Millions of people around the world have difficulty hearing. Hearing aids and cochlear implants help people hear better, especially in quiet places. Unfortunately, these devices do not always help in noisy situations like busy classrooms or restaurants. This means that a person with hearing loss may struggle to follow a conversation with friends or family and may avoid going out. We used methods from the field of artificial intelligence to develop “smart” hearing aids and cochlear implants that can get rid of background noise. We play many different sounds into a computer program, which learns to pick out the speech sounds and filter out unwanted background noises. Once the computer program has been trained, it is then tested on new examples of noisy speech and can be incorporated into hearing aids or cochlear implants. These “smart” approaches can help people with hearing loss understand speech better in noisy situations. Sound is created when objects vibrate. These vibrations travel through the air as sound waves. When sound waves reach our ears, they travel from the outer ear, down a tube called the ear canal, then they vibrate the eardrum and tiny bones in the middle ear, to reach the inner ear or The spiral-shaped part of the inner ear that contains the sensory organ of hearing.Around one in five people in the world has some degree of hearing loss. This adds up to around 1.5 billion people, so hearing loss is a huge problem with societal, health, and economic impact. The World Health Organization predicts numbers will rise to one in four people by 2050. Currently, 400,000,000 people have hearing loss serious enough to require hearing aids. There are many different reasons why hearing loss occurs, for example getting older, listening to too much very loud music, or due to illnesses or medicines that harm the ears. The most common type of hearing loss affects important processes in the inner ear that convert sound waves into nerve signals and this can cause difficulties understanding speech.Certain devices can help people with hearing loss. Depending on the amount of hearing loss, two main types of hearing devices are available. For most forms of hearing loss, A small electro-acoustic device to let people hear better.A small computer chip used in many electronic devices to process signals such as speech, sound, video, or other digital data.If hearing loss is severe, acoustic hearing aids may not provide enough amplification. In these cases, A small electronic device to let people hear via electrical stimulation of the auditory nerve in the cochlea.The rate at which a sound wave vibrates. Lower frequencies (slower vibrations) are heard as lower tones (like a man’s voice); higher frequencies are heard as higher tones (like a child’s voice).Even when using a hearing aid or a cochlear implant, it is often much more difficult for people to understand speech in noisy situations such as a busy classroom, workplace or restaurant. This means that a person with hearing loss might struggle to understand what friends or family are saying to them and may not want to take part in conversations.Difficulty hearing speech happens because hearing aids and cochlear implants cannot recover the fine details of sound that are needed to understand speech, so the sound information that reaches the brain is not as detailed as it needs to be. People with hearing devices may still find it difficult to hear pitch (how high or low a tone is) or whether a person’s voice rises at the end of a sentence to form a question. Some people with hearing aids or cochlear implants also find it hard to tell different sounds apart. This makes it particularly difficult for them to follow and to understand speech correctly when the background is noisy. Even the best hearing aids and cochlear implants cannot get rid of background noise in real-life situations.We are developing smart A set of rules to be followed for a process or operation, for example by a computer.In previous research, speech was separated from noisy background sounds based on fixed rules, similar to a recipe. These rules had to be followed strictly by the digital signal processor in the hearing device.This method only works for background sounds that are very different from speech. But in the real world, background noise often contains speech-like sounds, such as people chatting. For these situations, fixed rules do not work well. In the field of Methods to enable computers and algorithms to perform some intellectual tasks such as decision making, problem solving, communication, or perception.The specific algorithm we used is called an A computer algorithm inspired by the neural networks found in biological brains.At the beginning of the training, the DNN makes a lot of mistakes because it has no experience in cleaning up speech signals. But these mistakes are important and valuable, because we can use them to teach the DNN how to make decisions about which sounds to keep and which sounds to filter out. During training, the DNN builds its own set of rules, by adjusting the settings of its parameters step-by-step. By listening to thousands or even millions of examples of many kinds of noisy speech mixes, the DNN’s parameters are optimized to reduce the number of mistakes it makes when cleaning up speech. Once training is complete, the DNN algorithm can be added to the digital signal processors of hearing aids and cochlear implants .In our research, we trained DNNs to improve noisy speech as much as possible and then performed listening tests to study how well the algorithm worked for people that use hearing aids  and cochWe found that the DNNs improved the understandability of speech by up to 14% for people using hearing aids  [1] and Even a small amount of background noise causes difficulties for people with cochlear implants. So, the DNN algorithms we are building sometimes work better for people with cochlear implants than those with hearing aids because the algorithms do not need to filter out as much noise to make a difference. The DNN algorithms also work better if there is less noise to remove. Overall, the DNN algorithms perform well when speech is at least as loud as background noise, but they start to struggle when the noise is louder than the speech. DNNs successfully improved speech scores even when there were many people talking in the background, which is a clear improvement over previous algorithms, which did not work well in those cases.We also found that it is much easier for the DNN algorithm to improve speech signals when it has been trained on a particular voice or a specific background noise; it does not perform as well for completely different speech and noise recordings that were not part of the training. This can be seen as a strength of DNN algorithms, because we can fine-tune them to work better with specific voices or noises. However, it can also be seen as a weakness because the DNN algorithm may become too specialized and then would not help much in other listening situations, for example with other voices.For many people who have hearing aids or cochlear implants, listening to speech in noisy situations can be difficult and tiring. We are building smart algorithms based on artificial intelligence techniques that can be used to improve future hearing aids and cochlear implants, particularly to help get rid of background noises. The algorithms use artificial DNNs that become smarter by learning from their own mistakes during training with many thousands of noisy speech examples. These algorithms are then used to clean up speech recordings before presenting those recordings to people with hearing loss and measuring their speech scores. Our results showed DNNs resulted in improvements in how well people could hear speech in the presence of noise, and how much they liked the sound of that speech. These results, along with other studies , 5, show


In recent years there has been an increasing percentage of cochlear implant (CI) users who have usable residual hearing in the contralateral, nonimplanted ear, typically aided by acoustic amplification. This raises the issue of the extent to which the signal presented through the cochlear implant may influence how listeners process information in the acoustically stimulated ear. This multicenter retrospective study examined pre- to postoperative changes in speech perception in the nonimplanted ear, the implanted ear, and both together. Results in the latter two conditions showed the expected increases, but speech perception in the nonimplanted ear showed a modest yet meaningful decrease that could not be completely explained by changes in unaided thresholds, hearing aid malfunction, or several other demographic variables. Decreases in speech perception in the nonimplanted ear were more likely in individuals who had better levels of speech perception in the implanted ear, and in those who had better speech perception in the implanted than in the nonimplanted ear. This raises the possibility that, in some cases, bimodal listeners may rely on the higher quality signal provided by the implant and may disregard or even neglect the input provided by the nonimplanted ear. Indications for cochlear implantation have changed significantly since these devices were introduced to clinical practice. Initially, only patients with profound to total hearing loss were implanted, but as outcomes of cochlear implantation improved with the development of newer devices, electrodes, and stimulation strategies, it became more common for patients with residual hearing to receive a cochlear implant. In 2014, the FDA expanded cochlear implantation criteria and gave premarket approval for implantation in ears that have useable acoustic hearing in the low frequencies . In contInitial research on bimodal hearing indicated that even persons with severe to profound hearing loss in the nonimplanted ear could obtain bimodal benefit   14 of 18  14 of 1Some of these speech perception results may be driven by asymmetric hearing, defined as a situation where one ear receives (and conveys to the brain) much more information than the other one. Auditory asymmetries are known to have consequences that have been demonstrated in animal models and human listeners ,34,35. TWe first reported in 2011  an interParticipants were adults with post-lingual hearing loss who used a cochlear implant (CI) in one ear and a hearing aid (HA) in the contralateral ear . To be included in the study, documentation of hearing pre- and post-implant surgery was required for the implanted ear and the non-implanted ear. These longitudinal measures included documentation of unaided pure tone thresholds in the nonimplanted ear, as well as speech recognition performance pre- and post-implantation in the CI ear, the HA ear, and in the bimodal condition. An additional requirement was documentation that showed the hearing aid was functioning properly during testing. This requirement could be met in one of four possible ways: 1) in-the ear measures showing that hearing aid gain was adequate; 2) aided and unaided thresholds in the HA ear; 3) unaided speech perception scores in the HA ear pre- and post-implant ; or 4) indication from an audiologist or hearing aid technician that the HA was operating correctly during the evaluation.There were 132 subjects: 68 male and 64 female. The average age at implantation was 56.8 years . Data was collected at eight different testing sites. Speech testing consisted of words or sentences in quiet presented via audition alone  at 65 dB HL (unless otherwise noted) and zero degrees azimuth. See Speech perception scores before implantation were compared to post-implantation speech scores. This was done separately for three conditions: implanted ear only , nonimplanted ear only, and bilateral. These comparisons were made in two different ways: comparing pre-implant to post-implant scores averaged over all postoperative sessions, or just to the latest available postoperative score. These two ways of comparing preoperative to postoperative data yielded similar results. The statistical significance of pre- to postoperative differences was assessed at the group level using paired t-tests, or the Wilcoxon signed rank test when data failed to follow a normal distribution. Follow-up