Observer rats repeatedly witnessed familiar Demonstrators receive painful footshocks (six sessions). Results confirm that Observers freeze during the first testing session. The occurrence of this behaviour however gradually diminished as the experimental sessions progressed, reaching minimal freezing levels by the end of the experiments. In contrast, the appearance and continuous increase in the frequency of yawning, a behavior that was inhibited by metyrapone , might represent an alternative coping strategy, suggesting that the observer’s reduced freezing does not necessarily indicate a disappearance in the affective response to the Demonstrator’s distress.Witnessing of conspecifics in pain has been shown to elicit socially triggered freezing in rodents. It is unknown how robust this response is to repeated exposure to a cage-mate experiencing painful stimulation. To address this question, shock-experienced  Empathy, the ability to understand and share the feelings of others, can be conceptualized as a hierarchically organized multi-level capacity such that higher-level processes are built on top of more primal ones, like emotional contagion ;2. EmotiEvidence of emotional state sharing in rodents originates from studies showing socially-induced hyperalgesia , social Demonstrators undergoing footshocks to investigate whether and how behavior changed from day to day.So far, all the studies investigating emotional contagion in rodents have examined the behavior of animals after a single exposure or interaction with another in distress. Despite the increasing characterization of emotional contagion in rodents, it is still unknown how animals would respond following repeated witnessing of a conspecific in pain. How would animals respond following repeated testing with a conspecific in pain? This question is of methodological interest, as many experiments would require repeated testing of an individual, be it to investigate, using repeated measures the effect of drugs or to record neural activity and behavior over multiple exposures. Here, leveraging our rodent model of empathy , we expoObserver rats were exposed to familiar Demonstrators undergoing footshocks during six identical, semi-consecutive testing days. Each testing day contained a preshock baseline period and 5 shocks, during which the freezing of Demonstrators and Observers was scored . This interaction was driven by Day 1, as removing Day 1 rendered the interaction non-significant (p>0.05). A 6 Day repeated measures ANOVA on baselines showed differences between freezing levels of Demonstrators during the baseline period of Days 1 to 6 . Further, planned comparisons using paired sample t-tests of each shock period to baseline, revealed that on Day 1, Demonstrators froze more following each one of the five shocks than baseline  ANOVA revealed a significant Day x Epoch interaction  and Epoch . In contrast to Demonstrators, a 6 Day repeated measures ANOVA on baselines showed no significant differences between freezing levels during the baseline period of Days 1 to 6 (p>0.1). Planned paired sample t-tests comparing shock with baseline periods for each Day indicates diminished freezing after Day 4  while this was not the case on Days 4 to 6 . This reduction in freezing of Observers, could suggest that their sensitivity to the distress of the Demonstrator diminishes following repeated exposure.A 6 Days x 6 Epochs ANOVA on the freezing of er Day 4 . SpecifiObservers (st shock]) detected differences in yawning frequency between Days (χ2 (5) = 12.12, p = 0.033). Planned Wilcoxon posthoc tests comparing yawning frequency between Day 1 and Days 2 to 6 revealed that Observers yawned more during Day 6 compared to Day 1 (p = 0.017). The yawning response of Observers appeared to be specific to the distress of the Demonstrators, as a Wilcoxon signed rank test comparing the total number of yawns during the combined preshock periods of Days 1 to 6 to the total number of yawns during the shock periods of Days 1 to 6, showed that Observers yawned more during the shock periods (Z(6) = -2.2, p<0.05) , and showed on average a total of 9 yawns over the 6 days. Four rats were testing in the afternoon  and showed on average a total of 19 yawns. To ensure that the increase of yawns across Days was not due to an inadvertent shift in testing time, the total number of yawns on each Day was compared with the average testing time on that day, but the relationship was clearly non-significant . Importantly, the results of a separate experiment showing that yawning was significantly inhibited by pre-treatment with metyrapone (t(18) = 2.191, p = 0.042), suggests that yawning reflects heightened stress levels in the Observers  . Furtheron Day 1 . Moreovebservers .Observers towards the Demonstrators changed throughout the experiment, the percent of time Observers spent in the window zone  was quantified (st shock) revealed a main effect of Epoch . This indicated that Observers were drawn to spend a higher percent of time in the window zone following shock delivery. The absence of an interaction of Epoch and Day (p>0.05) however shows this effect to be constant across days, suggesting that the attention of Observers was captured by the Demonstrators receiving shocks in a way that was sustained throughout the experiment.To investigate whether the attention of antified . A repeaDemonstrators submitted to repeated and unavoidable foot shocks exhibit elevated freezing levels. Confirming previous findings [Observers display freezing in response to familiar Demonstrators enduring painful footshocks. As to our core question of whether these effects can be measured over repeated days, as necessary for designs that require repeated testing, our results show that freezing gradually diminishes as a consequence of repeatedly witnessing the Demonstrator receive painful stimulations. Results of a separate pilot experiment revealed that this reduction of freezing occurs even if a number of experimental factors are manipulated to reduce such habituation  than for within-subject designs. In addition, at first glance, our findings could suggest that the affective response of Observers to the distress of Demonstrators is progressively reduced throughout the testing sessions. The appearance of yawning however complicates this interpretation.findings ;11;12;16ituation . AlternaObservers was confirmed by the reduction of this behavior following administration of an anti-stress drug  on test days 5 and 6. This is in agreement, with results showing that administration of anxiogenic drugs to monkeys induces both anxiety-like behavior and yawning [Observers indicates a change in the type of arousal the Observers experience during the first testing days compared to the arousal in the last testing days. In contrast, the repeated daily shock exposure the Demonstrators undergo maintains a highly elevated but similar arousal type throughout the experiment, thus preventing yawning form emerging. However, this is highly speculative and further testing such as corticosterone measurements throughout the experiment would be necessary to confirm this hypothesis. In addition to the postulated role in stress, yawning has been linked to a variety of other functions, such as a thermoregulation [Observers suggests that the most parsimonious explanation for its emergence is that it is indicative of changes in autonomic regulation and reflects an affective response in the Observer to the distress of the Demonstrator. Together, these findings and the results showing that the Observers attention towards the Demonstrators is unaltered indicate that although the freezing response to witnessed distress changes over time, the affective response of the Observers may outlast socially triggered freezing and invite yawning as a coping mechanism. However, more data will be needed to fully understand the relationship between freezing, yawning and other stress related manifestations.Yawning is a phylogenetically old behavior, ubiquitously present across vertebrates –21. It i yawning , suggest yawning . Perhapsgulation ,29. Heread libitum. All experimental procedures were preapproved by The Institutional Animal Care and Use Committee of the Netherlands Institute for Neuroscience (IACUC-NIN-1211). Studies were conducted in strict accordance with the European Community’s Council Directive (86/609/EEC).Male Long-Evans rats (6–8 weeks old/ 250-350g) were obtained from Harlan Laboratories (Germany). Upon arrival animals were socially housed in couples and maintained at ambient room temperature , on a reversed 12:12 light-dark cycle (lights off at 07:00). Food and water were provided Observers and empathy testing. Behavior was video recorded throughout testing using a camera  placed ca. 1m in front of the setup onto miniDVDs for behavior scoring.Two adjacent compartments of equal dimensions  separated by stainless steel bars (6 mm in between bars) which allowed animals to smell, see, hear and touch each other. The compartment walls were made of transparent Plexiglas and the floor of stainless steel grid rods. One of the chambers was connected to a stimulus scrambler  for shock exposure of Observer or Demonstrator and allowed to acclimate to the colony room for 7 days. Rats were handled during the dark phase of the circadian cycle every other day for 3 minutes for a total of 10 days preceding empathy testing. Three days prior to experiment start, Observer-Demonstrator pairs were transported to the experimental room and placed in one of the compartments of the empathy testing apparatus for 20 minutes/day for 3 days. Between each pair, the testing apparatus was cleaned using dishwashing soap and 70% alcohol solution. Throughout the experiment  all Observers and Demonstrators were always placed in the same compartment of the testing apparatus.On arrival day, animals were randomly paired , assigned the role of Observers when witnessing Demonstrators experiencing shock [Observers. To maximize discriminability from the empathy-testing context, the walls were coated with black and white striped paper, the compartment was illuminated with bright white light and cleaned using 30% ethanol solution, followed by dishwashing soap with a neutral smell and 5% vinegar solution. During shock exposure, Observers were allowed to acclimate for 10 minutes and then delivered four mild footshocks . Compared to the Demonstrators, the Observers experienced a smaller number and intensity of shocks and a longer intershock interval to minimize the risk of fear conditioning and generalization during empathy testing. The pre-exposure test was conducted during the dark phase of the circadian cycle. Observers were then individually placed in a clean cage for 1 hour prior to return to their home cage.Prior foot-shock experience enhances freezing