The organization of night shift work affects sleep duration and quality. The aim of this study was to investigate the effects of the number of consecutive night shifts on sleep duration and quality among police officers with night shift work as part of their normal schedule.This quasi-experimental, within-subject crossover study included 73 police officers. All participants performed three work schedules: two, four and seven consecutive night shifts followed by the same number of recovery days, ie, day work or days off . Sleep assessed through sleep diaries and actigraphy after all night shifts and recovery days  was compared by use of repeated measures analysis.Participants experienced shorter sleep duration (with and without naps), more premature awakening, less difficulty falling asleep, and more non-refreshing sleep after night shifts compared with recovery days. Sleep duration and quality did not change with increasing number of consecutive night shifts. Sleep was shorter and of poorer quality after the last night shift in the 2+2 and 4+4 work schedule compared with the second and fourth night shift, respectively, in the 7+7 schedule.Sleep duration was reduced after night shift work and did not increase with more consecutive night shifts, which leads to accumulated sleep debt. Sleep duration was shortest and sleep quality was poorest after the last night shift in a series of night shifts. It is well established that night shift work causes reduced and disturbed sleep. Night shift workers report premature awakenings and insufficient sleep, yet ~50% of individuals experience a spontaneous and effortless sleep termination , 2. The Short sleep duration and poor sleep quality has, in studies with longitudinal designs, been associated with higher risk of diabetes  and cardThe overall aim was to contribute to the knowledge about how to optimally organize night shift work in order to reduce sleep deprivation and negative health consequences. We therefore investigated the effects of the number of consecutive night shifts on sleep duration and quality among police officers with night shift work as part of their regular schedule. During the study period, each participant was enrolled in three different work schedules: two, four and seven consecutive night shifts followed by the same number of recovery days, ie, day work or days off . The main aim of the study was fulfilled by answering the following questions: (i) Is sleep duration shorter and sleep quality poorer after night shifts compared with recovery days (independent of work schedule)? (ii) Does sleep duration increase and sleep quality improve with an increasing number of consecutive night shifts or recovery days? (iii) Is sleep duration shorter and sleep quality poorer after the last night shift in a series of night shifts regardless of the number of previous night shifts?This paper presents results from the project “In the Middle of the Night”. The National Committee on Health Research Ethics in Denmark approved the study (protocol number H-4-2012-155).Conducted with a quasi-experimental, within-subject crossover design, the study exposed the participants to three different work schedules: two night shifts followed by two recovery days (2+2), four night shifts followed by four recovery days (4+4), and seven night shifts followed by seven recovery days (7+7). The term recovery day were used for days that allowed sleep at night and included both day shifts (31%) and days off (69%). Day shifts were typically 07:00–15:00  and night shifts were typically 23:00–07:00 . The participants did not work any night shifts during the seven days preceding the start of the three different work schedules.Data were collected either in the period April–June 2013 or September–November 2013. Daylight saving time began March 27 and ended October 27 in 2013. None of the participants had data collection on these two dates and only three participants collected data after the end of daylight saving time. The three different work schedules lasted 26 days in total, and they could be distributed over no longer than three months. The order of the three different work schedules occurred in the order that suited the person in charge of the personnel-on-duty planning. This person was instructed to mix the different work schedules so they occurred in different orders and to let the different work schedules in the study begin on different weekdays. In accordance with the usual way of scheduling the shifts, the starting date of the different work schedules was planned so that it also suited the individual employee. This meant that of the 64 participants who performed all three work schedules, 18 (28%) started with the 2+2, 17 (27%) with the 4+4 and 29 (45%) with the 7+7.Throughout all phases of the study, there was intensive collaboration and strong support from both the management in the Danish police and employee representatives from the labor union. We recruited participants from the five police districts in Zeeland, Denmark. The inclusion criteria were that the participants had to be non-smoking male police officers with night shifts as a part of their regular schedule, but not working permanent night. The officers use self-rostering to plan their normal schedules. They are therefore not regular and may vary from person to person. In most cases, they include up to four consecutive night shifts, although seven consecutive shifts were in some cases allowed if the officer requested it. Before recruitment began, information meetings were held for the leaders, the people responsible for personnel-on-duty planning, and employee representatives. All districts were also offered an initial information meeting for potential participants, which were accepted by two police districts. Thereafter, an e-mail was sent to all potential participants in all districts with an invitation to participate in the study. A total of 121 police officers showed interest in participating in the study. Of these, a total of 73 received individual, detailed information about the project either face-to-face or on the phone and completed at least one of the three work schedules, and 64 completed all three work schedules. Reasons for dropping out were holidays or other fixed duties, change to a job without night shift work or family considerations.The participants completed a background questionnaire before starting their first work schedule in the study. From the questionnaire, we obtained information about tenure within the police force, night shift work experience, physical activity, self-rated overall health, general job satisfaction, and diurnal type.All participants scored their sleep quality upon awakening from their primary sleep on all 26 data collection days. Primary sleep was defined as the first sleep episode after the night shift or as sleep during the night after recovery days. Sleep was scored using a modified version of the Karolinska Sleep Diary (KSD) , 19. In Actiwatches  were worn on the non-dominant wrist during all 26 data collection days. Data were collected with a sampling rate of 30 Hz and 1-minute epochs were used to score sleep. Data were analyzed with ActiGraph Sleep Analysis . In and out of bed times were taken from the sleep dairies. The following variables were extracted: primary sleep duration (PSD), total sleep time per day including naps (TST), and sleep efficiency.Unless otherwise stated, we performed repeated measures ANOVA using the PROC MIXED procedure with a random intercept for each individual to account for within subject variation . SeparatIn the first set of analyses, sleep duration and quality after night shifts were compared with sleep duration and quality on recovery days independent of work schedule using the full dataset  (research question 1). In the second set of analyses, repeated measures linear regression analysis was used to evaluate if there was a change in sleep duration and quality with increasing number of consecutive night shifts or recovery days (research question 2). These analyses were performed separately for night shifts and recovery days. In this set of analyses, the last night shift and the last recovery day of each work schedule were excluded, ie, the second night shift and the second recovery day in 2+2 and the fourth night shift and the fourth recovery day in 4+4 etc. This was done because (later) analyses showed that sleeping behavior was different after the last night shift, and including these data in the analysis could mislead the interpretation of the effect of number of consecutive of night shifts on sleep. In the third set of analyses, we investigated if sleep duration and quality after the last night shift in a series of night shifts differed from other days (research question 3). We compared sleep duration and quality after the last night shift in the 2+2 and 4+4 work schedule with the second and fourth night shift, respectively, in the 7+7 work schedule (reference). All statistical analyses were done in the statistical software SAS for Windows 9.4 .The participants were aged 25–62 years with a mean age of 38 years; 22% had <3 years of night shift work experience, 38% had 3–10 years of night shift work experience, and 40% had >10 years of night shift work experience. Most participants rated their health excellent (31%) or very good (47%) and 78% were moderately or highly physically active in their leisure time . Of the Participants had shorter PSD  and TST  after the last night shift in the 2+2 work schedule compared with the second night shift in the 7+7 work schedule (research question 3). Participants also experienced more difficulty awakening  and more non-refreshing sleep  after the last night shift in the 2+2 work schedule compared with the second night shift in the 7+7 work schedule. Similar differences were observed after the last night shift in the 4+4 schedule compared with the fourth night shift in the 7+7 work schedule . There wIn this study of 73 police officers working 2+2, 4+4 and 7+7 work schedules, we found that participants experienced shorter sleep duration (with and without naps), more premature awakening, less difficulty falling asleep, and more non-refreshing sleep after night shifts compared with recovery days. Sleep duration and quality did not change with increasing number of consecutive night shifts, but reports of difficulty falling asleep, difficulty awakening, non-refreshing sleep, and disturbed sleep decreased with increasing number of consecutive recovery days. Sleep was shorter and of poorer quality after the last night shift in a series of night shifts.It has been argued that more consecutive night shifts cause adaptation of circadian rhythms leading to better and longer sleep during the day eg, among employees on oil rigs in the North Sea . In contWe have previously shown results regarding diurnal rhythms of hormones from the same study . The rhyPrevious studies have found that few consecutive night shifts (as part of a fast forward-rotating schedules) were associated with fewer sleep disturbances and difficulties than more consecutive night shifts (as part of a slowly backwards-rotating schedules) –6. In thOur findings – that night shift workers experience shorter sleep and find it easier to fall asleep and wake up although not feeling refreshed after night shift work – are congruent with previous studies , 2. The The study comprised a relatively large number of participants compared with previous field studies. In combination with the quasi-experimental  cross-over design and taking within subject variation into account in the statistical analyses, this is a strength of the study. We thereby enhanced internal validity by circumventing potential confounding from, eg, age and lifestyle factors, and accordingly we did not include these variables in the statistical analyses. The study was performed among experienced shift workers in a real-life setting, which enhances the external validity of the results. To avoid the risk of false positive findings due to multiple statistical testing, we considered P<0.001 to indicate statistical significance.Another strength was the handling of the general limitations with cross-over designs, ie, possible order effects and carry-over between the different work schedules in the study. The order effect was limited by instructing the schedule planner to mix the different work schedules so they occurred in different orders, which resulted in 28%, 27%, and 45% of participants starting with 2+2, 4+4, or 7+7 work schedules, respectively. Carry-over effects were minimized by having a seven-day wash-out period without night shift work before the first night shift in a series. For practical reasons and in order to resemble real-life scheduling, recovery days were allowed to include both days off and day shifts. The rationale was that both are day-time oriented as opposed to days with night shifts and because this is the standard way to schedule night shift work among the police in Denmark. Since the normal day shift for police officers begin at 07:00 hours, it may be argued that police officers need to get up before their natural wake up time of days with day shift and that differences in the proportion of day shifts and days off would therefore bias the results. However, the bias is expected to be limited as there was a fair distribution of day shifts across the different work schedules (particularly 4+4 and 7+7) and awakening time on day shifts and days off differed by only 37 minutes.Each work schedule was only performed once. As a consequence, it was only possible to study acute effects and not effects of long-term scheduling. However, effects of night shift work on sleep are generally acute and reversible, and we expect that the main potential differences between the three different work schedules in relation to sleep are covered with the present design. The study population consisted of male shift workers. Accordingly, we cannot be sure if the results extend to women, although similar biological mechanisms are likely. It may be speculated that permanent night work allows for better adaptation to daytime sleep because permanent night workers do not have to change their diurnal rhythms between night shifts . However, even permanent night workers are likely to being awake during the days on days off, eg, to be with family and for social reasons. Indeed, it has been shown that only a very small minority (0.3%) of permanent night workers show “complete” adjustment of their endogenous melatonin rhythm to night work . Yet, fuIn conclusion, we found that sleep duration was reduced after night shift work and did not increase with more consecutive night shifts. This leads to accumulated sleep debt with more consecutive night shifts. Furthermore, sleep duration was shortest and sleep quality particularly poor after the last night shift in a series of night shifts.


