Overall, choice order depended on the degree of relative preference among alternatives and a peak-first bias, providing evidence for prospective evaluation when choice order is self-generated.Animals typically must make a number of successive choices to achieve a goal: e.g., eating multiple food items before becoming satiated. However, it is unclear whether choosing the best first or saving the best for last represents the best choice strategy to maximize overall reward. Specifically, since outcomes can be evaluated prospectively (with future rewards discounted and more immediate rewards preferred) or retrospectively (with prior rewards discounted and more recent rewards preferred), the conditions under which each are used remains unclear. On the one hand, humans and non-human animals clearly discount future reward, preferring immediate rewards to delayed ones, suggesting prospective evaluation; on the other hand, it has also been shown that a sequence that ends well, i.e., with the best event or item last, is often preferred, suggesting retrospective evaluation. Here we hypothesized that when individuals are allowed to build the sequence themselves they are more likely to evaluate each item individually and therefore build a sequence using prospective evaluation. We examined the relationship between self-generated choice order and preference in rhesus monkeys in two experiments in which the distinctiveness of options were relatively high and low, respectively. We observed a positive linear relationship between choice order and preference among highly distinct options, indicating that the rhesus monkeys chose their preferred food first:  Animals live in the present but benefit from remembering the past and predicting the future. For goal-directed behavior, it has long been appreciated that many animals, including humans, consider future events, but discount their value based on the delay to receiving them, indicating that an immediate reward is more valuable than the same amount of reward in the future peak-end rule in which people judge previous events by averaging the peaks  and the end peak-end bias. Indeed, there are numerous examples in everyday life that appear to reflect this preference: from dessert at the end of meals to the climax of stories or performances normally occurring at the end.In fact, this retrospective evaluation process has been well-documented in people, leading to what has been called a Thus, outcome evaluation appears to be a decreasing function of distance in time from the present to both the future and the past. However, the conditions under which individuals use prospective or retrospective evaluation to guide behavior remain unclear. We hypothesize that the type of evaluation process evoked depends in part on the extent to which a series of events are organized into a unit or episode. When individual events are seen as parts of a larger episode, the evaluation process may be conducted on the episode as a whole once it is completed. In this case, memory is needed, and retrospective evaluation would occur. If, however, the events are seen as separate entities, prospective evaluation would take place, with each event being processed individually in order to maximize immediate reward.first one As a test of this hypothesis, we conducted a study with rhesus monkeys, which have proven to be an excellent primate model of human decision-making; and at the same time help delineate the evolutionary origins of these evaluation processes. It had previously been shown that when receiving a sequence of qualitatively distinct rewards, rhesus monkeys did not exhibit a clear peak-end bias, and if anything, appeared to show a peak-first sequence. Thus, for the current study, we asked, if given a sequence of choices without replacement, in which all items are initially available and an individual must select them in their preferred order, which evaluation process would be used? Subjects who determined the sequence retrospectively should have a peak-end bias and should choose in the opposite order of their preference, saving the best for last, whereas those who chose prospectively, should have a peak-first bias and should choose in the order of their preference, selecting their favorite first and so on. We found that all four monkeys exhibited strong peak-first order preferences, but only when there were large enough differences in preferences among the food items.Following the logic of items being evaluated prospectively if considered individually, we hypothesized that if the monkeys were offered four qualitatively distinctive food items, which they could obtain in any sequence and were allowed to generate the sequence themselves, they would evaluate each item in the sequence individually, leading to prospective evaluation and a preference for a peak-Animal care and use complied with all current laws, regulations, policies, and guidelines of the United States, the United States Department of Agriculture (USDA), the Public Health Service (PHS), and all procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Dartmouth College.Macaca mulatta) participated in Experiments 1 and 2: Hamlet, Caesar, Puck, and Titus. Hamlet, Puck, and Titus. The average age of the monkeys was 8.75±0.48 (mean ± s.e.m). They were housed in 32×27×68 (width x depth x height) inch cages  in a homeroom with automatically regulated temperature, ventilation, humidity, and lighting . The monkeys were intermittently housed in pairs and individually: at times when they engaged in confrontations, which is normal periodic behavior in young rhesus macaque males of similar size and temperament Four male rhesus macaques , supplemented with fresh fruit and vegetables, as well as various treats that included peanuts, cereal, and dried fruits .The Center for Comparative Medicine and Research (CCMR) at Dartmouth maintains a full-time animal care and veterinary staff that monitors the monkeys’ daily health and well-being. The monkeys were maintained at approximately 95% of their To allow for continued social stimulation, the subjects had direct visual contact with the other monkeys in the colony, the animal care staff, and experimenters. When pair-housed, they had direct physical contact with each other, and also when individually-housed, through a mesh grading divider between their cages. In addition, environmental enrichment included two or more enrichment items in their home cages at all times, daily playing of radio or videos in the room , and regular access to a larger enrichment cage (68×38×72 inch) in an adjacent room. The monkeys were brought to the testing room in custom-made chairs. The chairs were used (a) to minimize disruption in the test subjects’ daily routines, given that they were already acclimated to them from previous experiments; (b) to have precise control over the experimental testing conditions; and (c) to obtain clear, unbiased choice responses, with tray compartments (described below) at fixed positions relative to the monkey on every trial.Each monkey sat across from the experimenter in the chair. The chair loosely restrained the left arm of the monkey while allowing free movement of the right arm. An opaque plastic divider separated the experimenter and the monkey, thus preventing the monkey from seeing the face and upper body of the experimenter. An opening at the bottom of the divider allowed the experimenter to present a transparent plastic food tray to the monkey. The tray contained four separate compartments, which we labeled according to their position relative to the experimenter: left (L), middle left (ML), middle right (MR), and right (R). On a given trial, food was placed on a circular platform in a compartment so that the monkey could clearly see and easily select his choice of food item. Each compartment was covered by a transparent lid, which the monkey had to lift to gain access to a food item. The food items in Experiment 1 consisted of four different types of food, with all being generally yellow in color: shelled peanut halves (PN), yellow BioServ® Fruity Gems (FG), BioServ® banana-flavored dustless precision pellets (PL), and rice krispies®, or krisps (KR). The food items in Experiment 2 consisted of four differently colored  BioServ® Fruity Gems (FG).Both Experiments 1 and 2 consisted of a two-condition interleaved paradigm (described below) that studied ordered choice among four food items without replacement (Condition 1) and binary choice between two food items (Condition 2). Four different types of food  for Experiment 1 and four differently colored fruity gems for Experiment 2 were used. Because the binary choice condition allowed only one selection per trial, and the loss of the item not selected, we reasoned that choice in this condition would reveal preference. In the ordered choice condition without replacement, the monkeys would obtain all four food items regardless of order; and thus, selection order might or might not reflect preference. For example, an individual might select his favorite items first, or save them for last. The experiments, therefore, examined the specific relationship between choice order and preference.The conditions were interleaved in that each test session consisted of 1 trial of Condition 1  and 12 trials of Condition 2 . We interleaved the conditions to make sure choice order and preferences remained stable; and we conducted the ordered choice condition first to provide the monkeys with experience of all four food items before conducting the binary choice preference tests.In Condition 1, the ordered choice condition, a different food item was placed into each of the four compartments . The locTo measure food preferences, in Condition 2 we used the binary choice paradigm, in which two different food items were placed in the two middle containers (ML and MR), with one food item per container . To begiOnce the data was collected, for Condition 1, we calculated the choice rate of each item for a given choice order. The overall individual's choice order with respect to different items was determined by selecting an item that provides the highest choice rate for a given order. For Condition 2, we calculated the winning percentage of each food item, the percentage of winning in the binary choice paradigm. Then we obtained the rank order of individual preference by ranking individual's winning percentages of food items.F  = 408.0, p<0.001 for PN; F  = 148.8, p<0.001 for FG; F  = 7.4, p<0.01 for PL; F  = 7.7, p<0.01 for KR). This indicates that the four monkeys showed a similar choice order for each food item. Individually, all four monkeys exhibited significant choice orders for the different food items across sessions   = 204.0, p<0.001; Caesar: χ2(9)  = 192.0, p<0.001; Puck: χ2(9)  = 110.0, p<0.001; Titus: χ2(9)  = 158.7, p<0.001 ), indicating a stable choice order for each food item across sessions.In Condition 1, we tested the order in which the monkeys chose a food item among the four different food items without replacement. Overall, the monkeys predominantly chose the peanut half , fruity gem , pellet , and rice krisp  for their first, second, third, and fourth choices, respectively . There wF  = 54.8, p<0.001), indicating general food preferences in the monkeys.For Condition 2, we calculated the winning percentage of each food item using a binary choice paradigm that presented all possible combinations of two food items in order to measure preference between food items. 