of ng shock . The dayObserver-Demonstrator pairs were placed in their respective compartments for a maximum of 30 minutes. After 10 minutes of acclimation, Demonstrators received five footshocks . Following the last shock, animals were left in the testing apparatus for 2 minutes and then returned to their home cage. This procedure was repeated for 4 consecutive days, following 2 days of no test, and then 2 more consecutive test days. The original experimental design included a total of 8 testing days, with 2 resting days in the middle . However, in this experiment to prevent unnecessary discomfort, the experiment was stopped by test day 6, since the behavior of the animals reached a limit by this day, with Demonstrators exhibiting maximal freezing all the time and Observers displaying minimal freezing on test days 5 and 6. Testing schedule appears to play a minimal role in the behavior of Observers, as different schedules  have no effect on their behavioral response . Freezing behavior was defined as lack of movement (except movement due to breathing) for a period longer than 3 seconds. Every empathy test session was subdivided in two different periods: pre-shock and shock. The first period (pre-shock), corresponded to first 10 minutes immediately after the animals were placed in the test apparatus. The second period (shock) was further subdivided into five time bins: (1) from minute 10 to the first shock (baseline period), (2) from the 1st to the 2nd shock, (3) from the 3rd to the 4th shock, (4) from the 4th to the 5th shock and (5) after the 5th shock 2 minutes were added. For each period and time bin, freezing was scored as the percentage of time the animal spent freezing.Three experienced researchers manually scored the behavior exhibited by both Observers was scored throughout all tests. Since the occurrence of yawning was low, the frequency of this behavior was scored for 10 minutes following the 1st shock rather than individually for each intershock interval. To have a baseline period of a comparable length, yawning was scored during the first ten minutes prior to shock delivery. Lastly, the percent of time from the total test time that Observer animals spent in the area closest to the Demonstrator’s compartment (window zone- 12cm x 25cm area) was scored during the pre-shock and shock periods.Yawning was defined as a wide opening of the mouth sometimes accompanied by stretching and elongation of the body . The yawObservers and Demonstrators in Experiment 1 were analyzed separately using a 2-way repeated measures analysis of variance (ANOVA). The factors examined included test day (1 to 6) and Epoch  as the within factors. All posthoc analysis on freezing responses were conducted by using planned contrasts: (1) baseline of Day 1 was compared to baseline of Days 2 to 6 and (2) for each test day, freezing during each shock epoch (1 to 5) was compared to the baseline epoch. One pair was excluded of the analysis because the Demonstrator did not show any reaction to any of the shocks in two consecutive sessions.The freezing responses of Observers the test was divided into two Epochs: (1) the preshock/baseline  and (2) shock period . To investigate general differences between preshock and shock periods, the median yawning frequency was compared using Wilcoxon rank signed test across all test days. The frequency of yawning was normalized to the preshock period, which was achieved by subtracting the preshock frequency from the value observed during the entire shock period. Friedman rank sum test was used to examine differences between test days (repeated measures) in the yawning frequency. Post-hoc analyses were conducted by using planned comparisons between the first test day and all other test days. For the analysis of time spent in the window zone by Observers the test was divided into two Epochs: (1) the preshock  and (2) shock period . The percent of time Observer animals spent in the window zone was analyzed using a two way repeated measures ANOVA, with Day and Epoch (preshock vs shock) as the within factors.For the analysis of yawning by Observers, their familiarity with their Demonstrator varied based with 1,3 or 5 weeks of paired caging. Importantly however, no significant differences were observed in either the freezing levels or yawning depending on familiarity, allowing us to group the results. Following test days 1 to 4, animals were randomly split in two groups . The first group received a pretreatment with metyrapone on test day 5 and then underwent a normal test on day 6, while the second group experienced normal testing conditions on test day 5 and received the pretreatment with metyrapone on test day 6. Metyrapone was delivered to all animals 30 minutes prior to start of test. All animals received a 25mg/kg subcutaneous injection of Metyrapone , which was prepared by dilution in 0.9% saline solution. The number of yawns during the preshock and shock period of test days 5 and 6 were quantified. Then, for statistical analysis, a paired two-tailed t-test on the normalized number of yawns  following metyrapone injection was compared to the number of yawns following no injection.The glucocorticoid synthesis inhibitor, metyrapone  was used to investigate the effects of stress on yawning. For this experiment, a separate set of male Long-Evans rats  obtained from Janvier (France) was used. The overall experimental procedures including housing, acclimation, handling, habituation, pre-exposure and test were similar to those described above with some modifications. First, test time was between 08.30am to 12.30pm and testing order was randomized daily, with each animal tested at a different time each test day. Second, since these animals were part of an ongoing study that tested effects of familiarity on affective responses of S1 ARRIVE Checklist(PDF)Click here for additional data file.S1 FigObservers. For this purpose various changes were implemented. First, an additional Demonstrator was paired to each Observer. Second, Observers experienced an extra shock-exposure session halfway through the experiment. Third, the number of days in-between each empathy test was increased. Fourth, for extra habituation, Observer-Demonstrator pairs were placed on a second testing setup  30 minutes prior to each test and lastly; three different contexts  were used in addition to the Observers shock exposure context. (A) Schematic showing the timeline and illustration of the procedures conducted. On arrival day, animals (N = 24) were randomly distributed to one of eight groups, each group consisting of three animals . Acclimation and handling procedures were identical to those detailed for Experiment 1. In contrast to Experiment 1, three habituation sessions were conducted prior to each pair of tests. For each habituation session (triangles), Observers were first habituated with DEM1 and then with DEM2 (20 minutes/each). Observer animals experienced two shock exposure procedures (same as in Experiment 1), one the day before the first empathy test and the second one after the fourth empathy test. Each Observer experienced four alternating empathy tests (Test 1–8) with each one of the Demonstrators of their group for a total of eight empathy tests per Observer. Three different contexts were used throughout the tests. For Context A the test box was cleaned with dishwashing soap with neutral smell and then 70% ethanol, the setup was illuminated with red dim light and Observers were placed in the right compartment . For Context B, the test box was cleaned with hand washing soap with lemon smell and 30% vinegar, the walls were coated with black and white stripes printed paper, the setup was illuminated with white bright light and Observers were placed in the left compartment (tests 3 and 4). Lastly for Context C, the test box was cleaned with dishwashing soap with neutral smell, followed by application of rose oil, the walls were coated with white paper, the setup was illuminated with red light and Observers were placed in the right compartment (tests 5 and 6). Each empathy testing was conducted as described for Experiment 1 and pairs were left in a clean cage for 1 hour prior to returning to their home cage. (B) Graph of the freezing responses of Observers (gray line) and Demonstrators (black line) and yawning of Observers (red line). Graph depicts average freezing percent ± standard error of the mean (SEM) of 8 test days (1 to 8) each consisting of a baseline (2 minutes prior to 1st shock) and 5 shock periods: 1st to 2nd shock, 2nd to 3rd shock, 3rd to 4th shock, 4th to 5th shock and 5th plus 2 additional minutes, corresponding to numbers 1 to 5 in the graph. Black significance symbols are for Demonstrators while grey-colored symbols are for Observers. The background color in the rats test box represents the context in which the test was conducted. In addition, graph shows the cumulative  median yawning of Observers for test days 1 to 8 (red colored y axis on the right side). Two-way ANOVA results of the Demonstrators freezing revealed significant main effects in test day  and time period  as well as significant interaction between test day and time period . With one exception , baseline levels of all testing days were comparable to test 1 (p>0.05). Also, Demonstrators froze significantly less  or approximating significance  during the baseline period compared to shock periods of all tests except test 8. Significant main effects in the Observers freezing were detected between test days  and time periods , but no effect in their interaction (p>0.05). Observers displayed lower freezing responses during the baseline of tests 3, 6, 7 and 8 and during the shock periods of tests 5, 6, 7 and 8 compared to the first and second testing day . (C) Top left graph shows the total number of yawns displayed by Observers. A Friedman test revealed significant differences in normalized yawning between test days (χ2 (7) = 15.2, p<0.05). Posthoc comparisons (Wilcoxon) were made between the numbers of normalized yawns during shock period of test days 2 to 6  and normalized yawns during test day 1.These analysis showed higher number of yawns on test days 4 (p<0.05) and 8 (p = 0.066) compared to test day 1. Top right graph shows the percent of Observers that yawned at least once on each test day and the cumulative percent across test days of Observers that yawn (pink bars). All graphs show results during the preshock (gray) and shock (black) periods of test days 1 to 8. Numbers on top of the bars indicate the range of yawns displayed by the animals. *p<0.05, **p<0.01, ***p<0.005, t: p<0.07.This pilot was conducted to examine if manipulation of various experimental factors  would extend the freezing response of (EPS)Click here for additional data file.S1 Video(MP4)Click here for additional data file.