Sapajus apella) from their group enclosure to individual test cubicles. Experiment 1, which assigned different colored and geometric targets to each animal, rewarded animals for touching and following their assigned target into their test cubicle. The animals rapidly acquired the ability to identify their target, ultimately allowing them to cooperate by moving from one space to another voluntarily. Experiment 2 rewarded animals for spontaneously identifying their assigned target among other animals’ targets and novel targets. The animals chose their target more often than predicted by chance, although they did make some errors.Coercion and non-voluntary procedures can cause fear, anxiety and maladaptive behaviors for captive animals, which makes animal husbandry and care more difficult and reduces animal welfare overall. Positive reinforcement training  is shown to be beneficial in reducing animals’ fear responses and in encouraging voluntary cooperation across a wide range of species. This paper outlines two experiments in positive reinforcement training to understand the importance of individualized targets in facilitating the voluntary transfer of captive capuchin monkeys (Sapajus apella) in two experiments designed to facilitate movement from a group home enclosure to a test cubicle. In Experiment 1, each monkey was assigned an individualized target (a unique shape/color combination). In daily training sessions, the animal was rewarded with a click-sounding stimulus and a food reinforcer for (a) touching the target, (b) following the respective target into a test cubicle, and (c) touching progressively smaller targets until progressing to digitized images on a computer touch screen. All 10 animals learned to approach and touch their individual physical target in one or two sessions and were able to successfully transition this behavior to an image of their target on a touch screen, although they made more errors with the touch screen. In Experiment 2, the animals were presented with other animals’ targets and novel targets. The seven animals in this experiment all touched their target at higher-than-chance rates in Trial 1 without explicit discrimination training, but only five reached the learning criteria for the task  can decrease stress related to these procedures and increase desired behaviors. Our goal was to demonstrate the positive effects of “target training” on animal welfare by training 10 captive capuchin monkeys ( Animals in human care must often be moved from one location to another to facilitate enclosure cleaning, animal care, and veterinary treatment. Animals that are moved involuntarily can develop fear responses, and in turn, counterproductive behaviors and stress . HistoriPRT procedures give animals the opportunity to participate in experimental and husbandry procedures voluntarily, thereby mitigating the possibility of fear and/or anxiety and biological stress responses becoming associated with testing equipment, husbandry procedures, and specific personnel ,3. PositCallithrix jacchus), for example, have been trained to participate in their daily health care plans by following a perceptibly distinctive ‘target’ stimulus such as a plastic spoon to voluntary mount scales for weight checks [PRT programs with both visual and auditory  discriminative stimuli have proven useful in many areas of animal care with captive nonhuman primates ,10,11,12t checks . Target t checks . Vocal at checks .Evans and colleagues  applied Sapajus apella) at Franklin and Marshall College (F&M) into a new facility, comprising a group-housing enclosure with adjoining individual test cubicles in October 2007. Like the facility used by Evans and colleagues [The present study was prompted by the movement of the capuchin monkey colony  and vocal cues  while presenting individual targets made out of laminated construction paper with a unique hue/shape combination for each monkey. We used the gesture/vocal cue/targets first to transfer our animals back and forth between the group housing enclosure and individual test cubicles and subsequently to facilitate the animals’ in responding to computer touch screens. Throughout Experiment 1, each animal was presented only with his or her own target, so the degree to which particular features of the target acquired stimulus control was not assessed. Were the animals’ ‘target’ responses controlled by a single stimulus feature of their respective digitized target  or, alternatively, by a compound stimulus characterized by both hue and shape? In Experiment 2, we addressed the question of single or compound stimulus control directly by testing the discriminability of each animal’s own target against a diverse set of possible alternatives.The training protocols were approved by the Franklin and Marshall College Institutional Animal Care and Use Committee in 2008 (2008–2009 #1).In Phase 1 (Stages 1–4), each animal was initially reinforced in the group housing enclosure with a click-sounding stimulus, followed by a food reinforcer for approaching and touching his or her individual target .In Phase 2 (Stages 5 and 6), the animals were similarly reinforced for following his or her respective target into a test cubicle where they first generalized target-touching to differently-sized smaller targets presented behind either the mesh or glass cubicle doors (Stage 5). Finally, in Stage 6, the animals were presented with digitized targets on a computer touch screen.Sapajus apella)—three females aged 29, 10, and 7 years old, and seven males aged 13, 11, 9, 6, 5 , and 1 year old, respectively.The participants were 10 tufted capuchin monkeys  with a variety of fresh fruits, nuts, and vegetables. At the start of this study in 2008, all 10 animals were cared for without barriers between caretakers and animals  and wer contact ,18) but The animals were housed together in the Vivarium at Franklin and Marshall College. A diagram of the enclosure is presented in On the opposite solid sidewall of the enclosure were 8 pairs (two pairs per section—one upper and one lower) of guillotine doors  made from laminated construction paper . In fourIn Stages 1 and 2 of Phase 1, the experimenters presented the targets by hand to the animals as described in the Methods section. In Stages 3 and 4 of Phase 1, the targets were attached to the end of a white plastic PVC pole (1.35 m in length) by means of plastic zip-ties, presented as described below. In Stage 5 of Phase 2, each animal’s target also varied in size as described below. Primary reinforcers consisted of either craisins  or mealworms . A generic dog-training clicker was used as a secondary reinforcer to bridge the delay between correct responses and the delivery of the primary reinforcer.TM Luminance Meter LS-100  was used to equate the luminance levels of the digitized targets (candela per meter squared—(cd/m2)).In Stage 6 of Phase 2 training, each animal’s target was converted into a digital image and transposed into a 200 × 200-pixel square area using a Graphic Converter. During test sessions, these images were projected onto two 38-cm-diagonal touch screens  controlled by a Mac mini™ computer  mounted on a cart. A MinoltaThe window on the cubicle door (between the cubicle and the testing room) was raised during test sessions, and a Plexiglas™ grid template measuring 28 cm × 31 cm was affixed to the open cubicle door. An array of nine (3 × 3) rectangular holes (7.6 cm × 6.4 cm) that spatially matched nine positions on the computer screen were used to project a digitized target image. Black foam-board blinds attached to the front of the cubicles at the computer stations prevented each animal from seeing anything other than the touch screen.TM ENV-70, Fairfax, VT, USA) mounted on the cart dispensed the primary reinforcer—a single 190-mg pellet  into a plastic dish inside the cubicle via a 2.54-cm clear plastic tube.In Phase 2, a computer generated ‘click’ was substituted for the hand-held clicker used in Phase 1. An automatic pellet dispenser (Med AssociatesNew husbandry standard operating procedures (SOPs) were prompted by the capuchin colony’s transfer into new facilities in the Life Sciences and Philosophy Building’s vivarium. The animals were first collectively desensitized to their new housing by leaving the sliding doors within the group enclosure open, as well as the doors leading into and between the individual test cubicles. This allowed the animals to freely explore their new environment.The PRT sessions described in this manuscript began approximately 10 months after their arrival in the new facility. During the 10-month period before the study, the capuchins were desensitized to entering the cubicles and hand-feeding by leaving the doors between the housing area and the cubicles open and providing some food in the cubicle. Animals who were still hesitant to enter the cubicles and/or approach humans for hand feeding were provided small food items using a cup on a long stick until they eventually came into the cubicle for hand feeding. Trainers then desensitized the animals to the opening and closing of enclosure and cubicle doors using the verbal cue “Open” each time a door was opened.During the target training reported here, animals were often trained as a group with up to 6 trainers receiving animals as the doors opened between the group housing and the testing cubicles. Although we tracked whether a particular trainer/animal pair had an unproductive session and avoided such pairings in future sessions, animals were not paired with a consistent trainer and new trainers were introduced when the semester changed. Animals could station next to each other while learning to touch the target, shift over to another cubicle, remain in place on target while another animal shifted location, or learn target discriminations. Multiple sessions were carried out per day, ranging from 2 to 10 min. The testing cubicles were often open, allowing for interaction between animals and the caretakers and trainers. At the same time as the PRT procedures for target training, caretakers were also using PRT to train capuchins to move between adjoining cubicles in response to simultaneous vocal (“over”) and visual (dynamic pointing) discriminative stimuli.Phase 1 of target-oriented training consisted of four stages in which the correct response varied as described below. At each stage, an experimenter physically isolated an animal from the others in one section of the group’s enclosure via vocal and visual cues as described above. In an adjacent section of the enclosure, a second experimenter