Standard doses of conventionally fractionated radiation have had minimal to no impact on the survival duration of patients with locally advanced unresectable pancreatic cancer (LAPC). The use of low-dose stereotactic body radiation (SBRT) in 3- to 5-fractionshas thus far produced a modest improvement in median survival with minimal toxicity and shorter duration of treatment, but failed to produce a meaningful difference at 2 years and beyond. A much higher biologically effective dose (BED) is likely needed to achieve tumor ablation The challenge is the delivery of ablative doses near the very sensitive gastrointestinal tract. Advanced organ motion management, image guidance, and adaptive planning techniques enable delivery of ablative doses of radiation (> = 100Gy BED) when more protracted hypofractionated regimens or advanced image guidance and adaptive planning are used. This approach has resulted in encouraging improvements in survival in several studies. This review will summarize the evolution of the radiation technique over time from conventional to ablative and describe the practical aspects of delivering ablative doses near the GI tract using cone beam CT image (CBCT) guidance and online adaptive MRI guidance. Unresectable locally advanced pancreatic cancer continues to carry a grim prognosis with a median survival of 10–16 months even in the context of the significant improvement in chemotherapy options seen in the last two decades , and locp = 0.03) undermining the role for RT in the management of LAPC in the era of gemcitabine. It should be noted that the particularly intensive CRT regimen that resulted in poor compliance made the interpretation difficult. The other recent trial to compare chemoradiation to chemotherapy alone was conducted by the Eastern Cooperative Oncology Group (ECOG 4201). This trial compared gemcitabine-based chemoradiation to a total dose of 50.4Gy in 28 fractions followed by weekly gemcitabine to gemcitabine alone. Fields were reduced after 39.6 Gy. It randomized 74 patients before being stopped for poor accrual. A modest median survival benefit was seen in the chemoradiation arm (11.1 vs 9.2 months) [Standard radiotherapy options, commonly delivering 40 to 60 Gy in 1.8–2.0 Gy per fraction add minimal to no survival benefit for patients with locally advanced unresectable pancreatic cancer (LAPC) who have received chemotherapy. These doses were based on the tolerability of large field radiation to the stomach and duodenum in the 2D and 3-D era, and have been shown to provide a modest local tumor control benefit only. Five phase III randomized trials evaluated the role of standard doses of radiation delivered with concurrent chemotherapy to chemotherapy alone in the treatment of locally advanced pancreatic cancer with mixed results , 7, 8. Tp = 0.083). However, the use of chemoradiotherapy was associated with reduced rates of local disease progression , longer interval to re-initiation of therapy  and a trend toward improved progression-free survival . This was achieved with acceptable incremental toxicity, mainly nausea. Collectively these results show that conventionally fractionated chemoradiation up to 60Gy can produce a modest local control benefit, but only minimal, if any, effect on survival. The reason why a local control benefit is not translating into a survival benefit is likely multifactorial, and likely largely influenced by the high metastatic rate seen in this disease. However, another possibility is that for at least the subgroup of patients with predominantly locoregional disease progression, gains in local control have not been significant enough to make a difference in survival. This underscores the need for further dose escalation.The study with the greatest impact on clinical practice is the LAP 07 trial [Lack of a substantial benefit, coupled with the introduction of more active systemic regimens such as FOLFIRINOX  (5-fluorStereotactic body radiotherapy (SBRT) enables highly precise delivery of high doses of radiation to small tumor volumes by using image guidance. Increased precision coupled with evidence that a higher dose per fraction is associated with better local control has led to the emergence of 1- to 5-fraction regimens. Evidence across several tumor types suggests that doses of at least 100Gy BED need to be delivered for an ablative effect or > 90% durable local control. SBRT has gained wide acceptance for targets with little motion uncertainty such as spine or brain tumors. It is also an attractive option for tumors occurring in moving organs with parallel functional subunits, such as the lung or liver whereablation of a small volume of the surrounding normal liver or lung tissue carries no significant clinical consequence. In contrast, ablative doses delivered near an organ with serial functional subunits such as the gastrointestinal tract, are not possible without a risk of affecting organ function. This is particularly relevant for sites where organ motion creates a greater degree of uncertainty about the location of the target and sensitive structures at any given time. For pancreatic tumors, dose delivery is limited by the proximity of radiosensitive GI organs, primarily the duodenum, the jejunum and the stomach, and the uncertainty created by respiratory motion and day-to-day differences in luminal organ shape.Not surprisingly, early studies using ablative or near-ablative doses in 1–3 fractions were associated with significant early and/or late GI side effects Table . A phaseAnother early report of SBRT used 45 Gy in 3 fractions  in 22 patients [Although, comparison of hypofractionated regimens using the linear quadratic model may be somewhat inaccurate, collectively these early experiences showed that dose escalation using very hypofractionated regimens (1–3 fractions) is associated with excess toxicity, which in this setting may be further exacerbated by any set-up uncertainties including residual respiratory motion or using large GTV to PTV expansions to account for such uncertainties.p = 0.0014), reaching statistical significance due to the large numbers [To ensure safety, clinicians have adopted fractionation schemes of 25–33 Gy in 3 to 5 fractions, which amounts to only 54.78Gy BED using the standard linear-quadratic conversion. A prospective multi-institutional study using 33 Gy in 5 fractions in 49 patients receiving gemcitabine before (up to 3 weeks) and after SBRT, resulted in only minimal acute and late GI toxicity, 2 and 10%, respectively. Unfortunately, 1 year FFLP was only 78% with a median OS of 13.9 months, which is not significantly different from results seen with conventionally fractionated chemoradiation . However numbers . With thAny hope of improving outcomes in LAPC requiresdose escalation beyond 33Gy in 5 fractions. However, safe delivery of higher dose per fraction as a part of a 5-fraction regimen while respecting normal tissue constraints is only possible for a select few patients with tumors far away from the luminal GI tract. Relying on first principles of radiobiology, one way to achieve a higher effective total dose while maintaining an acceptable risk of toxicity is by increasing the number of fractions. Therefore, incorporating the precision of the SBRT technique into a more protracted course is one way to continue dose intensification in LAPC. Fractionation also has the added benefit of “randomizing” the internal day-to-day organ motion, making it less likely that unintended high dose will be delivered to a normal structure that may move closer to the target on any given day.Another conceptual change that would facilitate dose escalation is abandoning the goal of dose homogeneity with the tumor planning treatment volume (PTV). Dose homogeneity as a planning goal is largely a carry-over from more conventional planning approaches. Whenlarge treatment fields contained the target as well as the organs at risk, hotspots within the irradiatedvolume were undesirable. However, when the planning treatment volume is small and limited to the tumor, excluding all sensitive normal structures, a hotspot within that treatment volume has no detrimental effect. On the contrary, allowing a hotspot may improve the conformality of the high dose distribution thereby enabling dose escalation. Importantly, the center of a tumor is typically more hypoxic than the periphery and, therefore, more radioresistant. A hotspot within a more radioresistant portion of the tumor will only be of benefit. Thus our novel treatment planning strategy represents a three-part approach of 1) covering as much of the tumor as possible with an ablative dose while (2) placing supra-ablative hotspots in the center and (3) restricting the areas directly abutting the GI tract to safe doses used in conventional radiotherapy  dose painting, typically with 2 or 3 different planning target volumes (PTVs) . We predominantly use 15 or 25-fraction schedules based on the proximity to the luminal GI tract. For tumors located within 1 cm of the GI tract, we use a 25-fraction regimen, for tumors more than 1 cm away we use a 15-fraction regimen, with a 5-fraction regimen reserved for very select patients with no nearby GI structures. Typical regimens are listed in Table We simulate patients in the supine position with customized immobilization and arms elevated. Using the Varian RPM system, we obtain deep inspiration breath hold (DIBH) scans with diagnostic CT pancreatic protocol (150 mL iodinated contrast at 5 mL/s) with imaging at 45 s after the start of contrast administration and a second image obtained at between 1 min 30 s and 2 min after the start of the contrast bolus. This technique allows maximal contrast enhancement of the surrounding parenchyma around the tumor as well as arterial and portal venous enhancement on the first scan, which is usually used as the planning CT scan.high dose, a margin of 0-5 mm is added to the gross tumor volume (GTV) of the primary tumor, then all organs at risk (OARs) with an additional safety margin of 5 to 7 mm are excluded. The exact margin depends on the length of the interface of the tumor with the OAR, with greater margins utilized for cases with more extensive abutment. In this process, the edge of the PTV high dose will be separated from the PRVs by at least 2 mm, effectively preventing the high dose gradient from falling immediately adjacent to the sensitive organ where small amount of uncertainty can place the organ at risk within a very high dose region  with an additional safety margin from the high and ultra-high dose PTVs Fig. , Table 3ion Fig. b, d. To Management of both respiratory and day-to-day internal organ motion is paramount. At MSK we currently use DIBH respiratory gating using RPM Varian system with daily DIBH cone-beam CT (CBCT) image registration. The presence of fiducial markers or a metal biliary stent are required. DIBH is the solution for intra-fraction motion, and enables acquisition of high quality daily CBCT scans by eliminating motion artifact. CBCTs are used to verify the target position as well as day-to-day variation in the position of the adjacent luminal GI tract. The latter is