Osteoporosis is a bone disease that commonly results in a 30% incidence of fracture in hens used to produce eggs for human consumption. One of the causes of osteoporosis is the lack of mechanical strain placed on weight-bearing bones. In conventionally-caged hens, there is inadequate space for chickens to exercise and induce mechanical strain on their bones. One approach is to encourage mechanical stress on bones by the addition of perches to conventional cages. Our study focuses on the molecular mechanism of bone remodeling in end-of-lay hens (71 weeks) with access to perches. We examined bone-specific transcripts that are actively involved during development and remodeling. Using real-time quantitative PCR, we examined seven transcripts , RANKL (receptor activator of nuclear factor kappa-B ligand), OPG (osteoprotegerin), PTHLH (PTH-like hormone), PTH1R (PTH/PTHLH type-1 receptor), PTH3R (PTH/PTHLH type-3 receptor), and SOX9 (Sry-related high mobility group box)) in phalange, tibia and femur. Our results indicate that the only significant effect was a difference among bones for COL2A1 . Therefore, we conclude that access to a perch did not alter transcript expression. Furthermore, because hens have been used as a model for human bone metabolism and osteoporosis, the results indicate that bone remodeling due to mechanical loading in chickens may be a product of different pathways than those involved in the mammalian model. Skeletal bones and teeth are unique systems that rely on calcification of an extracellular matrix. Parathyroid hormone (PTH) regulates the inorganic calcium matrix in bone by stimulating the parathyroid hormone receptor (PTHR) . Upon PTActivation of bone remodeling occurs due to microfractures and mechanical loading, which is sensed by osteocytes. Although Transforming growth factor β (TGF-β) is necessary for osteoblast formation, during activation of bone remodeling, TGF-β production is inhibited . When PTSignificant bone diseases occur because of unbalanced bone remodeling. For instance, osteoporosis is a common and prevalent disorder resulting in the loss of bone mass and strength, increasing the probability for bone fractures . Many faLaying hens, responsible for the production of table eggs, experience a progressive loss of bone strength and mass as they age, due to the calcium requirements for eggshell formation. During the switch to medullary bone production, structural bone continues to undergo resorption, while the medullary bone is used as a calcium reservoir for eggshell formation. In conventionally-caged hens, the process of structural bone loss is accelerated due to the constraints of the housing unit used in many commercial operations, where the opportunity for exercise is limited . Non-cagOsteoporosis in white leghorns results in economic loss to the egg industry worldwide and is the cause of 20% to 35% of all mortalities during the egg laying cycle of caged hens ,9,10. Fuvs. the parathyroid hormone type-3 receptor (PTH3R), as well as the RANKL/OPG ratio, which can provide insight into the activity of osteocytes and osteoclasts in response to mechanical loading on the bone. We also examined transcript expression for Sry-related high mobility group box (SOX9) and PTH-like hormone , which are necessary for endochondrogenesis. We used two weight-bearing bones (tibia and femur) and a non-weight-bearing bone . The findings will be used to compare bone-remodeling pathways in chickens and humans  d humans .In our first hypothesis, we anticipate that the RANKL/OPG ratio will be decreased in chickens that have access to perches during their entire life cycle, as compared to hens that never have access to perches. The RANKL/OPG ratio expression provides detail into the overall level of osteoclast activity, where a higher ratio might occur during the resorption phase of bone remodeling and is associated with osteoclastogenesis and osteoclast activation; that is, OPG secretion by osteocytes decreases the RANKL/OPG ratio to inhibit the recruitment and activity of osteoclasts. SOX9 is downregulated in the hypertrophic cartilage and further during ossification of endochondral ossification. Because the bones of these 71-week-old hens are already ossified, our second hypothesis is that perch access will not affect the expression of SOX9.The third hypothesis we propose is that PTHLH will be involved in calcium regulation through either PTH1R or PTH3R. Although PTHLH activates both receptors, in zebrafish and chicken, it has been shown to preferentially activate the PTH3R in zebrafish ,29. We hFinally, we propose that bone turnover will be higher in weight-bearing bones, as compared to the non-weight-bearing phalange. Because cage-laying hens rely heavily on the musculoskeletal component of the leg to jump on and off the perch, we anticipate the greatest difference in transcript expression in the femur and tibia, as compared to the phalange. Because the phalange is not directly involved in the effects due to mechanical load (a perch) and medullary bone and diet supply the overarching source of calcium, we hypothesize that the phalange is likely to show no significant treatment effect. Overall, our findings indicate that the addition of a perch did not enhance the molecular mechanisms used for bone formation as a result of mechanical loading. This may be due to the already accelerated bone turnover in egg-laying hens. Thus, our data do not support the model, as we expected.http://web.expasy.org/translate/. GAPDH yielded an amplicon 210 bases with a 69 amino acid sequence match (100% identity). COL2A1 yielded an amplicon of 200 bases with a 66 amino acid sequence match (97% identity). SOX9 yielded an amplicon of 293 bases with a 97 amino acid sequence match (99% identity). PTHLH yielded an amplicon of 151 bases with a 50 amino acid sequence match (99% identity). PTH3R yielded an amplicon of 94 bases with a 29 amino acid sequence match (93% identity). OPG yielded an amplicon of 199 bases with a 46 amino acid sequence match (91% identity). RANKL yielded an amplicon of 298 bases with a 99 amino acid sequence match (99% identity). All primers and amplicons were validated by subcloning and DNA sequencing . FurtherThe first set of analyses were performed to determine if the RANKL/OPG ratio would decrease in hens provided access to perches throughout their life cycle as compared to no perch access or access to perches only during the pullet or laying phases. The ratio results indicated no effect due to treatment, bone or the interaction of treatment with bone . IndividOur hypothesis that SOX9 expression would be limited in ossified bone and not affected by perch access was supported by a lack of a treatment effect . Likewisi.e., was the PTH1R preferentially used for bone turnover). Expression of PTH1R was not affected by treatment, bone or the treatment by bone interaction  would respond differently to perch access compared to the leg bones. COL2A1 was utilized as an additional control gene. There was no effect due to treatment or the treatment and bone interaction ; howeverOsteoporosis in chickens is a serious disease that reduces livability, causing economic losses to the egg industry. To better understand the molecular mechanism of bone turnover, we compared seven factors that regulate weight bearing and non-weight bearing bones. All qPCR factors were subcloned and sequenced to demonstrate that we were analyzing the orthologs of intent. In addition, we designed our study to investigate chickens using the same mechanisms of bone remodeling as the mammalian model .We proposed that the RANKL/OPG ratio would decrease as a result of an increase in perching activity. In the mammalian model, exercise that induces mechanical strain prevents the recruitment and activation of osteoclasts by secreting OPG. In addition, RANKL secretion increases in humans when mechanical unloading is occurring . HoweverAnother deviation from the mammalian model may be due to the evolution of PTH receptors. Chicken PTH1R and PTH3R are co-orthologs to PTH receptors in teleosts. In mammals and teleosts, PTH and PTHLH activate the PTH1R nearly equivalently for calcium regulation. Because mammals only express a single ortholog of PTH1R, we theorized that the PTH1R would be the preferential receptor utilized for calcium homeostasis/regulation in adult laying hens. Previous studies have investigated the affinity of these receptors in chickens, but no molecular studies have examined these receptors during bone turnover. In our study, we were unable to determine differences in expression between PTH1R and PTH3R.SOX9 is directly involved in cartilage patterning and hypertrophic chondrocyte development , and thePTHLH performs several crucial roles in bone, such as promoting endochondral ossification, where the cartilage template is replaced by bone. Specifically, PTHLH has an anabolic effect by regulating endochondral bone formation by promoting the recruitment and survival of osteoblasts. PTHLH ligand activates the PTH receptors in mammals and inhibits the apoptosis of osteoblast. Similar to PTH, PTHLH extended secretion promotes RANKL secretion of osteoblasts and osteocytes. In our study, no difference in expression of PTHLH was observed as a result of perch access. The functional role of PTHLH during endochondrogenesis in aging hens may be limited.Medullary bone production may account for the unaltered RANKL/OPG ratio in the aging laying hen as a result of perch access, as this type of bone is unique to avians and crocodiles . Both thThere was a significant difference in the expression of COL2A1 in femur when compared to phalange across all treatment groups. COL2A1 is a collagen-specific protein produced in chondroblasts. The reason for the significant expression of COL2A1 in the femur as compared to the phalange of 71-week-old laying hens is unknown. It is reasonable to assume that this upregulation of COL2A1 in the femur as compared to the phalange is unlikely related to cartilage development, as these egg-laying strains of chickens sexually mature at 18 weeks of age, when the growth plate closes. The significant difference in COL2A1 between bones disqualified it as an additional control gene.We anticipated that phalange would not be significantly accessed as a calcium reservoir, because hens primarily use the calcium in feed and, if inadequate, then the medullary bone as a source of calcium for the formation of the eggshell. Thus, we expected that there would be a difference in transcript expression among the weight-bearing leg bones and the phalange. The phalange did not show any difference compared to the weight-bearing bones, which might be a result of wing flapping during perching. The phalanges, located in the wings, are likely experiencing mechanical strain, as they use their wings for balance when stepping onto and off the perch. In addition, the phalange of hens responded to perch access, as compared to controls with no adult perches, by demonstrating an increase in bone mineral density similar to the femur and the other wing bones .i.e., production of mRNA) is not always directly indicative of protein expression, which actually preforms the cellular function of regulating the transcription of other genes. As a result, we cannot confidently ascribe a function to the genes analyzed simply based on when and where the gene is transcribed [Mechanical loading in bone occurs in response to weight-bearing stress being placed onto the bone. Bone cells’ overall response to mechanical loading is to increase strength in order to manage