tests were conducted on subsets of the data to help tease out possible reasons for preoperative to postoperative changes in speech perception in the nonimplanted ear. Specifically, comparisons were made for the original dataset of 132 subjects who had pre- and postoperative scores in the nonimplanted ear and for the same group after excluding any evaluation session when correct hearing aid operation could not be ascertained (N = 122). By design, all datapoints obtained postoperatively in the nonimplanted ear were included if the pure-tone average (PTA) did not differ from the preoperative PTA by more than 25 dB. To better rule out the effect of progressive hearing loss, analyses of speech scores in the nonimplanted ear were conducted after removing datapoints showing an increase of more than 20 dB PTA. The remaining N was 120 for the preoperative to average postoperative comparison, and N = 118 for the preoperative to latest postoperative comparison. The process of elimination was repeated for datapoints showing a PTA increase of more than 10 dB, and the resulting numbers of the remaining subjects were N = 107 and N = 104, when comparing preoperative scores to the average postoperative and latest postoperative scores, respectively. Finally, this was also done after excluding datapoints showing a change of more than 5 dB in PTA. The resulting numbers of subjects in this case were N = 92 and N = 85 for the two types of comparison.https://osf.io/fha47/?view_only=3d5c2e239a64496481ea657215913469). This was done separately for each test. To determine whether two scores are significantly different, computer simulation was used to generate confidence intervals based on the number of observations used to obtain each score. Let n1 and n2 represent the number of observations from which those two scores were obtained, respectively. Next, consider all possible scores (as proportions) that can be obtained for these numbers of observations. For example, if n1 = 50, then the possible scores are 0, 0.02, 0.04, …, etc., and if n2 = 25, then the possible scores are 0, 0.04, 0.08, …, etc. Let p1(i) represent the set of possible scores for n1 where i = 1, 2, …, n1 + 1, and p2(j) represent the set of possible scores for n2 where j = 1, 2, …, n2 + 1. For every pair of possible scores from p1(i) and p2(j), one calculates a mean absolute difference score using computer-generated binomially distributed pseudorandom numbers for p1(i) and p2(j), repeated over many iterations. That is, on one iteration, two binomially distributed numbers are generated, one based on p1(i) and n1 and the other based on p2(j) and n2. The numbers are then converted to proportions by dividing by the respective number of observations, n1 and n2. The absolute value of the difference between these two scores is then recorded. This process is repeated for any number of desired iterations, e.g., 40,000. For this set of 40,000 absolute difference scores, one can calculate a z-score equal to the mean absolute difference divided by the standard deviation estimate across absolute difference scores. Having generated a z-score for each p1(i) and p2(j), one can find the values of p1(i) and p2(j) that produce z-scores exceeding a critical value . For p1(i), the values of p2(j), whose z-scores fall within +1.96 and −1.96, define the critical range.Pre- to postoperative comparisons were also conducted for each subject and condition  by generating critical difference tables as described by Carney and Schlauch . The criSome data from two of the sites were obtained using scores of correct keywords in sentences. The TAM test has between 114 and 125 scored words per list and the CUNY sentence test has 102 words per list, on average. In those cases, critical significance tables are used N = 40, which have been shown to be an appropriately conservative assumption for a sentence test like AzBio (which has 142 words per list) when scoring it using the percentage of correct keywords [The change in speech scores in the nonimplanted ear  was correlated with three variables of interest: the preoperative to postoperative change in pure-tone average, the speech score in the implanted ear, and the implanted ear advantage (difference in speech score between the implanted and the nonimplanted ear). The latter correlation was also calculated after partialling out the effect of the initial score in the nonimplanted ear. It was expected that the pattern of correlations might help tease out the effect of some possible causes of speech perception drops in the nonimplanted ear. A significant correlation between drops in speech scores and in pure-tone average in the nonimplanted ear would suggest that the progression of hearing loss might be at least partly responsible for decreases in speech scores. Conversely, a significant correlation between drops in speech scores in the nonimplanted ear and either speech scores in the implanted ear or the implanted ear advantage would suggest that the speech perception drops in question are at least partly due to a more central mechanism: namely, neglect of the poorer ear.Lastly, and for the sake of completeness, the change in speech scores in the nonimplanted ear was also correlated with several preoperative demographic variables: age at cochlear implantation, age at moderate hearing loss, age at profound hearing loss, preoperative aided and unaided PTA in both the implanted and the nonimplanted ear, whether cochlear implantation was done using a cochleostomy or a round window approach, whether the left or the right ear was implanted, whether a hearing was used in the ear to be implanted, years of education, and gender.This study was approved by the New York University Medical Center Institutional Review Board (S12-02951).p < 0.05) from preoperative scores for each subject. Green indicates that the postoperative score was significantly higher, red indicates that the postoperative score was significantly lower, and blue indicates no significant difference between preoperative and postoperative scores.The left panels, not surprisingly, show that speech perception in the implanted ear improved significantly for the vast majority of subjects . This expected result is more impressive when we consider that an additional 29 subjects could not be included because their preoperative score was listed as “could not test”, which almost always means that the subject in question did not have sufficient residual hearing in the ear to be implanted to understand any words without lipreading, so the score would have been zero. The same happened with speech perception in the binaural condition shown in the center panels: 62 out of 82 subjects showed statistically significant improvement when comparing to the latest datapoint, and this was the case for 61 subjects when comparing against the average postoperative score. For many of the remaining 19 subjects, it would have been impossible or at least very difficult to show a statistically significant improvement due to ceiling effects. Again, 41 subjects were not evaluated preoperatively in the binaural condition.p < 0.05) in their latest postoperative evaluation than they did preoperatively (right bottom panel) and 38 subjects did so when comparing their preoperative score to their average postoperative score. Twelve subjects obtained significantly higher scores in their latest postoperative evaluation compared to the preoperative one, and 11 showed a similar change when comparing their preoperative score to their average postoperative score. Remember that some of these “statistically significant” differences would have been observed even in the absence of any real change. Because 132 subjects were evaluated and a p-value of 0.05 was used as a threshold, we would expect an average of three subjects  to show significant increases and another three to show significant decreases. Thus, the number of subjects with postoperatively decreased scores in the hearing aid ear far exceeds what would be expected due to random variation in the absence of a real effect. This was true both at the site with the highest number of subjects  and in the rest of the sites, where this was true for 23 out of 79 subjects.A very different picture is seen when examining preoperative to postoperative changes in the nonimplanted ear (right panels). Here, we see that 41 subjects scored significantly lower (p < 0.001). Postoperative scores were significantly higher for the implanted ear and for the binaural condition, and lower for the nonimplanted ear. Because the results in the nonimplanted ear were somewhat unexpected, we also examined the preoperative–postoperative differences using increasingly strict criteria. First, we excluded ten subjects for whom proper hearing aid operation could not be confirmed. Then, we excluded subjects who showed more than 20, 10, or 5 dB of hearing loss in the nonimplanted ear during the course of the study. All preoperative-to-postoperative group differences were highly significant (p = 0.08). The middle panel shows the speech perception drop in the nonimplanted ear as a function of the implanted ear advantage . In other words, we examine the extent to which asymmetry between ears (with the implanted ear providing more information than the nonimplanted ear) may be associated with speech perception drops in the nonimplanted ear. That association seems to be supported by the correlation in the middle panel , but that may be a partially spurious effect driven by the fact that both variables are related to postoperative hearing aid scores. We address this issue in two ways. First, we found that the correlation becomes weaker but remains significant when partialling out the effect of postoperative hearing aid scores . Second, there was also a positive correlation between average postoperative CI speech scores and the speech perception drop in the nonimplanted ear  with age at cochlear implantation.None of the other preoperative demographic variables were significantly correlated with the drop in speech perception in the nonimplanted ear, although one of them came close. The drop in speech perception between the preoperative evaluation and the average postoperative score showed a weak (r = +0.106) nonsignificant correlation  gave the expected pre- vs. postoperative improvements in speech perception scores  . This reAlternatively (and this is a fourth possibility), it may be that speech perception decreases happened despite the continued use of acoustic input in the nonimplanted ear. One possible mechanism has to do with the adaptation that is required from postlingually deafened cochlear implant patients who have acoustic hearing in the nonimplanted ear. The frequency-place function imposed by a cochlear implant is typically different from the normal hearing frequency-place , and it The finding that drops in speech perception in the nonimplanted ear were correlated with implanted ear advantage  or even with the raw speech perception scores in the implanted ear suggests that the third or fourth possibilities discussed above  may underlie some of the observed changes. In other words, to the extent that speech perception in the implanted ear may be much better than in the acoustically aided ear, this may result in a central auditory process wherein the brain relies on the higher quality signal provided by the implant and disregards the input provided by the nonimplanted ear.A number of important caveats and suggestions for future research must be listed. Ideally, future studies should obtain more precise determinations of hearing aid fitting, particularly the gold standard of in-the-ear verification. Additionally, and given that both modern cochlear implants and hearing aids can log data about device use, it would be good to determine the extent to which the observed speech perception drops in the nonimplanted ear may or may not be related to the daily duration of hearing aid use, or the difference in daily duration of cochlear implant and hearing aid use.Taken together, the present results suggest that there are considerable individual differences in the way acoustic and electrical auditory input interact. Better understanding of this interaction may help guide future clinical practice in cochlear implant patients who have usable residual hearing.