used to select cases for adaptive planning as described below.It should be noted that despite using DIBH, some CBCTs will provide poor visualization of the luminal GI tract due to other sources of artifact such as luminal gas and peristalsis. However, the additional advantage of a multifraction regimen is that a small number of fractions where the doses to the GI tract are uncertain do not alter the risk profile of the overall treatment plan. Thus, such scans will not necessarily cause treatment delays as long as the position of the target can be verified. The process of using CBCTs to evaluate GI organ position is described in the subsequent section.Other methods of motion management and image guidance may be used depending on the availability of technology and proficiency of a particular center. These include gating, tumor tracking or abdominal compression for respiratory motion management. In addition to CBCTs, CT-on-Rails or MRI may be used for image guidance.. For all methods of image guidance, motion management will reduce artifact.Adaptive planning is used as a solution for non-random motion of the GI tract. We evaluate day-to-day organ motion using daily CBCT by projecting maximum point dose isodose line Table  as a strMost patients treated with ablative hypofractionated technique to date have received concurrent radiosensitizing chemotherapy. Current MSKCC standard is oral capecitabine twice daily on the days of radiation. Main toxicities include nausea, fatigue, diarrhea, and hand-foot symptoms. The exact contribution of radiosensitization to ablative radiotherapy is not known, but there are possible advantages to both locoregional and systemic disease control.Patients are simulated with a guided breath hold, supine, with one arm up or both arms down to ensure comfort during the treatment. IV contrast is used similarly as above.The most critical aspect of contouring is to identify the stomach, duodenum, small and large bowel within 3 cm of the CTV. The CTV includes the gross tumor, contoured generously to include the superior mesenteric artery and celiac artery when feasible. The CTV is expanded by 3 mm to create a PTV.Motion management and accuracy of treatment delivery are important in these high dose treatments. The MRI-linear accelerator used automatically processes 4 cine images per second, allowing the patient to be treated with guided breath holds or with gating on free breathing, based on patient comfort.Daily adaptive planning is the hallmark of this technique. On each fraction, the patient undergoes a couch shift to align the CTV based on simulation. At this time, the GI OARs are re-contoured within 3 mm of the PTV. A new plan is generated whenever the volume of each GI OAR exceeds 1 cc above 33 Gy for a 5-fraction treatment. Each fraction is evaluated separately, since technology does not yet exist to deformably map dose between different bowel loops.Treatment paradigms for locally advanced pancreas adenocarcinoma have evolved significantly over the last several years, primarily due to the expanded chemotherapy options. Likewise, there have been significant improvements in radiation therapy delivery techniques with the advent of SBRT.However the potential offered by these techniques has not been fully harnessed with the commonly used 1–5 fraction SBRT regimens. These technological advances enable delivery of radiotherapy doses that are at least 1.5 as potent as conventionally fractionated schedules or the commonly used low dose 1–5 fraction SBRT regimens, and are predicted to be ablative. Multiple single institution series show promising early results, and there are ongoing phase II studies investigating ablative radiation using a CBCT- and an MR-based approaches.


Safe human-dog relationships require understanding of dogs' signaling. As children are at particularly high risk of dog bites, we investigated longitudinally how children from 3 to 5 years and parents perceive and interpret dogs' distress signaling gestures. All participants were then taught how to link their perception of the dog with the correct interpretation of dogs' behavioral signals and tested again. Results show a significant increase in learning for children and adults, with them showing greater understanding of dogs' signaling after intervention. Better learning effects were found with increasing age and depended on the type of distress signaling of the dogs. Effects endured over time and it can be concluded that children and adults can be taught to interpret dogs' distress signaling more correctly. Awareness and recognition of dogs' stress signaling can be seen as an important first step in understanding the dog's perspective and are vital to enable safe interactions. Benefits of dog ownership include positive effects on human health and well-being and on child development and learning . WhWhile dog bite prevention programmes exist, and some address how to behave in public with unfamiliar dogs Given not only the popularity of dogs as pets, also the increasing popularity of animal-assisted interventions in educational settings as well as the application of pets in the classroom  showed *Power3; , we overer3; N = 01 of chiChildren took part in Test 1, 2, 3, and 4. Reasons for attrition in children are as follows: In Test 2, 3 children who took part in Test 1 did not complete Test 2 on the same day, hence were excluded from analysis. Test 3: Attrition of 20 children due to being ill, having moved school and being on holiday. Test 4: A slight gain of children occurred, as some who had missed Test 3 due to absence were back for Test 4.M = 3.4, SD = .32, range 2.8–3.9), 23 4-year-olds  and 39 5-year-olds . Of this sample, 37% had a dog.Overall, in the final sample entered into the data set, there are 88 children who took part in all testing sessions  only. Additional longitudinal parent testing was not possible due to limited funding. However, piloting had shown that adults showed clear improvements as they found the teaching phase to be a real “eye-opener.” Error rates dropped once they had realized what the behavior of the dog implied. The current study results confirm this and we have no reason to assume that adults with typical and intact memory capacity would forget this knowledge over time. Of the parents 27.5% were dog owners, these dog ownership figures for children and adults compare well with the national average of about 30% dog owners. Also, 47.5% of parents had been bitten by a dog, this is very similar to the 47% reported elsewhere . Thus, This study was carried out in accordance with the recommendations of University of Lincoln, School of Psychology Research Ethics Committee (SOPREC). The protocol was approved by the SOPREC. Written informed consent was gathered in accordance with the Declaration of Helsinki.The stimuli consisted of sets of 16 short video-clips portraying dogs with the full range of behavioral distress signals described in “Shepherd's ladder” , 73. TheDue to ethical considerations we did not show serious bites drawing blood, and parents had the opportunity to view the images beforehand and decide if they allowed their children to take part. Also, children received a thorough debriefing session after testing finished, so we could make sure children clearly understood the dog signaling. None of the children displayed any signs of distress during testing or after testing, none of the parents reported any detrimental effects back to the research team.Having piloted the video clips, we decided that the procedure worked best if we used 2 × 16 videos (2 per distress behavior)—we added 4 relaxed (happy) behaviors so that children would not get the impression that dogs are usually distressed, however, these items were not part of the intervention phase and children were not trained to recognized relaxed dogsVideos were clipped and resized using Bink and Smacker (RAD Video Tools): each video was 6,000 ms duration, 360 × 240 pixels, and with a data rate of 25 frames per second.Video clips were presented centrally on the monitor screen and displayed on a 15% greyscale background. Altogether, we used up to 4 different sets of videos in Test 1 (baseline), Test 2, 3, and 4 (see below). All video stimuli were assessed for their expression and approved by 3 internationally renowned dog behavior specialists.Audio recordings matching each of the visual stimuli were produced in a sound-proof professional audio-recording studio at the University. All recordings were carried out within one session so as to reduce variation in the voice of the speaker. The speaker was female and a native speaker of British English. Audio messages consisted of four features across all trials: an initial “Look” command, followed by a description of the dogs' behavioral signal to steer participants' attention, then a message of how the dog is feeling and lastly a message of safety instruction for the child. We have consulted closely with a consultant and dog behavior expert on the appropriate content of the verbal messages. Messages take the following character: (a) Attention getter (Look!), (b) highlighting the dog's signaling behavior, (c) followed by an explanation how to interpret the dog's behavior, (d) then a clear safety instruction for adapting their actions. An example of such a message is as follows: “Look! The dog is blinking its eyes. The dog is worried. You should leave the dog alone.” Audio files were cut and manipulated using Audacity version 2.0.1. Files were 1141 kbps, 2 channel and were used in .wav format.We used a child-appropriate 1-5 rating scale in which symbolic faces expressed either very happy (1), happy (2), just ok (3), unhappy/angry (4), and very unhappy/angry (5) emotions. Children had no problems using the scale.Children were tested in schools and nurseries in a quiet room. Videos were presented on a laptop and the experiment was programmed using the Lincoln Infant Lab Package 1.0 . ParticiChild participants took part in the study longitudinally; this included viewing an initial baseline phase of video stimuli (Test 1), immediately followed by a training phase of videos and then tested with novel videos (Test 2) afterwards to investigate if their knowledge had improved. Participants were then tested again 6- and 12 months later (Test 3 and 4) without any additional training to see if they had retained their knowledge. Hence, we have an integrated control group with each child being their own control (before and after learning and at the follow-up testing). In addition, we have further integrated controls in that 4-year-olds at testing start can be compared with 3-year-olds after 1 year (when they have turned 4 years of age). In the same way, the 5-year-olds at start of their testing can be compared with the 4-year-olds at testing point 1 year (when they have turned 5). Adults only took part twice on the same day  and results can therefore be compared before and after testing.Each participant viewed 20 trials. Each trial was made up of a 6,000 ms video displaying dog behavioral signals as described above. These were followed by a fixed choice user/child friendly rating 1–5 scale ranging from “very