the additional load. Within bone, mechanical loading alters fluid flow into the extracellular matrix, as well as increases the force on osteocytes. The sensed mechanical force is translated into a biochemical signal, which alters the cell membrane of osteocytes. The osteocyte’s response to mechanical loading is to reduce the recruitment of osteoclasts by downregulating the release of RANKL. Our results suggest that factors that induce mechanical stress in aging laying hens may not be the same as the associated factors used in aging mammals . Thus, tnscribed . Thus, ip = 0.45%) and grower  feed were used. The diet changed at 18 weeks of age, and chickens were switched to a laying hen diet . They remained on this diet to the end of the study. Diets were fed using the recommendation for nutrients by Hy-Line International (2009–2011) or the National Research Council (1994) [ad libitum for all birds. Feed was also provided on paper lining the cage floor for the first few days following hatching. Two drip nipple drinkers/cage provided a source of water [White leghorn females of the Hy-Line W36 strain were raised at Purdue University Poultry Research Farm using standard management and vaccination practices and under the guidelines approved by the Purdue University Animal Care and Use Committee  ,35. Infrl (1994) ,35. Feedof water ,35. A stof water ,35. Begiof water ,35.Our study contained four treatment groups defined by access to perches during the pullet and the laying phases. Treatment 1 (control) consisted of chickens with no access to a perch (0 to 71 weeks). Treatment 2 chickens had access to perches only during the egg-laying phase of the life cycle (17 to 71 weeks of age). Treatment 3 chickens had access to perches only during the pullet phase (0 to 16.9 weeks of age). Treatment 4 chickens always had access to perches (0 to 71 weeks of age). For the arrangement of metal round perches within the cage, see . There wn = 9 from each treatment). The chickens were euthanized using sodium pentobarbital followed by cervical dislocation [Thirty-six chickens from 71 weeks of age were used in this study , the bones were removed from the −80 °C conditions and manually crushed with pliers to form bone fragments. The bone fragments were further processed with a pestle in a stainless steel bowl to produce a bone fragment powder. For total RNA isolation, the bone powder was added to Trisure  following the manufacturer’s guidelines. The bone:Trisure mixture was homogenized using a PRO250 homogenizer, until all tissue was in solution, and subsequently, total RNA was precipitated with isopropanol. Following isopropanol precipitation and ethanol washing, the total RNA pellet was isolated and resuspended with 150–300 μL DEPC water, depending on size of pellet. To assess RNA quality and amount, the final concentration and purity of each RNA pellet were determined by nanospectroscopy. Following RNA isolation, DNase treatment was performed using RQ1 RNase-Free DNase  using the manufacturer’s guidelines. DNase-treated RNA was then converted to cDNA using reverse transcriptase and random primers .http://www.ncbi.nlm.nih.gov/tools/primer-blast/). All amplicons generated by qPCR were electrophoresed, excised, ligated into pGEMT-easy (Promega), transformed into TOP 10 E. coli cells , screened and sequenced with T7 and Sp6 primers, as previously described [Our previous quantitative RT-PCR methods were modified for this study as follows . Real-tiescribed ,36. The Following the manufacturer’s instructions, the qPCR used the Thermo-Fast 96 Detection plate and the DyNamo HS SYBR Green qPCR kit  on an Applied Biosystems 7300 Real-Time PCR machine. Each well contained 10 μL of 2× SYBRGREEN Dye Master Mix (Thermo Scientific), 5 μL of 40 ng of cDNA template . Each reaction was performed in triplicate. Five microliters of oligonucleotide primer pair master mix  for each transcript were added to the specific well. The total volume of each well was 20 μL. The qPCR was performed at 95 °C for 2 min followed by 95 °C for 10 s, 62 and 72 °C each at 30 s for 40 cycles. Following the Stage 3 step, the amplification was recorded. Each transcript was examined on separate plates allowing for the maximum amount of samples to be tested with all treatment groups measured.Ct values. The seven different genes of interest (GOI) were subtracted from GAPDH using Equation 1 [Ct) values involved transforming the data using GAPDH (2-change in Ct) [Ct values for each transcript was determined. Determining the relative quantity without efficiency correction was performed using Equation (2) [Glyceraldehyde 3-phosphate dehydrogenase  is expressed throughout all tissue used to normalize uation 1 . Analysie in Ct) . In shortion (2) .Statistical Analysis System (SAS) was used to analyze the relative quantity of each gene transcript. The relative quantity of each gene transcript was analyzed by mixed model repeated measures ANOVA  with four perch treatments, the repeated measurement factor bone  and the treatment × bone interaction as fixed effects, with bird as a random effect . SignifiThis study provided insight into the mechanism of chicken bone turnover in response to perch access . We were


SOX9 coding region. In this work we provide for the first time a causative mutation for the XXSR condition in the dog. Moreover this report supports the idea that the dog represents a good animal model for the study of XXSR condition caused by abnormalities in the SOX9 locus.Sexual development in mammals is based on a complicated and delicate network of genes and hormones that have to collaborate in a precise manner. The dark side of this pathway is represented by pathological conditions, wherein sexual development does not occur properly either in the XX and the XY background. Among them a conundrum is represented by the XX individuals with at least a partial testis differentiation even in absence of SRY. This particular condition is present in various mammals including the dog. Seven dogs characterized by XX karyotype, absence of SRY gene, and testicular tissue development were analysed by Array-CGH. In two cases the array-CGH analysis detected an interstitial heterozygous duplication of chromosome 9. The duplication contained the  Gonadal differentiation in mammals is initiated, controlled, and regulated by the coordinated action of several genes and hormones.During the last two decades many genes involved in this process have been identified SRY, the sex determination key gene SRY, that is in the XX embryos a different set of genes is activated, and the undifferentiated gonad becomes an ovary SRY role takes place in a short period of timeand ceases after the activation of SOX9. This gene is a main actor in testis differentiation and in several other embryogenetic fields The scepter of power remains firmly in the hands of SOX9 gene is surprisingly activated in the absence of SRYFOXL2 in goat SOX9SOX9 alterations in XXSR cases include duplications, triplications, and reciprocal translocations One of the most interesting issues is represented by the XX sex-reversal cases. In humans most of them do have the SRY gene that is transposed to the tip of Xp due a recurrent Non Allelic Homologous Recombination between of PRKX and PRKY in a particular Y haplotypic background SOX9 gene duplication.In this article, we report the molecular analysis of seven XX sex-reversal dogs and we clearly show, for the first time in literature, that two of them carry SRY gene. The other three cases, C61, C64, and C65, have been characterized in this study.Seven dogs from different breeds have been considered in this study: Four of these have already been described, while three are still unreported. Case C2, C9, C10, and C44 All clinical activities and surgical experiments on the dogs were carried out at the Veterinary Hospital of the University of Milan by veterinary surgeons. During the research no animals were sacrificed. The anesthetic and surgical protocol fulfilled the Federation of European Laboratory Animal Science Association’s recommendations and European Union legislation (Council Directive 86/609/EEC). Blood (1.5 ml) and gonad samples were collected for a routine medical procedure and stored for further analysis. Consistent with Italian regulation (D.L. 116/1992), the owners signed a voluntary consent, for their animals before undergoing surgery. This consent includes the possibility that the removed tissue may be used in scientific researches without economic interest.After surgical excision of the gonads, they were fixed in 10% neutral buffered formalin for at least three days. For histological examination, several slices of gonads were processed histotechnologically according to standard laboratory procedures, cut at 5 µm, and stained with hematoxylin and eosin SRY gene analysis was performed as reported Peripheral blood lymphocyte cultures were performed following the standard procedures Array-CGH was performed using a custom Agilent Canine Genome CGH Microarray 180 K  and processed as reported Sox9 duplications detected by array CGH were confirmed by Real-Time-qPCR with SYBR Green detection , using one non-polymorphic marker located within the duplicated region. The primers were designed by using the Primer3 Software online (http://frodo.wi.mit.edu/primer3/), with the following criteria: Amplicon size 80–200 bp, GC content of 20–80%, and melting temperature (Tm) of 59–61°C. The primer sequences are available on request. Real-time detection was performed using the Stratagene Mx3000P. The Real-Time-qPCRs were performed in triplicate for each reaction.The comparative CT method (ΔΔCT method) was used to discriminate between two and three allele copies of the DNA target sequence (Sox9) in the two dogs (resulted duplicated by a-CGH) relative to five normal control dogs DNA samples. The data have been normalized against two different reference sequences .SRY gene (not shown). Moreover, the histological analyses revealed the presence of testicular tissue in all the three cases, indicating that the male pathway was active during the fetal period in the absence of SRY. The testes of Case 61 are composed of testicular parenchyma with absence of the germline. In Cases 64 and 65 the right and left gonads are ovotestes -PCR (opy number variations (CNVs) in different dog breeds http://genome.ucsc.edu/index.html) to the human region chr17∶68,723,331–69,717,418 (genome assembly Hg19), located 500–600 Kb upstream of SOX9, which is suggested to be the human regulatory region critical for gonadal SOX9 expression In cases C10 and C44, the array-CGH analysis detected an interstitial heterozygous duplication of chromosome 9, of 577 Kb  . The dup(RT)-PCR  and it wSRY is the master gene of this process SOX9 represents the genetic factor that, activated by SRY, starts and regulates testis development. Although SRY is mammal-specific (with very few exceptions) SOX9 plays an important role in bird also SOX3 and SOX8 genes are also involved in the sex determination process SOX9 locus on HSA17q24.3, have been reported to be the causative mutations of the XX sex-reversal condition SOX9 coding region (CDS), but all are located 5′ to this gene. Indeed only the first reported SOX9 duplication includes the SOX9 CDS Variable-Number Tandem Repeat (VNTR) analyses, is at least 11.7 Mb long and is starts at 9.4 Mb 5′ and ends at 2.2 Mb 3′ of SOX9 CDS.Genes in the SOX family play a critical role in the sex determination process. Sox9 over expression is required to induce the testis development in a XX background.The mechanism