The purpose of this study was to examine the effects of fish oil (FO) on RMR, substrate oxidation, and skeletal muscle SERCA and NKA pump function in healthy older individuals. Subjects  were randomly assigned into groups supplemented with either olive oil (OO) (5 g/day) or FO (5 g/day) containing 2 g/day eicosapentaenoic acid and 1 g/day docosahexaenoic acid for 12 weeks. Participants visited the laboratory for RMR and substrate oxidation measurements after an overnight fast at weeks 0 and 12. Skeletal muscle biopsies were taken during weeks 0 and 12 for analysis of NKA and SERCA function and protein content. There was a main effect of time with decrease in RMR (5%) and fat oxidation (18%) in both the supplementation groups. The kinetic parameters of SERCA and NKA maximal activity, as well as the expression of SR and NKA proteins, were not affected after OO and FO supplementation. In conclusion, these results suggest that FO supplementation is not effective in altering RMR, substrate oxidation, and skeletal muscle SERCA and NKA protein levels and activities, in healthy older men and women.Omega‐3 polyunsaturated fatty acids (PUFAs) have unique properties purported to influence several aspects of metabolism, including energy expenditure and protein function. Supplementing with n‐3 PUFAs may increase whole‐body resting metabolic rate (RMR), by enhancing Na +/K+ ATPase activities.This figure resumes the main finding of the study which is that fish oil did not affect SERCA and Na Specifically, the SERCA pump transports 2 Ca2+ ions across the SR membrane, while the NKA pump transports 3 Na+ and 2 K+ ions across the plasma and transverse tubule membranes  Can = 12; 60–76 years) after a 12‐week period of fish oil (FO) supplementation (5 g/day), while no changes were observed in the olive oil (OO) control group  and fat oxidation (+19%) in community‐dwelling sedentary females  and females (n = 16) volunteered to participate in this study .Healthy physically active  older adult males  in the morning following an overnight fast and performed a familiarization trial where RMR and substrate oxidation were measured. After 1–5 days, a dual energy X‐ray absorptiometry (DXA) scan was performed. Subjects were subsequently matched for RMR, age, and body composition and were randomly assigned in a double‐blinded manner to the FO or OO supplement group. Participants then visited the laboratory in the morning at two different time points (during weeks 0 and 12) at the same time of day (±2 hr) for measurements of RMR and substrate oxidation. Fasted blood samples were taken immediately after these measurements. In addition, resting skeletal muscle biopsies were performed at weeks 0 and 12 in a subset of FO  and OO  participants.2.3n = 12, 8 females) supplemented with 5 capsules of OO  for a total of 5 g OO/day. The FO group  was supplemented with 5 capsules/day of Omega‐3 Complete , where each capsule provided 0.4 g of EPA and 0.2 g of DHA in triglyceride (TG) form, for a total of 2 g EPA and 1 g DHA/day. Subjects in both the groups were encouraged to take capsules throughout the day . To promote supplement compliance, subjects were only given 2–3 weeks of capsules at a time. Written and oral reminders were also given periodically to ensure capsules were consumed, and diet and exercise practices remained consistent throughout the study.The OO group (2.42) and carbon dioxide production (VCO2). Respiratory exchange ratio was calculated as VCO2/VO2. Whole body resting fatty acid (FA) oxidation, carbohydrate (CHO) oxidation, and RMR were calculated using the following equations . Participants were instructed to lie supine on a bed for ~30 min in a quiet, darkened room and were asked to breathe normally through a Hans Rudolph mouthpiece with nose clip during the last 18 min. Respiratory measurements were collected using a metabolic cart (AEI MOXUS II Metabolic System) and the last 10 min of data were analyzed for resting oxygen consumption , the cholesterol/HDL‐c ratio, glucose, and high‐sensitivity C‐reactive protein (hsCRP).For FA analysis, a second aliquot of blood was centrifuged to separate plasma and red blood cells (RBCs). These fractions were aliquoted and stored at −80°C until further analysis. The FA profile of RBC samples was measured by gas chromatography, as previously described  was measured using DXA (Hologic Discovery Wi). Participants were asked to refrain for 1 hr from food and drink, and were asked to lie on the scanning bed for ~6 min. X‐rays of two different energies were emitted from below the subject and were detected by a moving “arm” above, from head to toe. Prior to use, the DXA was calibrated using a standard calibration block of thermoplastic acrylic resin, according to the manufacturer's instructions.2.7Two to four resting muscle biopsies  were obtained under local anesthesia (2% lidocaine without epinephrine) from the vastus lateralis muscle, using the percutaneous needle biopsy technique described by Bergstrom . A porti2.7.12, 1 EGTA, 10 NaN3, 5 ATP, and 10 phospho(enol)pyruvate (pH 7.0 at 37°C). Before starting the reaction, 18 U/ml lactate dehydrogenase (LDH), 18 U/ml pyruvate kinase (PK), 5 μl homogenate, and 0.2 mM NADH were added to a cuvette containing ~1.5 ml of reaction buffer in the presence or absence of 40 μM ionophore . Assays were performed in duplicate at 37°C using a fluorometer  set with an excitation wavelength of 340 nm and an emission wavelength of 460 nm. SERCA activity was assessed by adding 15 μl of 10 mM CaCl2. SERCA activity increases with free Ca2+ concentrations ([Ca2+]f) until a plateau occurs, indicating maximal activity. The [Ca2+]f corresponding to each CaCl2 addition was determined using an online calculator  of NADH from a standard curve established with the same reaction conditions. SERCA activity was obtained by subtracting ATPase activity in the presence of 40 μM cyclopiazonic acid (which completely inhibits SERCA) from total ATPase activity at different [Ca2+]f.Total SERCA activity was measured using a fluorometric assay , the [Ca2+]f needed to obtain half‐maximal activity, and the Hill coefficient. To obtain the kinetic properties of the enzyme, SERCA activity was plotted against the negative logarithm of [Ca2+]f (pCa). The Vmax represented the peak value, whereas the pCa50 represented the pCa obtained from a sigmoid fit of the data, which yields 50% of Vmax. The Hill coefficient was determined through a nonlinear regression (variable slope) with Prism software  by using a portion of the curve that corresponded to between 20% and 80% of maximal activity, using the following sigmoidal dose–response equation:Y is the plateau, Ybot is the value at the bottom of the plateau and Ytop is the value at the top, log Ca50 is the concentration that gives a response halfway between Ybot and Ytop, and nH is the Hill coefficient.The kinetic properties of SERCA that were assessed included the maximal activity pyruvate, 3 ATP (pH 7.4), in the presence of 18 U/ml of PK and LDH, 25 μM of blebbistatin, and 0.3 mM NADH. Assays were performed in duplicate using a fluorometer  set with an excitation wavelength of 340 nm and an emission wavelength of 460 nm. Maximal NKA activity was measured by addition of 80 mM NaCl. The rate of NKA activity was calculated from the negative slope (fluorescence second−1) of NADH from a standard curve established with the same reaction conditions. NKA maximal activity was obtained by subtracting ATPase activity in the presence and absence of 2 mM ouabain (ouab), using the following equation:Y[Na] is the change in fluorescence in the presence of 80 mM Na, Y[ouab] is the change in fluorescence after addition of 2 mM ouab, and ‘m’ is the slope obtained from the standard curve. One participant from each supplement group was excluded due to an inability to accurately measure NKA activity.Measurement of NKA activity was performed with an enzyme‐coupled method using an ATP regenerating assay , SERCA2a , calsequestrin 1 (CSQ1) , CSQ2 , NKA α1 , NKA α2 , NKA β1 , NKA β2 , NKA β3 , and α‐tubulin . All samples for a given target were transferred onto a single membrane to limit variation. All samples were normalized to α‐tubulin.2.10SEMs). A two‐way repeated measures ANOVA was used to examine the effects of treatment group and time on body composition, blood measurements, RMR, and substrate oxidation. A paired t test was used to examine the independent effects of treatment on skeletal muscle ATPase activities and protein expression. Statistical significance was considered as p ≤ .05. GraphPad Prism, Version 7.0  was used for all statistical analyses and all data were checked for normality before analysis.All data are presented as means ± standard error of the means  and HDL‐c was increased (p < .05) in the FO group (Table p < .0001) after FO supplementation from 0.72 ± 0.05% to 3.43 ± 0.36%, and from 6.39 ± 0.24% to 8.28 ± 0.16%, respectively . However, there was a main effect of time with decrease in RMR (p < .01) and fat oxidation (p < .01) in both the supplementation groups  or FO  supplementation  has no effect on whole‐body RMR and substrate oxidation in healthy older adults. Further, we also demonstrated that FO supplementation did not affect skeletal muscle SERCA permeability, SERCA and NKA activities, or the content of SR calcium handling and NKA proteins. Altogether, these results demonstrate that RMR and skeletal muscle NKA and SERCA activities are not affected by n‐3 PUFA supplementation in older adults.n = 12; 60–76 years) showed a significant increase in RMR (14%) and fat oxidation (19%) following 12 weeks of FO supplementation , while the OO control group reported no change on either RMR or substrate oxidation  in the present study, BMI and body fat percentages of the female subjects were ~15% lower