202, p = 8.8*10−17, post-hoc Tukey’s test, Wistar/Alcohol vs. Wistar/Alcohol+Quinine: p = 3.8*10−9, Wistar/Alcohol vs. P Rat/Alcohol: p = 3.8*10−9, P Rat/Alcohol vs. P Rat/Alcohol+Quinine: p = 1.05*10−6). Therefore, approach initiation representation strength was depressed in compulsive rats drinking alcohol, while challenged drinking increased representation strength in non-compulsive rats and depressed it yet further in compulsive rats.Sipper approach initiation was the first physical action in the consumption process Fig. . We idenp = 5.7*10−15, liquid main effect: F = 92, p = 1.1*10−11, strain/liquid interaction: F = 133, p = 5.6*10−14, post-hoc Tukey’s test, Wistar/Alcohol vs. Wistar/Alcohol+Quinine: p = 3.8*10−9, Wistar/Alcohol vs. P Rat/Alcohol: p = 0.52, P Rat/Alcohol vs. P Rat/Alcohol+Quinine: p = 0.92). This drink representation increase may underlie Wistar rejection of quinine-alcohol, while the lack of drink representation change may allow continued compulsive drinking in P rats.We examined the neural population representation of drinking by comparing CS+ drink and CS+ no drink trials Fig. . PC traj51 , so we refer to this classification as putative. In non-compulsive rats, challenged drinking continued to produce elevated drink representation and suppressed intake in later compulsive-drinking tests  = 244, p = 4.0*10−18, post-hoc Tukey’s test, first Wistar/Alcohol vs. first Wistar/Alcohol+Quinine: p = 3.8*10−9, first Wistar/Alcohol vs. later Wistar/Alcohol: p = 0.44, later Wistar/Alcohol vs. later Wistar/Alcohol+Quinine: p = 3.8*10−9). The increase in drink representation during challenge  corresponded to increased firing in both putative excitatory  = 18.8, p = 1.5*10−5, post-hoc Tukey’s test, first Wistar/Alcohol vs. first Wistar/Alcohol+Quinine: p = 0.0015, first Wistar/Alcohol vs. later Wistar/Alcohol: p = 0.52, later Wistar/Alcohol vs. later Wistar/Alcohol+Quinine: p = 0.082) and putative inhibitory  = 7.27, p = 0.0076, post-hoc Tukey’s test, first Wistar/Alcohol vs. first Wistar/Alcohol+Quinine: p = 0.56, first Wistar/Alcohol vs. later Wistar/Alcohol: p = 0.99, later Wistar/Alcohol vs. later Wistar/Alcohol+Quinine: p = 0.018) neurons  = 21.7, p = 3.8*10−5, time main effect: F = 2.09, p = 0.026, test session/liquid interaction: F = 10.5, p = 0.0024), which corresponded to increased firing in putative excitatory neurons only . , though we did find significantly reduced consumption relative to baseline (one sample t-test) relative to p = 2.3*10−9) . CNO/DREADD did not alter locomotion , as well as when drinking alcohol during DREADD-mediated increased excitation following a CNO injection  = 4.24, p = 4.9*10−4, one sample t-test relative to p = 0.008, no significant differences between active DREADD and control). However, injection of vehicle  paired with challenged drinking decreased consumption in both DREADD and control animals to a similar degree as was observed in CNO+Qui test 1  = 0.96, p = 0.35), though not as substantially as was observed in Wistars . These results indicate the existence of a synergistic effect between injection and quinine adulteration that reduced consumption.Since increased firing during challenged drinking was associated with reduced intake in Wistar rats, we hypothesized that dmPFC excitation suppresses compulsive drinking. Therefore, we conducted a second experiment in a new cohort of 20 P rats using identical alcohol exposure, 2CAP training, and compulsive drinking test procedures Fig. . We expr−9) Fig. . HoweverWe propose that challenged intake in rats demonstrating compulsive alcohol drinking was driven by a strong seeking representation and weak representations of behavioral control variables, such as cues, approach initiation, and drinking Fig. . When grA striking result of these studies was the large increase in drink representation immediately after drinking onset in non-compulsive rats (Wistars) during challenged drinking. Furthermore, in these rats we found that excitatory and inhibitory neurons tended to increase their firing rate immediately after drink onset. This increase in drink representation strength and individual neuron firing response persisted through multiple compulsive drinking tests in non-compulsive rats. Conversely, compulsive rats (P rats) exhibited no change in drink representation or individual neuron firing rate during the first compulsive drinking test. During later compulsive drinking tests, compulsive rats exhibited a large increase in drink representation and excitatory neuron firing during both unchallenged and challenged drinking sessions.25) responds to unexpected negative outcomes 54 and that these large scale signals are caused by altered neural activity across many neurons55. Furthermore, neurons in ACC have been shown to respond to conflict during response inhibition in stop-go or stop-change tasks57. Therefore, we hypothesize that increased drink representation during challenged drinking in non-compulsive rats was the neural signature of conflict processing in dmPFC that contributed to decreased consumption.While these patterns of drink representation increases are similar, there are subtle differences in the conditions that produce these increases that point to possible explanations for dmPFC’s role in compulsive drinking. Non-compulsive rats exhibited increases in drink representation only during challenged drinking, when their expectation about the contents of the solution was violated, leading to response inhibition and decreased consumption. This behavior is similar to previous reports that the anterior cingulate cortex  in dmPFC in P rats. We found that activation of the DREADD did not eliminate but prevented the escalation of compulsive drinking. Therefore, we hypothesize that excitation of dmPFC excitatory neurons disrupted maladaptive processes necessary for the escalation of compulsive drinking. Indeed, recent research has shown that down regulation of SYT1 (a gene that influences synaptic transmission and plasticity) in prelimbic cortex increases compulsive alcohol consumption (assessed using quinine adulteration) and reduces neural excitability in Wistars with