presented the individual’s physical target on the mesh wall while uttering the verbal cue, “Target” see , left. TOn each trial, the animal was reinforced (clicker/food) for touching the target held in an experimenter’s hand at any one of a number of quasi-random positions on the mesh divider. Upon completing two successive errorless 10-trial blocks, an animal advanced to Stage 2.Each trial consisted of ten steps. The target was first presented on the mesh divider nearest to the enclosure wall in which the one-way mirror was mounted and then moved across 10 discrete locations to the opposing wall, where the guillotine doors leading into the test cubicles were mounted. At each location a target touch was reinforced as described above. However, if the animal failed to touch the target at any given location, the target was presented again at the immediately preceding location for up to two correction attempts. An animal advanced to Stage 3 following two consecutive errorless 10-trial blocks.The 10-step process within each trial was the same as in Stage 2 except that the target was now attached via a zip-tie to the end of the PVC TM pole  to indicate the monkey should keep following the target. Once the pole target reached the guillotine door on the opposite wall and the animal touched it, the first experimenter reinforced with both the clicker and food, removed the pole target, and cued a second experimenter to raise the guillotine door and present a duplicate pole target inside the testing cubicle. If the animal entered the cubicle and touched the duplicate target, the second experimenter reinforced that behavior with the clicker and food and moved the target to a new location within the cubicle. Following and touching the target one more time earned the animal a final clicker/food reinforcement, all without the use of the KGS. Hence, a total of 12 consecutive steps were required to complete a trial. Following two consecutive errorless sequences of all 12 steps, an animal advanced to Phase 2.TM, and targets shown on the touch screen computer.In Phase 2, the hand-held target and the pole target were considered interchangeable, and whichever was most convenient for a given training session was used to transfer the targeting behavior to targets of different sizes, targets behind PlexiglasTM portions of the test cubicle door. Animals did not easily transfer from touching the target itself touching the PlexiglasTM between them and the target. Initially, the monkeys tried to reach through the mesh beside the PlexiglasTM window to touch the target itself. It appeared the monkeys perceived a difference between reaching their fingers through the mesh to grab the paper target  were presented to the animals using a performance-determined titration procedure. Over two 10-trial blocks within each 20-trial session, an experimenter manually presented a target on the PlexiglasTM or mesh portion of the cubicle door in an alternating order . Each session began with the 100% sized target that was then titrated down from one size to the next after two successive correct trials with a given size. If the animal did not respond within 5 s, the target was increased in size by one step on the subsequent trial following a 15- to 30-s TO.Once an animal successfully completed four trials of alternating mesh and Plexiglas trials with the 25% sized target, the experimenter presented the target on only the Plexiglas portion of the cubicle door for the following 10-trial block. Once an animal met the criterion of 80% or greater correct initial responses over two consecutive sessions of two 10-trial blocks , then it advanced to Stage 6.TM template grid attached to the cubicle door backed by a clear plastic sheet. A computer touch screen on a cart was stationed approximately 25 cm back from the cubicle. Over a single 27-trial session, the experimenter presented each animal’s physical target by hand at one of the nine (3 × 3) grid locations in a quasi-random order. If the animal responded to the target within 5 s, it was reinforced as described previously, followed by a 5-s ITI. Failing to respond within 5 s, or responding to any other grid location, resulted in a variable 15- to 30-s TO.The monkeys were first desensitized to and reinforced for targeting with the PlexiglasTM grid was removed and the touch screen on the computer cart was placed flush against the grid. On each subsequent 27-trial session, a digitized target (200 × 200 pixels) appeared on the touch screen at one of the 9 grid locations in a quasi-random order and remained there for a maximum of 15 s. Hence, over 27 trials the animal’s target appeared at each grid position three times. If the animal touched its target within 15 s, the animal was reinforced by a computer-generated click bridge, followed immediately by a primary reward delivered manually through a small, human finger-sized 8-cm circular opening in the black foam board blind immediately to the left of the touch screen.Following the initial 27-trial active desensitization session, the plastic sheet backing the 3 × 3 PlexiglasIf the animal touched an incorrect grid position or failed to respond within 15 s to any grid position, the trial was terminated and followed by a 5-s ITI over the first five 27-trial sessions, as well as a 3-s ITI on any subsequent session. Following an initial-trial error, the digitized target appeared in the same grid position for up to three correction trials. If the monkey touched the target on a correction trial, it was reinforced, and the next computer-programmed trial was presented after the ITI. This was also the case if the animal failed to respond correctly after three correction trials.Touch = 1.7 sessions). Similarly, when learning to follow and touch the hand-held target (Stage 2), four animals responded without error and reached the performance criterion in the initial 20-trial session. However, only one of these four animals was similarly errorless in Stage 1. The remaining six animals met the Stage 2 criterion within two and five 20-trial sessions . Only one animal made an error in Stage 3 (follow and touch pole target) MPole of 1.1 sessions) and all the animals were errorless in Stage 4 (follow and touch target in cubicle). Hence, all animals reached this criterion in fewer trials than in Stages 1 and 2, F = 8.9, p = 0.05, partial Eta2 = 0.41 , three of the ten animals met the two consecutive, errorless 10-trial blocks criterion within the initial 20-trial session. The remaining seven animals met the criterion within two sessions  in use. The remaining animal (Lucky) responded without error when her smallest target was presented behind the mesh barrier. However, her performance decreased when it was presented behind the PlexiglasTM barrier, making three such errors  in Session 1, as well as one error in Session 3. Nevertheless, she reached the criterion in an additional five 20-trial sessions.Nine of the ten animals were errorless in reaching the performance criterion in the minimum possible two sessions when their respective individual target was systematically reduced in size, regardless of the barrier  = 1.48, 2 = 0.16 . MoreoveOverall, the results indicate that for all animals, the location, size, and manner of presentation of their respective target were not necessarily distinguishing characteristics of the task. Once an animal learned to touch its respective target, regardless of where it was presented in Phase 1 (Stages 1–2), all but one animal touched the target presented on the pole (Stage 3), and all ten animals were similarly errorless in following the pole target from the housing area into a cubicle (Stage 4). Similarly, only one had difficulty in generalizing the response across target sizes and recognizing the target through different barriers (Stages 5 and 6). Three of the four total errors occurred in the first session. The difference in learning between Stages 1 and 2, in which animals needed more sessions to acquire the requested task, compared to Stages 3 and 4, likely reflects that the initial training to touch the target was perceived by the capuchins as a new task, whereas generalizing from the hand-held target to the pole target and following the target into the cubicle was simply an extension of the task they had already learned. That is, once the animals had acquired stage 2 of following the target, it appeared to be quite easy for the animals to follow the target on the pole and also into the cubicle. The individual differences we observed could be due to the animal’s personality, age, and experiences, or simply reflect that animals learn at different rates . UltimatCould the increased error rate with the digitized targets be related to the absence of any extraneous cues previously associated with the experimenters? We think it unlikely, given the number of individuals testing the animals throughout all phases and stages of training. Instead, we note that in all the training stages preceding that with the digitized targets, an error was recorded only if an animal failed to touch the target within the specified time interval. However, the digitized task also recorded an error if the animals touched a section of the grid where the target did not appear or attempted to respond with both hands. The most common errors observed in the transition to the digitized targets were individuals ‘resting’ their hands on another part of the screen after a trial was initiated , touching the target with both hands, or touching an area of the screen that contained neither a target nor a distractor. As the equipment stayed in place, and animals could freely move, including leaning as they wanted, it is not surprising to see the types of errors they made. Although we cannot rule out increased errors resulting from the absence of experimenter cues, we believe the increased error rates associated with the digitized targets were more likely a consequence of this broader definition of an error. We recommend that future studies should consider using a ‘get ready signal’ to cue the animal that a trial is about to begin. Such a signal would cue the animal to stop touching the screen, finish eating, and attend to the screen prior to the start of the next trial, which could reduce the errors we most commonly observed. Alternatively, inserting an already-learned task  between discrimination task trials might also reduce errors in the discrimination task by increasing the capuchin’s overall success with the touch screen and therefore reinforcement rates.In Experiment 1, each animal was exposed only to its