2(3)  = 154.9, p<0.001; Caesar: χ2(3)  = 143.1, p<0.001; Puck: χ2(3)  = 126.0, p<0.001; Titus: χ2(3)  = 120.8, p<0.001). The food preferences were the same for three of the monkeys , and somewhat similar for the other monkey . All four monkeys preferred the peanut half most, then the fruity gem. Overall, there were strong food preferences for all individuals and small individual differences in less-preferred items.Individually, all four monkey showed significant preferences toward an option among the four alternatives in Condition 2 .Because all four monkeys showed the greatest preference for peanut halves and the second-greatest preference for fruity gems it indicates general preferences for these food items. This general preference suggests there are particular factors, such as caloric value, that may underlie preference. Thus, we tested whether the winning percentage was correlated with the caloric values of the food items : peanut half (66.9%); fruity gem (25.5%), pellet (5%), krisp (2.6%). The winning percentage for each food item was indeed significantly correlated with their caloric values   = 287.1, p<0.001), confirming that individual preference among ranks was significantly biased toward the favorite options.Although the individual monkeys exhibited similar preferences among food items, individual preference does not necessarily correspond to group preference . Thus, we defined rank as the order of winning percentage within individuals. Then, we compared the winning percentage with respect to rank to quantify the extent of preference toward an individual's favorite options . There wp<0.001) ; the fourth monkey, Puck, chose his first and second preferred items in order, but then flipped the choice order for the third and fourth preferred items . It is unclear why Puck tended to select the fourth preferred item (pellet) over the third (krisp) when choosing between them in the choice order condition, but selecting the third over the fourth in the binary choice condition. Although the difference in winning percentage between the third and fourth preferred items (17.4%) was less than the overall average difference (29.5%) or Puck’s average difference (27.6%), it, however, was not the smallest difference among the preferences (which was the difference between Caesar’s first and second preferred items: 16%).Comparing the two conditions, we found a direct linear relationship between choice order and the winning percentage of food items  . RegardiOverall, the monkeys exhibited a general peak-first bias, with at least the first and second favorites being selected in order.To help delineate the factors underlying preference and choice order, Experiment 2 tested color, which is considered a salient cue to primates with trichromatic color vision, such as rhesus monkeys F  = 2.9, p = 0.079 for red; F  = 0.27, p = 0.848 for orange; F  = 1.3, p = 0.330 for green; F  = 0.46, p = 0.715 for purple). This indicates that there was no generally preferred order with respect to color among the monkeys. Individually, three of the four monkeys exhibited no significant choice order for the different colors across sessions   = 5.3, p = 0.810; Caesar: χ2(9)  = 12.5, p = 0.188; Titus: χ2(9)  = 13.6, p = 0.138), indicating an unstable choice order with respect to color across sessions. In contrast, one monkey exhibited a significant choice order for color   = 21.0, p<0.013).In Condition 1, we tested the order in which the monkey chose the four differently colored but otherwise identical food items (fruity gems). The monkeys chose green (mean ± s.e.m = 28.1±6.5%) and purple (28.1±11.1%) most for their first choice in a two-way tie, purple most for their second choice (28.1±5.0%), red most for their third choice (35.4±9.2%), and red (28.1±2.8%) and green (28.1±6.1%) most for their fourth choice in a two-way tie . There wF  = 0.713, p = 0.563). Individually, however, all four monkeys had significant color preferences   = 13.3, p<0.01; Caesar: χ2(3)  = 21.6, p<0.001; Puck: χ2(3)  = 12.8, p<0.01; Titus: χ2(3)  = 20.1, p<0.001 )  . The colF  = 31.1, p<0.001), indicating that individual preference was significantly biased toward the favorite colors.Since there were large individual differences in specific color preferences, we defined rank as the order of winning percentage within individuals. Then, we compared the winning percentage with respect to rank to measure the extent of preference toward an individual's favorite option . There wp = 0.25)  .p = 0.61; Caesar: slope  =  5.42, Adj. R-Square  =  –0.21, p = 0.56; Puck: slope  =  6.94, Adj. R-Square  =  0.32, p = 0.26; Titus: slope  =  6.12, Adj. R-Square  =  0.20, p = 0.32). Thus, the monkeys did not exhibit an order preference with respect to color.Examining choice order in relation to specific color preferences individually, none of the monkeys exhibited a significant tendency to choose the most preferred item   = 14.7, p<0.001). Thus, although significant preferences were found among the food items in both experiments, the relative preference among options was significantly higher in Experiment 1 than in Experiment 2.Finally, to compare the relative preference among the choice options in Experiments 1 and 2, we calculated the average difference among winning percentages for each monkey in Experiment 1  and compared them to the same in Experiment 2 . The average difference in relative preference among the food items was significantly different between the two experiments  across the food items in Experiment 1 compared to Experiment 2. Thus, the relationship between relative preference and behavior—in this case choice order—is complex and apparently nonlinear in rhesus macaques. It will be important for future work to further characterize this nonlinear functional relationship to determine when and how relative differences become sufficiently salient to influence choice order However, when the relative differences were sufficiently salient, choice order was clear: when allowed to take four food items in any sequence, the monkeys chose the food items in order of preference, starting with their favorite first. In a study in which one choice response was followed by a sequence of outcomes , rhesus monkey order preferences were less clear, although if anything, they also tended to exhibit a peak-first preference In contrast, humans appear to evaluate sequences retrospectively, often exhibiting a peak-end preference Thus far, then, humans appear to exhibit a peak-end preference for outcome sequences, considering them as an episode, whereas rhesus monkeys, if anything, appear to exhibit the opposite. Whether this difference is due to species or methodological differences, however, remains unclear. For example, the human studies often use sequences composed of quantitative differences, which might be more conducive to sequence detection and formation; whereas, the rhesus study used qualitatively distinct food items. It will therefore be important to compare humans and non-human animals with more similar testing conditions, and in particular, more comparable items, which may influence sequence formation and the type of evaluation process used.If prospective versus retrospective evaluation is at least partly based on the extent individual events are combined into sequences, one might expect self-generated sequences to promote the evaluation of each choice outcome individually, which could explain why the current results from self-generated sequences more strongly revealed a peak-first preference as opposed to the experimenter-generated sequences in the Xu et al. (2011) study. It is therefore important to ask what order humans would produce if they generated the sequences themselves. Recently, Jeong et al. (under revision) conducted such an experiment with pieces of sushi. When choosing sushi pieces without replacement, two general populations of people emerged: some people selected their preferred items first, while others saved them for last. Different subpopulations of people with opposite preferences suggest that in addition to contextual factors, individual traits may also influence the preferred sequence order. A critical individual factor might be the degree of impulsivity versus self-control, given that there are clear individual differences among humans on this trait Comparing our results with those in the sushi experiment, our rhesus monkeys match the group preferring the peak-first sushi order. Indeed, evidence points to rhesus monkeys showing relative impulsivity, compared, for example, to great apes. When required to wait for a food item, rhesus monkeys rarely exceeded a 30-second delay, whereas chimpanzees and an orangutan waited roughly six times longer However, it has also been argued that the apparent riskiness of rhesus monkeys may be context driven Future work will need to further delineate the environmental conditions and individual factors determining the use of prospective versus retrospective evaluation. In general, goal-directed behavior involves the generation, maintenance and seeking of goals, which appears to promote prospective evaluation In more stable environments, however, sequences of events, actions and/or outcomes are more reliable, and linking (or ‘chunking’) them together into a sequence can provide efficient hierarchical memory storage and heightened predictability Finally, even though humans appear to have a peak-end bias for a series of events, including, e.g., personal experiences, performances, and stories, it does not necessarily mean that if given the opportunity to build the sequence themselves, they would do so. Here we demonstrated that rhesus monkeys showed a peak-first preference in self-generated choice order. Whether self-generated sequencing imposes a heavier burden on self-control and/or sequence formation is yet to be determined. To be sure, it is likely that different underlying processes, such as prospective and retrospective evaluation, impulsivity and self-control, and the ability to detect and formulate sequences, interact to determine our preferences, and more work including comparative analyses is necessary to identify the nature of these underlying processes.