underlying the XXSR condition in the presence of SOX9 duplications is still not clear, although it is clear that a SrySOX9 is initially expressed in both the developing gonads (XY and XX), but only in the XY gonads its expression increases greatly. This upward regulation is due to SRY activation, and later on, to an auto-loop reinforced by additional positive feed-forward signals (Fgf9). In the XX developing gonad the auto-loop is not able by itself to up-regulate Sox9 expression; moreover, female-specific genes repress additional feed-forward signals.Therefore all different Sox9 locus duplications must be organized to allow this possibility. The Sox9 transgenic mouse effectively develops the XXSR phenotype. Incidentally, in this case, the gene is under the regulation of a strong promoter, which is able to activate the Sox9 expression in the right place at the right time, also in the absence of The analyses of human duplications in XXSR suggest a model of action. In these subjects two CDS SOX9 doses are present , but the upstream region in one allele is duplicated, and this condition probably induces Sox9 over-expression.The perturbation of sex determination process may be caused either by gain of function (GOF) or Loss of function (LOF) mechanisms. In the first case male gene (i.e. SOX9 in human and probably in pig) are involved whereas in the second ones female genes are involved (i.e. RSPO1 in human and FOXL2 in goat). In addition, the mechanisms of LOF are often associated with more severe phenotypes that include other abnormalities. For this reason we believe that SOX9 GOF remains the most reasonable candidate mechanism to explain the remaining unexplained XXSR cases in the involved species.The dog represents a good animal model for the study of this disease because it shows relative high frequencies of XXSR cases and more precisely it can be a valuable model for the study of XXSR cases caused by SOX9 locus duplication.In addition, the dog could represent an alternative animal model to the mouse considering that it seems to be different from other mammals at least for: a) SRY expression SOX9 gene in the dog (CanFam3) seems to possess many assembly problems and consequently comparative analyses between this locus and the homolog locus in other species is quite difficult .Unfortunately the genome assembly around the


Supplemental Digital Content is available in the text The cut-point for diagnosing impaired fasting glucose (IFG) had been dispute, as reports about the associated clinical events are inconsistent. This meta-analysis evaluated the risk of coronary heart disease (CHD) in association with different criterion of IFG according to the American Diabetes Association (ADA) or the World Health Organization (WHO) Expert Group. We included prospective cohort studies with multivariate-adjusted data on IFG and CHD for analysis. The relative risks (RRs) of CHD were calculated and reported with 95% confidence intervals (95% CIs). Seventeen prospective cohort studies, comprising 527,021 individuals were included. The risks of CHD were increased in both participants with IFG defined as the ADA or WHO criterion . Subgroup analyses showed that in both definition of IFG, the risk of CHD was only increased in studies with possibility of enrolling patients with increased 2 hours plasma glucose (2-h PG), or in studies with inadequate adjustment, but not in studies excluded participants with increased 2-h PG or in those with adequate adjustment of other risk factors. Our meta-analysis demonstrates that the presence of IFG was significantly associated with future risk of CHD. The risk of CHD was increased when fasting plasma glucose was as low as 100 mg/dL according to the lower cut-point of IFG by the ADA criterion. However, the risk maybe confounded by the undetected increased 2-h PG or other cardiovascular risk factors. The reference lists of published articles and reviews on this topic were also checked to identify other eligible studies. The detailed search strategy used for PubMed is presented in online supplementary Table S1, http://links.lww.com/MD/A453. The strategy for the EMBASE database was similar, but was adapted where necessary.The search strategy was performed in accordance with the recommendations of the Meta-analysis of Observational Studies in Epidemiology (MOOSE) Group.6 Corresponding NFG comparator was defined as FPG < 100 or < 110 mg/dL, respectively.The inclusion criteria of studies for analysis were: prospective cohort studies involving adult participants (aged ≥18 years) with assessment of risk of CHD; blood glucose and other cardiovascular risk factors were evaluated at baseline; and adjusted relative risk (RR) and 95% confidence intervals (CIs) reported for events associated with IFG relative to normal fasting glucose (NFG). IFG defined as FPG of 100 to 125 mg/dL (IFG 100) or 110 to 125 mg/dL (IFG 110).13Studies were excluded if: data were collected from patients with a particular condition  but not general population; not accessed the risk of CHD in people with IFG compared with NFG; the risk of CHD in IFG was unadjusted for other risk factors; or reports were derived from the same cohort. If duplicate publications were identified as from the same cohort, only data from the most recent publication were used for analysis.14 which based on the assessment of bias for selection, comparability, and exposure/outcome, with a total score up to 9. In this meta-analysis, included studies were graded as good quality if they with a score ≥7, fair if they had less than 7 score.13 We also evaluated whether the studies were adequate adjusted for potential confounders .Two authors (TY and WL) independently conducted independent literature searches, reviewed the potentially articles, and abstracted data from eligible studies. The quality assessment was evaluated according to the Newcastle–Ottawa Quality Assessment Scale (NOS) for assessing the quality of nonrandomized studies in meta-analyses,We analyzed the RR of CHD in individuals with different definition of IFG. Subgroup analyses were conducted according to sex (women vs men), ethnicity (Asian vs non-Asian), specific end points , participant's age (average <50 vs ≥50 years), follow-up duration (<10 vs ≥10 years), possibility of enrolling patients with diabetes (yes vs no), and adjustment of risk factors (adequate vs un-adequate).16 The inverse variance method was used to combine the log RRs and SEs using random effects models. The I2 statistic was used to estimate between-study heterogeneity. Values of I2 > 50% were considered to indicate significant heterogeneity. The estimated RRs were calculated using random-effects models. The test for subgroup differences was calculated by Chi-square statistics.We extracted the most adjusted RRs and 95% CIs from each included studies and logarithmically transformed these values, calculated the corresponding standard errors (SEs) to stabilize the variance and normalize the distribution.P values were 2-tailed, and the statistical significance was set at 0.05. All analyses were performed with RevMan software .Publication bias was assessed by inspecting funnel plots for each outcome in which the natural log of RR was plotted against its SE. Sensitivity analyses were conducted by omitting one study at a time and recalculating the estimated RRs and CIs. 33 .33Oral glucose tolerance tests (OGTTs) were only performed in 6 studies and patients with increased 2 hours plasma glucose (2-h PG) were excluded for the analysis of risk in IFG.http://links.lww.com/MD/A453. Furthermore, according to the confounders adjusted, 7 studies did not meet our criteria for adequate adjustment26 and 10 studies were adequate adjusted for other potential confounders.33All studies were graded as good quality accessed by the NOS. The details of the quality assessment are presented in Supplemental Table 2, 33 There was moderate between-study heterogeneity in these studies (I2 = 33%). Meta-analysis using random-effects models showed that the risk of CHD was significantly increased in individuals with IFG 100 , and the risk of CHD was significantly increased in individuals with IFG 110  or IFG 110 group .Visual inspection of funnel plots suggested that there was no evidence of publication bias for either IFG 100 .The results of subgroup analyses are presented in Table P > 0.1, I2 = 0%).In subgroups analysis of IFG 110, the risk of CHD was also increased in studies with possibility of enrolling patients with increased 2-h PG , not in studies excluded participants with increased 2-h PG , in studies with inadequate adjustment , but not in those with adequate adjustment of other risk factors . However, there were no significant differences among all subgroups comparison . Many studies have shown that IGT was a stronger predictor of cardiovascular events than IFG.35 However, routine detection of IGT had been questioned due to the inconvenient use of OGTT and the results are not highly reproducible. Our results highlight the notion that OGTT could be required for further diagnosing individuals with IFG.36The 2003 ADA criterion of IFG had been criticized as it significantly increased the prevalence of IFG while without improvement of prediction for risk of CVD.37 Successful interventions in this large population could have a major public health impact. It had been proved that lifestyle is a fundamental management approach that can effectively prevent the progression from prediabetes to diabetes.38 Furthermore, recently data showed that lifestyle intervention in IGT can reduce incidence of cardiovascular and all-cause mortality.39 However, the evidence regarding CVD prevention in people with IFG is still absent.It has been estimated that, by the year of 2025, the number of people with prediabetes will be 472 millions.1 but most of the included studies did not adjust for subsequent blood glucose levels. So, the long-term risk of CHD in people with IFG was caused by the mild elevation of blood glucose or the future progression of DM remains unknown. However, it had been indicated that coronary atherosclerosis detected by intravascular imaging modalities is already ongoing in prediabetic status.40 Second, the adjusted confounders in the included studies were inconsistent and may be a potential source of bias in our study. However, it is interesting that, in both IFG 100 and IFG 110 subgroup analysis, the risk of CHD was increased in studies with inadequate adjustment, but not in those with adequate adjustment of other cardiovascular risk factors. These results reinforce the importance of detection of other cardiovascular risk factors in risk stratification of people with IFG.41The main strengths of our study are the very large sample size with general population included from prospective cohort studies. Detailed subgroup analyses also found interesting results that the risk of CHD associated with FPG may be confounded by the undetected increased 2-h PG and other cardiovascular risk factors. However, our study also has some limitations. First, individuals with IFG are more likely to progress to DM than those with normoglycemia,In conclusion, this meta-analysis showed that IFG was associated with an increased risk of CHD. The risk increased in people with FPG as low as 100 mg/dL. These results reaffirm the importance of screening for prediabetes using the ADA criteria. Furthermore, detection of 2-h PG and other cardiovascular risk factors are important for risk stratification in people with IFG. These informations are important for the prevention of DM and CVD.