longer alcohol drinking histories58. Overall, while this and previous studies22 have demonstrated that mPFC plays a functional role in compulsive drinking, this study also provided valuable insights into the neurocomputational phenomena underlying compulsive drinking. These results highlight the need for future experiments that simultaneously actuate and measure neural activity from neural ensembles of the dmPFC in animals drinking alcohol compulsively. These experiments will be capable of determining if the changes in computation described here are required for compulsive drinking.Based on the observed increase in excitation in non-compulsive rats during challenged drinking and transcranial magnetic stimulation studies in human substance use disorder patients28. Furthermore, differences between P rats and Wistars are likely related to alcohol consumption, given that P rats were bred from Wistars29. This common ancestry, along with the fact that Wistars can drink compulsively given sufficient drinking history32, indicates that Wistars and P rats share many similarities and are well-suited comparisons.With the strong correspondence of rat strain and the compulsive and non-compulsive phenotypes it’s important to consider whether the differences we observed were merely strain differences between Wistars and P rats unrelated to compulsive drinking. While this possibility cannot be entirely eliminated, we believe this is unlikely for several reasons. First, we detected differences in the neural firing behavior of a brain region known to be involved in decision-making and substance use disorders, and we detected these differences immediately before and during the expression of compulsive drinking, making it unlikely that these differences were merely genotypic artifacts unrelated to compulsive drinking. Furthermore, some of these differences were observed during epochs where no behavioral differences were observed between strains  or during epochs where the neural signal differences connected logically to behavior , indicating that these differences were not merely behavioral artifacts unrelated to compulsive drinking. In addition, there are several compelling reasons to use these strains for comparisons involving compulsive drinking. P rats are an established pre-clinical rodent model of genetic risk for AUDIt is possible that P rats are less sensitive to the taste of quinine in alcohol, which, if true, would likely influence the compulsive drinking patterns we observed. We showed that P rats find quinine aversive and prefer to drink alcohol that is unadulterated with quinine  rats (Indiana University School of Medicine) and male Wistar rats . All animals were shipped via ground transportation from breeding facilities to the laboratory, all within the city of Indianapolis. All animals were given food and water ad libitum in their home cages. All animal procedures were approved by the Indiana University – Purdue University Indianapolis School of Science Institutional Animal Care and Use Committee.The electrophysiology study utilized 24 P rats and 72 Wistars over 4 sub-cohorts. Following IAP and 2CAP training, 8 P rats and 8 Wistars were selected for surgery (two sub-cohorts of 4 Wistars and two sub-cohorts of 4 P rats). One P rat and one Wistar were excluded from these sub-cohorts due to damaged probes, resulting in a final yield of 7 P rats and 7 Wistars. Electrophysiological data for one Wistar was lost for its first ARD test, so it was excluded from the analysis of the first ARD test. Later ARD test data were intact, so they were included in those analyses.The DREADD experiment utilized 23 P rats. 20 animals were selected for virus surgery following IAP and 2CAP training . One DREADD animal was lost prior to the third from last test session.59. All animals were 12 weeks old at the start of IAP. 20% ethanol  in one bottle and tap water in another bottle was provided ad libitum in the animals’ home cages for 24 h periods on alternating days. Animals were weighed and bottles were placed on the cages on Monday, Wednesday, and Friday mornings  for 2 weeks. Bottles were pulled 24 h later and weighed to assess consumption. Animals were selected for 2CAP task training based on their average consumption across IAP  was used to acclimate animals to the taste and effects of alcohol44 task was used to assess voluntary consumption of 10% ethanol in tap water. All training sessions and DREADD testing sessions were conducted in standard rat shuttle chambers (MedAssociates) housed in custom sound attenuating boxes. Regular 2CAP sessions and baseline ARD testing sessions used 10% ethanol  in tap water. Quinine test sessions used a 10% ethanol solution adulterated with 0.1 g/L quinine (Fisher Scientific). Electrophysiological data were gathered in a custom made chamber to allow for sufficient space above the animal’s head for the headstage and wiring. We utilized an updated version of the 2CAP task that included negative conditioned stimuli (CS−). Two possible stimuli modalities were used: a solid 4s light on stimulus or a 4s stimulus composed of four 300 ms light on followed by 700 ms light off blinks. Positive conditioned stimuli consisted of one modality of stimulus presented directly above the sipper where fluid would become available. Negative conditioned stimuli consisted of the other stimulus modality presented above both sippers. The specific modality type assignment  was counter balanced across rats. The pseudorandom nature of the CS+ and CS− trials resulted in a slightly elevated likelihood (approximately 60% instead of 50%) for animals to receive a CS- trial following a CS+ trial and vice-versa. Each 2CAP session lasted approximately 1 h and consisted of 48 CS+ and 48 CS− trials. CS+ trials were separated by pseudorandomly selected intertrial intervals of 20, 28, 36, 44, 56, 68, 96, and 120 s. CS− trials randomly occurred within the intertrial intervals, though an exclusion period of 3 s at the start and end of CS+ trials prevented CS+ and CS− trials for occurring adjacently. Animals had a low likelihood to approach both sippers  or approach the sipper prior to the end of