happy” to “very unhappy/angry.” Participant ratings were recorded both electronically and verbally, and the rating scale stayed on the screen until the participant had made their choice. Duration of this phase was between 2 and 5 min.Participants viewed 32 trials . Each trial was made up of a 1,000 ms blank screen accompanied by the initial “Look” audio. This was followed by a 6,000 ms video displaying dog behavioral signals accompanied by the remainder of the audio sentence highlighting the dogs' behavioral stress. Duration of this phase is about 4–5 min.Participants were again presented with 20 trials . This was immediately followed by the fixed choice user/child friendly rating 1–5 scale as described above. This took between 2 and 5 min. Both, children and parents thoroughly enjoyed taking part.Note: In addition, half of the children always saw novel stimuli at each testing time, and the other half saw the novel set from Test 2 repeated at Tests 3 and 4. This was to explore if children learn differently with items that are novel each time as opposed to items that are novel at Test 2 and then reoccur, however, there was no statistical difference, hence, results below include both groups of stimuli.We initially calculated a repeated measures ANOVA with Gender , Dog Ownership (yes/no), Age Group  and Distress Signal Group  on the rating scores at different Testing times F = 7.84, p < .001, partial η2 = .16] with older children showing more correct results than younger children. A significant main effect of Distress Signal Group  also emerged, with children judging conflict escalating signals as different from conflict-avoiding and defusing signals, but not distinguishing between conflict-avoiding and defusing signals in dogs–post hoc tests with Bonferroni corrections (p < .0166) show that the following differences are highly significant: conflict-escalating vs. conflict-defusing (p < .001); conflict-escalating vs. conflict-avoiding (p < .0001); while children do not distinguish conflict-defusing vs. conflict-avoiding signals in dogs (p < .05).We found a highly significant main effect of Age  with children improving significantly from Test 1 (baseline measure before intervention) to Test 2 after intervention (p < .002). Children also show improved knowledge from Test 1 to Test 3 at 6 months (p < .0026) and from Test 1 to Test 4 after 1 year (p < .0006).We also found a highly significant main effect for Testing times  which demonstrated that the older the participants, the better they perform. Highly significant interactions of Age by Distress Signal , see Figure F = 6.02, p < .0001 partial η2 = .07] also emerged as well as a significant three-way interaction between Testing time, Distress Signal and Age  showing clear differences between conflict-escalating signals vs. conflict-defusing and avoiding signals, with children showing better performance with increasing age and improvement over time, especially in the conflict-escalating signal group.There was also a significant interaction between Age group and Testing Times  showing improved understanding after intervention and a highly significant main effect for Distress Signal group  highlighting differences between Distress Signal groups. Figure An ANOVA of Gender  by Dog Ownership (yes/no) by Distress Signal group  was calculated for Testing Times before and after intervention on rating scores. Gender and Dog Ownership yielded no significant results, therefore the analysis was calculated with Distress Signal group  and Testing Times before and after intervention. We found a highly significant main effect for Testing Time .We also found highly significant main effects on differences between the parents' and children's judgments of dog's behavior, with most mistakes occurring in the conflict-defusing and conflict-avoiding signal groups  and Distress Signal Group  as well as Testing Time . Significant interactions were shown for Testing Time by Age , Distress Signal by Age  and Testing Time by Distress Signal . Overall, children distinguish conflict-escalating signals better than conflict-avoiding and conflict-defusing signals. They show more correct answers with increasing age and improve after intervention, specifically in the conflict-escalating signal group. In this group, improvements are stable over time (up to 1 year). The 5-year-olds also improve in the conflict-avoiding signal group from before to after intervention, however, this effect is not enduring over time. Interestingly, despite the same rating categories 4 and 5 accepted for conflict-avoiding and conflict-escalating signals, children distinguished conflict-avoiding and conflict-escalating signals clearly (p < .0001). Overall, these results show significant differences over time and for the different distress groups, with older children giving more correct answers than younger children. See Figure We also calculated a repeated measures Anova of Gender  by Dog Ownership (yes/no) by Age Group 3, 4, 5) by Distress Signal Group  before and after Intervention  on correct answers. As there were no effects of dog ownership or gender, we ran the analysis with Age Group  by Distress Group  at the different testing times . The following main effects were found: A significant main effect for Age  with better results overall after intervention and a highly significant main effect for Distress Signal Group showing differences between distress signal groups are perceived . A highly significant interaction between Testing Time and Distress Signal also emerged  demonstrating higher rates of correct answers with higher distress as well as rates of correct answers rising from conflict-defusing via avoiding to escalating and all scores being higher after intervention. Results in overview in Figure We calculated a repeated measures Anova of Gender  by Dog Ownership (yes/no) by Distress group  for Testing Times before and after intervention on percentage of correct answers. Gender and Dog Ownership yielded no significant results, therefore the analysis was calculated with Distress Signal group  and Testing Times (before and after intervention) on percent correct responses. We found a highly significant main effect for Testing Times [Interestingly, if we calculate results on a stricter criterium, i.e., only count as correct for conflict-escalating those answers that said “very unhappy/very angry,” all main effects and interactions stay intact, however, performance of adults drops in the conflict-avoiding category to 40% - and in children to 35, 51-, and 60% respectively for 3-, 4-, and 5-year-olds after intervention.In addition to the quantitative data described above, we also would like to provide some additional observations. While we were working with the children, they often commented on the videos. The quotes below give an impression of children's thinking and reflect the most frequent comments, see Table These comments were frequently made and show that children often anthropomorphise dogs and try to find an explanation that would be appropriate to explain human behavior, but unfortunately does not fit the dog's signaling intentions.We would furthermore like to report, so far also only anecdotal comments of parents stating that they frequently provoked distress-signaling behaviors, for example, like lip/nose-licking in their dog, as the family found it funny. However, having learned about dogs' distress signaling in the intervention, the adults were upset that they and their children might have caused their dog distress and commented that they will change their (and their children's) behavior, thus contributing to a safer home environment for all and to dogs' welfare. Further research will need to be carried out to investigate this systematically.Results show that children and adults profit from the intervention and improve their knowledge of dogs' stress signaling significantly. When performing analyses over time we found that, overall, learning effects are still highly significant in children after 6 months and 1 year despite no training taking place in the meantime–thus, the intervention works successfully, even over the duration of 1 year.A closer look at the error results shows us the areas in which the intervention has worked most successfully, and also the areas in which we need to invest more training with children and parents alike. We have very good success teaching all age groups of children, even young children of 3 years, and parents the meaning of conflict-escalating distress signals. They learn to understand, recognize and correctly interpret the signals and the learning success is still evident after 1 year. This is an important success as dogs showing their teeth or snarling or biting, pose a significant risk to children if these approach the dogs displaying such signaling. We have good to moderate success in training especially older (5-year-old) children and parents on conflict-avoiding distress signals. However, the data also show that all participants, including adults, find the more subtle signals of dogs' distress hardest to judge. Here, after intervention, only adults show excellent improvements. More research is needed to analyse these signals and how they are perceived in detailed examinations of this in future studies.One could also question whether children's increase in knowledge is due to general learning and increase in maturity–however, the results of the 4- and 5-year-olds clearly contradict this as children who have taken part in the interventions (3-and 4-year-olds tested after 1 year when they turned 4 and 5 respectively) show significantly better results than the 4-and 5-year-olds at the start of the study (before intervention). Thus, our intervention has clearly improved their knowledge over time compared to the control group. To investigate the role of the intervention in light of children's learning and general maturity over time, it could be useful to devise larger studies with independent control groups, hence requiring significantly larger funding sources.Overall, it becomes evident from this data that it is possible to educate adults and children to understand dogs' distress signaling. Adults profited from the intervention throughout all distress categories and show clear and significant learning effects. Thus, it is advisable to teach dog signaling to parents, dog owners, dog trainers, veterinary students and the wider public. The short intervention is easy to use and leads to significant improvements in knowledge, recognition and interpretation straight away and with enduring effect.It has also become clear which areas need further attention and research–while our intervention works very well with adults and also with older children, it has to be adapted to improve especially the younger children's understanding, especially of the more subtle distress signals in dogs. Further research will need to explore how children process the signals and how to teach these signals best.Our background measures of dog ownership, SES/parental education showed that there were no effects of any of these factors–in other words, neither children's nor parents' performance was better