Pyropia haitanensis has a biphasic life cycle with macroscopic gametophytic blade (n) and microscopic filamentous conchocelis (2n) phase. Its gametophytic blades have long been believed to be mainly dioecious. However, when crossing the red mutant  with the wild type , the parental colors were segregated in F1 blades, of which 96.1% were linearly sectored with 2–4 color sectors. When color sectors were excised from the color-sectored blades and cultured singly, 99.7% of the color sectors appeared to be unisexual with an equal sex ratio. Although the sex of color sector did not genetically link with its color, the boundaries of both sex and color sectors coincided precisely. About 87.9% of the examined color-sectored blades were monoecious and the percentage increased with the number of color sectors of a blade. The gametophytic blades from each conchocelis strain produced by parthenogenesis of the excised color sectors were unisexual and unicolor, showing the same sex and color as their original sectors. These results indicate that most of the sexually reproduced Py. haitanensis blades are monoecious, and their sex is controlled by segregation of a pair of alleles during meiosis of conchospore, forming a sex-sectored tetrad. During the subsequent development of blades, one or two lower cell(s) of the tetrad contribute mainly to rhizoid formation, and rarely show their sexual phenotype, leading to reduced frequency of full sex phenotype of the meiotic blades. Moreover, the aberrant segregations of sex genes or color genes in a few of F1 blades were probably due to gene conversions, but there was no sex transfer in Py. haitanensis. Pyropia haitanensis (T. J. Chang et B. F. Zheng) N. Kikuchi et M. Miyata Pyropia in China Py. haitanensis has a biphasic life cycle that alternates between macroscopic gametophytic blades (n) and microscopic filamentous conchocelis (2n) phase Py. haitanensis is the same as that for Py. yezoensisPorphyra purpureaPy. haitanensis showed that the meiotic segregation of parental colors and the ontogenesis of color-sectored blades are very similar to those of Py. yezoensis and P. purpureaPy. yezoensis and Py. haitanensis were derived from a linear tetrad P. purpurea were derived from a nonlinear tetrad P. purpurea occurs during meiosis of conchospore, forming monoecious blades Py. haitanensis produced by sexual reproduction would be monoecious rather than dioecious Py. haitanensis blades has to be re-examined. In the present study, the segregation of two genetic markers (i.e. color and sex) was studied in F1 gametophytic blades produced in the cross between a red mutant  and the wild type  of Py. haitanensis. These data were used to better understand sex determination in this species.Therefore, the sex of PT–WT, W, ♂) was used as male parent. Its free-living conchocelis was obtained by the male-parthenogenesis 60Co-γ ray, and its vegetative cells were subsequently isolated enzymatically to regenerate into blades SPY–1, R, ♀) was picked out In the present study, a wild-type strain (−2 s−1 (14L∶10D). When the diameter of the conchocelis colonies reached about 1 cm, they were fragmented in a homogenizer, and inoculated into clean clam shells and cultured for growing shell-boring conchocelis (conchocelis shells). The inoculated shells were then incubated with the MES medium −2 s−1 (12L∶12D). After culture for 10 days, the shells were washed to clear superfluous conchocelis filaments on the shell surface, the culture medium was renewed, and the photon flux density was increased to 20 µmol photons m−2 s−1. A few weeks later, the culture temperature was increased to 25±1°C without change of other culture conditions. After another two weeks, the culture temperature was increased to 28±1°C, while the photon flux density was decreased to 10 µmol photons m−2 s−1 (10L∶14D) to induce formation of conchosporangia. Once conchosporangia formed, the conchocelis shells were transferred into a 250 mL Erlenmeyer flask containing 100 mL culture medium, and cultured with aeration in an incubator at 25±1°C under 40 µmol photons m−2 s−1 (10L∶14D). After 3–5 days of culture, conchospores released from the heterozygous conchocelis were collected, passed gently through a 50 µm nylon mesh filter, and cultured in Petri dishes containing the MES culture medium at 23±1°C under 40 µmol photons m−2 s−1 (10L∶14D) to obtain F1 gametophytic blades. The culture medium was refreshed every week.A male blade and a female blade were respectively selected from offsprings of the parental strains and co-cultured in a flask until zygotosporangia 1 gametophytic blades reached 2–4 mm long after cultured for about 3 weeks in Petri dishes, all blades were carefully collected using a single-side razor blade. From among them, 1400 blades were randomly selected and inspected under a microscope , and the color type and the number of sectors in each blade were recorded. The remaining blades were cultured in several flasks (2000 mL) with aeration at 25°C under 40 µmol photons m−2 s−1 (10L∶14D).When F−2 s−1 (10L∶14D) until the spermatangium or female gamete After 2–3 weeks of further culture, the color-sectored blades grew to be 3–5 cm long and their color sectors (0.5–2 cm long) could be clearly distinguished by naked eyes. Then, 30 color-sectored blades  were picked out and cultured singly in different flasks (500 mL) with aeration until each sector of the blade matured to confirm the sex and sexual boundary of the color sectors. In addition, before blades matured, 173 color-sectored blades with macroscopic color sectors  were picked out and all of color sectors were excised from them and cultured singly in different flasks (500 mL) with aeration at 25±1°C under 40 µmol photons mTo test the stability of sex heredity of color sectors, 50 color sectors (10 R (♀), 10 R (♂), 5 R′ (♀), 5 R′ (♂), 5 W (♀), 5 W (♂), 5 W′ (♀) and 5 W ′(♂)) were randomly excised from the immature color-sectored blades 3–5 cm long and were singly cultured. After 20–80 days of further culture, these color sectors underwent parthenogenesis and formed homozygous free-living conchocelis. After 3–4 months of further culture, the free-living conchocelis of each sector were respectively inoculated into clam shells to obtain shell-boring conchocelis as described above. The conchospores released from the mature shell-boring conchocelis were cultured into blade populations, and the sex and color of each population were examined when they matured as described above.2) test with Yates correction was applied to analyze the sex and color ratios in the F1 gametophytic blades at the 5% significance level. A student's t-test was applied to analyze the sex difference among 2-sectored, 3-sectored and 4-sectored blades. P value of<0.01 was considered significant.A chi-square  of them were color-sectored blades with 2–4 linearly arranged color sectors, and the rest were single-colored blades. The percentage of color-sectored blades with 2, 3 and 4 sectors was 54.4, 33.8 and 7.9%, respectively. Meanwhile, it was also found that each of the color sectors in a blade did not develop evenly. Generally, one or two basal color sector(s) stopped growth or grew very slowly, while one or two uppermost color sector(s) developed into the dominant part of a blade having a blade sector nearly with entire margin. As a result, when the blades grew to 3–5 cm long, the percentage of color-sectored blades decreased to 48.4%  while that of single-colored blades increased to 51.6%, as observed by naked eyes. However, when observed microscopically, it became clear that about 89.0% of the single-colored blades and 58.0% of the 2-sectored blades had one or two very small color sector(s) in the basal part.Four kinds of color sectors, two parental color sectors (W sector and R sector) and two recombinant color sectors (R′ sector and W′ sector) appeared in the Fperiment . R′ sect1 blades showed unusual color segregation. A few blades had five color sectors, such as R′+R+W′+R+W . However, the sex and color were not genetically linked. According to the sex of each color sector, the sex of 173 color-sectored blades was identified and shown in A total of 584 color sectors excised from 173 color-sectored blades  were cultured individually for sex analysis. Most of the color sectors matured after 15–70 days of further culture, but a few did not mature until 90–100 days of culture. Sex observation on these mature color sectors showed that 582 color sectors were all unisexual, male or female, except for 2 color sectors which linearly sectored with male and female parts. In those unisexual sectors, the ratio of female to male was 292∶290, nearly 1∶1. The ratio of parental sex sectors (R♀+W♂) to recombinant sex sectors (R′♂+W′♀) was in accordance with 1∶1 by statistical test , ♂+♀+♀+♂/♀+♂+♂+♀ = 17/11 , and ♂+♀+♂+♀/♀+♂+♀+♂ = 18/26   . StatistHowever, among the remaining 8 blades, 4 had four female sectors (♀+♀+♀+♀), 2 had four male sectors (♂+♂+♂+♂), 1 had one male and three female sectors (♀+♂+♀+♀) and the last one had color phenotype of R+W′+W+R′, although its W′ sector was sectored linearly with male and female sectors and had sex phenotype of R (♂)+W′(♂+♀)+W(♀)+R′(♂) . The rat1 blades produced homozygous conchocelis (strain) through parthenogenesis. Careful microscopic examination of over 1000 randomly collected gametophytic blades of each strain found that all blades of each strain were unisexual and unsectored, and had the same color and sex as their original sectors, indicating no sex transfer there.After 20–80 days of further culture, the singly cultured color sectors excised from color-sectored FPyropia (Porphyra) occurs during the conchospore germination, and produces a tetrad which subsequently develops into a genetically chimeric blade Pyropia used in the present cross-experiments was caused by