the CS+ Animals were selected for surgery based on their average consumption across 2CAP training sessions  with target coordinates of 3.2 mm A/P, 0.6 mm M/L, 4.3 mm D/V24 in the right hemisphere with a 7.5 degree angle in towards the midline. Movable drives were incased in antibiotic ointment and then affixed to the skull using dental cement. Ketofen (5 mg/kg) was administered and the animal was monitored for 7 days post-surgery.Animals selected for electrophysiological recordings underwent implantation surgery using procedures approved by the Indiana University Animal Care and Use Committee. Animals were anesthetized using isoflurane and held in a stereotax (Kopf). The scalp of the animal was shaved and cleaned with alcohol and betadine. Cefazolin (30 mg/kg) was administered IP and Bupivacaine (5 mg) was injected subcutaneously in the scalp. After removing the scalp and clearing the bone, bregma and lambda were located. Grounding screws were implanted posterior to lambda above the cerebellum and 6 mounting screws were implanted . A trephine was used to perform a craniotomy above medial prefrontal cortex. For all animals except one P rat, a multishank probe  was attached to a moveable drive and implanted with target coordinates of 3.2 mm A/P, 0.8 mm M/L, 3.0 mm D/V60 v2. Based on the manual identification of drinking trials and the tracking information, the drinking position of each animal was identified and the drinking initiation time was set as the first time point when the animal’s snout entered the drinking position (within 9 pixels (< ~1 cm) of the manually identified sipper location). Spike sorting was performed using Kilosort245. Moveable probes were lowered by approximately 0.1 mm the day before each ARD test baseline session. Typically, the probes were lowered after the rat ran a regular 2CAP session on Monday to prepare for ARD baseline and test days on Tuesday and Wednesday.Following recovery, rats were given two 2CAP sessions in a specialized electrophysiology chamber to reacclimate to the task and acclimate to the new chamber. Next, rats underwent at least 3 2CAP sessions with electrophysiology wires attached to acclimate to the presence of the wiring. Omnetics headstages were plugged in to the exposed connectors from the neural probes on the dental cement headcap. Electrophysiological data and task specific variables  from the Med Associates controller (MedPC v4.1) were recorded using Open Ephys v0.4.0 data acquisition systems. Video of the animal was recorded from above the chamber using a Logitech webcam (Logitech Webcam Software v2.80.853.0). Licks were recorded with piezo microphones (Korg) attached to sipper tubes. Animal tracking was performed offline using DeepLabCut61. Probe tracks were identified via glial fibrillary acidic protein (GRAP) staining visualized by Alexa Flour 555 and nucleus DAPI staining. There was a slight, but significant difference in mean medial/lateral placement of 0.11 mm between P rats and Wistars. There was a larger, significant difference in mean anterior/posterior placement of 0.81 mm between P rats and Wistars. To ensure that differences in recording sites could not explain differences in neural encoding, the correlation between neuron location (defined as the location of the shank on which the neuron was recorded) and neuron principal component coefficients for each stable principal component (see below) was calculated. Across all combinations of PC and the spatial dimension, the 62.Final positions of shanks were verified via histology  mean waveform  was administered and the animal was monitored for 7 days post-surgery. Virus expression was verified in all animals by histology . The scalp of the animal was shaved and cleaned with alcohol and betadine. Cefazolin (30 mg/kg) was administered IP and Bupivacaine (5 mg) was injected subcutaneously in the scalp. After removing the scalp and clearing the bone, bregma was located. Bilateral craniotomies were performed above dmPFC. Bilateral injections of 0.65 uL at 0.2 uL/min of virus were performed using Hamilton syringes and Harvard Apparatus pumps using the target coordinates of 3.2 mm A/P, 0.8 mm M/L, 3.0 mm D/VAnimals receiving the excitatory DREADD were given AAV5-CaMKIIa-hM3D(Gq)-mCherry (AddGene) and control animals received AAV5-CaMKIIa-mCherry (UNC Vector Core). Tests in which the DREADD was activated were conducted using 3 mg/kg Clozapine N-oxide  in 1% dimethylsulfoxide (DMSO) and saline IP injections approximately 30 min prior to session start.DREADD experiments took place in the same Med Associates shuttle chambers used for 2CAP training. Lick data was used to identify errors in consumption measures from bottle leaks using the following procedure. For each animal, number of licks and consumption for all baseline and test sessions were linearly fit. Then, the residuals for each session were calculated. The residuals for all sessions for all animals were then compared and residuals above three standard deviations were excluded. This resulted in 4 out of 197 test measurements being excluded.Data analysis of spike trains was conducted using the following steps  and no-approach CS+ trials. Trials were marked as approach trials if the animal approached either sipper during the access period. The approach initiation time was identified using a change point analysis. Let For the analysis of drinking, we focused on correct approach trials and not correct approach trials . We observed that animals that correctly approached  almost always licked the sipper at least once , though we were unable to resolve the precise lick time. Therefore, we refer to correct approach trials as drink trials. Let All trial means of interest were concatenated through time for each neuron , we determined the stability of the PC across the 500 iterations using the following method. Let PCs for each experimental group  were generated by projecting from only the 200 neurons from that group that were used in each of the 500 iterations. Let A leave one out analysis was performed by rerunning the analysis with every combination of one animal removed. Only minor qualitative differences were