The pseudoautosomal region (PAR) is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome. Recombination is a fundamental biological process, which shuffles genes between pairs of chromosomes during the production of eggs and sperm. After shuffling, the chromosomes consist of alternating sequences of genes from each parent, where the switches are the result of ‘crossovers’. Recombination is essential for eggs and sperm to receive the correct number of chromosomes, failure in which is an important cause of miscarriage, birth defects and mental retardation. Males have the particular challenge of recombining between the X and Y chromosomes. Unlike the other 22 chromosome pairs, the X and Y chromosomes do not match up, except for a small special region called PAR1, which must host a crossover. We investigate recombination in PAR1 by building a ‘map’ of where it occurs in African-American families. We use a variety of approaches, both analytical and experimental, to demonstrate the role of a protein called PRDM9 in marking crossovers in this region. PRDM9 has previously been shown to position crossovers on the other chromosomes, but a role in PAR1 was unexpected based on research in mice. We also show that the recombination map has changed in the evolutionary history of PAR1, both among human populations, and between human and chimpanzee. Pseudoautosomal regions (PARs) are segments of sequence homology between the X and Y (or Z and W) chromosomes, which are otherwise non-homologous. Uniquely, PARs are inherited in the same manner as autosomes, while also being partially linked with X-specific and Y-specific loci. They have a critical role in the successful progression of meiosis in mammalian males and in the heterogametic sex in many other plant and animal species In addition to their vital role in fertility, PARs contain genes in all mammals whose sequence has become available so far. The human PARs together contain at least 29 genes, with diverse roles in cell signalling, transcriptional regulation and mitochondrial function Studies in viable human sperm and pedigrees have shown that the recombination rate in PAR1 is consistent with one obligatory crossover per male meiosis, accompanied very rarely by a second crossover The second human pseudoautosomal region, PAR2, is much smaller at approximately 330 kb and specific to the human lineage, having likely arisen due to a translocation between the X and Y chromosomes Despite the critical role of PAR1 in fertility and disease, an understanding of its biology remains highly incomplete. In the reference human genome, the PAR1 sequence is not yet fully assembled, likely because of the exceptionally high GC-content and density of repetitive regions it contains. Since the publication of the X chromosome sequence Even less is known about recombination, which lies at the heart of PAR1 biology. For instance, it is not known how the extraordinarily high rate of recombination in this region is achieved biologically. In the autosomes, recombination clusters into short 1–2 kb segments known as ‘recombination hotspots’, which are flanked by regions with very low recombination rate An intriguing study Prdm9 variants have completely different autosomal recombination patterns, yet show similar recombination landscapes in and adjacent to the PAR region. Brick et al.Prdm9 may be positioning crossovers in the mouse PAR.The role of another key recombination protein, PRDM9, is also unclear in the PAR. Several lines of evidence have shown recently that PRDM9 positions sites of recombination in human and mice autosomes In this work, we aim to characterise the patterns of recombination in PAR1 to learn more about the biology of this region, and provide a resource for medical genetics research. We have built the most fine-scale genetic map containing directly identified crossovers to date in this region. This map contains more meioses, and an order of magnitude greater markers than the densest PAR1 map so far We have leveraged the genotype data of 220 markers from 135 African-American families with two or more children to build a new pedigree-based genetic map  in human cells . Specifically, we measured the binding of PRDM9 allele B, which is the human reference allele, and is predicted to have binding properties similar to PRDM9 allele A he motif , suggestFinally, we report an intriguing characteristic of the binding peaks in PAR1. Approximately 42% of PAR1 peaks contain close matches to the motif, which is consistent with the expected number of hotspots containing the motif in the autosomes The PRDM9 zinc finger array is highly variable in humans, with around 40 different alleles that have been identified so far P = 0.009). This suggests that recombination has evolved within the human lineage in PAR1, in a manner similar to the evolution observed in the autosomes.As shown in a previous section, African-American pedigree fathers have a significantly greater usage of the African (YRI) map than the European (CEU) map (P = 0.002 and 0.02 respectively) , African (YRI), East Asian (JPT+CHB) LD-based maps at the ChIP-seq binding sites of allele B, which is predicted to bind the 13-bp motif. As expected, the increase in rate in both the Asian and European maps near the binding sites is greater than that in the African map (ctively) . This sugene conversionbiased gene conversion (BGC). Several types of bias have been observed in different eukaryotes Programmed double-strand breaks leading to recombination may be resolved in one of two ways, as crossovers, which involve reciprocal exchange of chromosomal material, or as non-crossovers, which do not Fxy presents a particularly striking case study, indicating that GC-bias may operate in the mouse PAR as well. This gene has translocated from the non-recombining part of the mouse Y-chromosome to its PAR within the last 3 million years A broad range of evidence, across several eukaryotic taxa, indicates that bias towards GC bases is associated with recombinationWe investigated whether such a bias is observed in the human PAR1, both in the frequency of segregating sites and for the fixation of alleles leading to substitutions between human and chimpanzee. We reasoned that such a bias, if present, should act as an indirect marker of sites undergoing recombination in the two species, even in the absence of direct evidence on recombination sites in PAR1 in the chimpanzee. We investigated these patterns in (relatively) hot and cold regions of PAR1, and around copies of the 13-bp motif CCnCCnTnnCCnC, which marks peaks of recombination in PAR1 as shown above. Finally, we compared the distribution of GC-altering substitutions between human and chimpanzee to understand the evolution of recombination hotspots between the two species.Pan troglodytes), which again has 48% GC content in the PAR. We used 1000 Genomes data PAR1 in humans has a far higher GC content than the rest of the X chromosome (48% vs 39%)  to PAR1 . This su to PAR1 , consistnear the motif relative to that class of mutations in PAR1 as a whole. We observed that the elevation of the allele frequencies of GC mutations near the motif is extreme, and far stronger, over and above the rest of PAR1 . The signal is local to the motif, and weakens rapidly with distance away from it (It is significantly stronger within 25 bases of copies of the motif relative to PAR as a whole (P = 0.008), and also relative to within 500 bases of copies of the motif (P = 0.01)). The lowering of allele frequencies of AT mutations is also extremely strong near the motif relative to the rest of PAR1. The effect is strongest within 25 bp of the motif, and weakens with distance from it (P = 0.02 relative to PAR as a whole).We examined the role of PRDM9 by examining the allele frequency distributions of GCWe expect that, due to the much higher male recombination rate in PAR1, the GC-bias in PAR1 is driven mainly by male recombination. We confirmed this by comparing two regions with opposite trends in male and female recombination rates . Therefoet al.Brick P = 0.05). We note that this estimate is conservative since a subset of variants will have the wrong ancestral allele assigned due to polymorphism or errors in the chimpanzee . Another way to estimate the fixation bias close to the motif, in a conservative way, is to model the allele frequency distribution of derived GC alleles as a mixture of the PAR-wide allele frequency distribution of GC alleles, and a perfectly symmetric U-shaped distribution representing a situation where derived alleles are either newly arisen or completely fixed. Such an analysis indicates that 28.4% of motifs in the PAR are extremely active. This contrasts with an estimated 3% of motifs in Chr 20, which is consistent with previous autosomal estimates Within 25 bases of the 13-bp motif CCnCCnTnnCCnC , the fixIn the section above, we showed that recombination in PAR1 strongly accelerates the fixation of ATWe test this hypothesis by comparing human and chimpanzee PAR sequence. While no fine-scale genetic map is available for the chimpanzee PAR, we compare substitutions in the two species in regions which are hotspots in humans. Specifically, we consider substitutions in syntenic regions using a human-chimpanzee sequence alignment . If hotspots are the same in both species, we expect to see comparable numbers of To quantify the relationship between substitution and recombination rate, we modelled substitution rates using a linear model with recombination rate, GC content and CpG content as explanatory variables. We performed this analysis in 2 kb intervals, the approximate size of a hotspot However, while human recombination rate is strongly correlated with GC-biased transitions in humans, there is no evidence that it is correlated with GC-biased transitions in chimpanzee  in the sFinally, we investigated whether hotspot heat can be predicted using the observed substitution patterns. Current approaches, such as the building of LD-based maps, require multiple individuals from a species to be genotyped or sequenced. Since such data are currently not available for the PAR in most organisms, an ability to build recombination maps using only the reference sequence of closely related species could provide a preliminary method to analyse recombination. We found that the ‘optimal’ linear model using the human-chimpanzee divergence patterns  explains 23% of the variance in the LD-based map . While tIn this work, we have built the most fine-scale genetic map to date from directly inferred crossovers for the human PAR1. We used this map to validate, for the first time, the previously built LD-based genetic map in this region, which localises recombination to a resolution close to the size of a hotspot. We also show the existence of biological differences between LD-based maps in different populations. We hope that these resources will promote research in this gene-rich and fast-evolving region, which currently remains under-represented in both linkage studies and on genotyping chips used in large-scale disease association scans.Our analysis indicates that, in contrast with evidence currently available for the mouse In many ways, PAR1 has a recombination profile in male meiosis resembling a miniature autosome, with an elevated crossover rate near the telomere. However, we observe a key difference in that a relatively high rate appears maintained throughout most of the region, without recombination coldspots as seen in the autosomes. A clue to what might be going on is perhaps given by the examination of mutations near the positions of the 13-bp motif CCnCCnTnnCCnC in PAR1, which revealed extreme skews in frequency spectra with almost no high frequency mutations toward AT bases and a U-shaped distribution of mutations towards GC bases, particularly for mutations within 25 bp of the motif . RecombiPrdm9-independent hotspots in the mouse PAR. For example, it may be that a back-up mechanism, independent of PRDM9, exists to ensure crossover occurs in the PAR. This back-up mechanism might, speculatively, be identical in the two mammals, but play a much larger role in mouse meiosis than in humans. This seems plausible to us based on PRDM9 binding target characteristics in the two species – the human PRDM9 target is GC-rich Prdm9 alleles recognize much more AT-rich motifs We note that it is not clear our results are in contradiction with the finding of We have used genotype data from 135 previously published African-American pedigrees Each family had at