nuclear gene mutation(s), regardless it contains one or multiple mutated color genes, parental colors would surely segregate in each F1 blade, producing color-sectored blades, and the number of color sectors in a color-sectored blade would be 2 or 4. However, the single-colored blades and color-sectored blades with 3 sectors always appeared in F1 blades in the cross experiments between the color mutant and the wild type in Py. yezoensisPy. haitanensisP. purpureaPy. oligospermatangia and Py. katadae var. hemiphyllaIt was found that the meiosis in Py. haitanensis are derived from one or two upper cells of the tetrad, and one or two lower cell(s) of the tetrad stop development after several divisions, forming very small color sectors which contribute to rhizoid formation. In the extreme case, they even become only one cell of the rhizoid without division. Furthermore, microscopic observation of the basal sectors of the color-sectored blades indicates that when the blades were 2–4 mm long, the genotype of R, R′, W and W′ appeared in the basal sector with the same odds (data not shown). However, during the subsequent development of these young blades, the growth of the lowest sector was also arrested. In addition to a relatively low growth rate of the W and W′ sectors, they always become very small sector hardly discerned by naked eyes. While the probability of the lowest sector having genotype of R or R′ which grown into a larger sector in the adult blades due to the remarkable growth advantage of them was much higher than that of W or W′ sectors. Therefore, when the blades reached 3–5 cm long, almost all of the visual basal sectors were R or R′ , although their sex phenotype remains the same, mainly monoecious one. As a result, the percentage of monoecious blades should not differ significantly between the three- and four-sex-sectored blades, in consistence with our result . However, if the development of two lower cells in the tetrads is arrested, the tetrads would lose sex phenotypes of two sectors and develop into dioecious  blades (♂+♂ or ♀+♀) or monoecious blades (♂+♀ or ♀+♂). This probably is the main reason why the proportion of unisexual blades reached 30.2% among the two-sex-sectored blades  The present cross experiment indicated that the arrested development of lower cell(s) in the tetrad of Py. haitanensis would decrease the number of observable sex sectors and lead to misjudge their sex, and the upper cells of tetrads would divide to form the dominant part of blades in Py. haitanensisPy. haitanensis are not easy to be observed.The previous study found that 99.9% of wild blades of this species were dioecious and only 0.1% was monoecious when examined by naked eyes P. purpurea is the controlling factor of its sex. In the present study on Py. haitanensis, it was shown that (1) the segregation ratio of female and male was 1∶1; (2) almost every color sector of color-sectored F1 blades was unisexual; and (3) the boundaries for sex and color always coincided precisely. These results are in agreement with those obtained in the cross experiment of P. purpurea by Mitman and van der Meer Py. haitanensis is also controlled by meiotic segregation of a pair of alleles.Mitman and van der Meer Pyropia (Porphyra) was chimeric and composed of four meiotic products, theoretically, it should have no more than four sectors Aberrant segregations at ratios of 0∶4, 4∶0, 3∶1 and 2∶3 of the female and male in a few of four-sectored blades were observed in the present study. Because each blade of Py. haitanensis and fungi showed very similar characteristics in tetrad patterns, we analyzed them using the same method. As a result, we speculate that similar gene conversion events probably occur in Py. haitanensis during the meiosis, resulting in aberrant egregation of sex alleles in F1 blades (The red color mutant used in the present study was caused by nuclear gene mutations 1 blades . The abeMAT locus) with HO-induced cutting in yeast showed that among 1550 tetrads, 215(14%) had a ratio of 4α∶0a; 9 had 1α∶3a and 19 had 3α∶1a when the HO gene was expressed, while only a few aberrant segregations with a ratio of 1α∶3a at MAT locus when the HO gene was not expressed HIS4 locus Saccharomyces cerevisiae, mother cells but not daughter cells can switch mating type by HO-induced mitotic gene conversion Furthermore, studies on an artificial recombination-initiating region  of the tetrad mainly contribute to rhizoid formation. (2) Most gametophytic blades of Py. haitanensis produced by sexual reproduction are monoecious, and only a few are dioecious. (3) Gene conversions rather than sex transfer probably occur in Py. haitanensis.(1) The sex of


Although several cognitive processes, including speech processing, have been studied during sleep, working memory (WM) has never been explored up to now. Our study assessed the capacity of WM by testing speech perception when the level of background noise and the sentential semantic length (SSL) (amount of semantic information required to perceive the incongruence of a sentence) were modulated. Speech perception was explored with the N400 component of the event-related potentials recorded to sentence final words . During sleep stage 2 and paradoxical sleep: (1) without noise, a larger N400 was observed for (short and long SSL) sentences ending with a semantically incongruent word compared to a congruent word (i.e. an N400 effect); (2) with moderate noise, the N400 effect (observed at wake with short and long SSL sentences) was attenuated for long SSL sentences. Our results suggest that WM for linguistic information is partially preserved during sleep with a smaller capacity compared to wake. Previously, sleep has been considered to be a passive state favoring the recovery of energy. However, recent literature suggests that substantial cerebral activity allowing high cognitive processes occurs during this state. For instance, lexical and sentential semantic processing of auditory speech stimuli have been reported with event-related potentials (ERPs) during “slow wave sleep”, mostly sleep stage 2 (N2), and paradoxical sleep (R)  sentences that needed only the last few words of the context to be perceived as incongruent (referred to as “short SSL incongruent sentences”), and 2) sentences that needed more words of the context to be perceived as incongruent  final word of the sentence. Since the amplitude of the N400 is sensitive to the semantic congruency of a sentential context, the N400 to incongruent sentences was expected to decline (i.e. the difference between the N400 to incongruent and congruent sentences to be reduced) as the load on WM would increase. According to neuroimaging data Sixteen volunteers were tested . All participants were right-handed according to the Edinburgh Handedness Inventory Approche du rôle du contexte phrastique dans la reconnaissance lexicale auditive des mots, Doctoral Thesis, Marc Bloch University: Strasbourg, France, 1998) (see Supplementary Information). All these sentences: (a) had a cloze probability (based on the responses of 200 participants) higher than 20 (M ± SEM = 47.9±3.0) , (b) had a disyllabic noun target , (c) were presented at a natural speech speed, (d) had a duration of 2 to 3 sec (M ± SEM = 2.6±0.1), and (e) contained 7 to 13 words (M ± SEM = 8.8±0.2). The onset of the last word of the sentence  was estimated from the sentence's acoustic signal using visual  and auditory cues (listening to the truncated sentence and the target). Semantically incongruent sentences were built from the same material by reassignments of sentences truncated by the final word (the context) with different targets. One thousand one hundred seventy incongruent sentences were built by reassignment. Fifty sentences ending with a semantically congruent target word were selected from the corpus of an unpublished thesis of phonetics .A second pilot study was run to estimate the minimum load of sentential context that is required to be stored in WM in order to perceive each of the 1100 incongruent sentences as semantically incongruent (see Supplementary Information). The minimum number of words of the sentential context (i.e. the sentence truncated by its last word) that was required to perceive a sentence as semantically incongruent will be referred to hereafter as “sentential semantic length” (SSL). This number of words counted all types of words, whatever their syntactic function, i.e. including nouns, verbs, pronouns, and articles. An SSL unit may or may not be a unit of meaning or a chunk in WM. Although most of the sentential contextual words were units of meaning, a few other words (e.g. articles or pronouns) may or may not be perceived as units of meaning. Overall, we assume that long SSL incongruent sentences required more units of meaning or chunks stored in WM than short SSL incongruent sentences to be perceived as semantically incongruent.t (1078) = 1.96, p = 0.05]. The duration was 498,9±4.2 for short SSL incongruent sentences and 996.1±17.0 for long SSL incongruent sentences . The score of congruency measured in the first pilot study was 1.38±0.03 for short SSL incongruent sentences and 2.05±0.05 for long SSL incongruent sentences . The cloze probability  = 0.35, p = 0.72].The implication of the WM load/capacity during the perception of the incongruent sentences was tested with the measured SSL. To this aim, we separated the incongruent sentences into two groups (with short and long SSL) using a median split of the distribution of the SSL of all incongruent sentences . The number of words was 8.77±0.08 for short SSL incongruent sentences and 8.98±0.07 for long SSL incongruent sentences . Post hoc comparisons (ps<.05) indicated the following effects (independently of the DL):Between 250 and 500 ms, there was a Sentence Type x Antero-posterior interaction . Post hoc comparisons (ps<.05) indicated an N400 effect at DL0 for long (but not short) SSL sentences (−1.45 microV), no significant (p = .97) SSL effect, and no significant ERP effect at DL1 and DL3 .Between 300 and 450 ms, there was a significant Sentence Type x Degradation interaction . Post hoc comparisons (ps<.05) indicated a SSL effect (0.43 microV) at posterior sites. Other ERP effects were not significant .Finally, between 850 and 950 ms, we found a significant Sentence Type x Antero-posterior interaction . Post hoc comparisons (ps<.05) indicated the following effects:Between 400 and 550 ms, there was a Sentence Type x Degradation x Antero-posterior interaction . Post hoc comparisons did not reach significance . None of the interactions with Sentence Type reached significance .Between 600 and 750 ms, there was a main effect of Sentence Type . Post hoc comparisons (ps<.05) indicated the following effects:Between 400 and 550 ms, there was a Sentence Type x Degradation x Vigilance State x Antero-posterior interaction . Post hoc comparisons (ps<.05) indicated the following effects (independently of the State of Vigilance):Between 550 and 700 ms, there was a significant Sentence Type x Degradation x Antero-posterior x Hemisphere interaction . Post hoc comparisons (ps<.05) indicated the following effects:Finally, between 1100 and 1200 ms, we found a significant Sentence Type x Degradation x Vigilance State x Antero-posterior interaction [ps>.99) SSL sentences, and a SSL effect at all scalp sites (−0.85 microV), (c) at DL3: a negativity effect at central (−0.60 microV) and posterior sites (−0.89 microV) for short (but not long) SSL sentences, and a SSL effect at all scalp sites (0.87 microV).during wake: (a) at DL0: a negativity effect at central (−0.83 microV) and posterior sites (−1.31 microV) for long SSL sentences, a negativity effect at posterior sites (−0.77 microV) for short SSL sentences, and a SSL effect at central (−0.48 microV) and posterior sites (−0.54 microV), (b) at DL1: a negativity effect at all scalp sites (−0.77 microV) for long  SSL sentences, and a SSL effect at central (1.26 microV) and posterior sites (1.66 microV), (c) at DL3: a positivity effect at posterior sites (0.64 microV) for short  SSL sentences , and a SSL effect at posterior sites (−0.93 microV).during N2: (a) at DL0: a negativity effect at anterior (−0.94 microV) and central (−0.69 microV) sites for long SSL sentences, a positivity effect at anterior sites (0.63 microV) for short SSL sentences, and a SSL effect at anterior (−1.57 microV) and central sites (−1.00 microV), (b) at DL1: a negativity effect at central (−1.09 microV) and posterior sites (−1.34 microV) for short  SSL sentences, and a SSL effect at all scalp sites (−0.96 microV), (b) at DL1: a negativity effect at central (−0.74 microV) and posterior sites (−0.52 microV) for long SSL sentences, a negativity effect at central (−0.71 microV) and posterior sites (−0.75 microV) for short SSL sentences, and no significant SSL effect (ps>.99), (c) at DL3: a positivity effect at anterior (0.73 microV) and posterior sites (0.56 microV) for long SSL sentences, a negativity effect at posterior sites (−0.50 microV) for short SSL sentences, and a SSL effect at anterior (0.70 microV) and posterior sites (1.06 microV).during R: (a) at DL0: a positivity effect at central (0.77 microV) and posterior sites (0.93 microV) for short  for speech processing remained during sleep. Our findings were as follows:We found a larger N400 component in response to sentences  ending with a semantically incongruent word compared to a congruent word (N400 effect) during N2 and R. This finding replicates the results of Ibáñez et al. We showed for the first time speech-in-noise processing during N2 and R. Indeed, the N400 effect was observed during N2 and R in a mild noise condition (DL1). Furthermore, we found attenuated ERPs effects during N2 and R in a strong noise condition (DL3) that could be interpreted as a small N400 effects. These ERP effects at DL3 found during sleep but not during wake suggest that sleep and wake speech-in-noise processing seem to activate qualitatively different mechanisms.Using a gating paradigm that assessed the sentential semantic length SSL, see  and coupSleep ERP studies testing semantic processing have mainly used single word or word pair paradigms . A higher load on WM was required with long compared to short SSL sentences and with increasing sentential degradation. Thus, we were able to compare the N400 responses in response to sentences according to the load on WM.When sentences were perceived without noise (DL0), wake data showed a larger N400 effect to the long SSL incongruent sentences compared to the short SSL incongruent sentences. This difference suggests that long SSL sentences were more incongruent than short SSL sentences. However, our first pilot behavioral study (see Supplementary Information) suggests that the short SSL sentences were more incongruent than the long. Taking our N400 and behavioral data together, the difference of N400 between short and long SSL sentences would rather reflect a WM load difference between short and long SSL sentences rather than a difference of semantic congruency. This N400 difference at DL0 during wake (i.e. the control data) is not negligible and has to be taken into account for the interpretation of our data in all the other experimental (test) conditions.During N2 and R, a N400 effect was also found with long and short SSL sentences. As expected from Ibáñez et al. During wake, data at DL1 showed a similar pattern as data at DL0, with a larger N400 effect to long compared to short SSL sentences. Unlike wake data, N2 and R data showed a different pattern between DL1 and DL0.During N2, only the short SSL sentences showed an N400 effect, while long SSL sentences showed a larger positivity to incongruent compared to congruent sentences that might reflect a failure to fully process the sentences because of a too limited WM capacity. Indeed, the cognitive processing of the speech material may be impaired/disrupted, with for instance, an incomplete syntactic analysis of the sentential context. If this syntactic analysis is not completed, the final word of the sentence may not be perceived as a syntactically congruent word. During wake, when a word is perceived within a syntactically incongruent sentential context, a P600 is observed  was found at DL0 Figure 3These sleep (N2 and R) data differences between DL0 and DL1 would arise from a combined effect: the reduced WM capacity during sleep compared to wake together with the increased load on WM required to process degraded sentences. Indeed, speech-in-noise perception requires more WM capacity than speech perception without noise Although the study of Ibáñez et al. During wake, our data replicated Daltrozzo et al. Additionally, as the presumed N400 effects at DL3 during sleep were found only with long SSL sentences during N2 and only with short SSL sentences during R, qualitative differences of noise robust speech cognition between N2 and R may also exist, a conclusion in agreement with previous studies Provided that mechanisms of syntactic processing similar to the wake can be activated by the presentation of sentences during sleep, the larger positivity to long SSL incongruent sentences compared to congruent sentences found during R might reflect a P600 to a syntactic mismatch. Indeed, as mentioned above , which mobilize neuronal assemblies to scan for further information needed to build a “model of possible content” The SSL manipulation was designed on the basis of a gating paradigm (see Supplementary Information) with the aim to modulate the WM load. We checked the specificity of this parameter for WM load.The contextual constraint (i.e. the degree to which the context establishes an expectation for a particular upcoming word) is known to modulate the N400 and is generally estimated empirically by the cloze probability The number of words of the sentential context is obviously not independent from the SSL, i.e. the minimum number of words of the sentential context required to perceive a sentence as semantically incongruent: short SSL incongruent sentences had a context containing fewer words than long SSL incongruent sentences see . TherefoIn summary, we believe that: (i) contextual constraint and SSL were independent parameters. (ii) the SSL had two covariates: the number of words of the sentential contexts and the sentence duration.In addition to the SSL, the load on WM was manipulated with sentential degradation. Each sentence being presented in the three degradation conditions  across participants, the variation of load on WM across levels of degradation could not be confounded with contextual constraint, number of words of the sentential contexts, or sentence duration.To the authors' knowledge, the sound level cannot modulate the load on WM, and hence be a confounding parameter. Furthermore, the sound level did not vary between levels of SSL or levels of degradation, all sentences being presented at a normalized sound level  sentential processing remains during sleep Click here for additional data file.Table S1Averaged number of trials per experimental condition per participant.(DOC)Click here for additional data file.Figure S1Polysomnographic samples during wake (upper panel), sleep stage 2 (middle panel), and paradoxical sleep (lower panel). Electroencephalographic (EEG) signals from 18 Ag-AgCl electrodes  referenced to the nose, VE: Vertical Electrooculogram, HE: Horizontal Electrooculogram, EM: Electromyogram .(TIF)Click here for additional data file.Figure S210(microvolts2/Hz), horizontal unit: Hz).EEG spectra during wake (dotted line), sleep stage 2 (black solid line), and paradoxical sleep (gray solid line) Click here for additional data file.