individual target, leaving open the question of whether or not one stimulus dimension (shape or color) was more salient than the other. If not, and assuming the absence of a preference for novelty, then an animal should continue to choose her or his own target even when paired with that of another animal in which neither stimulus dimension matches his or her own. Alternatively, higher error rates might be anticipated if one or both of an animal’s stimulus dimensions overlaps with those of another animal. An analysis of error rates could then be evidence of a possible overshadowing stimulus control.Alternatively, absent any evidence of discriminative control by a target’s specific features, an animal might well choose randomly, regardless of whether or not its target did or did not overlap with the incorrect alternative. The aim of Experiment 2 was to test these two possibilities.Seven of the ten capuchins completed Experiment 2. Of the three that did not, one female had died, and another female was preoccupied with caring for her new-born infant. One male developed a habit of touching the touch screen targets with his mouth or head rather than his hand, leading to inconsistent recording of his responses by the computer program. Experiment 2 began approximately 14 months after the animals were moved to the new facility.Test sessions were conducted in either one of two test cubicles (at opposite ends of the cubicle row) to which the two Plexiglas grid templates (28 cm × 31 cm) and 38-cm-diagonal touch screens  were respectively affixed.TM model USB4000-VIS-NIR, 350–1000 Nm, Dunedin, FL, USA) and replicated in the touch screen format. The remaining 44 new ‘distractor’ target stimuli were created such that all six shapes and nine colors were combined, controlling for luminance  were measured with a spectrometer  were presented quasi-randomly on the touch screen at two of the nine grid positions delineated by the attached Plexiglas tm template. Within each 53-trial session, a subject’s training target (S+) was paired with one of the three types of possible S− stimuli described above. If the animal touched his or her target (S+) within 15 s she or he was reinforced with a computer generated ‘click’ bridge, followed immediately with a primary reward. However, if the animal touched the incorrect target (S−) the trial was terminated and followed by a 5-s ITI over the first five sessions and 3 s thereafter, given the animals’ promptness in engaging the task across the sessions.p < 0.001). Nevertheless, their overall Trial-1 success was influenced by similarities between their target and the S− distractor.In this experiment, the animals were required to discriminate their respective training targets used in experiment one from the other 53 possible shape/color target combinations by touching the former when a pair of stimuli appeared on the touch screen. On the initial trial, the monkeys correctly identified their respective targets  significantly more often than would be predicted by chance  = 7.45, n = 40 pairs, t(6) = 7.11, p < 0.001, leaving open the question as to whether one dimension was more salient  than the other. Trial-1 performance was significantly above chance if an S− distractor differed from the S+ target in color but not shape  = 3.27, p = 0.017). However, correct Trial-1 performance was not significantly above chance if the S− distractor was of the same color as the S+ but differed in shape  = 1.63, p = 0.155). This dichotomy in results indicates that the capuchins attended primarily to color and not shape to identify their respective targets. Interestingly however, mean error rates were similar in cases where the target and distractor were not only the same color, but also when the target and distractor were the same shape. Nevertheless, the variability among individuals was much higher when both the target and distractor were the same color .Despite starting out with better-than-chance performances that significantly improved across trials , five ofExperiment 2 revealed that all the animals discriminated their targets from those of the other animals despite never having been explicitly trained to do so. Colors were randomly assigned to the shapes, and it could be that the color in combination with a shape influenced the strategy that the animals were using, discriminating on the basis of shape or color. Individual monkeys that were using color to identify their target , could potentially have performed better if the shape had been salient to them. Shapes like the ones featured in In the present study, all the animals successfully generalized their performances at each stage to digitized targets presented on a touch screen in Phase 2, Stage 6, over an average of 5.6 and a maximum of twenty 27-trial sessions across two weeks of testing. These results compare more than favorably with those reported earlier by Evans and colleagues , who useA variety of factors likely contributed to our capuchins’ faster training. Several aspects of our PRT procedures were chosen to maximize training speed: desensitization to the cubicles and hand feeding preceding PRT, the use of individual targets, more frequent and shorter training sessions , the usOnce using the touch screen, the capuchins in our study were also able to successfully discriminate their target from distractors without explicit training. However, some individuals exhibited chance performance when asked to differentiate their target from a distractor that was the same color but a different shape. This suggests that some of the individuals were attending to color, rather than shape, when identifying their target. Even though the capuchins had not been asked to choose among the targets prior to Experiment 2, they were able to observe some training sessions with other individuals and may have learned how their target differed in appearance from other targets. Two animals (Hector and Izzy) used targets that were exactly the same color and others may have appeared similar because capuchins’ color perception differs from that of humans, and male capuchins may be dichromatic . The colIn conclusion, group housing greatly enriches the lifestyles of nonhuman primates ,27. When


Alcohol drinking alters stressresponse to predator odor via BNST kappa opioid receptor signaling in male mice. Published 21 July 2020eLife paper based on error in methods and data reporting, which they identified following publication, that cast doubt on the conclusions.The authors are retracting this Following publication several co-authors  came to the senior author (TK) with concern about an error in the published data set. The initial error identified was the inclusion of an incorrect group for measurement of blood ethanol content . Following the identification of this initial error, the first author (LH), co-authors  and senior (TK) deeply examined the results and identified several more errors. Notably, these errors occurred across three major domains:1) Inclusion of female mice where only male mice were reported to be used . Specifically:Figure 1D: Blood Ethanol Concentrations. The current figure legend states, "Blood ethanol concentrations (mg/dl) correlated with EtOH intake (g/kg/2 hr) in a subset of mice." Figure 1 shows male C57BL/6J mice, but these blood ethanol concentrations were from female Pdyn-GFP mice from a different study.Figure 2: One female Dyn-GFP mouse was included in this figure.Figure 4: Two of the IA mice are female, Three of the Water/TMT mice are female.2)Inclusion of mice with drinking outside the stated alcohol drinking range  and withdrawal dates : Specifically:Figure 2: The Figure shows Pdyn-GFP mice drank alcohol for 6 weeks, but Pdyn-GFP mice that drank EtOH up to 8 weeks were included in the analysis. In addition, mice were included with withdrawal times ranging from 4 to 14 days.Figure 4: The Figure shows Pdyn-GFP mice drank for 6 weeks, but as above experiment we identified that several mice that drank for up to 7 weeks. In addition, the withdrawal time points were broader  instead of the reported 7–10 days post-EtOH recording time.Figure 6: The Figure shows Pdyn-GFP mice drank for 6 weeks, but as above, several mice that drank 7 weeks were included in the analysis. Additionally, the withdrawal time points were broader  instead of the reported 7–10 days post-EtOH recording time.Figure 6 Supplemental 1: The withdrawal time points were outside the stated range 3)Errors in the data presented in several of the behavioral figures. The data plotted in several of the published figures did not match the raw data values obtained directly from the ethovision tracking files . When the raw data obtained from ethovision was re-analyzed, there were multiple changes in statistical significance, leading to an overall change in the interpretation of the results. Specifically:In Figure 1F,G,H, we identified that the norBNI treated groups were swapped, with the water norBNI mice being shown as IA norBNI mice, and the IA norBNI mice being shown as water norBNI mice. The statistical differences across groups were not different.In Figure 1G: 2 values for TMT contact were different between the raw values provided by ethovision and the values plotted in prism. When the raw values were analyzed there was no longer a statistical difference between groups. In addition, in the published manuscript, the factors of drinking and TMT were swapped in the analysis.Figure 3A: 7 values for TMT contact were different between the raw values provided by ethovision and the values plotted in prism. When the raw values were analyzed there was no longer a statistical main effect.Figure 5I: 2 values for TMT contact were different between the raw values provided by ethovision and the values plotted in prism, noted that this was due to hand-scoring. With 1 of these differences the values do not match between excel and prism. When the values from excel are plotted and analyzed (including hand scored corrections) there is a main effect of drinking on TMT contact, but no other significant differences.Figure 7K: 5 values for TMT contact were different between the raw values provided by ethovision and the values plotted in prism. 1 of these values is due to a mouse being analyzed twice. When the values from excel are plotted, there is are no longer any statistical differences between groups.The changes that would be required to correct the paper are sufficiently extensive that its overall conclusions must currently be considered to be in doubt. All of the authors are in agreement that retraction is the appropriate course of action.