than those reported in females from the previous study by Logan & Spriet (After the 12‐week supplementation period, we observed decrease in RMR and fat oxidation, and no change in CHO oxidation, with both OO and FO. These results suggest that n‐3 PUFA supplementation had no effect on RMR and substrate oxidation, consistent with recent investigations which reported no change on RMR and substrate oxidation after n‐3 PUFA supplementation in healthy young individuals (Jannas‐Vela et al., & Spriet . As RMR In‐line with previous work (Eslick, Howe, Smith, Priest, & Bensoussan, 2+ uptake/release across the SR membrane (Dulloo, Decrouy, & Chinet, While evidence is mounting to suggest that n‐3 PUFAs do not regulate whole‐body energy metabolism in humans, it is possible that the molecular changes affecting RMR after FO supplementation cannot be detected by the current methods of indirect calorimetry (Jannas‐Vela et al., We also observed that NKA maximal activity and protein levels were not affected after FO supplementation. This result contradicts previous findings in rodents showing that heart microsome membrane DHA content was positively associated with maximal NKA activity Hulbert, . It is pWe recently showed distinct incorporation of n‐3 PUFAs into human skeletal muscle plasma and mitochondrial membranes after FO supplementation, where the sarcolemma was less responsive than whole muscle and mitochondria (Gerling et al., The rationale to suggest that n‐3 PUFAs could increase energy expenditure were based on past studies in rodents that reported a positive association between DHA content in cell membranes and increased RMR Hulbert, , increasIn conclusion, this study has demonstrated that supplementation with FO in healthy, active older adults had no effect on RMR and whole‐body substrate oxidation. In addition, supplementation with FO did not affect SERCA and NKA activities or the expression of SR calcium handling and NKA proteins in skeletal muscle. Altogether these results show that FO does not affect RMR, and skeletal muscle SERCA and NKA activities and protein levels in healthy older adults.The authors declare no conflict of interest.It should say:SJ‐V, GJFH, GPH, DMM, and LLS contributed to the conception of the study. SJ‐V, SLK, DTC, KAW, GJFH, GPH, and LLS assisted with measurements. SJ‐V, GPH, and LLS analyzed and interpreted the data, and wrote the first draft of the manuscript. All authors read and approved the final manuscript.


Existing evidence indicates that both iron deficiency anemia and sickle cell anemia have been previously associated with hearing loss. However, human data investigating the association between anemia and auditory threshold shifts at different frequencies in the adolescent, adult and elderly population are extremely limited to date. Therefore, this cross-sectional study used the dataset from the US National Health and Nutrition Examination Survey from 2005 to 2012 to explore differences in low- or high-frequency hearing thresholds and hearing loss prevalence between participants with and without anemia. A total of 918 patients with anemia and 8213 without anemia were included. Results indicated that low- and high-frequency pure tone average were significantly higher in patients with anemia than that in those without anemia in the elderly, but not in adult or adolescent population. In addition, the prevalence of low-frequency hearing loss but not high-frequency hearing loss was also higher in patients with anemia than in those without anemia in the elderly population. After adjusting various confounders, multiple regression models still indicated that patients with anemia tended to have larger threshold shift. In conclusion, anemia was associated with auditory threshold shifts in the elderly population, especially those vulnerable to low-frequency hearing loss. Approximately 466 million people worldwide have disabling hearing loss in 2018 . HearingIn a Taiwan population-based study, Chung et al. found that the odds ratio (OR) for having a previous IDA diagnosis among patients aged ≥18 years with sudden sensorineural hearing loss was 1.34 , 1.11–1.61), which was most pronounced among those aged ≤44 years compared with controls  .Although IDA and SCA mechanisms that lead to hearing loss differ, hemoglobin may play a common and critical role in the deterioration of hearing function. In addition, typical changes in age-related hearing loss usually start with a hearing loss on high frequencies , and thehttps://wwwn.cdc.gov/nchs/nhanes/). This survey includes information about questionnaires, demographic data, laboratory tests, and physical examinations, among others. All the study participants in the NHANES are sampled from the residents in the United States. Its research protocols were approved by the Research Ethics Review Board of National Center for Health Statistics. All participants provided written informed consents. Because we only used de-identified secondary data from NHANES, this study was exempted from full review by the Institutional Review Board.The data used in this cross-sectional study were derived from the NHANES in the United States  during the test were also excluded. All audiometry examinations were carried out by professionally trained audiologists from the National Institute for Occupational Safety & Health using the interacoustics model AD226 audiometer (with standard TDH-49 headphones and Etymotic EarTone 3A insert earphones). The hearing threshold test in this study was conducted on both right and left ears at 0.5, 1, 2, 3, 4, 6, and 8 kHz across an intensity range of −10 to 120 decibels (dB). Full audiometric protocols and procedures are available online.In the relevant analyses of this study, PTA thresholds of hearing at 0.5, 1, and 2 kHz were identified as the low-frequency PTA (low PTA) and PTA thresholds of hearing at 3, 4, and 6 kHz were identified as the high-frequency PTA (high PTA) . AdditioTo eliminate the potential effects of some confounders and investigate the actual relationship between anemia and HL, participants’ age group, sex, race, hypertension, diabetes, coronary heart disease, heart failure, and stroke were considered in the regression models. In this study, the race was categorized as non-Hispanic white, non-Hispanic black, or others . In addition, participants were considered to have medical history of hypertension, coronary heart disease, heart failure, and stroke if participants self-reported to have medical history about these diseases according to their physicians. Moreover, patients self-reported to have diabetes by physicians or other health professionals or receive diabetic medications were identified as diabetes cases.p-value of 0.05 was used to determine the statistical significance of this study.All analyses were performed with the SAS system . This study used the chi-squared tests to explore differences in age group, sex, race, hypertension, diabetes, coronary heart disease, heart failure, and stroke, LFHL, HFHL, HL, between participants with and without anemia. Independent t-tests were further conducted to compare differences in low PTA and high PTA between participants with and without anemia. Additionally, regression models were carried out to estimate the effects of anemia on PTA thresholds of hearing. A two-sided p < 0.001), sex (p < 0.001), and race (p < 0.001) between the two groups. In addition, the prevalence of hypertension (p < 0.001), diabetes (p < 0.001), coronary heart disease (p < 0.001), heart failure (p < 0.001), and stroke (p < 0.001) between participants with anemia and controls without anemia was significantly different.This study consisted of 918 participants with anemia and 8213 without anemia. p < 0.001). LFHL was observed in 42.59% of participants with anemia and in 32.66% of controls without anemia (p < 0.001). Furthermore, the HFHL was found in 61.44% of participants with anemia and in 51.88% of controls without anemia (p < 0.001).Because age is an important factor in the development of HL, p < 0.001) and 2.33 for the left ear (p < 0.001), and β coefficients of the low PTA were 1.53 for the right ear (p < 0.001) and 2.25 for the left ear (p < 0.001) in the overall population. Additionally, β coefficients of the high PTA comparing participants with anemia to controls without anemia were 3.76 for the right ear (p = 0.002), and 4.03 for the left ear (p = 0.001) and β coefficients of the low PTA were 3.55 for the right ear (p < 0.001) and 4.64 for the left ear (p < 0.001) in the elderly population. Additionally, this study performed the regression models to investigate the relationships between PTA and anemia according to different sexes and ethnic groups  [Previous studies indicated that IDA is a potential risk factor for ischemic stroke , and pat = 0.27) . HoweverPresbycusis, or age-related HL, is a common disorder characterized by symmetrical progressive loss of high-frequency hearing over the years. The World Health Organization estimates that >500 million people aged >60 years worldwide will suffer significant impairment from presbycusis by 2025 [This study had several limitations. First, all data in NHANES are cross-sectional, and thus this study cannot establish the causal relationship between anemia and hearing threshold shift. Second, we cannot assess environmental, occupational, or recreational noises around the participants included in this study. Previous reports have highlighted the significance of noise-induced HL from both work and recreational activities ,27. ThirIn conclusion, our study demonstrated that anemia was associated with LFHL in the elderly population. However, further larger epidemiologic studies need to be conducted to confirm the effects of anemia on auditory threshold shifts in different ethnic groups and countries.