Maternal mortality in referral hospitals in Mali and Senegal surpasses 1% of obstetrical admissions. Poor quality obstetrical care contributes to high maternal mortality; however, poor care is often linked to insufficient hospital resources. One promising method to improve obstetrical care is maternal death review. With a cluster randomized trial, we assessed whether an intervention, based on maternal death review, could improve obstetrical quality of care.The trial began with a pre-intervention year (2007), followed by two years of intervention activities and a post-intervention year. We measured obstetrical quality of care in the post-intervention year using a criterion-based clinical audit (CBCA). We collected data from 32 of the 46 trial hospitals  and included 658 patients admitted to the maternity unit with a trial of labour. The CBCA questionnaire measured 5 dimensions of care- patient history, clinical examination, laboratory examination, delivery care and postpartum monitoring. We used adjusted mixed models to evaluate differences in CBCA scores by trial arms and examined how levels of hospital human and material resources affect quality of care differences associated with the intervention.For all women, the mean percentage of care criteria met was 66.3 (SD 13.5). There were significantly greater mean CBCA scores in women treated at intervention hospitals (68.2) compared to control hospitals (64.5). After adjustment, women treated at intervention sites had 5 points’ greater scores than those at control sites. This difference was mostly attributable to greater clinical examination and post-partum monitoring scores. The association between the intervention and quality of care was the same, irrespective of the level of resources available to a hospital; however, as resources increased, so did quality of care scores in both arms of the trial.ISRCTN46950658The QUARITE trial is registered on the Current Controlled Trials website under Each day, 800 women worldwide die during pregnancy or of childbirth-related causes. Most maternal deaths occur in sub-Saharan Africa . GloballWhile elevated facility maternal mortality indirectly suggests deficiencies in quality of care, little work in the region has directly measured the problem. Anthropologists working in five West African capital cities have documented shocking departures from quality obstetrical care. These ranged from gross neglect to physical violence against patients . AdditioRecently, there has been substantial interest in employing maternal death review (MDR) to improve obstetrical quality of care and reduce maternal mortality -10. MDR by improving quality of care, through better obstetrical practice. For the trial, we randomly allocated the ALARM intervention to referral hospitals in the region. To avoid contamination between health professionals, and because the intervention targets teams of professionals, a cluster design was deemed more appropriate than an individual randomized controlled trial.In September, 2007, the QUARITE cluster randomized trial was initiated to assess the effect of a multifaceted intervention, the ALARM International Program, on reducing facility maternal mortality . The halThe QUARITE trial began in September 2007 and was completed in December 2011. It consisted of a one-year pre-intervention period, a two-year intervention period, and a final year post-intervention period. A detailed description of the trial design and objectives has been described elsewhere . The triThe QUARITE trial took place in referral hospitals in Mali in Senegal. Referral hospitals are evacuation sites for complicated deliveries and in theory, should have sufficient infrastructure to provide comprehensive EmOC including caesarean section and blood transfusion . In bothAs this is a cluster design, eligibility criteria apply to both the hospital and patient levels of analysis. To be eligible for the QUARITE trial, the referral hospital needed to have comprehensive EmOC capacity  and at least 800 births per year. We focused on high volume hospitals in order to have enough maternal deaths  and because the health system is organized in a pyramidal fashion. That is, lower order health centres refer cases of complication up to higher level, larger facilities that, in theory, should be able to able to treat life-threatening obstetrical complications. QUARITE’s eligibility criteria have been described in detail elsewhere, and a total of 46 of the 49 eligible referral hospitals were included in the trial .We sampled the charts of two populations of women. Providers were not aware of which charts would be sampled until the day of data collection which was after all care had been provided to the obstetrical patient. The first sample, called the consecutive sample, included the last 20–25 births having occurred at a hospital as of September 30, 2010. It was a random snap shot of the population of women attending a given hospital on an arbitrary date and included some of the women in the second sample. The second sample, called the complicated sample, included only women with severe pre-eclampsia and/or eclampsia and post-partum haemorrhage (defined below). We only looked at pre-eclampsia and haemorrhage, because these were the two most frequent and lethal complications of women treated in trial hospitals, according to data collected during the baseline year of QUARITE. Women in the complicated sample were selected within a three-month sampling frame: July-September, 2010 in Senegal and September-November, 2010 in Mali. We aimed for 10–15 complications per hospital, starting in September and working backwards in Senegal  until the desired sample size was achieved or the sampling frame was exhausted. The dates of the sampling frames differ slightly because of when data was collected and the availability of records for audit .To locate cases of severe (pre-) eclampsia/ eclampsia and post-partum haemorrhage, we used three data sources: the delivery register, the patient medical chart, and the QUARITE trial data sheet . Prior to QUARITE trial commencement, staff at both intervention and control sites received training on the diagnosis of obstetrical complications according to international guidelines. This was done to ensure consistency in the definitions of complication used across sites.severe pre-eclampsia: mention of severity, convulsions, albumin laboratory scores of +++, and vomiting. For post-partum haemorrhage, in addition to an express diagnosis of the complication, we looked in depth at patients with antenatal haemorrhage  continuing into the post-partum period. We looked for indicators of postpartum haemorrhage such as: an important postpartum drop in blood pressure and/or post-partum blood pressure below 90/50 [For this sub-study, we assessed the care of patients with severe pre-eclampsia/ eclampsia and post-partum haemorrhage. To locate cases of severe pre-eclampsia or eclampsia, we searched the three forms of medical records described above. We looked for a diagnosis of pre-eclampsia, toxaemia, or eclampsia, as recorded by a healthcare professional . Since many pre-eclampsia patients had mild to moderate pre-eclampsia, we also looked for the following indications of ow 90/50 ; a postpow 90/50 , retaineow 90/50 . The casThe ALARM International Program was developed by the Society of Obstetricians and Gynaecologists of Canada. It combines clinically-oriented and evidence-based outreach visits with facility-based MDR. Specifically, the intervention included the following activities: 1) A six-day workshop to train and certify opinion leaders (one physician and midwife from each intervention site) in EmOC best practices, audit techniques, and sexual and reproductive rights; 2) the creation of a multidisciplinary audit committee  at each site; 3) commencement of a once-monthly audit cycle according to WHO guidelines ; 4) the results forthcoming). Secondary objectives of the trial included: 1) reducing stillbirth and neonatal mortality; 2) reducing severe maternal morbidity; 3) improving quality of care and 4) increasing health professional job satisfaction [The primary objective of the trial was to reduce facility maternal mortality . Secondary outcomes for this study were: scores for different dimensions of quality, quality scores for severe pre-eclampsia and eclampsia, and quality scores for postpartum haemorrhage.Quality of care was assessed with a form of chart abstraction known as criterion-based clinical audit (CBCA) . When usThe CBCA that we used has been described extensively elsewhere  and is bThe CBCA contains 26 unweighted criteria that measure 5 dimensions of care: patient history, clinical examination, laboratory examinations, labour management (partograph), delivery care and postpartum monitoring. These sections apply to all women sampled. A quality of care score was attributed to each woman based on the number criteria met. For example, if 20 criteria were attained during the audit of a given medical record, then the score for that record would be 20/26 or 76.9%. In accordance with previous studies [We used Hayes and Moulton’s (2009) formula for cluster randomized trials with a quantitative endpoint . Our endCentres were included on the basis of formal, informed consent on the part of the hospital director and the person in charge of maternity services. After a one-year pre-intervention data collection phase, each hospital was randomly assigned (August 2008) to either an intervention group, in which ALARM was implemented, or a control group.The participating hospitals were stratified into six strata corresponding to the combination of two countries  and three hospital types: hospitals in the capital, regional hospitals, and district hospitals outside the capital. We attempted to ensure optimal balance between the hospitals assigned to the intervention and the control groups in terms of their number and size (number of deliveries per year). Therefore, within each stratum, we first ranked the hospitals with respect to size, and then used blocked randomization, with each block of size two, containing two hospitals with adjacent ranks, i.e., of similar size. All participating hospitals were randomized simultaneously, after their list was provided, which eliminated any risk of allocation bias.Patients attending the study facilities were blinded to group assignment. For obvious reasons, those administering the intervention and health professionals implementing the intervention were not blinded.All statistical analyses were conducted in SPSS 17.0. We used t-tests to assess if there were statistically significant differences in CBCA scores according to arms of the QUARITE trial (ALARM versus control). We used mixed