We used Fisher test to compare categorical variables between cases and controls, while we used logistic regression model to evaluate which risk factor was associated with preeclampsia. Although there was no statistically significant difference between the two groups, we found different percentages of two of the polymorphisms considered (rs1045642 and rs2032582 in the gene ABCB1). Despite these results, our work may be helpful for future research to better understand the pathogenesis of preeclampsia.Pregnancy can be considered as an allogeneic transplant and preeclampsia can be seen as a failure of the acceptance mechanisms of this transplant as occurs in acute organ transplant rejection. Some genetic polymorphisms may be involved in its pathogenesis. Since the kidney is one of the organs mainly involved in preeclampsia, our study attempted to determine the frequencies of single nucleotide polymorphisms of DNA (SNP) in 3 genes (adenosine triphosphate-binding cassette sub-family B member 1 (ABCB1)/multi drug reactivity 1 (MDR1) gene, interleukin 10 gene and tumor necrosis factor α gene) which are targets of immunosuppressive therapies and related to acute renal rejection. The study was an observational, monocentric, case-control study. We enrolled 20 women with severe preeclampsia and 10 women age-matched with regular pregnancy. Continuous variables were compared by the Student’s  Preeclampsia is a major cause of maternal, fetal and neonatal mortality as well as maternal and neonatal morbidity. It is characterized by new-onset hypertension accompanied by new-onset proteinuria. The diagnostic criteria of preeclampsia are listed in Furthermore, the onset of this pathology can affect several organs, first of all the kidney, and some of these women can worsen up to kidney failure.Pregnancy can be considered as an immuno-accepted allogeneic transplant. For a successful pregnancy the functional balance between T helper phenotype 1 (TH1) and T helper phenotype 2 (TH2) is crucial. Current evidence suggests that the success of fetal tolerance is based on an immunological deviation to a TH2-induced antibody response instead of predominantly TH1-induced response . The tolIn this context preeclampsia can be considered as the failure of this system and pathogenesis is related to a still unclear mechanism involving the immune system and kidney.Until now there are no predictive tests to identify which patients will present an increased risk of developing severe preeclampsia at an early stage. Therefore, the identification of genetic profiles with greater or lesser risk of developing this syndrome may be a useful contribution.The aim of our study was to determine the allelic and genotypic frequencies of some single nucleotide polymorphisms (SNP) of DNA in 5 genes (adenosine triphosphate-binding cassette sub-family B member 1 (ABCB1)/multi drug reactivity 1 (MDR1) gene, genes coding for interleukin 10 (IL-10) and for tumor necrosis factor α (TNF-α)), which are targets of immunosuppressive therapies used in acute rejection in kidney transplantation and could therefore be involved in the pathogenesis of preeclampsia.This is an observational, monocentric, case-control study. The study protocol was approved by the Ethics Committee of the Udine University Hospital on July the 13th, 2016 with approval number 44/16.We enrolled thirty patients who went to Gynecology and Obstetrics Unit of the Udine University Hospital between September 2016 and June 2017. We divided these patients into two groups: the first group (case group) was composed by twenty pregnant women who were enrolled during hospitalization for severe preeclampsia (according to the ACOG 2019 criteria listed in The other group (control group) was composed by ten pregnant women matched for age with the case group, who had a regular pregnancy defined as pregnancy without any fetal or maternal problems that could complicate its regular course .All women included in this study had signed the informed consent of participation.All data was collected in an anonymous database.For all women, we collected two peripheral blood samples (12 mL divided into two test tubes each one containing 6 mL of blood in EDTA). All blood samples were processed in the International Renal Research Institute Vicenza (IRRIV) Laboratories of the Department of Nephrology, Dialysis and Transplantation, San Bortolo Hospital Vicenza.On the base of published literature, we analyzed 5 SNPs located in three different genes: rs1800872 in the IL-10 gene; rs1800629 in the TNF gene; rs1045642, rs1128503 and rs2032582 in the ABCB1 gene.The genetic analysis was achieved by DNA extraction and purification, DNA qualitative and quantitative evaluation, polymerase chain reaction (PCR), agarose gel electrophoresis, fragment purification, Sanger sequencing reaction, purification of the sequences and capillary electrophoresis. DNA was extracted from each frozen blood sample using the NorDiag Arrow instrument with the Blood DNA 200 Extraction Kit disposable cartridge . The instrument uses a magnetic bead-based extraction method. The NanoPhotometer N50 Touch  instrument, based on a spectrophotometric technology, was used for DNA quantitative and qualitative evaluation. The necessary parameters were adjusted before preparing the reaction mix for the PCR: primer design and annealing temperature (Ta). The primers were designed using the Primer3 program , as shown in PCR was performed using the standardized reaction mix AmpliTaq Gold 360 MasterMix , which contains a specific Hot Start Taq polymerase enzyme. The protocol  was as follows: initial denaturation: 10 min at 95 °C; 35 amplification cycles of denaturation: 30 s at 95 °C, annealing: according to the SNPs, extension: 40 s at 72 °C; final extension cycle: 10 min at 72 °C. The PCR products were verified by electrophoresis in 2% agarose gel (Starpure Agarose melted in Tris Borate EDTA buffer) stained with Midori Green Direct . We used Illustra Exo-ProStar 1-Step  for purification of the fragments, according to the instructions of the manufacturer. The Sanger sequencing reaction was performed using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the instructions of the manufacturer. The products of the reaction were purified using Centri-Sep Columns , according to the instructions of the manufacturer. The purified sequences were denatured using HiDi deionized formamide (Applied Biosystems): the denaturation protocol consisted of 5 min at 95 °C. At the end we analyzed the sequences using capillary electrophoresis with the AB 3500 Genetic Analyzer (Applied Biosystems). The obtained sequences were evaluated using the Variant Reporter Software v1.1 (Applied Biosystems).t-test for independent variables or using the Mann-Whitney test depending on their distribution. We used Fisher test to compare categorical variables between cases and controls, while we used logistic regression model to evaluate which risk factor was associated with pre-eclampsia. For each SNP the heterozygosity (HET) was determined and the test for Hardy-Weinberg equilibrium (HWE) was performed in the control groups and in the cases by the Chi-Square Goodness-of-Fit Test. The measure of heterozygosity (HET) indicates how many individuals are heterozygous for a given locus and represents an index of genetic variability. Allelic frequencies and genotype frequencies were determined for each SNP. Genetic associations were explored using ‘per-genotype’ analysis, ‘per-allele’ analysis and ‘linear trend’ analysis using the SAS version 9.1.4 program . Statistical significance was evaluated by the two-tailed t-test (p < 0.05).Continuous variables were expressed as median and Interquartile Range (IQR) or as mean ± standard deviation (SD), depending on their distribution, while categorical variables were described as frequencies. Continuous variables were compared by the Student’s Analyzing the demographic characteristics of our population, no statistically significant differences emerged between the two groups. As shown in Regarding parity in the case group the majority of women were nulliparous (12/20), while all women in the control group were not at their first pregnancy. Considering risk factors, one woman in the case group was affected by diabetes mellitus and another one in the same group by gestational diabetes. None reported familiarity with preeclampsia. In the case group only 3 patients were affected by hypertension before pregnancy. In the same group there were 3 twin pregnancies and 5 women got pregnant with assisted reproductive technologies .Caesarean section was performed in 18/20 women in case group while 9/10 women in the control group had a vaginal delivery. Regarding prematurity, in the control group none delivered before 37 weeks while in the case group 16/20 women had a preterm delivery. ABCB1 gene: rs1045642, rs1128503 and rs2032582. We found rs2032582 mutation in 15/20 of women of the case group and 5/10 of control group; the polymorphism rs1128503 was present in 11 cases and 6 controls and rs1045642 was found in 7 cases and 6 controls. Regarding the TNF gene, we analyzed the polymorphism rs1800629 that was present in 6 cases and 4 controls. As last we studied the polymorphism 1800872 of the IL-10 gene: 10 women of case group resulted positive for this mutation and 5 women from of control group. No statistically significant difference was found between the group of the cases and that of controls, but we found different percentages of two of the polymorphisms in the gene ABCB1: 75% of women in the control group showed SNP rs2032582 and only 50% in the case group; SNP rs1045642 was present in 39% of women in the controls group vs 60% in the case group.Genetic analysis showed heterogeneous results as shown in Early diagnosis of preeclampsia is essential to be able to set an adequate treatment and prevent the most serious complications of this disease such as eclampsia and intrauterine growth restriction. For this reason, the knowledge of the pathogenetic mechanisms underlying this condition and the identification of reliable hallmarks for early diagnosis is crucial.We tried to identify an allele variant specifically associated with preeclampsia that could be an interesting marker to identify patients who present an increased risk of developing severe preeclampsia at an early stage.Since we compared the pregnancy to a transplant and one of the targets of preeclampsia is the kidney, we decided to consider the genetic polymorphisms involved in the mechanisms of kidney transplant rejection. These SNPs are involved in immune response, in particular they are targets of the immunosuppressive therapy in kidney transplant ,6,7.In this study we analyzed five single nucleotide polymorphisms: one SNP of the IL-10 gene, one SNP of TNF gene and three SNPs of ABCB1 gene. These SNP were implicated in the response to immunosuppressive therapies in acute renal transplant rejection ,7,8,9,10We analyzed their allelic and genotypic frequencies in pregnant women in order to determine a possible association with preeclampsia. Despite our study didn’t find any statistically significant results, the difference found in two of the analyzed polymorphisms of the ABCB1 gene (SNP rs2032582 and SNP rs1045642) could be interesting.Furthermore, the ABCB1 gene encodes a P-glycoprotein belonging to the superfamily of the ATP-binding cassette (ABC) transporters which make up the bloodbrain barrier and also the placental barrier. This protein has protective functions as it participates in the elimination of metabolites, toxins and some drugs . Number According to the study of Scalzotto et al.  the SNP As far as fetal outcomes are concerned, the data on prematurity should be highlighted: in our population sixteen out of twenty babies were born before 37 weeks in the preeclamptic group, while in the physiological pregnancy group all babies were born at term. This data is in agreement with the literature pointing out preeclampsia as a risk factor for prematurity ,16.Although our analysis did not report statistically significant differences between the two groups regarding the polymorphisms of the analyzed genes, our work may be helpful for future research to guide the choice of polymorphisms possibly implicated in the pathogenesis of preeclampsia.We must also emphasize that it is the first study investigating these genes as possible risk factors implicated in the pathogenesis of preeclampsia.As we know, preeclampsia has a multifactorial pathogenesis , and theOne of the limitations of our study is the small sample analyzed and this fact could condition the lack of significance. Probably expanding the sample of subjects analyzed we could obtain statistically significant results for the two polymorphisms just mentioned.Furthermore, one of the advantages of our study is the use of a matched-with-age control group which was able to reduce the bias of age.Polymorphisms’ analysis is an expanding field and future research could focus on this analysis not only to identify earlier susceptible subjects to the development of this disease but also to personalize therapies and to predict which patients will not respond to conventional therapies.