Since then, 368 papers containing the term ‘telocyte’ have been published, 261 of them in the last five years. These numbers underscore the growing interest in this cell type in the scientific community and the general acceptance of the name telocyte to indicate this interstitial cell. Most of these studies, while confirming the importance of transmission electron microscopy to identify the telocytes with certainty, highlight the variability of their immune phenotypes. This variability was interpreted as due to (i) the ability of the telocytes to adapt to the different sites in which they reside; (ii) the distinct functions they are likely to perform; and (iii) the existence of telocyte subtypes. In the present paper, an overview of the last 10 years of literature on telocytes located in the gut will be attempted, confining the revision to the morphological findings. A distinct chapter will be dedicated to the recently hypothesized role of the telocytes the intestinal mucosa. Through this review, it will be shown that telocytes, despite their variability, are a unique interstitial cell.Ten years ago, the term ‘telocyte’ was introduced in the scientific literature to describe a ‘ The oval shaped body carries a clear nucleus containing a large nucleolus. The cytoplasm is basophilic. Under the transmission electron microscope (TEM), fibroblasts show a large Golgi apparatus, extended rough endoplasmic reticulum, and several mitochondria. The membrane is devoid of caveolae and basal lamina. They never form cell-to-cell contacts and are responsible for the extracellular matrix (EM) synthesis. The exact identity and role of the fibrocytes are under debate: are they quiescent fibroblasts or, instead, correspond to the telocytes (TCs) [mast cells are round or oval shaped and contain a central nucleus and numerous basophilic and metachromatic granules enclosing molecules that are able to regulate the exchanges between blood and tissue. Dispersed adipocytes are often seen in the loose connective tissue, especially in large mammals and humans. They are unilocular adipocytes whose large body is filled by a single fat vacuole containing triglycerides. The heterochromatic nucleus is restricted to the periphery, as are the other organelles. Under the TEM, adipocytes show a prominent smooth endoplasmic reticulum and numerous pinocytotic vesicles involved in lipid biosynthesis and transport. These cells are surrounded by a basal lamina and by reticular fibers. Adipocytes are support cells carrying energy store, cushioning, and padding functions.There are many types of cells in the loose connective tissues. Some of them are autochthon, while others migrate in these tissues to perform their functions. Examples of the former case are fibroblasts, fibrocytes, mast cells, and adipocytes. The es (TCs) . The masThe migrating cells are the macrophages and other immune cells.All of the above-mentioned cells are also called interstitial cells (ICs) or stromal cells. All of them originate from the mesenchyma.myoid cells  apparatus contains almost all the above-listed ICs as well as other particular ICs: the interstitial cells of Cajal , other roblasts ) while oroblasts  interstiroblasts ,8).The term TC was proposed based on the cell unique TEM features ,9. The ‘Since  was publTo date, the identity of TCs is still being debated and, even in the most recent literature, an overlap between them and other interstitial cells is frequent. Most of the difficulties in giving a definitive uniqueness to TCs depend on two elements: (i) the absence of a selective immune marker; and (ii) the unknown or, only partially known, role(s) these cells play in the organs. Probably, the two elements are related, namely, the TCs express different immune markers according to the role(s) they play. Currently, there are two markers that, although not exclusive for this cell type, are commonly considered reliable to label the TCs, CD34 and PDGFRα.Starting from these immunohistochemical (IHC) data, some researchers have chosen to call the TCs, ‘PDGFRα-positive cells’, and this definition is often used in scientific reports. However, we consider this nomenclature to be limited for some reasons. First, and most significant, not all the TCs are PDGFRα-positive ,11; secoIn the gut muscle coat, the TC network runs parallel or even intercalates with that formed by the ICCs ,16,17,18Unlike ICCs, TCs are also present in the submucosa where their network envelops the local ganglia, forms tight meshes in the loose connective tissue, embraces the muscularis mucosae, and terminates in the axes of the intestinal villi  bordering the gastric glands, the intestinal crypts, and the entire profile of the epithelium ,20. To nIn the submucosa, fibroblasts and fibrocytes are also present, but only TCs are organized in the network. In the mucosa, the TC network lines myoid cells, fibroblasts, and, possibly, Myo. However, only TCs are PDGFRα- and CD34-positive ,17. It iIn summary, the TCs are always organized in networks and, most likely, this condition is mandatory to perform their functions.The most obvious function of this network in the gut is to be a scaffold that guarantees mechanical and rheological support to the connective components during peristalsis and maintains the integrity of the ganglia  during bfibroblasts and TCs coexist in the interstitium of almost all organs [TCs could also have the role of presiding over the 3-D organization and maintenance of the extra-cellular matrix (ECM) usually produced by fibroblasts. Indeed, in adult life, l organs  and, soml organs ,24. Final organs ,26.smooth muscle cells, SMCs).In the gut, TCs interact with a large variety of connective cells by establishing functional communications and cell-to-cell contacts ,16,17,27In the stroma of the gut and of many other organs, TCs are often closely apposed to immune cells , and in the narrow spaces between them, small exosomes are present. It has been assumed that the TCs have a role in immune-regulation and immune-surveillance. Shortly, the TCs, by exocytosis vesicles, could release soluble chemoattractant molecules along their processes, acting as guides for the immune cells, and either by their contacts or exosomes, present tissue derived antigens to these cells ,12,14,27macrophages that share an anti-inflammatory phenotype [Although macrophages belong to the immune cell class, they deserve a separate discussion because of the peculiar role attributed to these cells in the muscle coat of the GI apparatus. In fact, recent studies have demonstrated the existence of GI resident henotype  and whoshenotype . These Ghenotype ,31. By uhenotype .(i)The TCs might be adult stromal mesenchymal cells able to differentiate in ICCs ,26. Nota(ii)ICCs are constantly and often richly innervated and participate in the control of the muscle wall activity as intermediaries of neuronal actions ,36,37,38(iii)TCs and ICC are involved in the SIP  syncytium (see below).In the gut muscle coat, the TC and ICC networks are parallel and sometimes intermingled, forming cell-to-cell contacts with each other  15,16,1,116,17. The TCs form several types of contact with the SMCs (27). As above-mentioned, the TCs might mediate the macrophage actions on the SMCs, intercalating between one another. Furthermore, the finding that the TCs established cell-to cell contacts with both the SMCs and the ICCs , has broInterestingly, although synaptic-like contacts between nerve varicosities and TCs were not observed under the TEM, it has been demonstrated that TCs express purine receptors P2Y1 and apamin-sensitive SK3 channels and respond to agonists and antagonists to these receptors ,41,42,43The presence and roles of the TCs in the gut mucosa need to be separately treated because of the peculiar function(s) TCs might play at this level and because of the necessity to also clarify the TCs’ identity, nomenclature, and distribution in the mucosa.muscularis mucosae.The mucosa is made by the columnar epithelium plus the annexed glands and the underlying thin layer of loose connective tissue, the Lamina Propria (LP), which, despite its thinness, hosts nerve fibers, blood and lymphatic vessels, and numerous and assorted cells . The mucosa is separated from the submucosa by the As reported above, the submucosal TC network continues up to the mucosa and forms an elaborate 2-D structure lining the epithelium in a strategic position for the maintenance of epithelium integrity  and functionality (absorption). In the following, the roles hypothesized for the mucosal TCs are reported.new’ cell type present in the intestinal LP.In the last decade, the presence of thin and elongated cells organized in networks in the LP of intestinal villi has attracted the attention of researchers. Using fine methodologies, Furuya et al. , describfibroblasts [fibroblasts organize in a network [fibroblasts and TCs [Undeniably, ascertaining the proper identity of each cell type located in the LP is challenging because of the presence of numerous cells in a thin space. Discriminating among them under a light microscope is often difficult, unless a selective marker is available. More recently, Kurahashi et al. ,20, usinroblasts  are the  network  is consi and TCs . Moreove and TCs ,48. All The possibility that TCs could induce cell differentiation and/or preserve the stem cell status of the precursors was primarily considered for the myocardium ,50,51,52All these articles, while confirming previous findings ,59, add To correctly recognize the identity of the subepithelial nursing cells, it is useful to mention the results obtained by Kurahashi et al. ,20 and bDifferences in the expression of the labeling for the TCs are not a novelty and these differences have been