models, which take into account clustering by hospital, to examine predictors of CBCA score. Because of the limited number of clusters in most CRTs, it is not unusual to find imbalances in covariates between trial arms . Thus, aFor each site, we collected information on material and human resources allowing us to score the structural capacity of each centre. This score, called the Complexity Index Score, was derived from an instrument used in the WHO Global Survey on Maternal and Perinatal Health . It is cBecause the Malian and Senegalese health systems are organized differently, we also assessed for effect modification by country. Similarly, we assessed for effect modification by Complexity Index Score, hypothesizing that ALARM would be most effective at sites with greater resources.for this study on quality of care, we only analyzed 32 of the 46 study hospitals.Figure This is an intention to treat analysis. In each trial arm, there were 16 clusters . We analyzed data from two samples: the consecutive (N = 658) and complicated samples (N = 209). Figure Table Observations in the consecutive sample were well-distributed according to the intervention (48.5% at ALARM sites and 51.5% at control sites) and by location . In the complicated sample, there were slightly more observations in the control arm than the ALARM arm , the mean CBCA score was 66.30 (SD 13.46) and the mean Complexity index score was 68.27 (SD 10.21). For the complicated sample (N = 209 patients), the respective means were 69.11 (SD 12.42) and 68.54 (SD 9.36). For 27 (4.1%) women we could not calculate a CBCA score due to missing information in the consecutive sample. A slightly higher proportion (6.7%) could not be calculated for the complicated sample.In both samples, unadjusted analyses indicate that there were significantly greater mean CBCA scores in women treated at ALARM hospitals compared to women treated at control hospitals. This difference was mostly attributable to greater patient scores for clinical examination at admission and post-partum monitoring scores Table .We also looked at differences in the proportions of women receiving good quality care (greater than 70% criteria attainment) versus moderate to poor care (70% or fewer criteria attained). In the consecutive sample, significantly more women at ALARM sites received good quality care compared to women at control sites . In the complicated sample, a similar difference between ALARM and control sites was observed .For the subsections of the CBCA questionnaire pertaining to the direct complications of severe pre-eclampsia/ eclampsia and post-partum haemorrhage, there were no significant differences in total scores between arms of the QUARITE trial. Overall, criteria attainment for these sections of the questionnaire was very low Tables  and 5. HWe used the larger consecutive sample to assess predictors of CBCA score. Table Figures In Mali, a ten-point increase above the average Complexity index score has a slightly more important effect on CBCA score than the ALARM intervention (6% improvement for Complexity index score versus 5% for ALARM). In Senegal, however, a 10-point increase above the average Complexity index score has a less important effect on CBCA score than ALARM (1% improvement for Complexity index score versus 5% for ALARM).This was a sub-study of data from the QUARITE cluster randomized controlled trial. In this study, we attempted to parse out potential mechanisms of effect for the ALARM intervention. By doing so, we hoped to better understand how the intervention worked and what aspects of the intervention can be modified for future quality improvement efforts. Analyses from this study indicate that women treated at hospitals with the ALARM intervention have modestly greater mean CBCA scores. This suggests that the intervention is having the desired effect; it is associated with better obstetrical quality of care. Further, 15% more women are receiving good care (70% or greater criterion attainment) at ALARM hospitals when compared to control hospitals.Results indicate that the intervention mostly improves clinical examination at admission and postpartum monitoring. There is also a trend suggesting that ALARM may be associated with increased use of magnesium sulphate in women with severe pre-eclampsia/ eclampsia. Finally, there was significantly greater administration of oxytocin for women with post-partum haemorrhage at ALARM sites and a greater proportion of women with post-partum haemorrhage at these sites were recorded as having had the placenta removed manually.This study looks at the potential mechanisms of a complex quality improvement intervention and points to areas for future focus, specifically laboratory tests and birth and delivery care. It goes beyond simply assessing efficacy. By attempting to better elucidate what worked and why, we “look more closely inside the ‘black box’ of an intervention” ( p792), iThere are several strengths to this study. To our knowledge, this is the first validated CBCA to be used in a resource-limited setting to measure obstetrical care . This waWe sub-sampled women with direct complications that are both prevalent and have high case-fatality rates in West African hospital settings . We wereThere are limitations to this study. By not sampling all 46 hospitals in the QUARITE trial, we may have broke-down the initial randomization. For this reason, we considered potential confounders at both the hospital and patient levels. Given that hospitals in our sample were well-balanced by trial arm and by level  and that patient variables, such as age and parity, were not confounders, suggests that we did not introduce bias with the smaller sample. However, the proportion of district-level hospitals was greater in those sites not sampled Table  and resuAn additional limitation is related to the sample size for both of the direct obstetrical complications sampled. It is possible that we would have observed a significant result for the administration of magnesium sulphate at ALARM sites had we had a larger sample of clusters in both trial arms. There was very large variability at both the patient and hospital levels for the treatment of these complications. This reduced precision around the effect estimate and thus, our ability to detect a significant result. A larger sample would have allowed us to better interpret the laboratory results, as very few women received recommended laboratory tests for both complications. It should be noted that no patients in Mali were recorded as having tests of bleeding time or coagulation rate. Estimators for these criteria are thus very unstable.Finally, the greatest weakness of CBCA is that it is dependent on what is recorded. In applying this tool, one assumes that what is recorded in the medical chart has actually been done and what has not been recorded, has not been done. This assumption may be flawed. For example, in Senegal, the name and qualification of the person who assisted the birth was recorded about a quarter of the time and in 80% of these cases, information was only recorded when the woman was seen by a medical doctor. Incomplete recording practices in Senegal may partially explain why CBCA scores were significantly lower there, compared to Mali. Nonetheless, charting is itself an indicator of quality of care. Good charting is necessary because the medical team following a woman may change over the course of a day and important decisions, such as when to conduct a caesarean section, are best made with the aid of tools such as the partograph .The ALARM intervention appears to improve certain aspects of the care process , but other aspects  need further programmatic targeting. There is evidence suggesting that the program improves the care of women with post-partum haemorrhage, but that it is less effective for cases of severe pre-eclampsia/ eclampsia. Resource limitations, particularly in Mali, may be slightly more influential in determining the care of these patients  than interventions targeting medical practice. Overall, these analyses demonstrate that future efforts in the region need to continue to target medical practice, but that a greater effect can be expected if resource availability is also improved.CBCA: Criterion based clinical audit; MDR: Maternal death review; EmOC: Emergency obstetrical care.The authors declare that they have no competing interests.CP, AD, and MVZ were involved in the design, analysis, and writing of this manuscript. CP, MT, and AD were involved in data instrument development. CP and MT were involved in data collection. All authors revised and approved the final manuscript. All authors had full access to all of the data  in the study and can take responsibility for the integrity of the data and the accuracy of the data analysis.QUARITE trial research groupSteering Committee: Alexandre Dumont, Pierre Fournier, Michal Abrahamowicz, William Fraser, François Beaudoin, Slim Haddad.Data management: Anna Koné, Drissa Sia, Papa Dambé.Data Security and Monitoring Committee: José Villar (Chair), Christiane Welffens-Ekkra, Allan Donner.Participating institutions and Staff: Society of Obstetricians and Gynaecologists of Canada : André Lalonde, François Beaudouin, Astrid Buccio, Jean-Richard Dortonne, François Couturier, Gilles Perrault, Pierre Levesques; CRCHUM : Pierre Fournier, Slim Haddad, Anna Kone, Drissa Sia, Caroline Tourigny; Institut de recherche pour le développement : Alexandre Dumont, Mamadou Yatoudème Ndiaye, Papa Dambé; Centre de Recherche du CHU Sainte-Justine : Sylvie Cossette, Carole Gariépy, François Beaudoin, William D. Fraser; CAREF : Mouhamadou Gueye, Mamadou Kani Konate; HYGEA : Idrissa Diop, Amadou Sow. Participating hospitals in Senegal: Centre Hospitalier Universitaire Abass Ndao: Prosper Bamboky, Keita Soma Diallo; Hôpital Principal: Pierre Dionne, Lô Asta; Centre Hospitalier Régional El Amadou Sahir Louga: Voulimata Fall, Maty Diop Guèye, Fatoumata Dedhiou; Centre Hospitalier Régional de Tambacounda: Maréme Soda Samba, Fatou Cissé Seck; Centre de Santé de Référence de Kédougou: Sène Doudou, Aïssatou Samba; Centre de Santé de Référence de Bakel: Yaya Baldé, Astou Guèye Thiombane; Centre de Santé de Référence de Goudiry: Julien Manga, Aïssata Sy Ndiaye, Absa Sene; Centre de Santé de Référence de Koungel: Mamadou Sarr, Diahaby Aminata Sylla; Centre Hospitalier Régional de Thiès: Fatou Rachel Sarr, Mbaye Khady Sarr; Centre Hospitalier Régional de Diourbel: Malick Gueye, Mbaye Binetou Diatta; Centre Hospitalier Matlaboul Fawzeïni: Mouhamadou M Seck, Niane Coumba Sarr Guéye; Centre Hospitalier Régional de Saint-Louis: Lamine Diouf, Oumou Kalsoum Fall; Centre de Santé de Référence de Richard Toll: El Hadji Lamine Dieye, Fall