Daily fiber intake can increase the diversity of the human gut microbiota as well as the abundance of beneficial microbes and their metabolites. Whole-grain wheat is high in fiber.This manuscript presents a study protocol designed to understand the effects of different types of wheat on gastrointestinal tract microbes.Human adults will consume crackers made from three types of wheat flour . In this study, participants will alternate between crackers made from refined soft white wheat flour to those made from whole-grain soft white wheat and whole-grain soft red wheat flour. Survey and stool sample collection will occur after 7-day treatment periods. We will assess how wheat consumption affects gastrointestinal bacteria by sequencing the V4 region of 16S rRNA gene amplicons and the inflammatory state of participants’ intestines using enzyme-linked immunosorbent assays. The butyrate production capacity of the gut microbiota will be determined by targeted quantitative real-time polymerase chain reaction.We will report the treatment effects on alpha and beta diversity of the microbiota and taxa-specific differences. Microbiota results will be analyzed using the vegan package in R. Butyrate production capacity and biomarkers of intestinal inflammation will be analyzed using parametric statistical methods such as analysis of variance or linear regression. We expect whole wheat intake to increase butyrate production capacity, bacterial alpha diversity, and abundance of bacterial taxa responsive to phenolic compounds. Soft red wheat is also expected to decrease the concentration of inflammatory biomarkers in the stool of participants.This protocol describes the methods to be used in a study on the impact of wheat types on the human gastrointestinal microbiota and biomarkers of intestinal inflammation. The analysis of intestinal responses to the consumption of two types of whole wheat will expand our understanding of how specific foods affect health-associated outcomes.DERR1-10.2196/29046 An individual’s gut microbial community is colonized by trillions of microorganisms . These mBifidobacterium spp., Lactobacillus spp., and Bacteroidetes [Dietary intake is strongly associated with the composition of the gut microbiota -9. This roidetes -15.As ultraprocessed foods have become a common component of the Western diet, resulting in a loss of whole grains and fiber, the gut microbial composition has become enriched with proinflammatory microbes that correlate with metabolic risk factors and which contribute to noncommunicable chronic diseases such as obesity and type 2 diabetes ,16,17. CGrains are a staple of the Western diet; however, grains exist in many forms. Thus, it is important to consider the types of grains being consumed ,22. The Clostridium difficile [Similarly, in humans, differences in the gut microbiota have been observed among different geographic groups consuming their culturally habitual diets ,24. Thusifficile . Recent ifficile ,13.Whole grains are rich in carbohydrates accessible to the gut microbiota, which bacteria can use for food and energy . ConsequBacteroidetes in the gut [Additional components of whole-grain wheat that convey health benefits are polyphenols . Polyphe the gut ,20. Thus the gut . As diffTo fully understand the impact of whole-wheat products on humans, we must understand the effects of different types of wheat  on human gastrointestinal tract microbes. However, previous studies have ignored these differences in wheat types. Therefore, we describe a protocol in which we will feed human adults crackers made from three different types of wheat flours and assess how cracker consumption affects (1) the gastrointestinal microbial communities and (2) the inflammatory state of the intestines. We hypothesize that whole-grain soft white wheat flour will promote different microbes compared with whole-grain soft red wheat flour. In addition, we expect that the whole-grain soft red wheat treatment will result in decreased intestinal inflammation compared with the refined white wheat flour treatment. The successful completion of the proposed research will improve our understanding of the unique effects of different types of wheat on the human gut microbiota and the inflammatory state of the gastrointestinal tract.. During each of the two test periods, 80 g of crackers made from whole-grain soft white wheat flour or whole-grain soft red wheat flour will provide each participant with approximately 10.7 g of total dietary fiber per day (This wheat intervention study will characterize the variable response of gut bacteria to supplementation in adult humans. Treatments will include (1) refined soft white wheat flour, (2) bran-containing whole-wheat flour of Michigan soft white wheat, or (3) bran-containing whole-wheat flour of Michigan soft red wheat. All 3 flour samples will be provided by King Milling Company . Each of the two whole-wheat interventions will be administered over a 1-week period, with a baseline week (refined soft white wheat flour) before the intervention begins and a washout week (refined soft white wheat flour) between the two test periods . Wheat f per day . At the This study will be conducted during the summer in East Lansing, Michigan, on the campus of Michigan State University and will enroll 30 adults. Subjects will be recruited via flyers hung in buildings around the Michigan State University campus as well as through the Michigan State University–paid research pool . To be iThe dietary intervention will consist of a 4-week period of wheat consumption in the form of wheat crackers. Each week, participants will be given 7 bags of crackers (1 bag for each day of the week), containing approximately 80 g of baked crackers per bag, to be eaten throughout the following week. Each 80 g cracker allotment will provide approximately 10.7 g of total dietary fiber for crackers made from either whole-grain soft white flour or whole-grain soft red flour. The nutrients and calories of the 80 g cracker allotment consumed per day are listed in day 1 to day 7; then, 7 bags will be placed in one larger bag to be distributed to each participant.Crackers for each treatment week will be made in bulk to yield 260 daily portions (7 portions per participant plus extra), with approximately 80 g of baked crackers per daily portion per person. The cracker was formulated as 100 g flour (approximately 13% moisture), 1.5 g of iodized salt, 2 g of sugar, 6 g of vegetable shortening, and 49 to 50 g of tap water. The proportions of flour, salt, and sugar, according to the formulation stated above, will be added into the bowl of a Hobart mixer (model A-200) and whisked to combine the dry ingredients. Fat will be added to the dry ingredients and mixed until the fat particles are reduced in size to approximately 1 to 2 mm. Water will then be added and mixed until a firm dough forms. The amount of water (approximately 48 g) will need to be adjusted to ensure that all flour is fully hydrated. The dough will then be portioned by dividing it into 127 g aliquots based on the expected yield of 80 g of baked crackers per 127 g of dough (determined in preliminary testing). Each 127 g portion will be flattened between two pieces of parchment paper using a dough sheeter set to 3/32 inch. The top sheet of the parchment paper will be removed, and the dough will be cut into approximately 1- to 2-cm-wide strips. Crackers from each portion of dough will be baked in a carousel oven for approximately 11 to 12 minutes at 400 °F until browned. Crackers will be cooled on a rack on the workbench before being placed into individual resealable portion bags labeled As this study involves adding approximately 300 calories of carbohydrate-containing food to the daily diet, participants will receive flyers  informinBifidobacterium and Lactobacillus, and butyrate production capacity of the gut microbiota will be assessed after each week of treatment. The abundance of Bifidobacterium and Lactobacillus will be determined from the sequencing results. The butyrate production capacity will be determined by quantitative real-time PCR assays targeting the bacterial butyrate kinase and butyrate transferase genes [At the conclusion of each week, stool samples will be collected for analysis. The primary outcome measure will be the alpha diversity of the gut microbiota. This will be measured using the sequencing data obtained from the participants’ stool samples after DNA extraction and polymerase chain reaction (PCR) amplification of the V4 region of the 16S rRNA gene. The secondary outcome measure will be biomarkers of gastrointestinal inflammation, as measured by protein concentrations of calprotectin and lipocalin-2 in protein extracts of the stool samples. Furthermore, the microbiota beta diversity, abundance of se genes .Background characteristics of the study participants will be recorded during recruitment when each participant will complete a short questionnaire. In addition, current weight, health status, 24-hour dietary intake, and water source will be collected each week along with a fiber intake questionnaire (described below) recording the amount of fiber-containing foods consumed throughout the week. A 24-hour recall will be implemented as this has been previously reported to supply sufficient information to interpret microbiota data from participants’ fecal samples . ParticiIndividual diet diversity scores will be calculated for each participant at each time point. The use of the diet diversity score provides a more general measure of the variety of dietary intake than specific food consumption data. This scoring system counts the number of food groups consumed by an individual. Data will be supplied based on the 24-hour intake data provided in response to this prompt, “What did you eat and drink in the past day? ” as well as their answers to a checklist of 34 food and supplement items where participants are prompted to “Place an 'x' in the box next to anything that you ate in the past 24 hours.” The food checklist was designed to capture the intake of the foods listed in the diet diversity scoring tool document of the Food and Agriculture Organization of the United Nations [The PhenX fiber intake protocol will be used to assess additional dietary fiber intake . Each weStool samples will be extracted, prepared for sequencing of the 16S rRNA gene, and sequenced as described in references -38.but) and butyrate kinase (buk). Primer sets, But_RosEub, But_G.prausn, and Buk, previously designed by Vital et al [2 to 107 will be used to create a standard curve. All wells will be filled with 7.5 μL SYBR Green, 3.5 μL ddH2O, 1 μL F primer, 1 μL R Primer, and 2 μL template. The thermocycling program to be performed is as follows: 2 minutes at 50°C, 10 minutes at 95°C, 45 seconds at 95°C, 45 seconds at annealing temperature for the specific primer, and 45 seconds at 72°C for 40 cycles. A one-way repeated-measures analysis of variance (ANOVA) will be performed on each set of data to compare the relative abundance and presence of the following butyrate synthesizers: Faecalibacterium prausnitzii, Eubacteria spp., Roseburia spp., and Clostridium