least two children, and at least one parent genotyped. Crossovers were identified using an adaptation of the Lander-Green algorithm To increase power to detect crossovers near the pseudoautosomal boundary, we have included 100 SNPs from the X chromosome . FathersThe algorithm estimates the posterior probability of crossover in each SNP interval across all parents. To build a male map, we add the probability of crossover for each SNP interval for all fathers, and divide by the total number of male meioses. We repeat this process for mothers to produce a female map. We post-process the cumulative posterior probability distribution of crossover over all SNP intervals for each parent to identify individual crossovers .The male and female genetic maps are provided in The HapMap2 population-averaged LD-based map for PAR1 was downloaded from:https://mathgen.stats.ox.ac.uk/impute/impute_v1.html#DownloadPopulation-specific recombination maps were kindly provided by Colin Freeman from the Wellcome Trust Centre for Human Genetics, Oxford University.LiftOver tool PRDM9 B-allele was synthesised and cloned into a transient expression vector  alignment available from Ensembl. The alignment was restricted to regions with accurate expected LD-based map rates . After this, the alignment contains approximately 1.2 Mb of sequence. For this analysis, we divided PAR1 into 2 kb regions, and included only those regions for analysis where at least 1 kb of the sequence was not repeat-masked and aligned without deletions or missing data on either lineage. A small number of regions were observed with total human/chimpanzee divergence greater than 5% and up to 11%. They were strongly clustered and represented clear outliers in the divergence distribution. These were filtered out from the analysis as they are not representative of PAR1 in general, and because we suspect that they represent mismapped or misaligned regions.A stepwise search was performed to predict recombination rate using a linear model. The Aikake Information Criterion (AIC) was used to perform model selection and minimize overfitting. The full set of explanatory variables considered were the GC-content fraction, CpG content fraction and divergence rates for each of Informed consent was provided by all the individuals participating in the study, and was approved by all of the institutions responsible for sample collection.Figure S1Broad-scale pedigree-based maps for PAR1. Sex-specific pedigree-based genetic maps smoothed to 250 kb to reveal broad-scale trends. The male map (blue) shows a decreasing overall trend away from the telomere, while the female map (red) shows an increase away from the telomere. The male map also shows a modest increase in rates close to the pseudoautosomal boundary. Vertical black tick marks show marker positions. Repeat content is calculated at a 10 kb scale. Physical coordinates are in build 36.(PDF)Click here for additional data file.Figure S2Recombination rate in PAR1 near copies of CCnCCnTnnCCnC in L2 elements. The presence of the canonical human 13-bp motif CCnCCnTnnCCnC predicts a strong local increase in recombination rate in the LD-based map. This plot shows rates around the 4 instances of L2 elements containing an exact match to the motif and where rates could be estimated .(PDF)Click here for additional data file.Figure S3Recombination rate in PAR1 around PRDM9 binding sites identified by ChIP-seq. Rate in the HapMap2 population-averaged LD-based map in the vicinity of ChIP-seq binding locations of the PRDM9 reference allele  in PAR1 for: (a) binding locations containing at least one close match to the PRDM9 binding motif (b) binding locations without a close match to the motif.(PDF)Click here for additional data file.Figure S4The frequency spectra of derived alleles in Chr 20. The frequency spectra of derived alleles in Chr 20 shows a U-shaped distribution, and an excess of high frequency GC-increasing mutations, relative to GC-reducing and GC-neutral mutations. However, the differences between the mutations are much greater in PAR1 .(PDF)Click here for additional data file.Figure S5Comparison of the allele frequency distributions of AT(PDF)Click here for additional data file.Figure S6within 50 bp of the motif CCnCCnTnnCCnC in two 500 kb regions of the PAR, relative to those mutations throughout the respective regions (including both transitions and transversions). Recombinogenic activity of the motif is at least as high in the more+ telomeric region of PAR1 as it is in the region closest to the pseudoautosomal boundary.Comparison of the allele frequency distributions of AT(PDF)Click here for additional data file.Figure S7PAR1 genetic map estimated using the allele frequency spectra of derived alleles in human populations. A map estimated using a linear model based on 70th percentile of the derived allele frequency of AT(PDF)Click here for additional data file.Table S1Resolution of crossovers identified using African-American pedigrees.(PDF)Click here for additional data file.Table S2Male and female broad-scale rates in PAR1.(PDF)Click here for additional data file.Table S3Differences in male broad-scale rates in PAR1.(PDF)Click here for additional data file.Table S4Differences in female broad-scale rates in PAR1.(PDF)Click here for additional data file.Table S5Linear model for predicting recombination rate from sequence features.(PDF)Click here for additional data file.Dataset S1Markers in the pedigree-based map. Columns 1 and 2 are the rsID and Build 36 positions of the SNPs respectively. Columns 3–6 show whether the SNPs were included in each of the constituent studies, with “1” representing inclusion, and “0” not.(TXT)Click here for additional data file.Dataset S2Male and female pedigree-based maps. Column 1 is the Build 36 position, and columns 2 and 3 are the cumulative genetic distance in Morgans up to the marker in column 1.(TXT)Click here for additional data file.Dataset S3Paternal Crossover locations. Columns 1 and 2 are the start and end points of paternal crossovers in build 36.(TXT)Click here for additional data file.Dataset S4Maternal Crossover locations. Columns 1 and 2 are the start and end points of maternal crossovers in build 36.(TXT)Click here for additional data file.Dataset S5PRDM9 binding sites. Binding sites inferred by a ChIP-seq protocol in human cells. Columns 1 and 2 are the start and end points of inferred binding peaks in Build 37. Column 3 is the p-value of the peak call.(TXT)Click here for additional data file.Text S1Supplementary text. Details of pedigree map-building work, and the ChIP-seq experimental protocol to measure PRDM9 binding in human cells.(PDF)Click here for additional data file.


The primary aim of this study was to evaluate and compare the effectiveness of two specific Non-Invasive Brain Stimulation (NIBS) paradigms, the repetitive Transcranial Magnetic Stimulation (rTMS), and transcranial Direct Current Stimulation (tDCS), in the upper limb rehabilitation of patients with stroke. Short and long term outcomes  were evaluated. We measured, at multiple time points, the manual dexterity using a validated clinical scale (ARAT), electroencephalography auditory event related potentials, and neuropsychological performances in patients with chronic stroke of middle severity. Thirty four patients were enrolled and randomized. The intervention group was treated with a NIBS protocol longer than usual, applying a second cycle of stimulation, after a washout period, using different techniques in the two cycles (rTMS/tDCS). We compared the results with a control group treated with sham stimulation. We split the data analysis into three studies. In this first study we examined if a cumulative effect was clinically visible. In the second study we compared the effects of the two techniques. In the third study we explored if patients with minor cognitive impairment have most benefit from the treatment and if cognitive and motor outcomes were correlated. We found that the impairment in some cognitive domains cannot be considered an exclusion criterion for rehabilitation with NIBS. ERP improved, related to cognitive and attentional processes after stimulation on the motor cortex, but transitorily. This effect could be linked to the restoration of hemispheric balance or by the effects of distant connections. In our study the effects of the two NIBS were comparable, with some advantages using tDCS vs. rTMS in stroke rehabilitation. Finally we found that more than one cycle (2–4 weeks), spaced out by washout periods, should be used, only in responder patients, to obtain clinical relevant results. Motor and cognitive impairment are frequent aftermaths of brain damage after a stroke. Many authors reports cognitive deficits in 12–56% of stroke patients and reduced performances in several cognitive domains in 32%  are a reproducible electrophysiological response to an external stimulus , representing the brain activity associated with various cognitive processes such as selective attention, memory, or decision making. Interestingly, ERP can be valuable in the diagnosis of cognitive impairment and are able to track the cognitive changes during the follow-up in stroke patients  techniques have been proposed as support of standard cognitive and motor rehabilitation. The application of NIBS in stroke rehabilitation arises from the observation that cortical excitability can be modulated after electrical or magnetic brain stimulation. It can be reduced or enhanced  and Long Term Depression (LTD) like mechanisms  and transcranial Direct Current Stimulation (tDCS) are the most used NIBS techniques in rehabilitation .A randomized clinical trial divided into three studies was designed to explore the following issues:A longer NIBS stimulation could be beneficial in stroke rehabilitation?What are the differences between rTMS and tDCS in stroke rehabilitation?NIBS motor stimulation effects can modulate or be modulated by patients' cognitive status?In the first study we evaluated if a cumulative effect, mediated by an offline improvement (consolidation or savings), was clinically detectable. We also evaluated the differences between a first priming cycle with rTMS followed by tDCS and first priming with tDCS followed by rTMS.In the second study we compared the effects of the two techniques to test if brain plasticity effects could depend on the type of NIBS. In the third study, we searched for a possible link between motor and cognition changes, evaluating if cognitive measures changed in patients with motor improvement differently from the patients without motor improvement.Thirty four consecutive patients , interventional, with a factorial design  received 10 daily sessions of rTMS for 2 weeks and after a washout period (at least 6 months) 10 daily sessions of dual-tDCS + MT for 2 weeks.The first intervention group  received dual-tDCS + MT and then rTMS, after washout.The second intervention group  received 10 daily sessions of sham-tDCS + MT for 2 weeks.A control group (The primary outcome measure was the Action Research Arm Test (ARAT) a quantitative upper extremity function test. The endpoint for a successful intervention was set, considering the Minimal Clinically Important Difference (MCID/MCD), in a range between 3.5 and 5.7 points  auditory evoked response potentials (ERP) and neuropsychological paper and pencil tests (NPS).Time frames for the outcome measurements and interventions administration are shown in Figure Interventions were administered at weeks 2–3 and at around weeks 26–27 (6 months and 2–3 weeks). At baseline (T0) ARAT, ERP and NPS were assessed, at T1 (4 weeks) ARAT, ERP and NPS, at T2 (3 months) ARAT, at T3 (6 months) ARAT and NPS, at T0′ (6–11 months and 1 week) ARAT, ERP and NPS, at T1′ (7–12 months) ARAT, ERP and NPS, at T2′ (9–14 months) ARAT, at T3′ (12–17 months) ARAT and NPS.The study was realized in a clinical setting so we decide to apply both stimulations  in the same patients to compare the safety and effectiveness of the techniques intra-patient, overcoming the problem of the sample's heterogeneity.Demographic or clinical variables did not significantly differ between the patients that received the interventions and the control group . It has four subscales: grasp (6 tasks), grip (4 tasks), pinch (6 tasks), and gross movements (3 tasks). In the first three subscales the tasks consist in grasping, moving and releasing different objects , the last scale consists of three large movements . Summing the scores, ARAT ranges from 0 