if, for example, they owned a dog, had a higher SES/education. Instead, performance was independent of these factors.There was also no difference between children seeing novel stimuli in all test phases or the same stimuli again. This is useful to know for the future creation of interventions as we can now be confident that we do not need to increase the amount of novel stimuli to be shown in order to train and assess children on dog body signaling.Finally, children's utterances illustrated how they perceived—and misinterpreted—dogs' body language. Further quantitative as well as qualitative research in this area is warranted and could help develop additional dog bite prevention tools.By assessing if our intervention works, we have undertaken the first step toward preventing misunderstandings and risk escalation due to addressing the current lack of knowledge and replacing it with knowledge that is stable over time. In the case of conflict-escalating signals, all participants showed significant improvements in knowledge over time.Knowledge of stress signaling (step 1) and Recognition and correct interpretation of stress signaling in context (step 2) are to Adapt the action. Having created awareness of the situation, insight to act accordingly should follow (step 3). Finally, Repeat recognition of future triggers and contexts and avoidance of risk (step 4) need to follow to effectively implement the taught knowledge. Further research will have to assess how to achieve these aims best.Further steps next to teaching children and parents In particular, future studies should address how best to implement the above so that beyond recognizing and understanding the signals, specific human actions and contexts wherein the dog presents these signs are recognized. It will also be useful to investigate if parents—or other educators—can guide and educate children to be aware of specific risk contexts. Concerning parental supervision, it would be interesting to find out to what extent they supervise child and dog and stop children from engaging in risky contexts with their dog in the first place. Furthermore, it would be beneficial to follow up in how far the welfare of family and dog are compromised after escalations have happened as well as investigate the role of professional help from a veterinary behavioral specialist.Finally, and importantly, we assume that dogs will benefit from children and adults having been taught how to read their distress signals. This increased understanding will mean that dogs are better understood, and if humans apply their knowledge appropriately this will lead to greater wellbeing of the dog living within a family household.This project is the first to offer an intervention to enhance children's and adults' abilities to interpret dog signaling correctly.We investigated how children perceive and categorize dogs' body language and interpret their signals and we then trained them and were able to improve their knowledge, recognition and interpretation skills.We showed very good results in improving the potentially very dangerous misunderstandings of dogs' conflict-escalating distress and threat signals. For example, a snarling dog showing teeth which children often misinterpreted as a happy dog, can now be corrected–children showed significant improvements that were stable over time. We have shown successfully that we can significantly improve all participants' abilities to recognize and understand these signals and enable all participant groups to avoid escalating risk situations–our intervention works especially well for these high risk situations. This is especially useful as—if such escalation occurs—it should be stopped to avoid risk of dog bite incidents and continued stress to the dog. Crucially, as our intervention furthers understanding of conflict-defusing and conflict-avoiding signals, hopefully, this may help to avoid risk escalation.We have revealed the extent of children's and adults misinterpretation errors for the first time, and we have shown areas in which children and adults make most errors. We have also shown that we can teach adults and children successfully to learn, recognize and interpret the signals correctly.With this new knowledge we enhance the currently scarce scientific database on children's and adults' interpretation abilities of dog signaling. We can now also address not only the most dangerous misinterpretations, but also commit ourselves to creating awareness of the less well understood and most frequently misunderstood signaling behaviors of dogs in order to avoid escalation of risk. The materials used can be further developed into an awareness raising intervention that is more widely usable for children and adults. For future effective prevention the above mentioned steps of implementation need to follow and, in turn, also be assessed as to their effectiveness.In sum, we have now got a solid knowledge base about how children and adults look at and perceive dogs and (mis)interpret their behavior.Our study was able to close these particular knowledge gaps, establish the necessary knowledge for the first time and therefore significantly advance the scientific knowledge in this area. Our study was also able to show that we can teach dog signaling successfully, and it outlines the current limitations.Veterinarians will profit from these results insofar as they can help to raise awareness of the existing knowledge gaps in both adults and children.Our study can also serve as an example of good practice in that we have evaluated the learning effects of the intervention cross-sectionally and longitudinally, as well as using additional measures.In the future, integrated research projects including child psychology, veterinary, medical, educational and other social sciences can be developed as a result of these efforts and produce research with impact on One Health-related injury prevention challenges.KM conceived and designed the research project. TD fed back on the project proposals, contributed to their improvement and contributed the majority of dog videos. As behavior specialist, she also carefully assessed and commented on the video pool and helped select appropriate videos. KM and VB contributed to all aspects of the research itself from planning to testing to data analysis and writing up results. TD also contributed to initial design and planning and to writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Students in need of mental health care face many barriers including cost, location, availability, and stigma. Studies show that computer-assisted therapy and 1 conversational chatbot delivering cognitive behavioral therapy (CBT) offer a less-intensive and more cost-effective alternative for treating depression and anxiety. Although CBT is one of the most effective treatment methods, applying an integrative approach has been linked to equally effective posttreatment improvement. Integrative psychological artificial intelligence (AI) offers a scalable solution as the demand for affordable, convenient, lasting, and secure support grows.This study aimed to assess the feasibility and efficacy of using an integrative psychological AI, Tess, to reduce self-identified symptoms of depression and anxiety in college students.In this randomized controlled trial, 75 participants were recruited from 15 universities across the United States. All participants completed Web-based surveys, including the Patient Health Questionnaire (PHQ-9), Generalized Anxiety Disorder Scale (GAD-7), and Positive and Negative Affect Scale (PANAS) at baseline and 2 to 4 weeks later (T2). The 2 test groups consisted of 50 participants in total and were randomized to receive unlimited access to Tess for either 2 weeks (n=24) or 4 weeks (n=26). The information-only control group participants (n=24) received an electronic link to the National Institute of Mental Health’s (NIMH) eBook on depression among college students and were only granted access to Tess after completion of the study.P=.03), whereas those in the control group did not. A statistically significant difference was found between the control group and both test groups 1 and 2 for symptoms of anxiety as measured by the GAD-7. Group 1 (P=.045) and group 2 (P=.02) reported a significant reduction in symptoms of anxiety, whereas the control group did not. A statistically significant difference was found on the PANAS between the control group and group 1 (P=.03) and suggests that Tess did impact scores.A sample of 74 participants completed this study with 0% attrition from the test group and less than 1% attrition from the control group (1/24). The average age of participants was 22.9 years, with 70% of participants being female (52/74), mostly Asian , and white . Group 1 received unlimited access to Tess, with daily check-ins for 2 weeks. Group 2 received unlimited access to Tess with biweekly check-ins for 4 weeks. The information-only control group was provided with an electronic link to the NIMH’s eBook. Multivariate analysis of covariance was conducted. We used an alpha level of .05 for all statistical tests. Results revealed a statistically significant difference between the control group and group 1, such that group 1 reported a significant reduction in symptoms of depression as measured by the PHQ-9 (This study offers evidence that AI can serve as a cost-effective and accessible therapeutic agent. Although not designed to appropriate the role of a trained therapist, integrative psychological AI emerges as a feasible option for delivering support.International Standard Randomized Controlled Trial Number: ISRCTN61214172; https://doi.org/10.1186/ISRCTN61214172. Approximately 20 million college students suffer from mental illness in the United States alone . More thMental health care solutions such as computer-assisted therapy (CAT) have been shown to be a less-intensive and more cost-effective method to deliver empirically validated treatments for depression and anxiety ,5. AlthoMoreover, 1 study revealed that nearly 70% of patients expressed interest in using mobile health  apps to self-monitor and self-manage their mental health . With 1 The objective of this study was to assess the efficacy of using the integrative psychological AI, Tess, to reduce symptoms of depression and anxiety in an engaging way. Tess was designed to deliver personalized conversations based on the expressed emotions and mental health concerns of participants, not to replace trained therapists. Tess focuses on language as the most explicit form of communication, with the proposition that communication between people reveals individual conceptualizations of specific emotions (unpublished data ) 16]..16].This study compared outcomes from 2 to 4 weeks of using integrative psychological AI (Tess) with an information control group  in a nonclinical college population. It was hypothesized that engaging in conversations with Tess would lead to greater improvement in symptoms relative to the information control group. In addition, we predicted that the duration of time in which participants interacted with Tess would impact the level of symptom reduction. To