related to the diverse functions the cells might play. For example, Vannucchi and coworkers showed the presence of three subtypes of TCs in human urinary bladder lamina propria based on either IHC or TEM observations and each subtype was supposed to play a proper role . KurahasEstablishing the right identity of the nursing cells is fundamental both from the biological and pathological point of view. In the latter case, it is important to consider that in inflammatory, degenerative, and tumor GI diseases, events such as proliferation, differentiation, and apoptosis play a major role. Therefore, the identification of the cells involved in the regulation of these events could be crucial to direct the pharmacological intervention ,59.TCs are ubiquitous and unique cells whose name is highly suggestive of their ultrastructural peculiarities and TEM is the best instrument to identify these cells with certainty.In the GI apparatus, all the TCs express the CD34, as also confirmed by immune-TEM, and PDGFRα; these two markers are reliable to identify the TCs among those IC that share a similar shape and territory. Furthermore, immunohistochemical findings indicate that TCs neither express c-Kit nor αSMA; nevertheless, attention must be paid when the TCs are cultured in vitro. PDGFRα is the most widely used labeling to identify the TCs and several authors call the TCs “PDGFRα- positive cells”. The main limitation of this nomenclature is that not all the TCs express this marker.TCs always form networks and this organization is probably essential to carry out the many roles attributed to these IC. Many of these roles are probably common to all locations where TCs have been featured. However, being able to identify them in the intestine is fundamental for a better understanding of the physiology of gastrointestinal functionality and the etio-pathogenetic mechanisms underlying many gut diseases.TCs are the mechanical support during gut movements.TCs organize the ECM, compartmentalize, and restrain the other IC  inside their meshes.TCs take several types of cell-to-cell contacts with almost all the other IC and, through the production of exosomes, might interact with all the cells present in the gut wall, neurons included.TCs express receptors and contain molecules involved in the neurotransmission and might participate in the control of the GI functions in terms of absorption and motility.TCs might be mesenchymal stem cells as precursors of the ICCs (and possible of other IC);TCs lining the gut epithelium are likely involved in controlling the proliferation and differentiation of cryptal stem cells behaving as nurse cells.In summary:


As the population ages, increased prevalence of cognitive and sensory impairments may pose growing public health challenges. Among the nine modifiable risk factors for dementia, the highest percentage (9%) of dementia cases are attributed to hearing impairment. While much research has examined the relationship between hearing impairment and cognition, almost none has translated these relationships into a meaningful, life course metric: how many years of life individuals can expect to live with both impairments and how hearing impairment affects years lived with cognitive impairment. Our study fills this gap by using Bayesian multistate life table methods applied to nine waves of the Health and Retirement Study (1998-2014) to estimate years of life to be spent (1) with/without hearing and cognitive impairment, and (2) with/without cognitive impairment, conditional on having versus not having hearing impairment. Preliminary results for aim 1 reveal that at age 50, individuals will live 18.9 (18.7-19.2) years healthy, 4.3 (4.2-4.5) years hearing impaired but cognitively intact, 4.2 (4.0-4.3) years hearing unimpaired but cognitively impaired, and 2.3 (2.2-2.6) years with both impairments. Women will spend more years healthy, hearing unimpaired but cognitively impaired, or with both impairments; men will spend more years hearing impaired but cognitively intact. People with more education will spend more years hearing impaired but cognitively intact; people with less education will spend more years hearing unimpaired but cognitively impaired or with both impairments. Our study is one of the first to investigate the implications of hearing impairment for years of cognitively impaired life.


Potassium voltage-gated channel subfamily q member 4 (KCNQ4) is a voltage-gated potassium channel that plays essential roles in maintaining ion homeostasis and regulating hair cell membrane potential. Reduction of the activity of the KCNQ4 channel owing to genetic mutations is responsible for nonsyndromic hearing loss, a typically late-onset, initially high-frequency loss progressing over time. In addition, variants of KCNQ4 have also been associated with noise-induced hearing loss and age-related hearing loss. Therefore, the discovery of small compounds activating or potentiating KCNQ4 is an important strategy for the curative treatment of hearing loss. In this review, we updated the current concept of the physiological role of KCNQ4 in the inner ear and the pathologic mechanism underlying the role of KCNQ4 variants with regard to hearing loss. Finally, we focused on currently developed KCNQ4 activators and their pros and cons, paving the way for the future development of specific KCNQ4 activators as a remedy for hearing loss. The lensequence  [11]. ThKCNQ1 are known to cause heart diseases, including long QT syndrome (MIM 192500), and Jervell and Lange-Nielsen syndrome (MIM 220400) in autosomal dominant and recessive manners, respectively . In. In pore region mutation, which results in unresponsiveness to KCNQ activators [+ channels or variants in the two cytoplasmic termini of KCNQ4 to investigate the in vivo efficacy of channel openers. In addition, the effectiveness of KCNQ4 activators needs to be examined in mouse models of noise-hearing loss.Although some compounds could activate KCNQ4 in vitro, none of them were potent in inducing the native I in OHCs . The mol in OHCs ; howevertivators ,42. TherKCNQ4 mutation might also be an option for clinical application of KCNQ4 activators in deafness treatment with advantages of reducing the cost and shortening the time when compared to de novo drug discovery. In addition, the half-life duration and bioavailability of drugs targeting the activation of KCNQ4 would also be required to satisfy clinical degrees.More importantly, KCNQ4 activators should be validated in clinical trials, as there is no ongoing clinical trial targeting hearing loss by KCNQ4 activators currently. Target population, such as individuals with genetic hearing loss or noise-induced hearing, should be carefully selected. Both the therapeutic window and convenient application methods are important issues to be discussed in clinical trials. Drug repurposing and optimization for applicable specific


In cocultures of human platelets and coronary artery smooth muscle cells (CASMC), we found that platelets induced morphologic changes and enhanced the migration of CASMC. The exposure of platelets to Aspirin [an inhibitor of cyclooxygenase (COX)-1] reduced the generation of TXA2 and prevented the morphological and functional changes induced by platelets in CASMC. Platelet-derived TXA2 induced COX-2 and enhanced prostaglandin (PG)E2 biosynthesis in CASMC, a known mechanism promoting neointimal hyperplasia. COX-2 induction was prevented by different antiplatelet agents, i.e., Aspirin, the TP antagonist SQ29,548, or Revacept (a dimeric soluble GPVI-Fc fusion protein). The administration of the novel antiplatelet agent Revacept to C57BL/6 mice, beginning three days before femoral artery denudation, and continuing up to seven days after injury, prevented the increase of the systemic biosynthesis di TXA2 and reduced femoral artery intima-to-media area and the levels of markers of cell proliferation and macrophage infiltration. Revacept might serve as a therapeutic agent for percutaneous coronary angioplasty and stent implantation.Neointima hyperplasia is a crucial component of restenosis after coronary angioplasty. We have hypothesized that enhanced generation of platelet-derived thromboxane (TX)A Thromboxane (TX)A2, a primary product of arachidonic acid metabolism in activated platelets via the cyclooxygenase(COX)-1 pathway, promotes migration and proliferation of vascular smooth muscle cells(VSMC)5. TXA2 operates through the binding to TXA2 receptors (TP)6, which by coupling to Gα 12/13 lead to YAP/TAZ activation; they are the major downstream effectors of the Hippo signaling pathway7. Injury-induced vascular proliferation is reduced in mice lacking the TP receptor or treated with a TP antagonist8. In patients undergoing PTCA, enhanced systemic biosynthesis of TXA2  was largely suppressed by low-dose Aspirin10, thus suggesting that this biochemical change reflects platelet activation at the site of vascular damage10.Collagen exposure at the vascular damage site leads to platelet adhesion and aggregation mainly via the Glycoprotein (GP) VI signaling1. These events constitute the neointimal hyperplasia, which contributes to postprocedural lumen loss. In animal models, peak neointimal growth is observed 28 days following bare-metal stents placement, whereas in humans, it is identified at 6–12 months11.Restenosis is an excessive response of the coronary artery to damage during angioplasty. It consists of platelet activation, inflammatory cell recruitment, VSMC proliferation, and migration to the intima. VSMC change their phenotype from contractile to synthetic, which promotes the extracellular matrix synthesis3, plays a role in restenosis progression12. COX-2 is markedly upregulated in vascular