to 57; higher scores indicate better upper extremity functionality. It is a reliable, valid, and standardized functional assessment tool with good to excellent psychometric characteristics . The patient was placed in a comfortable seated position, controlled by a technician in order to prevent drowsiness and limb movements. Nineteen standard scalp electrodes were applied to the scalp in accordance with the 10–20 International System . Impedance was < 2 kΩ in each active lead before the starting of the recording, and the reference was obtained by averaging the channels. Data were collected for each subject and digitized at a sampling rate of 256 Hz, with a band pass filter of 0.1–70 Hz and a notch filter to remove the main electrical noise in each channel. EEG was recorded for 5 min periods at rest (baseline). Electrooculography was simultaneously recorded with two electrodes placed near the left eye to detect and reject ocular movement artifacts from EEG data offline. ERP was recorded using the auditory oddball paradigm. The subjects were asked to react by counting target stimuli appearing rarely (r) amongst a series of more common stimuli (f) administered bilaterally by stereophonic earphones at 100 dB. Rare stimuli consisted in a 1500 Hz pure tone and frequent stimuli consisted in a 1000 Hz pure tone, presented in random order and with a mean r/f ratio of 1 r every 6 f. The recorded signal was cut appropriately (the time window started 200 ms before and ended 1100 ms after the stimulus) and ERP were averaged separately for the rare and frequent stimulus (using a mean of 20 stimuli), and the latencies of endogenous N200 and P300 components were evaluated according to the international recommendation  involving the following domains: verbal short-term memory, visuospatial learning, working memory, verbal learning, attention and frontal executive functions, and general cognitive impairment. Parallel and equivalent forms were used for all tests; we used standardized tests with normative values for the Italian population  was administrated using a PowerMAG 100 device , at 80% resting motor threshold, for 15 min (900 stimuli) over the intact M1 with an eight-shaped coil. The coil was placed on M1, aiming for cortical area coding for hands' movement.Single pulse TMS was used to determine bilateral M1 hot spots for the first dorsal interosseus (FDI) muscles defined as the place onto the scalps where the motor evoked potential (MEP) was maximum. MEP where obtained with the 120% of the minimum intensity required eliciting electromyography activity of at least 50 μV peak-to-peak amplitude in ≥50% of pursued trials (≥3/6) with the muscle at rest.We registered surface electromyography with the Neurowerk EMG  moving the coil in a grid of 0.5-cm steps medial, lateral, posterior, and anterior until the point of the maximum MEP was ascertained. The procedure was repeated iteratively until the hot spot was identified. The distances between C3/C4 and TMS hotspots were noted bilaterally for all patients. Cortical targets were identified using SofTaxic neuronavigation system  along sessions.2 pads (one anode and one cathode) soaked with saline solution. The tDCS was applied with the cathode onto the controlesional M1 and the anode onto the perilesional M1, the stimulation was online together with mirror-box therapy (MT). The anode (stimulating activity) was placed on the damaged hemisphere in the area corresponding to C3 or C4 position in the 10–20 systems, while the cathode (inhibitory activity) was placed in the analog position on the opposite hemisphere in a dual-tDCS design. The intensity of the stimulation was set at 1.5 mA and the duration of the tDCS was set at 20 min.tDCS was administrated using a HDC Stim device , via two 5 × 5 cmIn our sample the mean distance between hotspots and C3/C4 was 2.1 ± 1.7 cm for the unaffected hemisphere and 2.4 ± 1.5 cm for the lesioned hemisphere; around 67% of the hotspots were inside the areas covered by pads.MT consisted in the optical illusion of bimanual movements created by a box with a mirror in the middle, it has been ideated by Ramachandran et al.  to treatThe box consisted in a wooden enclosure separated in two sections by a mirror. The patients had to insert his/her hand through the holes situated on the side of the box and could watch the normal hand while performing the requested gestures. The sensation of the movement of the plegic hand was generated when the patients looked at the reflection of the normal hand during the exercises. During the tDCS application the patient had to execute 3 series of 25 repetitions of 6 different movements . The exercises were changed completely between the first and the second week of intervention.Only tDCS stimulation was paired with MT to enhance its specificity to reach the level of focal rTMS targeting the hand area. In fact, one way to gain specificity is to have a precise simulation of the delivered power on a small anatomical area of interest  when needed, otherwise we adopted non parametric tests . For post-hoc comparisons we used Tukey HSD or Simes corrections (respectively for parametric and non-parametric tests).We adopted parametric statistics  are still significant after a Bonferroni correction (p < 0.017) for multiple comparisons.As we tested multiple hypotheses with three different studies we also controlled if the significative results  and one between factor . In addition, we compared, with the same model, the sham group and the subgroup of responders, defined as patients that get an improvement in ARAT score after the first stimulation, to look if responders reached the MCID/MCD. Finally, we used a repeated measures ANOVA  and one between factor (priming stimulation tDCS or rTMS) to look if the second cycle could be useful and if the order of priming stimulation was relevant on outcome.Similar model were used for ERP and NPS with appropriate repetitions over time frames.To compare ERP we used the repeated measures ANOVA  and one between factor . In addition, we used the repeated measures ANOVA  and one between factor (priming stimulation tDCS or rTMS).To compare NPS we used the repeated measures ANOVA  and one between factor . In addition, we used the repeated measures ANOVA  and one between factor (priming stimulation tDCS or rTMS).To compare tDCS and rTMS induced changes for ARAT, ERP, and NPS intra-patients in the real stimulations groups we used a repeated measures ANOVA with time  and type of NIBS (two levels) as factors.We also tested if rTMS and tDCS had a similar level of specificity looking at the profiles of ARAT subscales that, in our hypothesis, should be improved in a similar way by the two stimulations.We also looked if responders had some differences in the cognitive measures (ERP and NPS) at baseline or after one or after two cycles of stimulations using repeated measure ANOVA with time  and responders vs. no-responders (between) as factors.p = 0.212).ARAT did not differ at baseline . Considering the P300 grand mean, latencies were over a normative cut off, determined on the basis of published data .At baseline, there were significant differences for the N200 and P300 in the study group. Indeed the sham group had shorter latencies compared to other groups , group  or their interaction .We did not find any significant effect on ARAT scores for time  and time by group , but not for group . It is evident that the effect of time and the interaction is due to the responders that reached the MCID/MCD range .The responders subgroup included the 44% of the treated patients, while only 20% of sham patients improved their ARAT score; all responders improved also after the second intervention and the gains were stable in the 75% of cases after 6 months. Most (90%) non-responders did not improve after the second intervention. When comparing responders vs. sham  but not priming stimulation  or interaction , the post-hoc analysis showed that there were higher values at the end of the second cycle of NIBS .ARAT had a very similar progression for both groups and the two sequential protocols Figure , reachinF = 41.9, p < 0.001, η2 = 0.2) and P300  latencies. The electrode was not significant for both N200  and P300 . The intervention was significant on both N200  and P300 . A significant interaction term time by intervention was found for N200  and P300 . Results survived Bonferroni correction (p < 0.016). Other interactions terms were all not significant for N200 and P300.Time had a significant effect on both N200  and tDCS+rTMS  groups compared to sham .In the F = 5.9, p = 0.006, η2 = 0.2), as was time by intervention interaction , but not for intervention . In the post-hoc analyses rTMS+tDCS and tDCS+rTMS groups had a similar stable improvement that the sham group did not show.Time was only significant for the copy of figure with immediate recall , for the dual-tDCS group  and for the rTMS group . Time was a significant factor for the other three tests only in the dual-tDCS group: copy of figure with delayed recall , attentional matrices , and perseverations in the Nelson's MCST test . See Table post-hoc tests and average differences. Only some results survived Bonferroni correction (p < 0.016).Time had a significant effect only for four tests: the copy of figure with immediate recall was significant for the sham-tDCS group  have been excluded, in order to reduce confounding factors, but focal deficits were detected in some patients, mostly with left hemisphere lesions Tables , 2. This5 have beThe interventions were safe and tolerated Table  and had Motor improvement in hands' functionality, measured with ARAT, was observed after NIBS treatment in a large percentage of patients (44%), but not after sham Figure . The effERP endogenous components (N200 and P300) reflect perceptual and cognitive processing and can play an important role in testing stroke patients . On the other hand, ERP were not useful to predict long term outcome and to identify responders or as a surrogate quantitative marker of the effect of NIBS.The cognitive improvement was prevalently observed in dual-tDCS + MT for tests that are mainly influenced by the visuospatial domain . While some results were also found in TMS or in sham + MT they were not stable at follow-up as were instead in dual-tDCS + MT. This could be interpreted as an effect of neural plasticity that strengthened and stabilized the MT rehabilitation training. This result is not surprising as MT could enhance spatial coupling, as previously argued by Michielsen et al. Indeed, they hypothesized that the mirror illusion could increase the tendency of one limb to take on the spatial properties of the other . Consequently, cortical stimulation targets have been chosen in order to improve plasticity of cortical and sub-cortical motor networks and not specifically of cognitive networks. Nevertheless, a transient cognitive improvement has been observed after each NIBS technique. These observations demonstrate that the actual knowledge of physiological mechanisms underlying NIBS techniques is still very limited. In fact, many different cortical targets have been stimulated with many different NIBS techniques  to improve cortical plasticity in cognitive rehabilitation protocols on attention domain, but results are contradictory , so it was impossible to provide a precise functional map of the damaged cortical and subcortical areas.There were some differences in N200 and P300 at baseline in the different groups.rTMS was underpowered compared to tDCS as in the second arm two interventions (tDCS + MT) were administered, but it was not feasible to use MT online with TMS;The poor knowledge of physiologic mechanisms could limit the interpretations of our results.The present study allows some practical considerations, useful for neuro-rehabilitation:First the impairment in some cognitive domains cannot be considered an exclusion criterion for rehabilitation with NIBS.Second, NIBS generally improved ERP, but transitorily.Third, attentive processes depend on different cortical areas and may improve with brain stimulation, also on M1, perhaps because of restoring the hemispheric balance or by distant connections effects.Finally, NIBS effects were comparable, but there are some advantages of using tDCS vs. rTMS in stroke rehabilitation. More than one NIBS cycle (2–4 weeks) should be used in rehabilitation to obtain clinical relevant results after a washout period only in responder patients.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.