assess this, participants in the test group were randomly selected to participate in 1 of the 2 groups, which received either 2 or 4 weeks of unlimited access to Tess.Participants were recruited using a flyer  distribuConfirmed participants were randomized via a computer algorithm that automatically generated a number between 0 and 2 . All parThis study was retrospectively registered under the trial number of ISRCTN61214172, and involved a nonclinical population of college students. Refer to the Participants indicated their consent to the study’s terms via a checkbox (e-signature) on a closed form  sent viaThe third author of this paper served as an external representative to support data collection and analysis, which was provided by X2AI Inc. All data, including usage, was deidentified so as to protect the privacy of participants and was reported in aggregate only.Tess is a psychological AI chatbot designed by X2AI Inc to deliver brief conversations in the form of integrative mental health support, psychoeducation, and reminders. Tess serves as a therapeutic tool or resource that can be used as an adjunct to therapy that supports an integrative approach and is not intended to replace the role of a therapist.Tess was inspired by the first conversational AI, ELIZA, which examined natural language communication between people and machines in the 1960s  doesn’t necessarily provide specific ways I can better myself.”And that Tess was able to:coach [the participant] through a difficulty.These comments reinforce the potential for psychological AI to remove barriers and stigma and operate as an adjunct to traditional therapeutic methods.This study reveals that AI offers a cost-effective and accessible mental health solution and may serve as a scalable tool to complement traditional treatment methods. Although integrative psychological AI is not designed or intended to replace the role of a trained therapist, Tess emerges as a feasible option for delivering emotional support. The results support and expand on findings from a previous randomized controlled trial  and demo


Over 75% of patients  with coeliac disease in the UK have not been formally diagnosed.To determine if case-finding of coeliac disease is better than random testing in primary care.A pragmatic study looked at all referrals across a 12-month period (December 2013–November 2014) for coeliac serology testing and the indications for testing across 38 GP practices in a well-defined geographical area in North Yorkshire. There was further follow-up for an additional 12 months to determine conversion of positive serology to duodenal biopsy.All serology samples sent into York Hospital biochemistry department during the study period were analysed for the indication for testing. Positive results were cross-referenced for duodenal biopsies over the following 12 months on the York Hospital pathology database.Case-finding of coeliac patients in primary care is no better than random testing of the population. Only 71% of patients with positive serology went on to have a duodenal biopsy in the following 12 months.More education of the population and of primary care physicians is needed around the indications for checking for coeliac disease. It may be that primary care is not the best place to case-find patients with coeliac disease. It is known that only approximately 25% of cases of coeliac disease in the UK have been diagnosed. Opportunistic case-finding by primary care is one of the methods of diagnosing more patients. It is not known how successful this strategy is for checking for coeliac disease. Conclusions from this study are likely to be applicable to other chronic diseases that rely on opportunistic case-finding.1 that is, actively searching for people at risk of the disease from a larger population for a specific purpose, such as flu vaccination for those with chronic obstructive pulmonary disease (COPD). The process of case-finding can be systematic  or opportunistic . With regards to opportunistic case-finding, there are a number of barriers to this strategy being successful. Two significant barriers are the at-risk groups coming into contact with healthcare professionals, and the healthcare professionals being aware that they have an at-risk patient with whom they should discuss testing for the associated disease. Coeliac disease is an example of such a disease in which case-finding is recommended, as large-scale studies suggest a prevalence of approximately 1%.2 In coeliac disease, the case-finding is usually an opportunistic process, often occurring in primary care, when patients present with symptoms or other conditions with which coeliac disease is associated. To help healthcare professionals in the UK with this process, the National Institute for Health and Care Excellence (NICE) has produced clinical guidelines for identifying at-risk groups of people with coeliac disease, in whom testing for coeliac disease should be carried out.4There are a number of diseases that have no, few, or protean symptoms, yet expose patients to potential risks. Owing to constraints such as finite resources, or expensive or invasive tests, it is not possible for all such diseases to be screened for within the general population. In this circumstance, case-finding is recommended;5 Adopting a gluten-free diet still remains the sole intervention for coeliac disease.6 However, in the UK only 24% of those who have coeliac disease have been diagnosed,7 which means that there are estimated to be nearly half a million people who are unaware that they have coeliac disease; hence, the need for case-finding.Coeliac disease is a chronic immune-mediated enteropathy predominantly affecting the small bowel, which is precipitated by exposure to dietary gluten in genetically predisposed people.7 To help understand why this might be the case, a retrospective study of coeliac serology requests from a defined geographical area over a 12-month period was undertaken.The Yorkshire and The Humber region has the third lowest quoted incidence of coeliac disease (out of seven regions) in England.2 Repeat requests and those patients in whom known coeliac disease was given as an indication were excluded from analyses. All positive biopsies from the second part of the duodenum (D2 biopsies) were obtained from the York Hospital pathology database and were cross-referenced to obtain the crude rate of referral for D2 biopsy. York Hospital pathology department serves a population of approximately 500 000.Data were obtained from the York Teaching Hospital biochemistry department over a 12-month period (December 2013–November 2014) for all requests for immunoglobulin A anti-tissue transglutaminase antibodies (anti-tTG), a recognised serological marker for coeliac disease.coeliac disease:recognition andassessment ofcoeliacdisease, was the most up-to-date guideline available to clinicians.3 This guideline, published in May 2009, suggested a number of signs, symptoms, or conditions in which patients should be offered testing for coeliac disease and those which should be considered for testing . The indications were broadly grouped into 'NICE-approved requests', where the indication was one that NICE stated made patients eligible to be offered or considered for testing, and 'non-NICE approved requests', where the indication fell into neither of these categories.2 contingency table with Yates’ correction. Where a correlation between variables was analysed, Spearman’s Rank correlation test was used. A two-tailed P value of <0.05 was considered significant.Categorical data were analysed by means of a χThere was a total of 15 183  anti-tTG requests: 11 321 from primary care and 3862 from secondary care. Of the 15 183 requests, there were 14 494 unique requests, of which there were 10 984 (75.8%) unique requests from primary care. Of these 10 984 unique requests from primary care, 8584 (78.2%) were NICE-approved. There were 97 requests in children, and 1814 requests for coeliac serology gave no clinical details (12.5% of unique requests). A list of all the clinical indications for checking coeliac serology, as recorded by the York Hospital biochemistry department, is given in Box 2.P = 0.2). Of the 124 patients from primary care , 89 (71.8%) went on to have a D2 biopsy within 12 months . Of those biopsied, the positive predictive value for coeliac serology testing was 93.6%. Of the five patients who had positive serology but negative biopsies, two had ‘diarrhoea’ as an indication, with ‘abdominal pain’, ‘weight loss’, and ‘rash’ all being the indication for one further patient each . A total of 95 (76.6%) of the positive requests from primary care were NICE-approved. Of those with positive serology without a previous diagnosis of coeliac disease, the most common indications for serological testing were 'anaemia' (13.0%), 'no details given' (11.3%), 'diarrhoea' (10.5%), and 'abdominal pain' (9.2%). 'Osteoporosis', 'osteomalacia', 'fracture', or similar was given as a clinical indication for 0% of the requests in which a positive serology was found . Percentage of total practice list size tested for coeliac serology during the 12 months varied from 0.04% to 9.39%, with percentage testing positive varying from 0% to 4.17%  yielded a positive result compared with 50 (1.3%) of the requests from secondary care  of requests from primary care had a NICE-approved indication for coeliac serology testing and 95 (1.1%) of these requests revealed positive serology. A total of 1216 (11.0%) requests from primary care specified a non-NICE approved indication on the request and 12 (1.0%) of these requests revealed positive serology . There were 7795 requests from primary care with an indication that was considered to be 'classic coeliac disease' ; see R = 0.38, P = 0.02) between percentage of practice population tested and positive serology . Similarly, looking at subcategories of NICE-approved requests  there was no correlation between percentage of requests within a given NICE-approved subcategory and positive serology (P>0.05).There was a weak, albeit statistically significant, correlation , gynaecological conditions, and metabolic bone disease. It is likely that both these factors  are playing a role in the low level of case-finding seen in this study. This also raises the question of how valuable the NICE guidelines actually are with regards trying to identify at-risk groups in primary care (as pick-up rates were no different between NICE-approved and non-NICE approved requests), as well as how the guidelines are interpreted by primary care clinicians. There must also be concern that 29% of patients who tested positive do not seem to have had a D2 biopsy (at least within 12 months of their positive blood test).The most striking conclusion from this study is that, with an overall positive predictive value of 1.1%, the rate of case-finding in coeliac disease in primary care is no higher than the expected prevalence; that is, it is no better than random testing of patients. This could imply that those patients who are at increased risk of coeliac disease do not attend their GP practice . Awareness campaigns, such as