inflammation, such as atherosclerosis and balloon-injured arteries14. Pharmacological inhibition or specific deletion of COX-2 in VSMC reduces vascular neointima hyperplasia in response to mechanical injury14. COX-2 mediates these effects by enhancing the generation of prostaglandin (PG)E2 that contributes to vascular restenosis pathogenesis through the activation of the PGE2 receptor subtype EP3α/β and its signaling pathways cAMP/protein kinase A and phosphatidylinositol 3-kinase12. Moreover, genetic disruption of microsomal PGE2 synthase-1 attenuates neointima formation after vascular injury15. However, the pharmacological inhibition of vascular COX-2 by the use of selective COX-2 inhibitors (named coxibs) is not recommended in this setting for their cardiovascular hazard16.COX-2, an inducible enzyme that mediates the generation of prostanoids in inflammation17 through their adhesion to VSMC, at the site of the vascular damage, facilitated by subendothelial collagen exposure, and to the release of soluble factors, such as lipid mediators derived from arachidonic acid 18.In the early phase of restenosis, platelets are the chief cellular players19. Using fluorescence-labeled glycoprotein VI (GPVI)-Fc in ApoE-deficient , the mechanical-induced carotid injury was associated with increased GPVI-Fc-binding to injured carotids compared to intact carotids20.Revacept, a novel antiplatelet agent in clinical development, is a soluble form of the platelet GPVI receptor that binds specifically to collagen at vascular damage sites, thus inhibiting platelet adhesion and aggregation without affecting general hemostasis in humans2 is the trigger of the induction of COX-2-derived-PGE2, a pathway involved in the development of neointimal hyperplasia12. Then, we tested whether Revacept attenuates neointimal formation and prevents enhanced systemic TXA2 biosynthesis in mice after arterial injury. Our findings suggest that the inflammatory response to vascular damage after coronary angioplasty might be mitigated by avoiding platelet-vessel wall interaction and preventing enhanced TXA2 biosynthesis using Revacept.In cocultures of human platelets and coronary artery smooth muscle cells (CASMC), we addressed the hypothesis that platelet-derived TXA2 in air, in the culture system BulletKit (Lonza) [containing SmBM  and SmGM-2 (Smooth Muscle Cell Growth Medium-2) SingleQuots supplements] and 5% fetal bovine serum (FBS). Then, CASMC (0.8 × 105 cells) were seeded in a six-multiwell plate coated with collagen type I from rat tail in 2 ml of culture system containing 5% FBS. After 48 h (h), the medium was changed with Dulbecco’s modified Eagle’s medium (DMEM) containing 0.75% bovine serum albumin (BSA)  and 1% polymyxin-B sulphate (Sigma-Aldrich) and human washed platelets (0.5 × 108 cells in 100 μl), freshly isolated from concentrated buffy coats , as previously described21, were added. This study was carried out following the recommendations of the Declaration of Helsinki. Healthy volunteers (23–45 years) who had not taken any nonsteroidal antiinflammatory drug (NSAID) in the 2 weeks before blood donation were enrolled. Informed consent was obtained from each subject. All experimental protocols were approved by the Blood Center , local Ethics Committee of "G. d’Annunzio" University of Chieti-Pescara. Separate experiments (n) were performed using different buffy coats.CASMC (at passage 5)  were cultured in flasks coated with collagen type I from rat tail , at 37 °C in a humidified atmosphere of 5% CO5 cells) were cultured alone or cocultured with platelets (0.5 × 108) for 8 h; then, cells were washed twice with phosphate buffer solution (PBS) pH 7.4 and fixed in acetone/methanol (40:60) for 20 min at room temperature. Cells were blocked with a filtrated solution of BSA (3%) in PBS for 30 min at room temperature. Cells were incubated overnight at 4 °C with polyclonal antibodies anti-COX-1 or anti-COX-2  and anti-α-SMA . Cells were then washed three times with PBS and incubated with the secondary antibodies, donkey anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 546  for 1 h at room temperature. Cells were washed three times with PBS and incubated for 5 min with 4′,6-diamidino-2-phenylindole  to label nuclear DNA. Finally, cells were washed and mounted in slides with Diamond antifade mounting media (Life Technologies). Slides were observed with a Zeiss LSM 510 meta microscope , and confocal images were analyzed using Zeiss LSM 5 series software (Carl Zeiss).CASMC ] were added to CASMC for 1 h, and then, the medium was changed, replaced with the platelet cell suspension, and incubated for 20 h.The selective COX-1 inhibitor Aspirin , the selective COX-2 inhibitor Rofecoxib , and the TP antagonist SQ 29,548 (Sigma-Aldrich) were dissolved in dimethylsulfoxide . Rofecoxib (0.3 μM) or SQ 29,548 (10 μM), or DMSO were added to CASMC for 30 min before the addition of platelets. In other experiments, platelets were pretreated with Aspirin (100 μM) or DMSO before the incubation with CASMC; briefly, platelet-rich plasma was incubated with Aspirin (100 µM) or DMSO for 30 min at room temperature; then platelets were isolatedg for 5 min, and stored at − 80 °C until assayed for prostanoid levels.CASMC or platelets cultured alone or cocultured were lysed in 1%Triton and 1 mM phenylmethylsulphonyl fluoride (Sigma-Aldrich) and stored at − 80 °C until assayed by Western blot. In some experiments, the supernatants were collected, centrifuged at 700×23. Briefly, after 20 h of platelet-CASMC cocultures, platelets were washed away. Cells, seeded on a 6-multiwell dish, were scraped using a 200 μl pipette tip, simulating a wound, incubated in DMEM 0.75% BSA, and monitored periodically by light microscope up to 24 h. Image processing was performed using Image J 1.44 software , and the percentage of cell-free area and cell covered area were calculated using the analysis particle tool.Cell migration of confluent CASMC cultured alone or with platelets, pretreated, or not with Aspirin, was evaluated by scrape wounding assay, as previously described2 and 6-keto-PGF1α , PGD2, PGE2, and PGF2α, were measured in cell culture media by previously described and validated immunoassay techniques24.TXB23. Anti-COX-2, anti-COX-1 polyclonal antibodies , GAPDH polyclonal antibody , or anti-β-actin polyclonal antibody  were used23. Quantification of optical density (OD) of different specific bands was carried out using Alliance 4.7 and Alliance 1 D software  and normalized to the OD of β-actin or GAPDH.Cell lysates were mixed with sodium dodecyl sulfate (SDS)(Sigma-Aldrich) sample buffer and heated to 95 °C for 5 min. Proteins (15 μg) were analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE)Male C57BL/6 mice  were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. . All animals were maintained and used according to the veterinary guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Chinese Academy of Sciences, Shanghai (China). Experimental protocols were approved by the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Chinese Academy of Sciences, Shanghai (China). Every effort was made to minimize the number of animals used and any pain or discomfort experienced by the animals.26. A contralateral  sham surgery was used as a control by performing the protocol without the wire injury. Right femoral arteries were exposed by blunt dissection and subjected to denudation. Briefly, arteries were exposed by blunted dissection, dilated by topical application of one drop of 1% lidocaine hydrochloride (Sigma-Aldrich) and transverse arteriotomy was performed in the muscular branch; a straight spring wire  was carefully inserted into the femoral artery for more than 5 mm toward the iliac artery and left in place for 1 min to denude and dilate the artery. Then, the wire was removed, and blood flow in the femoral artery was restored. Animals were randomly assigned to two different treatment groups. One group (n = 10) received Revacept, the protein obtained by fusing the extracellular domain of GPVI with the human immunoglobulin (IgG) Fc domain19 (at the dose of 2 mg/kg/day), via tail-vein injection, for three days before bilateral femoral artery denudation was performed. The treatment was continued up to day 10. Control group mice (n = 10) were treated with recombinant human IgG1 Fc (at the dose of 2 mg/kg/day)  via tail-vein injection. The compounds were dissolved in a solution of PBS/mannitol(4%)/sucrose(1%). The concentration of Revacept and Fc were selected based on previously published studies27. For all surgical procedures, the animals were ventilated with isoflurane and oxygen to maintain anesthesia throughout the experiment, and anesthetic depth was assessed by observing the reflex responses to paw pinches. Histology and immunohistochemistry were performed on specimens collected at day 32 . Moreover, 24 h-urine collections were performed on day 0 (baseline), day 3, day 7, and 32  for the assessment of 11-dehydro-TXB2 (TXM) levels, as a marker of the systemic TXA2 biosynthesis, mainly derived from the activity of platelet COX-129.The femoral artery wire injury model was described previously elsewhere12. Briefly, the intimal area is the area encircled by the internal elastic lamina minus lumen area. The medial area is calculated as the area encircled by the external elastic lamina minus intima area. The intima-to-media ratio was calculated as the intimal area divided by the medial area. For immunohistochemistry, tissue sections were incubated with primary antibodies against Ki67 , CD68 , followed by incubation with horseradish peroxidase(HRP)–conjugated secondary antibody . After three washings with PBS, samples underwent 3′-Diaminobenzidine  staining, hematoxylin restaining, dehydration, and cover glass mounting. Slides were observed with a Zeiss Axiovision microscope (Carl Zeiss) and Image J 1.44 software was used for the analysis.At the end of the in vivo experiments , all animals were euthanized and intracardially perfused with 10 ml of 4% paraformaldehyde . Femoral arteries were fixed with 4% PFA for 10–15 min and then embedded in paraffin. Paraffin-embedded Sections (7 μm) were stained with hematoxylin and eosin , and the intima-to-media area (I/M) ratio was assessed as previously described2, were measured by previously validated immunoassay techniques29. The urinary TXM levels were corrected for creatinine excretion and reported as ng/mg of creatinine.Two-hundred μl of 24 h-urine collection samples were extracted, and the levels of the TXM, i.e., an enzymatic urinary metabolite of TXBP values < 0.05 were considered statistically significant.All values were reported as mean ± SD (standard deviation) in the text, while shown as mean ± SEM (standard error of the mean) in the Figures; the n values (indicating the number of separate experiments) were reported in Figure legends. Statistical analysis among two groups was performed by Student's t-test; for 3 and more groups, ANOVA (one-way or two-way) or Mixed-effects analysis for repeated measures  were carried out using GraphPad PRISM software . 