observed. Furthermore, a randomized analysis was performed by shuffling assignments for the trial types of interest . PC stability and PC separations were substantially reduced, indicating that the signals observed herein represent true neural population signals associated with the decision-making signals of interest.63 using the same underlying data structures that were used for the PCA  results of these comparisons. We followed these ANOVAs with post-hoc Bonferroni corrected two-sided t-Tests , we utilized a three-way ANOVA to detect main effects of rat strain, liquid type, and time, as well as strain*liquid and strain*liquid*time interactions. In the main text, we report only significant , with the exception of PC separation plots and their summary bar graphs where we show standard deviation (std). We chose to show standard deviation for these graphs because the sample size was controlled by the number of subsamples used in the PCA method in those cases. In all figures, we identify in the caption the type of method used for each error bar or fringe to avoid confusion.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Given the suboptimal acceptance of iCBT for depression, finding ways to increase the acceptance and uptake is crucial for its dissemination. Moreover, it remains unknown to what extend the Unified Theory of Acceptance and Use of Technology (UTAUT) could aid the design of DTC in psychological service.To explore whether the regulatory processes theorized in the UTAUT  could be modified and mediate the change of acceptance of iCBT.This randomized controlled trial recruited 219 individuals with at least mild level of depression. Upon completion of pre-assessment, participants were randomly allocated to an intervention (IG) and a control group (CG). The IG received a 7-minute UTAUT-driven promotion video, while the CG received a video of same length on general psychoeducation. Both groups completed a post-assessment.Repeated measures ANOVA revealed a significant time by group effect on treatment acceptance. The video in IG was perceived to be clearer and more persuasive. Mediation analysis showed that the intervention effect was mediated by increase in perceived performance expectancy of iCBT, and the indirect effect was conditional on dispositional help seeking stigma.Pre-treatment acceptance of iCBT can be improved by brief DTC promotion video. The finding casts light that performance expectancy was the most modifiable and mediatable regulatory process on iCBT acceptance, although such relation could be attenuated by high help-seeking stigma. In sum, DTC marketing could aid implementation of Internet-based interventions, effort in stigma reduction should continue to encourage uptake of effective treatment.No significant relationships.


Many patients present to Paediatric Rheumatology clinic, (53%) with chronic pain related to Hypermobility. Pre-Covid, an evidenced based hypermobility class was developed and run by the Allied Health Professional team for children and young people (C & YP), and their parents/carers. These 6 classes were group-based and included education and exercise therapy but were stopped due to the pandemic. Feedback gained was good from the class, although reported issues included reduced school attendance, accessibility due to location, parental time off work, financial and time implications of travel, and unsuitability of group interventions for CYP with social anxiety and autism disorders.A digital platform (Vimeo) was used to present Hypermobility education and exercise content created by the Paediatric Rheumatology team. The video aims to address these barriers, allowing more equality of access for all CYP; digital access allows CYP and parents to re-visit this information at any time, providing ongoing support for long term management of this condition. The digital platform is being developed to maximize the effectiveness of therapy resources and to improve the patients experience to promote self-management of their hypermobility.Funding was applied for via the Transformation service. Meeting with the hospital videographer enabled the AHP team to devise a template for the video including education on what is Hypermobility, on the role of exercise - targeted versus general, the benefits of exercise, supportive footwear, pain management, pacing, sleep hygiene, and hand function advice to manage hypermobility. The class exercise programme including hand therapy was videoed using a model.The video was filmed in half a day. It was filmed using an unscripted question- and- answer format. The Paediatric Rheumatology MDT were filmed in the video. The digital platform is currently with our videography team for further edit. Further signposting to established resources will be included in the Vimeo resource.Following a final edit, this resource will be used for this patient population group. Feedback from CYP and parents, along with other health professionals is planned.On receipt of the first edit, multi-source feedback from parents/carers, CYP and other health professionals is planned via Microsoft Teams. It is hoped to show the video via Teams and seek anonymized feedback on the Teams chat function. This feedback will be discussed by the Paediatric Rheumatology MDT and will inform the final edit of the Hypermobility video resource.Further data from Vimeo analytics will be gathered e.g. how many times video watched, how long video is watched for, and what devices are being used.It is hoped that the Hypermobility Video will help support CYP and families in the long-term management and pain resulting from this condition.It is hoped that the use of new technologies and innovative ways of delivering care can enable health professionals to implement clinical support, improve self-management of chronic pain related to hypermobility, whilst improving equitable patient access to therapy across the North-West.Multi-source feedback will help inform the final edit of the video resource. Ongoing evaluation and analysis of Vimeo data will inform whether the resource is a useful adjunct in long term management of pain arising from Hypermobility.