that recently undertaken by Coeliac UK, have shown that with advertising, pop-up campaigns, and point-of-care testing, the percentage of people seeing their GP subsequently being diagnosed with coeliac disease is as high as 17%.The data show a near 200-fold difference in percentage of practice population tested for coeliac disease between the highest and lowest testing practices. It was noted that a number of the lowest testing practices are on the periphery of the geographical area, so this could simply reflect those practices sending more blood requests to other laboratories outside the area. Two of the highest testing practices were close to garrisons within York; therefore, by definition could be expected to have higher turnover rates of patients within any given 12-month period. Further information about drivers of serology testing rates in the different practices would be helpful in this regard. An interesting footnote is that there were 689 duplicate samples taken over the course of the study. This could be argued to be an unnecessary cost to the local health economy .10 Similarly, it would also be interesting to know how many patients on a 'self-imposed' gluten-free diet either declined or were not offered coeliac serology testing as they were not prepared to reintroduce gluten back into their diet (and thus the testing would not be meaningful). Also, data on patients who are in the increased risk categories of developing coeliac disease have not been captured, nor any data on whether: (a) they did not attend their GP practice owing to lack of awareness on their behalf; or (b) did attend their GP practice but were not identified as being at increased risk. The main strengths of this study are that it was undertaken in a relatively small geographical area (approximately 25 miles diameter) in which the vast majority of blood results from primary care come through the study laboratory; the size of the study ; the breakdown of the data by individual GP practice; and follow-up of at least 12 months on all patients. Indeed, in the 12-month period covered by this study, it seems 7% of the York population were tested for coeliac disease.The main limitation of this study is that it relies on what the laboratory has recorded as the indication for undertaking coeliac serology testing. Information recorded by healthcare professionals as indications for testing is often not robust and does not usually give a clear insight into the overall thinking of the healthcare professional requesting the test. For example, the authors would like to know how many patients were tested for coeliac disease simply because a patient requested the test .11 The first strategy involved serial testing of consecutive subjects aged <75 years being offered a point-of-care test (POCT) for coeliac disease. The second strategy involved offering only those with symptoms or associated conditions a POCT, but there was a systematic search for patients with this second strategy. In this study, 1.6% of POCT were positive with the first strategy and 30.5% with the second strategy. Between May 2015–August 2016, Coeliac UK held pop-up events in seven British cities and tested almost 500 people with a POCT test if they had associated symptoms on filling out a simple questionnaire.8 A total of 17% of people had a positive test result using the POCT and were referred to their GP for further investigation, but whether these patients did indeed see their GPs and the outcome of this follow-up is not known. A study from Catalonia using point-of-care testing in a smaller area than this study (a population of approximately 100 000) and only 350 patients were consecutively recruited. This study had a pick-up rate of 1.14%.12 This was twice previous pick-up rates in this area, although 34/58 of the primary care physicians participating in this study undertook a pre-study questionnaire to assess their knowledge of coeliac disease (the other 24 were invited but declined). Previous studies in primary care in the UK suggest that patients with undiagnosed coeliac disease have more consultations with their GPs in the 5 years prior to diagnosis, implying the opportunity to case-find is there.13As far as the authors are aware, there are no other studies that have looked at case-finding for coeliac disease in adults in primary care in the UK. There was a very recent study from Sicily that looked at two separate strategies for testing in primary care.4 in 2015 and publicity campaigns by Coeliac UK), and with other diseases where case-finding is used as a strategy . This would enable common themes to be seen, from which lessons could be learnt. It may be that as point-of-care testing becomes available for coeliac disease, and if the requirement for at least some adults not to have to undergo biopsy to confirm diagnosis of their coeliac disease ,14 that case-finding strategies can be more successfully implemented.It is recommended that further studies assess whether the findings in this study are repeated in other parts of the UK with regards to coeliac disease (particularly as there have been new NICE guidelines published with regards to coeliac disease


From the collected heart sounds, relative power of the frequency band, energy of the sinusoid formants, and entropy were extracted. PAH subjects were differentiated by applying the linear discriminant analysis with leave-one-out cross-validation. The entropy of the first sinusoid formant decreased significantly in subjects with a mean pulmonary artery pressure (mPAp) ≥ 25 mmHg versus subjects with a mPAp < 25 mmHg with a sensitivity of 84% and specificity of 88.57%, within a 10-s optimized window length for heart sounds recorded at the 2LICS. First sinusoid formant entropy reduction of heart sounds in PAH subjects suggests the existence of a vowel-like pattern. Pattern analysis revealed a unique sound signature, which could be used in non-invasive screening tools. Increased blood pressure in the pulmonary artery is referred to as pulmonary hypertension and often is linked to loud pulmonic valve closures. For the purpose of this paper, it was hypothesized that pulmonary circulation vibrations will create sounds similar to sounds created by vocal cords during speech and that subjects with pulmonary artery hypertension (PAH) could have unique sound signatures across four auscultatory sites. Using a digital stethoscope, heart sounds were recorded at the cardiac apex, 2 When left untreated, pulmonary artery hypertension (PAH) is a progressively fatal disease . ImpactiMany investigators in the field of non-invasive PAH screening methodologies strive to detect the auscultation of an abnormally loud pulmonary component (P2) of the second heart sound (S2). These investigations have led to phonocardiographic exploration and the seeking of time-domain associations between the pulmonary artery pressure (PAp) and S2 ,9,10,11.nd left intercostal space (2LICS), however, was not sufficient to capture PAH [nd left intercostal space (2LICS) site. The analysis of this pattern revealed a unique sound signature, which could be applied to a non-invasive method to diagnose PAH. However, these results were obtained over a sample size of 27 subjects using only two sites: 2LICS and apex. This led us to further investigate two additional sites: 2nd right intercostal space (2RICS) and 4th left intercostal space (4LICS) to (1) determine if these two new sites also express the same reduction in entropy of the first sinusoid formant, (2) compare all four sites to identify the optimal site that is associated with reduced entropy indicating the existence of an organized pattern, and (3) examine the pattern existence over a larger sample size to validate our findings.Quantitative information on the frequency domain of heart sounds is explored in this paper, with the aim of differentiating between subjects with and without PAH . The relture PAH . Recent ture PAH  showed tNormal speech patterns have a unique signature related to vocal cord vibration that can be used to make a distinction between certain traits . We postulated that unique sounds could be detected from the vibrations that originate in the movement of the pulmonary valve leaflets and pulmonary artery, in a manner similar to vocal cords. Through these sounds, a unique sound signature for PAH patients can be defined. Through formant (defined as the frequency resonance of sound and concentrations of energy that are prominent in a sound spectrogram) detection, we can ascertain the sound frequency spectrum . First aWe postulated that pulmonary circulation vibrations would create sounds similar to the sounds generated by the vocal cords during a speech, and that subjects with PAH will have a unique sound signature. The University of Alberta (UofA) Research Ethics Board approved the study. Informed and written consents were obtained from subjects  or guardians. In this section, a new, speech-based, and numerically efficient algorithm is proposed to detect PAH in heart sounds based on the entropy of its formant. The structure of the proposed algorithm is shown in Subjects undergoing right heart catheterization (required for managing an underlying cardiac condition) were approached. No subjects had an abnormal or prosthetic valve.TM Littmann® 3200 electronic stethoscope  was used to record heart sounds and Zargis CardioscanTM software  was used for storing recorded heart sounds (*.wav mono audio format). Recorded heart sounds were obtained over 20 s with a sampling frequency of 4000 Hz. Heart sounds were recorded sequentially at the 2nd left intercostal space (2LICS) and 2nd right intercostal space (2RICS), 4th left intercostal space (4LICS), and over the cardiac apical impulse. For signal analysis and optimization, MATLAB 2010b  was used.The PAp was measured directly and collected simultaneously with heart sounds which were obtained via fluid filled catheters. A 3MPAH, in adults and children, is defined as a mean pulmonary artery pressure (mPAp) ≥ 25 mmHg and a pulmonary artery wedge pressure (PAWp) or left atrial pressure (LAp) ≤ 15 mmHg measured at cardiac catheterization in subjects at rest ,18,19. TEntropy holds different meanings across different disciplines, therefore, we will define entropy in the context of this project. Entropy is defined as a measure of the disorder of the heart sound pattern. A lower entropy value suggests the existence of an organized heart sound pattern, while a higher entropy value indicates no existence of pattern. t-tests were used to discover which recording site  was more informative for the diagnosis of PAH. Classification for two groups was carried out based on whether or not each subject’s mean PAp was ≥ 25 mmHg or < 25 mmHg. All subjects had a PAWp < 15 mmHg. Heart sounds were analyzed by extracting the entropy of the first four sinusoids of the heart sound frequency bands. Two-sample th order linear-predictive-coding (LPC) analysis was applied to each window, which finds the coefficients of an 8th order linear predictor (finite impulse response filter) that predicts the current value of the heart sound segment based on past samples. The four coefficients