30. CASMC cultured alone appeared as spindle-shaped cells  and α-SMA [typical of a contractile phenotype]23. As shown in Fig. 2 generation) showed a significantly lower migratory capacity versus CASMC cultured with platelets untreated with Aspirin, at 8–24 h  . In CASMC cultured alone, PGE2  were the most abundant prostanoids . In the coculture of platelets and CASMC, PGE2 and TXB2 were significantly enhanced . To verify the COX-1 or COX-2 origin of the increased biosynthesis of TXB2 and PGE2 in the coculture of CASMC and platelets, we assessed the impact of the exposure of platelets to Aspirin which was washed away before the incubation with CASMC (to target only platelet COX-1) or Rofecoxib (a selective COX-2 inhibitor). As shown in Fig. 2 detected in platelet-CASMC cocultures was entirely prevented by the treatment of platelets with Aspirin, suggesting that enhanced TXB2 derived from platelets. This is further sustained by Rofecoxib inability to affect TXB2 production in the coculture  to mice prevented the increase of TXA2 biosynthesis in response to vascular injury as compared with mice treated with recombinant human IgG1 Fc (2 mg/kg/day) (control group). The experimental design of the study is reported in Fig. We used the femoral artery wire injury modelP < 0.05) increased at 3 days after injury versus the values detected before vascular damage. At 28 days after injury (remodeling phase), TXM values were comparable to those measured at pre-injury condition  (not shown). As shown in Fig. P < 0.05) versus Fc control at three days after vascular injury , we studied the expression levels of proteins, which are markers of macrophages infiltration and cell proliferation, such as CD68 and Ki67, respectively, in paraffin-embedded section of femoral arteries. The vascular injury was associated with an increased expression of CD68  average reduction of I/M ratio by 41.36% versus the Fc-control group.In this model of neointima hyperplasia induced by mechanical damage, we evaluated the effect of the administration of Revacept on the arterial media thickness in paraffin-embedded sections of femoral arteries. As reported in Fig. 34. Platelets adhere to extracellular matrix proteins exposed in the injured vasculature and release numerous soluble mediators, including TXA2. It acts in an autocrine or paracrine manner to stimulate adjacent platelets, thus generating more TXA2 and amplifying other agonist action6. TXA2 also enhances lymphocyte and macrophage functions37 and stimulates the biosynthesis of extracellular matrix proteins38. For all these actions, TXA2 might play a crucial role in initiating and accelerating intimal hyperplasia development.Platelet activation represents an early response to vascular damage26 in C57BL/6 mice, we assessed the time-course of the systemic biosynthesis of TXA2 by measuring the urinary levels of TXM, an enzymatic metabolite of TXB2, which is mainly derived from activated platelets29. Our results show a rapid increase in the systemic biosynthesis of TXA2 in response to vascular damage, which returned to pre-injury values at a later phase on neointimal hyperplasia . The enhanced generation of platelet TXA2 may induce numerous cellular events that contribute to neointimal hyperplasia. By performing coculture experiments between platelets and CASMC, we have shown that platelet-derived TXA2 contributes to COX-2 induction and enhanced PGE2 generation by CASMC. We exploited Aspirin capacity to cause an irreversible inhibition of platelet COX-1-dependent TXA2 generation, persisting when the drug is washed away. The incubation of CASMC with Aspirin-treated platelets mitigated the induction of COX-2. Comparable results were obtained by TP blockage.In a femoral artery wire injury model12 have previously shown that COX-2-derived PGE2 promotes vascular neointimal hyperplasia in response to mechanical injury. Moreover, COX-2-derived PGE2 regulates polarization and directional migration of vascular smooth muscle cells via the activation of the PGE2 receptor subtype EP3α/β12. Here, we show that platelets enhanced the migration of CASMC; this response was mitigated by the selective inhibition of platelet COX-1 by Aspirin.Zhang et al.2, which contributes to enhanced COX-2-dependent PGE2 biosynthesis. Our findings imply the effectiveness of pharmacological inhibition of platelet TXA2 biosynthesis by Aspirin to restrain the development of injury-induced vascular neointimal hyperplasia. However, the results of clinical trials with Aspirin suggest a small reduction in the restenosis after coronary angioplasty but a significant reduction in the frequency of ischemic complications39. The weakness of these studies is that different doses of Aspirin were used; furthermore, the drug capacity to cause a virtually complete suppression of platelet COX-1-dependent TXB2 biosynthesis was not verified40. The possible beneficial effect of low-dose Aspirin in patients undergoing percutaneous coronary intervention through the inhibition of vascular inflammation triggered by activated platelets remains to be verified in appropriate clinical studies.Collectively these results suggest that platelets are activated by the interaction with vascular smooth muscle cells and extracellular matrix proteins, such as collagen, and release TXA2‐dependent aggregation responses and amplifying these responses by released ADP41. Conversely, Revacept combines inhibitory effects on both collagen-mediated platelet adhesion and subsequent aggregation at the site of vascular injury19. These concomitant inhibitory effects on platelet function by Revacept are proper to limit the consequences of vascular damage. We show that the administration of Revacept mitigated vascular neointima hyperplasia in a femoral artery injury mouse model. This effect was associated with reduced vascular expression of markers of macrophages infiltration and cell proliferation. Revacept administration prevented the rapid increase of the systemic biosynthesis of TXA2 after vascular injury derived mainly from platelets10. These data support the hypothesis that platelet activation in response to vascular injury represents an early event contributing to the inflammatory response involved in vascular remodeling.Aspirin exerts antiplatelet effects by inhibiting TXAInterestingly, the interaction of platelets with CASMC in vitro led to morphological changes typical of a synthetic phenotype with downregulation of α-SMA and upregulation of COX-2. These changes were associated with the enhanced migratory capacity of CASMC. Revacept prevented the induction of COX-2 by the interaction of platelets with CASMC.2 induces morphological changes and enhances their capacity to proliferate and migrate, thus contributing to tissue fibrosis23. Moreover, we have shown that the interaction of platelets with colon cancer cells translates into the overexpression of COX-2, a hallmark of malignancy, and the induction of marker genes of epithelial-mesenchymal transition21. Inhibition of platelet-cancer cell interactions by Revacept prevented platelet-induced COX-2 induction and changes of epithelial-mesenchymal transition markers21.Collectively our findings sustain the distinct roles of platelets beyond their fundamental participation in primary hemostasis. As previously shown in cocultures with myofibroblasts, platelet-derived TXAThe main limitation of the present study is that the effect of Revacept on neointimal formation was not compared with Aspirin given alone or co-administered with Revacept. Low-dose Aspirin could cause additional efficacy by preventing the amplification of platelet response induced by other platelet agonists, in addition to collagen. This is an important point that needs to be explored in a specific study.19, constrains the release of TXA2 from activated platelets. These distinct effects played by Revacept make it a promising therapeutic strategy to prevent restenosis in patients with coronary artery disease treated with percutaneous transluminal coronary angioplasty and stent implantation. Importantly, Revacept was found to affect platelet function in the absence of bleeding complications19. A phase II randomized, double blind trial  is ongoing to assess the efficacy and safety of Revacept in patients undergoing elective percutaneous coronary intervention  to collagen exposed in areas of damaged endotheliumSupplementary Information.