Canines have a high incidence of mammary tumors, which is a major problem in the veterinary field. Approximately half of canine mammary tumors are malignant and metastasize to nearby lymph nodes. In this study, we analyzed the expression levels of RAD51, a DNA homologous recombination repair-related molecule, in canine mammary tumor cell lines derived from the primary tumor CHMp and metastatic tumor CHMm, which were established from the same individual. The RAD51 expression level in CHMm was higher than that in CHMp. However, CHMp was more resistant than CHMm to irradiation and formed functional Rad51 foci, which form at DNA lesions for homologous recombination repair. The cell cycle regulator p21 was expressed upon radiation exposure in CHMp cells, but not in CHMm cells, indicating that CHMm did not induce cell cycle arrest after irradiation. These results suggest that the radioresistance of CHMm, with high RAD51 expression, is lower than that of CHMp.Due to the high incidence of mammary tumors in dogs, it is important to elucidate the pathogenesis of these tumors in veterinary medicine. Radiation therapy is often used to treat mammary tumors that target DNA lesions. RAD51 is a key molecule that repairs DNA damage via homologous recombination. We examined the relationship between RAD51 expression and radiosensitivity in mammary tumor cell lines. CHMp and CHMm from the same individual were selected based on the differences in RAD51 expression. The radiosensitivity of both cell lines was examined using MTT and scratch assays; CHMm, which has high RAD51 expression, showed higher sensitivity to radiation than CHMp. However, the nuclear focus of RAD51 during DNA repair was formed normally in CHMp, but not in most of CHMm. Since irradiation resulted in the suppression of cell cycle progression in CHMp, the expression of p21, a cell cycle regulatory factor, was detected in CHMp after 15 Gy irradiation but not in CHMm. These results indicate that functional expression is more important than the quantitative expression of RAD51 in canine mammary tumor cells in response to DNA damage. Escherichia coli to higher mammals and plays an important role in maintaining genome stability  (p53RE-pNL1.1) (Promega). To measure endogenous p21 transcriptional activities, CHMp and CHMm cells were transfected with the p53RE-pNL1.1. The cells were harvested 48 h after transfection, and luciferase activity was measured using the Nano-Glo Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was normalized to the value of luc2 activity from the co-transfected pGL4.51[luc2/ CMV/Neo] (Promega).The p53 response element in p21WAF  and human cytomegalovirus in the pTet-splice vector  [p < 0.01 indicated a statistically significant difference.Data are expressed as mean ± standard deviation. Analysis of variance with Tukey’s post hoc test was used when multiple comparisons were required. A value of 0 and S phases  and CHMm (derived from metastatic foci), which were established from the same individual, showed remarkable differences in both GS phases .50 of radiation exposure of CHMp was 4.61 Gy and that of CHMm was 4.66 Gy, which were similar; however, high dose irradiation of 10 or 15 Gy resulted in lower survival rates for CHMm  consisted of 495 base pairs (bp) and was predicted to encode 164 amino acids. Canine p21 protein showed 82% homology with the human p21 protein (GenBank NP_000380.1). Canine p21 contained a putative cyclin-dependent kinase inhibitor domain A.To confirm the cross-reactivity of the anti-human p21 antibody with canine p21, western blotting was performed using anti-Halo tag and p21 antibodies. Halo-tagged p21 was detected at the same size as anti-Halo and anti-p21 antibodies; thus, the anti-human p21 antibody was demonstrated to have cross-reactivity against canine p21 B.To verify why CHMm cells with high RAD51 expression were vulnerable to DNA damage, we examined p21 expression in cells induced by DNA damage. In both CHMp and CHMm cells irradiated with 15 Gy, RAD51 expression was unchanged before and 24 h after irradiation; however, p21 expression increased only in CHMp A. SimilaThe p53 tetramer response element, along with the human cytomegalovirus minimal promoter sequence, was cloned upstream of the sequence encoding Nano Luc , and theMammary tumors are a common disease in dogs, and a variety of treatment options are available ,33, incl0 and S phases synchronized using double thymidine block revealed that the expression levels varied among cell lines, but no differences were observed between cell cycles. This is because RAD51 does not exhibit increased protein quantity upon DNA damage, but functions by congregating at the site of damage [50 up to 5 Gy irradiation was observed. However, at 10 and 15 Gy irradiation, the survival rate decreased in CHMm cells having higher RAD51 expression. When cells were scratched 72 h after 15 Gy irradiation, the recovery rate at 24 h was significantly higher for CHMp. These results indicate that CHMm with higher RAD51 expression is more radiosensitive than CHMp with lower RAD51 expression.Comparison of RAD51 expression in eight cell lines derived from mammary tumors using western blotting in Gf damage ,38. In tTo investigate the intracellular dynamics of RAD51, which is highly expressed in CHMm cells, we investigated RAD51 nuclear focus formation in cells irradiated with 15 Gy. In CHMm, an internuclear RAD51 was stained deep red at a higher ratio, which was more significant after irradiation. In contrast, the number of CHMp cells with RAD51 internuclear foci increased significantly after irradiation. RAD51 foci after the induction of DNA damage were visualized by RAD51 assembly at DNA damage sites and are an indicator of DNA damage repair by HR ,40. DeepWhen cells are exposed to DNA damage, tumor suppressor genes, such as p53, are expressed, resulting in cell cycle arrest . FACS anBecause irradiation suppressed cell cycle progression in CHMp, but not in CHMm, we examined the intracellular dynamics of p21, a p53 downstream molecule, and a major cell cycle regulator during DNA damage. Since the canine p21 homolog had not been cloned, it was newly cloned in the present study and the cross-reactivity of