acetobutylicum.Quantitative real-time PCR will be performed on the genomic DNA from each sample to determine the levels of butyryl-CoA: acetate CoA-transferase  and calprotectin  will be purchased from Epitope Diagnostics, Inc. First, the fecal samples will be aliquoted and stored at −80°C until protein extractions are performed. Then, approximately 100 mg of fecal material will be aliquoted into a conical tube, and the exact weight of the sample will be recorded. Each sample will be extracted separately with each of the two extraction buffers. Using a serological pipette, 4 mL of extraction buffer per 100 mg of stool will be aliquoted into a conical tube for each sample. Each tube will be gently vortexed and incubated at room temperature on an orbital shaker for 30 minutes. The protein extracts will then be aliquoted into microfuge tubes and stored at −80°C until ELISAs (enzyme-linked immunosorbent assays) are performed. The ELISA kits will be purchased from R&D Systems (a Bio-Techne brand) for human calprotectin  and human lipocalin-2 . Fecal extracts will be diluted at 1:25 or 1:100 in sample diluent for calprotectin assays and 1:20 or 1:100 in sample diluent for neutrophil gelatinase-associated lipocalin assays.Statistical analysis of differences between pre- and posttests will be assessed using ANOVA or Friedman Wilcoxon rank-sum tests . MultivaEnterotypes will be manually assigned based on clustering within quadrants in a Bray-Curtis principal coordinates analysis plot overlaid with the most abundant taxa driving community differences, as in Wu et al , using tpositive responder if alpha diversity  is greater at time points B and D (whole wheat) than at both A and C (refined white wheat). However, if this change is not observed, participants will be considered nonresponders. After post hoc assignment of response groups, differences in overall alpha diversity between positive responder and nonresponder groups will be tested with ANOVA (if parametric) or Wilcoxon ranked-sum (if nonparametric). The normality of data will be determined using the Shapiro-Wilk test. Differences will be tested within each time point. Beta diversity differences between response groups will be determined at each time point using PERMANOVA and PERMDISP. Continuous demographic variables  will be compared with a two-tailed t test if parametric and a Wilcoxon rank-sum test if nonparametric, with normality confirmed using the Shapiro-Wilk test. The Fisher exact test will be used to determine the independence of sex, smoking exposure, and race regarding response to treatment. Differences in genera present at >1% average relative abundance will be tested between response groups using a negative binomial model in the MASS package [If only a subset of participants is found to respond to the treatment, response groups will be determined by an individual’s change in alpha diversity metrics across the treatments. A participant will be considered a  package .The study procedures were approved by the Michigan State University institutional review board (IRB #00002638) in spring 2019. Sample collection occurred in summer 2019. The results of this research are expected to be published in late 2021.Bifidobacterium, Lactobacillus, and butyrate-producing bacteria that are present following each treatment. These analyses will elucidate how different types of wheat may affect the gastrointestinal tract microbiota.The primary aim of this study is to measure the alpha diversity of gut microbiota following each week of cracker treatment to determine whether whole-wheat consumption increases alpha diversity. Additional aims are to measure the levels of gastrointestinal inflammation as well as Only recently have we begun to understand the mechanism by which whole grains promote health ,16,21,45We anticipate that the results of this intervention study will have implications for both wheat producers and consumers. Recent trends suggest an increase in the number of consumers who avoid wheat products as part of new diets that emphasize carbohydrate reduction, despite the unclear benefits of avoidance in those without celiac disease . In factAn important strength of this study is the composition of the crackers. The aim of the study is to test the effect of specific types of wheat; thus, it is important to note that we have created a cracker formulation that is composed almost entirely of the wheat of interest with few additional ingredients. The simple recipe we have developed for the crackers will improve confidence that the differences observed in the microbial community and inflammatory biomarkers are owing to the consumption of the wheat in the study crackers rather than a different ingredient in the treatment food. The quick turnaround from the cracker intake to sample collection also helps strengthen the study. Stool samples will be collected on the final day of each week's cracker intake, ensuring that little time has elapsed between the last cracker intake and stool sample collection. This is important for microbiota analyses as the temporal intake of food is tightly associated with microbial composition . In addiA potential limitation of our study may be participant retention. Participants will be expected to consume approximately 80 g of crackers every day for 4 weeks, so it is important that they enjoy the taste of the crackers to ensure their compliance and continuance in the study. However, the study participants may tire of the taste of the crackers. A high dropout rate could hinder our ability to detect statistically significant differences.Whole grains alleviate dysbiosis in the gut microbiota; however, little is known about the impact of different wheat types on this phenomenon ,18,20. A


This paper reports a transwell insert-embedded microfluidic device capable of culturing cells at an air-liquid interface (ALI), mimicking the in vivo alveolar epithelium microenvironment. Integration of a commercially available transwell insert makes the device fabrication straightforward and eliminates the tedious device assembly processes. The transwell insert can later be detached from the device for high-resolution imaging of the cells. In the experiments, the cells showing type-I pneumocyte markers are exploited to construct an in vitro alveolar epithelium model, and four culture conditions including conventional liquid/liquid culture (LLC) and air–liquid interface (ALI) cell culture in normal growth medium, and ALI cell culture with inflammatory cytokine (TNF-α) stimulation and ethanol vapor exposure are applied to investigate their effects on the alveolar epithelium barrier function. The barrier permeability is time-lapse monitored using trans-epithelial electrical resistance (TEER) measurement and immunofluorescence staining of the tight junction protein (ZO-1). The results demonstrate the functionalities of the device, and further show the applications and advantages of the constructed in vitro cell models for the lung studies. The lung is one of the most important organs in a human body, and its key function is to perform oxygen transportation from the atmosphere into the bloodstream to oxygenate entire body through the circulatory system. Lungs can also help to release carbon dioxide from the bloodstream to the atmosphere . The gasIn order to study effects of atmospheric exposure and diseases on the primary lung functions, animal experiments are commonly exploited. However, due to complex biological compositions (over 40 cell types in a lung) and physical environments , it is challenging to investigate the underlying mechanisms and pathways of lung dysfunction. In addition, without representative in vitro cell culture models to reconstitute in vivo microenvironments, it is infeasible to elucidate the effects of single factors on the pneumocytes . As a reMicrofluidic cell culture devices have recently obtained, increasing attention due to its desired advantages, including—capability of controlling culture microenvironments in spatial and temporal domains, and mimicking physiological arrangements of cells in vivo ,16,17,18Despite various microfluidic cell culture devices mimicking the cell arrangement in lungs, they suffer several disadvantages retarding their practical usage, especially for atmospheric exposure study. Most of the existing devices are constructed with multiple layers and complicated structures requiring sophisticated fabrication processes. The multiple layer architecture limits the imaging capability due to the requirement of long working distance objectives. Moreover, the devices are not optimized for introduction of vapor contents to conduct atmospheric exposure experiments mimicking stimulation during respiration. The slender microfluidic channel geometry limits the passive diffusion efficiency for the vapor samples in contact with the cultured epithelial cells. As a result, active actuation pumping may be required to deliver atmospheric samples for the experiments, which makes experimental setup complicated for practical usage in biological labs and scaling up of the device for multiple tests challenging.In order to construct an in vitro alveolar epithelium model culturing cells at an air–liquid interface (ALI), porous membranes need to be integrated within the devices ,24. The To ensure the functionalities of the device for vapor exposure on the ALI cell culture, an alveolar epithelial cell line with type-I pneumocyte-like properties is exploited to be cultured in the device in the experiments. In order to compare the effects of culture formats, conventional liquid/liquid culture (LLC) and ALI culture are performed. In addition, an inflammatory cytokine and ethanol vapor are also used to stimulate the cells cultured at the ALI to mimic a pathological epithelium and an epithelium exposed to hazardous VOC atmosphere, respectively. During the experiments, time-lapse trans-epithelial electrical resistance (TEER) measurements across the formed alveolar epithelium cultured under different conditions are performed. TEER measurement offers a quantitative approach to measure the tight junction expression between cells that plays an important role in solute transportation through the paracellular space ,25,26. TIn order to culture the cells at the air–liquid interface (ALI) and investigate the responses of the cells exposed to different atmospheric environments, a microfluidic cell culture device with an embedded commonly used transwell insert is developed in this paper. The microfluidic device is made of an elastomeric material, polydimethylsiloxane (PDMS), due to its cell culture compatibility, optical transparency, and gas permeability ,28,29. Tw/w). The top layer is made by carefully pouring the PDMS prepolymer on a silicon wafer on which a transwell insert with 0.4 µm pore  is temporarily attached. The wafer is silanized with 1H,1H,2H,2H-perfluorooctyltrichlorosilane  in a desiccator for more than 30 min at room temperature to prevent undesired bonding of PDMS to the wafer before the process. In order to prevent the prepolymer flowing into the transwell insert to block the porous membrane, deionized water is filled into