Ndèye Niang; Centre Hospitalier Départemental de Ourossogui: Charles Fall, Mame Laina Diattara, Astou Traoré; Centre Hospitalier Départemental de NDioum : Sidy Dieye, Ousmane Thiam, Kadiata Seck, Coumba Mbow, Aminata Dime; Centre Hospitalier Régional de Ziguinchor: Guy Boukar Faye, Badiane Aida Gaye; Centre de Santé de Référence de Sédhiou: Kalidou Konté, Fatou Ndoye; Centre Hospitalier Régional de Kolda: Aïdara Seynabou Sylla, Jacqueline F Ngom; Hôpital Général de Grand YOFF: Mariéme Fall, Aïssatou Diouf; Centre Hospitalier Régional El Hadji Ibrahima Niass: Dembo Girassy, Maye Seck Ly; Centre de Santé de Référence Nabil Choucaire: Seynabou Beye, Djipméra Fatou Ndiaye; Centre de Santé de Référence Youssou Mbargane: Dieynaba Ndao Ndiaye, Khady Fall; Centre de Santé de Référence Ndamatou Touba: Tacko Seck Leye ; Hôpital de Pikine: Codou Séne, Amsatou Cisse. Participating hospitals in Mali: Centre de Santé de Référence de Fana: Salif Sidibé, Aïssata Daba Traoré; Centre de Santé de Référence de Dioila: Broulaye Diarra, Korotoumou Doumbia; Centre de Santé de Référence de Markala: Bintou Coulibaly, Bintou Cissé; Hôpital Régional de Kayes: Mahamadou Diassana, Drissa Konaté, Dienfra Diarra; Centre de Santé de Référence de Niono: Moussa Modibo Diarra, Soundiè Fané; Hôpital Régional de Ségou: Haka Kokain, Souma Boiaré; Centre de Santé de Référence de San: Aliou Bagayogo, Diamilatou Diallo; Hôpital régional de Mopti: Famakan Kané, Pierre Coulibaly, Fatoumata Tolo, Fatoumata Dolo; Centre de Santé de Référence de Bougouni: Daouda Goïta, Bintou Sidibé; Centre de Santé de Référence de Koutiala: Moustapha Coulibaly, Sadio Tounkara, Mariam Beledogo; Hôpital régional de Sikasso: Mala Sylla, Cécile Dembélé, Rokia Touré; Centre de Santé de Référence de Kadiolo: Emilien Diarra, Ramata Fofana; Centre de Santé de Référence de Yanfolila: Aliou Coulibaly, Kadiatou Samaké; Centre de Santé de Référence Commune I de Bamako: Modibo Soumaré, Haoua Ba, Salimata Coulibaly; Centre de Santé de Référence Commune II de Bamako: Lassana Sissoko, Penda Fané, Hamsa Maïga; Centre de Santé de Référence Commune IV de Bamako: Moustapha Touré, Fanta Koné; Centre de Santé de Référence Commune V de Bamako: Oumar Soumana Traoré, Assitan Dembélé; Centre de Santé de Référence Commune VI de Bamako: Aminata Cissé, Feue Aminata Yattara, Katahit Zeneba; Centre Hospitalier Universitaire Point G de Bamako: Maïga Bouraïma,Kadiatou Traoré; Centre de Santé de Référence de Koulikoro: Hamadou Coulibaly, Oumou Konaté; Centre de Santé de Référence de Nioro du Sahel: Youssouf Coulibaly, Maïmouna Kassibo, Binta Kontao; Centre de Santé de Référence de Macina: Moussa Kanté, Diakité Diallo, Aminata Koné.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2393/13/24/prepub


Bile stones represent a highly prevalent condition and abnormalities of the biliary tree predispose to stone recurrence due to development of biliary stasis. In our study, we assessed the importance of an altered bile duct course for stone formation.1,307 patients with choledocholithiasis in the absence of any associated hepatobiliary disease who underwent endoscopic retrograde cholangiopancreatography (ERCP) between 2002 and 2009 were analysed. The angle enclosed between the horizontal portion of the common bile duct (CBD) and the horizontal plane was measured (angle α). Oblique common bile duct (OCBD) was defined as a CBD with angle α<45°.103 patients (7.9%) were found to harbour OCBD and these were compared to 104 randomly selected control subjects. Compared to controls, OCBD patients were (i) significantly older ; (ii) more frequently underwent a cholecystectomy (p = 0.02) and biliary surgery (p = 0.003) prior to the diagnosis and (iii) more often developed chronic pancreatitis (p = 0.04) as well as biliary fistulae (p = 0.03). Prior to and after ERCP, OCBD subjects displayed significantly elevated cholestatic parameters and angle α negatively correlated with common bile duct diameter . OCBD subjects more often required multiple back-to-back ERCP sessions to remove bile stones (p = 0.005) as well as more ERCPs later on due to recurrent stone formation (p<0.05).OCBD defines a novel variant of the biliary tree, which is associated with chronic cholestasis, hampers an efficient stone removal and predisposes to recurrence of bile duct stones. Gallstones represent a highly prevalent condition in the Western world, affecting up to 20% of the population Endoscopic retrograde cholangiopancreatography (ERCP) is the treatment of choice for symptomatic CBD stones In the present study, we describe a new entity of the biliary tree, which was termed the oblique common bile duct (OCBD) and is characterised by a horizontal common bile duct course. OCBD was found to be associated with chronic cholestasis, hampers an efficient stone removal and predisposes to recurrence of bile duct stones.The study was approved by the Human Subject Committee of Ulm University, which allowed the retrospective analysis of the data and permitted us to approach the selected patients to obtain further data. An informed written consent was obtained from all subjects who were part of the control and study groups and from whom additional clinical data have been obtained.In this retrospective study, we analysed all patients who underwent endoscopic retrograde cholangiopancreatography (ERCP) in the Department of Internal Medicine I at University Medical Centre Ulm between January 2002 and December 2009 . All subjects with choledocholithiasis with the absence of any associated hepatobiliary disease such as primary sclerosing cholangitis, bile duct stenosis, hepatobiliary malignancies or diverticula were included .Maximum CBD diameter and other parameters of the “oblique” CBDs were measured manually on the ERCP radiograph and corrected according to the scope diameter. In patients who underwent more ERCPs during the course of the study, the first ERCP was used to determine CBD parameters and to find out whether patients harboured an “oblique” CBD. All data were expressed as means±SD or as median values and quartiles. Statistical analysis was performed with a two-tailed Chi-square, Fisher’s exact, Student’s Between 2001 and 2009, a total of 2,459 patients underwent an ERCP at the University Medical Centre Ulm. Among them, 1,307 met the study inclusion criteria and 103 (7.9% of the study subjects) presented with an “oblique” CBD OCBD; . Of noteTo determine the events leading to OCBD, past medical history was analysed in OCBD patients and a control group consisting of 104 subjects randomly selected from the remaining study population . Prior tTo analyse how the presence of OCBD affects the development and resolution of biliary disease, we analysed the lab values before and after the ERCP procedure . SurprisIn order to analyse to what extent the bile duct morphology affects the course of biliary disease, OCBD subjects were divided into severe (angle α≤15°) and moderate subgroups  of OCBD and to find out, what additional hits may contribute to development of clinically apparent condition in these subjects.We also analysed the consequences of OCBD. Our data indicate that it is associated with (i) development of chronic cholestasis and (ii) chronic pancreatitis; (iii) more severe acute cholangitis; (iv) more difficult gallstone removal and with (v) recurrent gallstone formation. The most likely explanation of these findings is that OCBD subjects experience an impaired bile flow, which represents an established risk factor not only for gallstone development but also for the development of chronic pancreatitis A reduced bile flow might be due to a compromised motility of the biliary tract or due to a biliary obstruction. Given that CBD does not significantly contribute to biliary motility, further studies should analyse whether OCBD associates with gallbladder and/or sphincter of Oddi dysfunction, which represent the major reasons for biliary dysmotility The observation that OCBD subjects require multiple ERCPs is of particular clinical relevance. First of all, OCBD subjects need more ERCPs to completely remove all stones, a finding which is likely due to a challenging biliary morphology. In addition to that, “late ERCPs” were more frequent in the OCBD subjects and their frequency was significantly above the data reported in the literature In summary, OCBD defines a new entity of bile duct abnormality, which is associated with chronic cholestasis, hampers an efficient stone removal and predisposes to recurrence of bile duct stones. Further studies are needed to clarify the pathogenesis of this syndrome and possible treatment strategies. To accurately determine the angle α, these trials should include a magnetic resonance imaging of the bile ducts with a precise three-dimensional reconstruction. Thereby, these studies will enable us to define a cut-off value for angle α which is associated with significant clinical outcomes.Figure S1Examples of the radiographs depicting the oblique common bile duct. Of note, panel (A) depicts a patient in a non-standardised half left position which was used to evaluate the oblique choledochus for further stones. Panel (B) represents the X-ray of the same patient taken in a standardised face-down position, thereby confirming the presence of an oblique bile duct syndrome.(TIF)Click here for additional data file.Table S1Study cohort. CBD, common bile duct; SD, standard deviation; 1p<0.00001(DOCX)Click here for additional data file.Table S2Angle α in consecutive endoscopic retrograde cholangiopancreatographies. Angle α was defined as the minimal angle between the horizontal plane and the CBD. ERCP, endoscopic retrograde cholangiopancreatography.(DOCX)Click here for additional data file.Table S3Lab values in subjects who underwent cholecystectomy prior to endoscopic retrograde cholangiopancreatography. Median values are shown. Calculation of statistical significance is based on a two-tailed Mann-U-Whitney test. AP, alkaline phosphatase; GGT, gamma glutamyl transferase; Bili, bilirubin; AST, aspartate transaminase; ALT, alanine transaminase; AP, alkaline phosphatase; d, day; wk, week; mo, month; ERCP, endoscopic retrograde cholangiopancreatography; OB, “oblique” bile duct; Co, Controls. 1p = 0.03; 2p = 0.006; 3p = 0.0001; 4p = 0.09; 5p = 0.03; 6p = 0.01; 7p = 0.06; 8p = 0.05; 9p = 0.02; 10p = 0.007; 11p = 0.003; 12p = 0.02(DOCX)Click here for additional data file.Table S4Impact of OCBD morphology on cholestatic liver injury during ERCP. ALT, alanine transaminase; AP, alkaline phosphatase; AST, aspartate transaminase; Bili, bilirubin; CBD, common bile duct; OCBD, “oblique” common bile duct; GGT, gamma glutamyl transferase; SD, standard deviation 1p<0.0001(DOCX)Click here for additional data file.