the anti-human p21 antibody against canine p21 was confirmed. Structural comparison of canine p21 with human p21 showed high amino acid sequence homology (82%) and conservation of the functional domain of the cyclin-dependent kinase inhibitor. Same-size bands were observed using both anti-Halo and anti-human p21 antibodies against Halo-tag fused canine p21 using western blotting, confirming the cross-reactivity of anti-human p21 antibodies to canine p21. Using this antibody, observation of p21 expression in CHMp and CHMm 24 h after 15 Gy radiation showed that p21 expression was increased in CHMp, but not in CHMm. Furthermore, 1 or 5 μM doxorubicin treatment, which induces DNA damage through reactive oxygen species generation , increasNo studies have investigated the radiosensitivity and DNA damage repair mechanisms of canine mammary tumor cell lines by focusing on RAD51 expression profile. Among CHMp and CHMm derived from the same individuals with large differences in RAD51 expression, CHMm with high RAD51 expression was more radiosensitive at high doses of irradiation. It is possible that in CHMm with high RAD51 expression, the rate of RAD51 foci formation during irradiation is low compared to that in CHMp, resulting in inefficient HR. There was also no cell cycle arrest after irradiation in CHMm cells, because p21, a cell cycle regulator, was not expressed. The present study was based on analysis of only one pair of cell lines derived from the same individual. To determine the universal function of overexpressed RAD51 in dogs, more cell types are needed for subsequent investigations. In the future, studies on molecules surrounding RAD51, such as p53, which is involved in p21 expression, and BRCA2, which is essential for the functional expression of RAD51, should be expanded to establish a foundation for elucidating the mechanism of mammary tumorigenesis and developing novel therapeutic strategies in dogs.


A total of 109 subjects completed the study. The HD2 group had greater amounts of TWI  and higher and lower contributions of total drinking fluids and water from food to TWI, respectively, than the LD1, LD2, and HD1 groups (p < 0.05), but the amounts of water from food did not differ significantly among the four groups . Participants in the HD2 group had higher amounts of water than participants in the LD1, LD2, and HD1 groups (p < 0.05); SSBs were the second top contributor of total drinking fluids, ranging from 24.0% to 31.8%. The percentage of subjects in optimal hydration status increased from 11.8% in the LD1 group to 58.8% in the HD2 group (p < 0.05). The HD2 and HD1 groups had 212–227 higher volumes of urine than the LD1 and LD2 groups (p < 0.05). No significant differences were found in the plasma biomarkers (p > 0.05), with the exception of higher concentrations of K in the HD1 group than in the LD1 group (p < 0.05). Subjects with higher amounts of total drinking fluids had better hydration status than those with lower total drinking fluids, but not better drinking patterns. Habitual total drinking fluids did not affect the plasma biomarkers.The purposes of this study were to explore the drinking patterns, and urinary and plasma hydration biomarkers of young adults with different levels of habitual total drinking fluid intake. A cross-sectional study was conducted among 111 young male athletes in Beijing, China. Total drinking fluids and water from food were assessed by a 7-day, 24-h fluid intake questionnaire and the duplicate portion method, respectively. The osmolality and electrolyte concentrations of the 24-h urine and fasting blood samples were tested. Differences in groups LD Maintaining adequate water intake is crucial for human beings. If one’s water intake is less than the output of the water, the body may be in a state of dehydration. Research has demonstrated that dehydration can impede cardiovascular functions and even cognitive performance ,2. It taStudies show that up to 50% of women and about 60% of men do not meet the recommendations for total drinking fluid intake of the European Food Safety Agency (EFSA) . FurtherStudies have shown that the types of fluids consumed, including water and other beverages, were different among different age groups; milk consumption was higher in children, whereas tea, coffee, and alcoholic beverage consumption was a little higher among adults . MoreoveMaintaining the adequate water intake or the optimal hydration status is crucial for the optimization of physical and cognitive performances. Moreover, it has been revealed that the comparing with the exogenous carbohydrate intake, the hydration status was more important for athletes doing push-to-the-finish cycle in the heat . DespiteThe purposes of this study were, firstly, to explore the differences in drinking patterns among young athletes, and secondly, to investigate the hydration biomarkers of young adults with different levels of habitual total drinking fluid intake in free-living conditions. With this research, we aim to contribute to the provision of a science-based education on fluid intake for young athletes.A cross-sectional study was designed, and 111 young adult males were recruited in Beijing, China. Inclusion criteria were as follows: healthy adult male college students aged 18–25 years with a regular exercise training plan (more than 5 moderate-intensity exercises per week). Exclusion criteria were as follows: those with chronic diseases, such as oral cavity, endocrine, kidney, gastrointestinal tract, and metabolic diseases; sports injury; cognitive impairment; and those who have taken drugs or vitamins and other health products within 1 month.n = t2p (1 − p)/e2, the maximum sample size is 101. Considering the drop-out rate, set at 10%, 111 subjects were needed in this work.In similar study, more than 75% of the random urine samples of 4.4% of the subjects were of optimal hydration status , with thThe study spanned 7 consecutive days, including five weekdays and two weekends. On the first study day, anthropometry including height, weight, and waist circumference, were measured. As for recording fluid intake from water and other beverages, all subjects were instructed several times to successfully