the transwell insert before pouring the prepolymer. The bottom PDMS layer is fabricated using the replica molding process. For the proof-of-concept experiments, the microfluidic channel is designed with a width, length, and height of 3, 30, and 1 mm, respectively. The channel dimensions can be further adjusted as microfluidic channels to simulate in vivo physical and pathological microenvironments under dynamic flow conditions. In the process, a laser-cut 1-mm thick acrylic layer with channel geometries attached onto a silicon wafer using a double-side tape is exploited as a mold, and the mold is also silanized before the process. The PDMS layers are cured at 60 °C for more than 4 h in an oven. After curing, the PDMS layers are then peeled from the silicon wafers, and the inlet and outlet of the microfluidic channel are punched using a biopsy punch with a diameter of 2 mm on the top layer.The fabrication process of the device is based on the well-developed soft lithography process, as shown in w/w) is first spun on a silanized silicon wafer as an adhesive layer. The bottom PDMS layer is then placed on the adhesive layer with the channel pattern surface facing down to coat the prepolymer on the surface. The two layers are then aligned and bonded with each other by placing the assembled device in a 60 °C oven for more than 16 h. The thin PDMS adhesive layer provides reliable leakage-free bonding between the layers and does not block either the porous membrane or the microfluidic channel. To assemble the device, a thin layer of PDMS prepolymer, prepared by mixing base and curing agent with a ratio of 3:2  with type-I pneumocyte-like properties  were used ,30. With5 cells is pipetted into the transwell insert for the experiments. The cells are cultured for 2 days (Day 0 to 2) in the liquid environment with the growth medium to form confluent epithelium before changed to different culture conditions, and the medium is exchanged every day during the experiments.The stocks of hAELVi cells are cultured in huAEC medium  according to the protocol provided by the supplier. 2) and temperature (37 °C).After a 2 day-formation of the epithelium, four cell culture conditions are performed for 3 days (Day 2 to 5) to investigate cellular responses under different culture formats, cytokine stimulation, and volatile organic compound (VOC) exposure in the cell experiments. The four conditions as shown in In order to observe the cell compatibility of the device and cell viability under different culture conditions, fluorescence imaging-based cell viability assays are performed in the experiments. In the experiments, the live and dead cells cultured in the device are stained using calcein AM (4 mM)  and ethidium homodimer-2 (EthD-2) (2 mM) , respectively. The nuclei stain, NucBlue Hoechst 33342 , is also used for cell counting and analysis. The stained cells are then imaged using an inverted fluorescence microscope .Transmembrane electrical resistance offers a quantitative approach to real-time measure the integrity of the tight junctions that govern solute transport across the paracellular space of live endothelial and/or epithelial monolayers. As a result, trans-epithelial electrical resistance (TEER) measurement is exploited to investigate the barrier function of the epithelium formed in the devices under various conditions in a time-lapsed manner. 2 in the experiments. For each cell culture condition, three independent experiments are conducted for statistical analysis. Statistical analysis is performed by ANOVA followed by Holm–Sidak tests.The cell culture area is the area of the porous membrane in the transwell insert, which is 0.33 cmIn order to compare the TEER measurement results and the tight junction formation between the hAELVi cells cultured under various conditions, immunofluorescence staining and imaging of the tight junction protein, zonula occludens-1 (ZO-1), are performed in the experiments at the same time points as the TEER measurements. The cells are first washed using DPBS twice, and then fixed using 4% paraformaldehyde (PFA) for 15 min. After fixation, the cells are washed using DPBS three times. The cells are then treated with a blocking buffer composed of 0.5% bovine serum albumin (BSA) and 0.025% saponin in DPBS for one hour. The cells are then incubated with a ZO-1 antibody  in the blocking buffer (1:100) at 4 °C overnight. The cells are stained with CF 543  in the blocking buffer (1:400) for an hour. The nuclei of the cells are also stained using DAPI  in DPBS. The stained cells are imaged using a confocal microscope  in the experiments.The viability of the cells cultured in the microfluidic devices are characterized at Day 5 to confirm the cell compatibility of the device and investigate the effects of the culture conditions on the cell viability. The TEER measurements are performed after the two-day cell culture in the devices. 2) at Day 5 which is about 14.1% higher than that obtained from the LLC experiments, which is about 218.1 (Ω·cm2). When the cells are cultured at the ALI with the addition TNF-α stimulation from the basal side, the TEER value remains similar from Day 2 to 5 without significant increase (between 15.4 and 45.3 Ω·cm2) and becomes lower than that cultured in the growth medium without additional stimulations. The average value is 45.3 (Ω·cm2) at Day 5, which is about one fifth of the value obtained in the LLC experiments. The results suggest that the inflammatory cytokine, TNF-α, can alter tight junction protein expression and increase the junction permeability of the epithelium. As a result, it is confirmed that the experimental setup can be exploited to quantitatively characterize the barrier function based on the TEER measurement.In order to compare the TEER value variation of the cell layers cultured under different conditions, the calculated TEER values measured from Day 2 to 5 are plotted in 2) at Day 5 that is even higher than that obtained from the experiments conducted using normal growth medium without additional stimulations (both LLC and ALI). The higher TEER values suggests that there is a possible increase of tight junction protein expression, which may lead to lower epithelium permeability, after the cells are exposed to the ethanol vapor. The results also confirm that the device can be exploited to study cellular responses of epithelium exposed to VOC vapor mimicking hazardous atmosphere conditions.Interestingly, when the cells cultured at the ALI exposed to the ethanol vapor with an average concentration of 87.1 ppm c, the TE2), and the value obtained from the ALI culture experiments without additional stimulations is significantly higher (14.1%) than that obtained from the control experiments. With the cytokine stimulation, the average TEER value of the TNF-α-stimulated cell layers cultured at the ALI is 45.3 (Ω·cm2), which is approximately 79.2% and 81.8% lower than those obtained in the LLC and ALI experiments performed with the growth medium, respectively. In contrast, the average TEER value of the cell layers cultured exposed to the ethanol vapor at the ALI is 358.1 (Ω·cm2), which is approximately 64.2% and 43.9% higher than those obtained in the LLC and ALI experiments performed with the growth medium, respectively.In order to statistically compare the epithelium formed in the device under different culture conditions after experiments, the TEER values obtained at Day 5 in the experiments are plotted, as shown in For comparison, the immunofluorescence staining of the tight junction protein, ZO-1, is also performed on the hAELVi cells cultured under the different conditions at the same time points as the TEER measurements, and the captured confocal fluorescence images are shown in For the cells cultured at the ALI, the tight junction protein (ZO-1) expression slightly increases from Day 3 to 4 when the cells are stimulated by the inflammatory cytokine TNF-α (Device 3) and remains similar from Day 4 to 5. In addition, the ZO-1 expression presents at only some of the cell interfaces, which is less than the expression observed in Device1 and Device2. In contrast, the tight junction protein expression keeps increasing from Day 3 to 5 when the cells are exposed to the ethanol vapor (Device 4), and presents at almost all the cell/cell interfaces. The immunofluorescence staining results also agree well with the TEER measurements on the cells under cytokine treatment and VOC exposure conditions, suggesting the impairing and enhancing roles of TNF-α and ethanol vapor on the tight junction formation of the alveolar epithelium, respectively.The results show that when the cells are cultured in normal growth medium in different formats, the ALI culture can promote the epithelium tight junction formation for the specific hAELVi cells resulting in higher TEER value and ZO-1 expression . With thIn this paper, a transwell insert-embedded microfluidic cell culture device is developed to study alveolar epithelium barrier function based on an in vitro cell culture model. The transwell insert embedded architecture makes it feasible to culture the cells at the ALI for atmospheric environment exposure experiments. In addition, the device fabrication process is straightforward, without tedious assembly of multiple layers. Although the real-time imaging of the cells with high magnification ratio is infeasible due to the height of the bottom microfluidic channel and limited transparency of the membrane, the insert can be later detached from the device at the end point of the experiments for high resolution imaging without considering limited working distance of the objective. It is noted that the transwell insert cannot be reassembled to the microfluidic device due to possible leakage and additional undesired force application on the cells cultured on the porous membrane. The device is exploited to culture commercially available human alveolar epithelial cells to investigate epithelium barrier function under various culture conditions based on TEER measurement and immunofluorescence staining in a time-lapse manner. In the experiments, conventional LLC and ALI cell cultures are performed to compare the effects of culture format on the barrier function. The results show that the ALI culture can promote tight junction protein expression and reduce the barrier permeability. In addition, two types of stimulations—inflammatory cytokine and ethanol vapor exposure—are conducted on the cell culture at the ALI, and the results show the impairing and promotion of the barrier permeability by the cytokine stimulation and ethanol vapor exposure, respectively. The TEER measurement provides time-lapse observation capability on the same batch of cells. Consequently, the developed device can provide a practical platform to construct in vitro models for barrier function study under various physiological and pathological conditions. In addition, alveolar epithelial and endothelial cells can be co-cultured in the channel and on the porous membrane, respectively to construct in vitro blood-air barriers. The channel dimensions in the device can be adjusted to simulate in vivo dynamic flow conditions.