with the highest magnitudes were then converted to frequencies and magnitudes and stored in a data file. The resulting recordings each had an associated data file that included the eight parameters (four frequencies and four associated amplitudes) measured in each 32-millisecond window. This window captured information sufficient to track the change of the major formants in the original sound file over time. A synthesis routine [S in the below equation. The entropy of a sinusoid as the power of the log-transformed spectrogram was calculated as follows:L is the length of the processed heart sounds segment, f is the formant order: 1, 2, 3, or 4, k is the recording site: apex, 2RICS, 4LICS, or 2LICS, and E is the speech spectral feature for a specific recording site.Extraction of unique heart sound signatures (defined as informants) was carried out using sine wave replicas. Through this process, sound patterns were distilled down to key elements through the removal of extraneous aspects. Heart sound recording tracks were transformed into sine wave replicas, as seen in speech analysis . These s routine  was thent-test to compare the means, with p-value ≤ 0.05 considered significant. After calculating the speech spectral feature (entropy of the sinusoid formant) for each of the four recording sites for 60 subjects, the speech spectral feature set contained 60 values: 35 values calculated from subjects with mPAp < 25 mmHg and 25 values from subjects with mPAp ≥ 25 mmHg. To demonstrate the significance of the mean of the samples within the speech spectral feature set, we compared the values by applying a two-sample L. This allowed for accurate classification. Between 1 and 20 s window lengths were tested systematically. For each window length, the speech spectral feature was calculated and then averaged over all disjointed segments of length L within the duration of the 20-s heart sound recording. Note, the window length optimization was carried out on the most informative recording site as described in Stationary heart sounds could theoretically be used as a whole to calculate the speech spectral feature (entropy of the first sinusoid formant), however, typically, these sounds are highly non-stationary and noisy. Given a heart sounds’ stationary nature, features can vary over time, thus making feature estimation from the entire 20-s signal no longer meaningful. A search was conducted over heart sound segment recordings to accommodate for potential non-stationarity recordings and to identify an appropriate window length, defined here as n splits was then determined.For subject classification (PAH and non-PAH) an applied linear discriminant analysis (LDA) was used, based on the entropy of the first sinusoid formant of the heart sound of the most informative recording site. Classification performance was evaluated with LDA through leave-one-out (LOO) cross-validation, where each subject provided one case. Training sets were created by taking all cases except one, which was held out as a disjointed training set. Each training sets’ classification accuracy was determined by the single held-out test. The average accuracy over all For each recording, the speech spectral feature (entropy of the first sinusoid formant) was calculated. Since there were 60 subjects, the speech spectral feature set contained 60 values: 35 values calculated from subjects with mPAp < 25 mmHg and 25 values from subjects with mPAp ≥ 25 mmHg.t-test was applied to establish the mean and median significance of the samples within the speech spectral feature set; here, the p-value ≤ 0.05 was considered significant. This procedure was repeated to determine the most informative window length for each feature over a range of window lengths of heart sounds . The two-sample p-values were small merely due to the stochastic fluctuations, rather than due to the systematic difference between subjects with mPAp < 25 mmHg and ≥ 25 mmHg. Consequently, the p-values needed to be appropriately corrected as implemented in [Given that three different features were considered and that various window lengths settings were also considered simultaneously, it is plausible that a few ented in ,25. To cented in  can be uented in .Two statistical measures were used for the output of the LDA analysis: sensitivity, calculated from the formula t-test to have a power of 90% if the significance level was 0.05.Finally, we conducted a post-hoc power analysis on the speech spectral feature calculated from the most discriminative recording site, to ensure that our sample size (60 subjects) was sufficient to draw meaningful conclusions. The power calculation was carried out using a two-sided Recordings were collected from 60 subjects . In Group 1, 35 subjects had a mean PAp < 25 mmHg (range 5–24 mmHg), and 25 subjects from Group 2 had a mean PAp ≥ 25 mmHg (range 25–93 mmHg). All subjects had a mean PAWp or left atrial pressure < 15 mmHg. No recordings or subjects were omitted from the analysis. Hemodynamic and clinical details of subjects are included in The middle panels of The top panel of p-value for distinguishing between subjects with and without PAH. p-value was the lowest (3.31 × 10−9) compared to those of the other three recording sites. We investigated which out of the four sinusoid formants of heart sounds was more informative in all recording sites. As shown in p-value to emphasize the optimal length value.We conducted a search over segments of the 2LICS heart sound recordings to identify the most appropriate window length for capturing a unique pattern (informative sinusoid formant). The significance, after statistical post-correction, was achieved after systematically testing a range of window lengths of the processed heart sound recordings (see window optimization section). As shown in The entropy of the first sinusoid formant with an optimal 10-s length of heart sounds recorded at the optimal 2LICS recording site of subjects with mean PAp < 25 mmHg was significantly lower than that of subjects with mean PAp ≥ 25 mmHg, as shown in We conducted LDA through LOO cross-validation on the recordings at the 2LICS with the optimal 10-s window. The entropy of the first sinusoid formant of the heart sounds incurred four false positive and four false negative results; see also In subjects with PAH (mean PAp ≥ 25 mmHg), entropy of the first sinusoid formant (within an optimized 10 s window length of the heart sound recordings at the 2LICS) was decreased significantly, with a sensitivity of 84% and specificity of 88.57%; see also The reduced entropy of heart sounds in subjects with PAH suggests the existence of an arranged pattern within the heart sounds. Using a non-invasive recording device, this pattern could be used to diagnose PAH using a non-invasive recording device. It can be seen in TM Littmann® 3200 digital stethoscope and 3M, Inc., Denmark electronic stethoscope, were included. This suggests that this analysis of the heart sounds in the frequency domain is robust. Higher fidelity sensors would likely improve the sensitivity and specificity value of the submitted results. In clinical settings where time is at a premium for health care practitioners, a 20 s short recording time for the collection of diagnostic data is helpful and practical, especially in pediatric clinics. The cooperation of infants and children is unpredictable and lasts for a limited amount of time, and quickly capturing much needed and valuable diagnostic information is an asset. All recordings collected from the 3MOur signal analysis did not focus on timing, detection, or the splitting of S2 intervals between aortic and pulmonary components. There are significant challenges with the traditional PAH clinical indicators, such as seen with the differentiation between the aortic and pulmonary components of the S2 and the splitting interval ,9,12,13.It is interesting to note that the recording site that best distinguishes patients with a mean PAp ≥ 25 mmHg from subjects with a mean PAp < 25 mmHg was the 2LICS, which is the traditional area for auscultation of pulmonary artery events.In human spoken words, the vowel formants appear as “harmonic stacks” that mark the arrival of the glottal pulse train, and the spacing of the harmonics is clearly much wider in the high-pitched vs. the low-pitched vowels . Figure We found in the case of PAH that the vowel-like sound has a very prominent first formant harmonic around 300 Hz; however, there is little sound energy between 200 and 700 Hz. This phenomenon has been similarly found in the human /e/ and /i/ vowels . ResultsInvestigation of PAH was the main focus of this research, perhaps looking into different heart valve diseases using the same methodology would be interesting. We may be able to find different vowel-like patterns for each abnormality. Note, in our study here we did not have any subjects with abnormal or prosthetic valves.p-value, which is the entropy of the first sinusoid formant derived from the heart sounds  was 4.56 × 105. The power calculation of the speech spectral feature from the 2LICS suggests that the representative sample size of the population should be ≥6 subjects for a power of 90% when the significance level is 0.05, which indicates that our sample size (60 subjects) was sufficient to draw meaningful conclusions. A larger sample size is needed to confirm the findings of this study. We studied 60 subjects. We conducted a post-hoc power analysis on the speech spectral feature with the smallest post corrected overall unds see . The meaProspective recordings with the investigators blinded to the patients’ diagnoses are also required in future studies. However, the use of LDA and LOO to analyze the findings removes investigator bias considerably. Since we had a small sample size, it was difficult to investigate whether the vowel-like pattern was more salient in adults or infants. However, this is one of the points that needs to be addressed in future research in this area.The entropy (of the first sinusoid formant) of heart sounds in PAH subjects, recorded via a handheld digital stethoscope and recorded simultaneously with direct pulmonary artery pressure measurements, was significant and revealed a mean PAp ≥ 25 mmHg, yielding a classification sensitivity of 84% and specificity of 88.57%. An optimized window length of 10 s, within a 20-s recording of the heart sounds at the 2LICS, was applied. Results suggest that heart sounds contain organized vowel-like patterns similar to the organized patterns found in speech analysis. This pattern is a unique sound signature produced by the hypertensive pulmonary artery and right ventricle. More work is needed to generalize the findings, such as conducting similar research on a larger sample size, allowing for a more informed collective decision based on multiple heart sounds. We suggest that the entropy of the first formant be used as a screening tool, as results show potential for providing insight into screening for PAH cases, when compared to healthy subjects. Another point for consideration is trying different digital stethoscopes to confirm the scalability of the findings over different devices.