record the related information on the self-designed 7-day, 24-h fluid intake questionnaire. In addition, over the 7 consecutive days, all the foods that the subjects ate were weighed and recorded for three consecutive days , as described in our previous study . FurtherTotal water intake included the total drinking fluids and water from food (mL).A 7-day, 24-h fluid intake record questionnaire was used to assess the total drinking fluids, which was designed by the investigator, with high validity, as described before ,16,17,18All foods that the subjects ate for the three days were weighed before and after subjects ate to calculate the amount of water from food. Water from food was assessed with the duplicate portion method. Moreover, all the samples of food were collected by the investigators and sent to the laboratory to be stored immediately. Samples of all foods were measured according to the national standard of GB 5009.3-2016 , and theThe indoor and outdoor temperature and humidity were recorded each day for 7 days . The outdoor temperature and humidity were 24.2 °C ± 5.8 °C and 29.5% ± 15.8%.2). Height, weight, and waist circumference were measured twice by the trained investigators with standard procedure ,34. The The 24-h urine samples, including the first morning urine, were collected by the subjects using self-designed containers by the investigators. Each urine sample was asked to be collected by the participants and sent to the investigators, and then the investigators brought the samples of urine to the laboratory to be stored. All the urine samples were stored at +4 °C in a special refrigerator for samples before being measured. When the urine samples were sent to the laboratory, the investigators recorded the weight of the samples using the desktop electronic scale , to the nearest 0.1 kg. Urine osmolality, urine specific gravity (USG), pH, urea, and creatinine, urine electrolyte concentrations  were tested with standard procedure, which was described in our previous study . OptimalFasting venous blood samples were collected with standard procedure by trained investigators to measure the concentrations of Na, K, and Cl, and testosterone, cortisol, creatinine, and copeptin.The concentrations of electrolyte  were tested, as described before in our previous study . The tesParticipants were asked to record the information of the physical activity during the 7 days on the questionnaire, which was self-designed by the researchers, including the time, the type, and the place of the activities. Furthermore, the training program of the participants was provided to the researchers by the coaches that guided them. The types of the sports were aerobics, table tennis, tennis, football, track-and-field, and others. 1 (low drinker 1), LD2 (low drinker 2), HD1 (high drinker 1), and HD2 (high drinker 2), according to the quartiles of total drinking fluids of the subjects . One-way ANOVA, Kruskal–Wallis H-tests, and chi-squared tests were used to compare the differences among the four groups. Differences between each two groups were compared using SNK (p < 0.05). The significance level was set at 0.05 (p < 0.05). The SAS 9.2 software  was used for statistical analysis. Data were presented as mean ± standard deviation, and median or interquartile ranges if the data were normally distributed or not, respectively. Subjects were divided into four groups, LDp > 0.05).In total, 111 subjects were recruited, and 109 of them completed the study, giving a completion rate of 98.2%. p < 0.05). Subjects in HD2 had 1433–1620 mL, 1170–1203 mL, and 733–853 mL more than other athletes in groups LD1–HD1 in the amounts of total drinking fluids and TWI (p < 0.05), respectively. There were no significant differences in the amounts of water from food among the four groups (p > 0.05). According to the proportions of total drinking fluids in TWI, they were different among the four groups compared with each other (p < 0.05), which ranged from 56.9% in group LD1 to 72.7% in group HD2. Refer to the water from food, accounting for 27.3–43.1% of TWI from group LD1 to group HD2, these levels also differed significantly among the four groups (p < 0.05). Subjects in group HD2 had a 20.4% higher and 20.4% lower contribution of total drinking fluids and water from food to TWI than those in group LD1, respectively (p < 0.05).TWI increased simultaneously, with the intake of the total drinking fluids increased (p > 0.05). The amounts of water differed significantly among the four groups (p < 0.05), and increased with higher total drinking fluids. However, no statistically significantly differences were found in the consumptions of beverages including the tea, milk and milk products, or alcoholic beverages among the four groups , except the amounts of SSBs and other beverages . For SSBs, the consumption in the LD1 group was lower than in other groups (p < 0.05). Furthermore, the contributions of water, tea, milk and milk products, SSBs, or alcoholic beverages to total drinking fluids did not differ significantly among the four groups , except the contributions of other beverages (p < 0.05). Surprisingly, the SSBs were the second top contributor to total drinking fluids after water in all the groups, ranging from 24.0% to 31.8%. Regarding the drinking patterns, which included the amount of fluids intake and the types and contributions of fluids, the main contributor to total drinking fluids was water in all the four groups, accounting for 61.2–69.7%, and there were no significant differences among the groups (p > 0.05), neither in the contributions of water from different foods, nor in the contribution of staple food, dishes, soup, porridge, and snacks , as shown in Regarding the water from food, subjects in HD groups had similar water from food intake with those in LD groups. In all four groups, the main source of water from food was dishes, with staple food following by. However, there were no statistically significant differences in the amounts of water from staple food, dishes, soup, porridge, and snacks in the four groups . The proportions of subjects with optimal hydration status were higher in HD2 and HD1 than that in LD1 and LD2 (p < 0.05).p > 0.05), except