We previously showed that in the myocardium of infants with heart defects, mRNA is alternatively spliced as compared to control tissues. We also demonstrated that alternatively spliced genes were concentrated in the pathways that control heart development. Furthermore, we showed that modifying expression of scaRNAs alters mRNA splicing in human cells, and zebrafish embryos. Here we present evidence that SNORD94 levels directly influence levels of methylation at its target region in U6, suggesting a potential mechanism for modifying alternative splicing of mRNA. The potential importance of scaRNAs as a developmentally important regulatory mechanism controlling alternative splicing of mRNA is unappreciated and needs more research.Understanding the regulation of development can help elucidate the pathogenesis behind many developmental defects found in humans and other vertebrates. Evidence has shown that alternative splicing of messenger RNA (mRNA) plays a role in developmental regulation, but our knowledge of the underlying mechanisms that regulate alternative splicing are incomplete. Notably, a subset of small noncoding RNAs known as scaRNAs  contribute to spliceosome maturation and function through guiding covalent modification of spliceosomal RNAs with either methylation or pseudouridylation on specific nucleotides, but the developmental significance of these modifications is not well understood. Our focus is on one such scaRNA, known as SNORD94 or U94, that guides methylation on one specific cytosine (C Congenital heart defects are structural problems in the heart that are present at birth that affect normal function and blood flow through the heart . Nearly Chromosomal defects and single gene disorders are known to cause some CHDs, often in the context of multisystem diseases . GeneticGATA4, MBNL1, MBNL2, DICER, DAAM1, and NOTCH2), and showed that the alternative splice isoforms of these genes were abnormal in TOF patients compared to control, and they were in fact more similar to fetal tissue splice isoforms [During embryonic development, spatiotemporal signaling between the first and second heart field gives rise to the left and right ventricles and conotruncal outflow tract, which is critical for correct development of the vertebrate heart . The genisoforms . We hypoMany human diseases are the result of aberrant pre-mRNA splicing and therefore understanding splicing on a molecular level is of medical relevance . SeveralThe spliceosome is a multimegalodalton ribonucleoprotein complex comprised of several subunits  that carries out specific mRNA processing including splicing of introns and exons according to the specific needs of the cell . During 62)on spliceosomal RNA (snRNA) U6 for 2’-O-methylation [62 on snRNA U6 lies within the stem-loop structure of the catalytic site of the activated U6 RNP, potentially contributing to the catalytic site structure and function and influencing the spliceosome’s ability to splice correctly.The 2’-O-methylated sites tend to reside in functionally important regions in ribosomal RNA, potentially influencing ribosomal structure and function . Within hylation . The 2’-Previously, it has been shown that snoRNA expression is different in the right ventricular tissue of children with TOF, with 135 snoRNAs being significantly differently expressed from control tissue, most of which having decreased expression in TOF samples . SNORD9462 on snRNA U6 to establish a link between levels of SNORD94 and levels of C62 methylation. These data may help decipher the underlying biochemistry essential for correct spatial and temporal gene expression patterns in key regulatory pathways in the developing vertebrate heart.In the same snoRNA expression study, it was also shown that U6 had reduced expression by 3.2-fold in all 16 TOF samples as compared to control . This dr62 in our samples, we followed the approach used by Dong et al. to quantify 2’-O-methylated cytosine [62) causes increased stalling of the reverse transcriptase resulting in less long fragments that leads to a relatively greater ΔCt  and the other was unmethylated. We used 0.1ng RNA for each RT reaction . We compared the cycle change for long fragments in low vs normal dNTP concentrations (ΔCt long fragments) and found that the difference is significantly greater when methylation is higher. We also found that there exists a nonzero ΔCt when no methylation is present, showing there is some loss in overall RT efficiency in low dNTP concentration. t value for the difference in long fragment quantity between normal and low dNTP concentrations with synthesized fragment with either 100% methylation at C62 on U6 or 0% methylation (1.0 cycle difference corresponds to 2-fold change).We validated the technique for quantifying methylated cytosine by using synthesized sequences corresponding to the sequence from human U6. RNA oligos were custom synthesized by IDT  and corresponded to the sequence of whole human U6. One oligonucleotide was methylated at Cd and effect-size r for each group, which are measures for the power of the difference in treatment groups with small sample size. [d (d>0.8) is considered a “large effect” of treatment on the experimental group as compared to control, and r is another effect-size measure that represents the percentile of the treated group in relation to the control group [d value and r values correspond to greater effect-size of the treatment as compared to control [We analyzed the data from all groups using t tests for the difference between groups. Because of the small sample sizes in each comparison group, we also calculated the Cohen’s le size. , 22. Lar62 in RNA isolated from tissues and cells. We compared the amount of methylation present in the target region of U6 in the right ventricular tissue of infants with TOF compared to right ventricular tissue from infants with normally developing hearts. We found that the patients with TOF had a significantly reduced amount of methylation in right ventricle tissue as compared to age-matched normally developing infants   .in vitro assessment of altered levels of SNORD94, we found that knockdown of SNORD94 in primary cardiomyocytes derived from a normally developing heart caused a significant decrease in methylation at C62 as compared to untreated cardiomyocytes from the same starting sample   (In our ference) . Finallyference) . Within 62 contributes the most to the difference in long fragment between groups with higher known mC62, as it did with the IDT synthesized oligopeptide validation. The loss of long fragment seen is likely slightly affected by methylation at C77 and C60 because of the proximity of C60  to our nucleotide of interest and the downstream location of C77. More research is needed to systematically determine the contribution of each methylation point to the overall loss of long fragment, with particular regard to C60 as the scaRNA that modifies it does have reduced expression in RV tissue from infants with TOF[60 is small because we observed significant changes in long fragment loss when only SNORD94 expression was changed in vitro.Of note, there are other known sites of 2’-O-ribose methylation within spliceosomal RNA U6 see . These m with TOF. However62 within spliceosomal RNA U6. These findings taken together suggest a dysfunction in an important regulatory pathway that is responsive to scaRNA expression levels. Directly stated, our accumulated results suggest that dysregulated scaRNA levels contribute to defective heart development by disturbing proper alternative splicing of mRNAs. This study focused on one specific scaRNA producing one biochemical modification, but more research is necessary to determine the interplay of multiple scaRNAs on spliceosomal integrity. Of note, there are other posttranscriptional modifications present on U6 as mentioned in the introduction, and there may be dynamic relationships between these modifications and the structure and function of the U6 snRNA within the spliceosome so these relationships need to be investigated as well. However, we note that these were not targeted in our knockdown and overexpression experiments and thus should have remained constant with no net effect on these analyses.Alternative splicing has a significant impact on developmental pathways in multiple organ systems, including the heart. We have shown that methylation in the target region of SNORD94 on U6 is reduced in right ventricle tissue of children with TOF as compared to control. We have also confirmed that changing levels of SNORD94 in cell culture produces a corresponding change in methylation at COur previous studies provided evidence that modest dysregulation of scaRNA expression impacted mRNA alternative splicing and development , 8, 23. https://www.lifenethealth.org, Virginia Beach, VA), a non-profit regenerative medicine company that provides bio-implants and organs for transplantation [Our subjects included 16 nondysmorphic infants  with idiopathic TOF but without chromosome abnormalities (22q11.2 deletion) , 23. Comantation . The conPrimary cell cultures were derived from right ventricle (RV) tissue of infants with TOF. The RV tissue was immediately immersed in DMEM  plus 10% fetal calf serum  and 1% penn/strep (Gibco). The tissue was minced and most of the media was removed, leaving only enough to keep the tissue from drying out. After 24 hours, additional media was added, and cells were growing robustly after 3 to 4 days. Media was exchanged every 48 hours. In addition, we obtained a primary neonatal cardiomyocyte cell culture derived from normally developing human neonatal cardiac tissue from Celprogen . These cells were also grown in DMEM plus 10% fetal calf serum and 1% penn/strep.The SNORD94 expression vector used in this study was that which was used in Patil et al, 2015 and was a generous gift from Dr. Tamas Kiss . The scaWe used an antisense LNA oligo  to suppress the SNORD94 in primary cardiomyocytes as has been done previously in immortalized cell cultures , 25, 26.RNA from tissue or cell culture was extracted from the whole cell pellet using a mirVana total RNA isolation kit (Invitrogen) according to the manufacturer's instruction.It has been previously shown that reducing concentration of dNTP to 4μM or below in the cDNA synthesis reaction can induce a stop at a 2’-O-ribose methylated nucleotide on RNA , 20. We 62. However, there is some reduction in efficiency of reverse transcription in the presence of low dNTP concentration, so there will be a nonzero change in long fragment quantity in the absence of methylation. We used the difference in cycle number (ΔCt) as a measure of methylation; a greater ΔCt represents a larger amount of mC62 due to a greater loss of long fragment in low dNTP concentration as compared to high concentration. An increased level of methylation in an RNA sample results in a reduced amount of long fragments produced in low dNTP concentration during the RT reactions. These changes during RT will result in a larger ΔCt in qRT-PCR.The RT reaction stalls at 2’-O-ribose methylated cytosines (mC) when dNTPs are in low concentration. Thus, the loss of the long fragment in low as compared to normal dNTP concentrations will reflect the amount of mCS1 Raw Data(XLSX)Click here for additional data file.