the concentration of K, as shown in No statistically significant differences were found in the concentrations of Na, Cl, copeptin, testosterone, cortisol, and creatinine among subjects in the four groups (The present study is the first to investigate the differences among athletes with different total drinking fluid in China. The results of the present study show that the consumptions of total drinking fluids, TWI, and water from food of subjects were with 447–654 mL, 148–359 mL more, and 219–256 mL less than the results of survey conducted among young adults in China, respectively . This coIn the current study, among the four groups, when the total drinking fluids increased significantly, the TWI also increased significantly, and the percentages of total drinking fluids in TWI were increased. Simultaneously, the contributions of water from food to TWI were decreased, from 47.1% to 27.3%, much lower than reported in studies on young adults in Baoding, China, and adults from four cities of China ,36. Surp2 and LD1. In both the volume and osmolality, the LD1 group had statistically significant lower urine volume coupled with higher osmolality than the HD2 group, similar to the results of studies in China [2 group than the LD1 group. This indicates that subjects with higher total fluid intake had better hydration status than those with lower total drinking fluid intake. The proportion of subjects with optimal hydration status was only 15.6%, which is much lower than that of young male and female adults in China [The results of the study demonstrated several differences in urinary biomarkers between participants with HDin China  and Franin China . Furtherin China , revealiin China . Recent in China . This hiin China . Researcin China . Hence, Hormones potentially impact the performance and the status of health among athletes . AccordiAs is known, copeptin is associated with type 2 diabetes and heart diseases . The preThe circulating concentration of cortisol is monitored by the hypothalamus pituitary adrenal axis and the neuroendocrine feedback circuit, which can be activated by physiological stimuli including stress and exercise. The increase of the serum cortisol has been linked with hypohydration in some strenuous conditions. In our study, no significant differences in the plasma cortisol were found between subjects with different levels of total drinking fluid intake, which is similar with the results of other studies. The research supported the results showed that cortisol levels remained unchanged after exercise-induced dehydration, resulting in an average weight loss of 1.3% to 1.6% . AnotherTo the best of our knowledge, this is the first study to document the concentrations in plasma cortisol among athletes in free-living conditions even without acute dehydration. Our results confirmed the previous findings, expanding the knowledge for young adults regarding the effects of different levels of total drinking fluids among athletes with free-living conditions. The proportion of subjects with higher total drinking fluids was 40% higher than participants with lower total drinking fluids. Moreover, these data suggest that there is a threshold value of different levels of total drinking fluids on plasma cortisol among young athletes. The study has some strengths and weakness. As for the strengths, firstly, the total drinking fluid intake was evaluated by a 7-day, 24-hour fluid questionnaire, which is an effective and reliable method of estimating total drinking fluids in other studies before . MoreoveSubjects with higher total drinking fluid intake had better hydration status than those with lower, but not better drinking patterns, even though the former had higher TWI than the latter. Furthermore, habitual total drinking fluids did not affect the plasma biomarkers. Interventions should be taken to encourage athletes to consume a sufficient amount of total drinking fluids and a healthier choice of beverages, in order to maintain optimal hydration.


The last five decades have witnessed an inordinate number of advances in the diagnosis and management of congenital heart defects (CHDs), as reviewed elsewhere . These aIn the first paper, Dr. Pop from the University of Medicine Pharmacy Sciences and Technology of Tirgu Mures and the Tirgu Mures Emergency Institute for Cardiovascular Diseases and Heart Transplant, Tirgu Mures, Romania, compares different contrast agents  used for computed tomographic angiography (CTA) studies in infants referred for aortic arch evaluation [In the next paper, Dr. Singh from the Baylor College of Medicine/The Children’s Hospital of San Antonio, San Antonio, TX, USA, reviews “as low as reasonably achievable” (ALARA) in pediatric electrophysiology laboratory . Dr. SinIn the third paper, Dr. Divekar and his associates from the University of Texas Southwestern Medical Center/Children’s Medical Center, Dallas, TX, USA, and the Cleveland Clinic Children’s Hospital, Cleveland, OH, USA, review transcatheter device therapy and how to integrate advanced imaging with invasive procedures in the management of CHD . These aIn the subsequent paper, Dr. Arar and his colleagues at the University of Texas Southwestern Medical Center/Children’s Medical Center, Dallas, TX, USA, review the role of cross-sectional imaging in pediatric interventional cardiac catheterization . This paIn the next paper, Dr. Betancourt and her associates from The Children’s Hospital of San Antonio/Baylor College of Medicine, San Antonio, TX, USA, review the utility of a three-dimensional printed model in the biventricular repair of complex CHDs . They reIn the final paper of this series, Dr. Sharma and associates from The Children’s Hospital of San Antonio/Baylor College of Medicine, San Antonio, TX, USA, and the Cohen Children’s Medical Center, New York, NY, USA, discuss advances in the prenatal management of fetal cardiac disease . They stGiven the small number (N = 6) of papers included in this Special Issue, not all the advances that happened in the last 50 years could be discussed. Nonetheless, the authors of these papers and we hope that the reviewed material is useful to the reader and help them to provide better care for their patients.